WO2019214604A1

WO2019214604A1 - Crispr/cas effector protein and system

Info

Publication number: WO2019214604A1
Application number: PCT/CN2019/085826
Authority: WO
Inventors: 赖锦盛; 周英思; 朱金洁; 张湘博; 赵海铭; 宋伟彬
Original assignee: 中国农业大学
Priority date: 2018-05-07
Filing date: 2019-05-07
Publication date: 2019-11-14
Also published as: CN112105728B; CN112105728A

Abstract

The present invention relates to the field of nucleic acid editing, and in particular, to the technical field of clustered regularly interspaced short palindromic repeat (CRISPR). A Cas effector protein, comprising a fusion protein of said protein and nucleic acid molecules encoding same. Provided are a complex and a compound for nucleic acid editing (e.g. gene or genome editing), comprising the Cas effector protein or the fusion protein, or the nucleic acid molecules encoding same. Also provided is a method for nucleic acid editing (e.g. gene or genome editing), which uses the Cas effector protein or the fusion protein.

Description

CRISPR/Cas效应蛋白及***CRISPR/Cas Effect Proteins and Systems

技术领域Technical field

本发明涉及核酸编辑领域，特别是规律成簇的间隔短回文重复(CRISPR)技术领域。具体而言，本发明涉及Cas效应蛋白，包含此类蛋白的融合蛋白，以及编码它们的核酸分子。本发明还涉及用于核酸编辑(例如，基因或基因组编辑)的复合物和组合物，其包含本发明的蛋白或融合蛋白，或编码它们的核酸分子。本发明还涉及用于核酸编辑(例如，基因或基因组编辑)的方法，其使用包含本发明的蛋白或融合蛋白。The invention relates to the field of nucleic acid editing, in particular to the field of regularly clustered short palindrome repetition (CRISPR) technology. In particular, the invention relates to Cas effector proteins, fusion proteins comprising such proteins, and nucleic acid molecules encoding the same. The invention also relates to complexes and compositions for nucleic acid editing (e.g., gene or genome editing) comprising a protein or fusion protein of the invention, or a nucleic acid molecule encoding the same. The invention also relates to methods for nucleic acid editing (eg, gene or genome editing) using a protein or fusion protein comprising the invention.

背景技术Background technique

CRISPR/Cas技术是一种被广泛使用的基因编辑技术，它通过RNA引导对基因组上的靶序列进行特异性结合并切割DNA产生双链断裂，利用生物非同源末端连接或同源重组进行定点基因编辑。CRISPR/Cas technology is a widely used gene editing technology that specifically binds to target sequences on the genome by RNA guidance and cleaves DNA to generate double-strand breaks, using biological non-homologous end joining or homologous recombination. Gene editing.

CRISPR/Cas9***是最常用的II型CRISPR***，它识别3’-NGG的PAM基序，对靶标序列进行平末端切割。CRISPR/Cas Type V***是一类近两年新发现的的CRISPR***，它具有5’-TTN的基序，对靶标序列进行粘性末端切割，例如Cpf1,C2c1,CasX,CasY。然而目前存在的不同的CRISPR/Cas各有不同的优点和缺陷。例如Cas9,C2c1和CasX均需要两条RNA进行向导RNA，而Cpf1只需要一条向导RNA而且可以用来进行多重基因编辑。CasX具有980个氨基酸的大小，而常见的Cas9，C2c1,CasY和Cpf1通常大小在1300个氨基酸左右。此外，Cas9，Cpf1，CasX，CasY的PAM序列都比较复杂多样，而C2c1识别严谨的5’-TTN,因此它的靶标位点比其他***容易被预测从而降低了潜在的脱靶效应。The CRISPR/Cas9 system is the most commonly used type II CRISPR system, which recognizes the PAM motif of 3'-NGG and blunt-ends the target sequence. The CRISPR/Cas Type V system is a newly discovered CRISPR system in the past two years. It has a 5'-TTN motif for viscous end cleavage of target sequences, such as Cpf1, C2c1, CasX, CasY. However, the different CRISPR/Cas currently exist have different advantages and disadvantages. For example, Cas9, C2c1 and CasX require two RNAs for guide RNA, while Cpf1 requires only one guide RNA and can be used for multiple gene editing. CasX is 980 amino acids in size, while common Cas9, C2c1, CasY and Cpf1 are usually around 1300 amino acids. In addition, the PAM sequences of Cas9, Cpf1, CasX, and CasY are more complex and diverse, while C2c1 recognizes the rigorous 5'-TTN, so its target site is easier to predict than other systems and thus reduces potential off-target effects.

总之，鉴于目前可获得的CRISPR/Cas***都受限于一些缺陷，开发一种更稳健的、具有多方面良好性能的新型CRISPR/Cas***对生物技术的发展具有重要意义。In summary, in view of the fact that the currently available CRISPR/Cas system is limited by some defects, the development of a more robust and multi-faceted new CRISPR/Cas system is of great significance to the development of biotechnology.

发明内容Summary of the invention

本申请的发明人经过大量实验和反复摸索，出人意料地发现了一种新型RNA指导的核酸内切酶。基于这一发现，本发明人开发了新的CRISPR/Cas***以及基于该***的基因编辑方法。The inventors of the present application have unexpectedly discovered a novel RNA-directed endonuclease after extensive experimentation and repeated exploration. Based on this finding, the inventors developed a new CRISPR/Cas system and a gene editing method based on the system.

Cas效应蛋白Cas effector protein

因此，在第一方面，本发明提供了一种蛋白，其具有SEQ ID NOs：1-18任一项所示的氨基酸序列或其直系同源物、同源物、变体或功能性片段；其中，所述直系同源物、同源物、变体或功能性片段基本保留了其所源自的序列的生物学功能。Accordingly, in a first aspect, the invention provides a protein having the amino acid sequence set forth in any one of SEQ ID NOs: 1-18, or an ortholog, homolog, variant or functional fragment thereof; Wherein the ortholog, homologue, variant or functional fragment substantially retains the biological function of the sequence from which it is derived.

在某些实施方案中，所述蛋白具有Cas效应蛋白活性。在某些实施方案中，所述蛋白是CRISPR/Cas***中的效应蛋白。In certain embodiments, the protein has Cas effector activity. In certain embodiments, the protein is an effector protein in the CRISPR/Cas system.

在本发明中，上述序列的生物学功能是指Cas效应蛋白活性，包括但不限于，与导向RNA结合的活性、核酸内切酶活性、在导向RNA引导下与靶序列特定位点结合并切割的活性。In the present invention, the biological function of the above sequence refers to Cas effector activity, including but not limited to, binding to a targeting RNA, endonuclease activity, binding to a specific site of a target sequence under the guidance of a targeting RNA, and cleavage Activity.

在某些实施方案中，所述直系同源物、同源物、变体与其所源自的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性，并且基本保留了其所源自的序列的Cas效应蛋白活性(例如，与导向RNA结合的活性、核酸内切酶活性、在导向RNA引导下与靶序列特定位点结合并切割的活性)。In certain embodiments, the ortholog, homolog, variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least compared to the sequence from which it is derived. 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, and substantially retains the Cas effector activity of the sequence from which it is derived (eg, Activity that binds to the targeting RNA, endonuclease activity, activity that binds to a specific site of the target sequence and cleaves under the guidance of a targeting RNA).

在某些实施方案中，所述直系同源物、同源物、变体与SEQ ID NOs：1-18任一项所示的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性，并且基本保留了其所源自的序列的Cas效应蛋白活性(例如，与导向RNA结合的活性、核酸内切酶活性、在导向RNA引导下与靶序列特定位点结合并切割的活性)。In certain embodiments, the ortholog, homolog, variant has at least 80%, at least 85%, at least 90%, compared to the sequence set forth in any one of SEQ ID NOs: 1-18, At least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity and substantially retains the origin of the sequence Sequence of Cas effector activity (eg, activity binding to targeting RNA, endonuclease activity, activity that binds to a specific site of the target sequence and cleaves under the guidance of a targeting RNA).

在某些实施方案中，所述蛋白来自选自下列的物种：Sulfuricurvum sp、Omnitrophica WOR_2、Smithella sp和Agrobacterium sp。在某些实施方案中，所述蛋白为包含于选自下列的物种的CRISPR基因座(CRISPR locus)中的Cas效应蛋白：Sulfuricurvum sp、Omnitrophica WOR_2、Smithella sp和Agrobacterium sp。在此类实施方案中，所述蛋白具有SEQ ID NOs：5-18任一项所示的氨基酸序列或其直系同源物、同源物、变体或功能性片段；其中，所述直系同源物、同源物、变体或功能性片段基本保留了其所源自的序列的生物学功能。在某些实施方案中，所述直系同源物、同源物、变体与SEQ ID NOs：5-18任一项所示的氨基酸序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性，并且基本保留了其所源自的序列的Cas效应蛋白活性(例如，与导向RNA结合的活性、核酸内切酶活性、在导向RNA引导下与靶序列特定位点结合并切割的活性)。In certain embodiments, the protein is from a species selected from the group consisting of Sulfuricurvum sp, Omnitrophica WOR_2, Smithella sp, and Agrobacterium sp. And X. In such embodiments, the protein has the amino acid sequence set forth in any one of SEQ ID NOs: 5-18, or an ortholog, homolog, variant or functional fragment thereof; wherein the homologous The source, homologue, variant or functional fragment substantially retains the biological function of the sequence from which it is derived. In certain embodiments, the ortholog, homolog, variant has at least 80%, at least 85%, at least 90% compared to the amino acid sequence set forth in any one of SEQ ID NOs: 5-18 At least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, and substantially retains its origin The Cas effector activity of the sequence (eg, activity binding to the targeting RNA, endonuclease activity, activity that binds to a specific site of the target sequence and cleaves under the guidance of a targeting RNA).

在某些实施方案中，本发明的蛋白包含选自下列的序列，或由选自下列的序列组成：In certain embodiments, the protein of the invention comprises a sequence selected from the group consisting of: or consists of a sequence selected from the group consisting of:

(i)SEQ ID NOs：1-18任一项所示的序列；(i) the sequence set forth in any one of SEQ ID NOs: 1-18;

(ii)与SEQ ID NOs：1-18任一项所示的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)的序列；或(ii) a substitution, deletion or addition of one or more amino acids compared to the sequence set forth in any one of SEQ ID NOs: 1-18 (

eg

1, 2, 3, 4, 5, 6 a sequence of 7, 7 or 9 amino acid substitutions, deletions or additions; or

(iii)与SEQ ID NOs：1-18任一项所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性的序列。(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% of the sequence set forth in any one of SEQ ID NOs: 1-18 a sequence of at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

(i)SEQ ID NO：1或2所示的序列；(i) the sequence shown as SEQ ID NO: 1 or 2;

(ii)与SEQ ID NO：1或2所示的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)的序列；或(ii) a substitution, deletion or addition of one or more amino acids compared to the sequence set forth in SEQ ID NO: 1 or 2 (

eg

1, 2, 3, 4, 5, 6, 7 a sequence of 8, 8 or 10 amino acid substitutions, deletions or additions; or

(iii)与SEQ ID NO：1或2所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性的序列。(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96 with the sequence set forth in SEQ ID NO: 1 or 2. %, at least 97%, at least 98%, or at least 99% of sequences of sequence identity.

在某些实施方案中，本发明的蛋白具有SEQ ID NO：1或2所示的氨基酸序列。In certain embodiments, the protein of the invention has the amino acid sequence set forth in SEQ ID NO: 1 or 2.

(i)SEQ ID NO：3或4所示的序列；(i) the sequence of SEQ ID NO: 3 or 4;

(ii)与SEQ ID NO：3或4所示的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)的序列；或(ii) a substitution, deletion or addition of one or more amino acids compared to the sequence set forth in SEQ ID NO: 3 or 4 (

eg

(iii)与SEQ ID NO：3或4所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性的序列。(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96 with the sequence set forth in SEQ ID NO: 3 or 4. %, at least 97%, at least 98%, or at least 99% of sequences of sequence identity.

在某些实施方案中，本发明的蛋白具有SEQ ID NO：3或4所示的氨基酸序列。In certain embodiments, the protein of the invention has the amino acid sequence set forth in SEQ ID NO: 3 or 4.

(i)SEQ ID NO：5或6所示的序列；(i) the sequence of SEQ ID NO: 5 or 6;

(ii)与SEQ ID NO：5或6所示的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)的序列；或(ii) a substitution, deletion or addition of one or more amino acids compared to the sequence set forth in SEQ ID NO: 5 or 6 (

eg

(iii)与SEQ ID NO：5或6所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性的序列。(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96 with the sequence set forth in SEQ ID NO: 5 or 6. %, at least 97%, at least 98%, or at least 99% of sequences of sequence identity.

在某些实施方案中，本发明的蛋白具有SEQ ID NO：5或6所示的氨基酸序列。In certain embodiments, the protein of the invention has the amino acid sequence set forth in SEQ ID NO: 5 or 6.

(i)SEQ ID NOs：7-18任一项所示的序列；(i) the sequence set forth in any one of SEQ ID NOs: 7-18;

(ii)与SEQ ID NOs：7-18任一项所示的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)的序列；或(ii) a substitution, deletion or addition of one or more amino acids compared to the sequence set forth in any one of SEQ ID NOs: 7-18 (

eg

(iii)与SEQ ID NOs：7-18任一项所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性的序列。(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% of the sequence set forth in any one of SEQ ID NOs: 7-18 a sequence of at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

在某些实施方案中，本发明的蛋白具有SEQ ID NOs：7-18任一项所示的氨基酸序列。In certain embodiments, the protein of the invention has the amino acid sequence set forth in any one of SEQ ID NOs: 7-18.

衍生的蛋白Derived protein

本发明的蛋白可进行衍生化，例如被连接至另一个分子(例如另一个多肽或蛋白)。通常，蛋白的衍生化(例如，标记)不会不利影响该蛋白的期望活性(例如，与导向RNA结合的活性、核酸内切酶活性、在导向RNA引导下与靶序列特定位点结合并切割的活性)。因此，本发明的蛋白还意欲包括此类衍生化的形式。例如，可以将本发明的蛋白功能性连接(通过化学偶合、基因融合、非共价连接或其它方式)于一个或多个其它分子基团，例如另一个蛋白或多肽，检测试剂，药用试剂等。A protein of the invention can be derivatized, for example, linked to another molecule (e.g., another polypeptide or protein). In general, derivatization (eg, labeling) of a protein does not adversely affect the desired activity of the protein (eg, activity binding to the targeting RNA, endonuclease activity, binding to a specific site of the target sequence under the guidance of a targeting RNA, and cleavage Activity). Thus, the proteins of the invention are also intended to include such derivatized forms. For example, a protein of the invention can be functionally linked (by chemical coupling, gene fusion, non-covalent attachment or otherwise) to one or more other molecular groups, such as another protein or polypeptide, a detection reagent, a pharmaceutical reagent Wait.

特别地，可以将本发明的蛋白连接其他功能性单元。例如，可以将其与核定位信号(NLS)序列连接，以提高本发明的蛋白进入细胞核的能力。例如，可以将其与靶向部分连接，以使得本发明的蛋白具有靶向性。例如，可以将其与可检测的标记连接，以便于对本发明的蛋白进行检测。例如，可以将其与表位标签连接，以便于本发明的蛋白的表达、检测、示踪和/或纯化。In particular, the proteins of the invention may be linked to other functional units. For example, it can be ligated to a nuclear localization signal (NLS) sequence to increase the ability of the proteins of the invention to enter the nucleus. For example, it can be linked to a targeting moiety to render the protein of the invention targeted. For example, it can be linked to a detectable label to facilitate detection of the proteins of the invention. For example, it can be linked to an epitope tag to facilitate expression, detection, tracing, and/or purification of the proteins of the invention.

缀合物Conjugate

因此，在第二方面，本发明提供了一种缀合物，其包含如上所述的蛋白和修饰部分。Thus, in a second aspect, the invention provides a conjugate comprising a protein and a modified moiety as described above.

在某些实施方案中，所述修饰部分选自另外的蛋白或多肽、可检测的标记或其任意组合。In certain embodiments, the modified moiety is selected from another protein or polypeptide, a detectable label, or any combination thereof.

在某些实施方案中，所述另外的蛋白或多肽选自表位标签、报告基因序列、核定位信号(NLS)序列、靶向部分、转录激活结构域(例如，VP64)、转录抑制结构域(例如，KRAB结构域或SID结构域)、核酸酶结构域(例如，Fok1)，具有选自下列的活性的结构域：甲基化酶活性,去甲基化酶,转录激活活性,转录抑制活性,转录释放因子活性,组蛋白修饰活性,核酸酶活性,单链RNA切割活性,双链RNA切割活性,单链DNA切割活性,双链DNA切割活性和核酸结合活性；以及其任意组合。And X. (eg, a KRAB domain or a SID domain), a nuclease domain (eg, Fok1), having a domain selected from the group consisting of methylase activity, demethylase, transcriptional activation activity, transcriptional repression Activity, transcription release factor activity, histone modification activity, nuclease activity, single-strand RNA cleavage activity, double-stranded RNA cleavage activity, single-strand DNA cleavage activity, double-strand DNA cleavage activity and nucleic acid binding activity; and any combination thereof.

在某些实施方案中，本发明的缀合物包含一个或多个NLS序列，例如SV40病毒大T抗原的NLS。在某些示例性实施方案中，所述NLS序列如SEQ ID NO:73所示。在某些实施方案中，所述NLS序列位于、靠近或接近本发明的蛋白的末端(例如，N端或C端)。在某些示例性实施方案中，所述NLS序列位于、靠近或接近本发明的蛋白的C端。In certain embodiments, a conjugate of the invention comprises one or more NLS sequences, such as the NLS of the SV40 viral large T antigen. In certain exemplary embodiments, the NLS sequence is set forth in SEQ ID NO:73. In certain embodiments, the NLS sequence is at, near, or near the end of the protein of the invention (eg, the N-terminus or the C-terminus). In certain exemplary embodiments, the NLS sequence is located at, near, or near the C-terminus of the protein of the invention.

在某些实施方案中，本发明的缀合物包含表位标签(epitope tag)。这类表位标签是本领域技术人员熟知的，其实例包括但不限于His、V5、FLAG、HA、Myc、VSV-G、Trx等，并且本领域技术人员已知如何根据期望目的(例如，纯化、检测或示踪)选择合适的表位标签。In certain embodiments, the conjugates of the invention comprise an epitope tag. Such epitope tags are well known to those skilled in the art, examples of which include, but are not limited to, His, V5, FLAG, HA, Myc, VSV-G, Trx, etc., and those skilled in the art know how to achieve the desired purpose (eg, Purify, test or trace) Select the appropriate epitope tag.

在某些实施方案中，本发明的缀合物包含报告基因序列。这类报告基因是本领域技术人员熟知的，其实例包括但不限于GST、HRP、CAT、GFP、HcRed、DsRed、CFP、YFP、BFP等。In certain embodiments, a conjugate of the invention comprises a reporter gene sequence. Such reporter genes are well known to those skilled in the art, and examples include, but are not limited to, GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, BFP, and the like.

在某些实施方案中，本发明的缀合物包含能够与DNA分子或细胞内分子结合的结构域，例如麦芽糖结合蛋白(MBP)、Lex A的DNA结合结构域(DBD)、GAL4的DBD等。In certain embodiments, the conjugates of the invention comprise a domain capable of binding to a DNA molecule or an intracellular molecule, such as a maltose binding protein (MBP), a DNA binding domain of Lex A (DBD), a DBD of GAL4, and the like. .

在某些实施方案中，本发明的缀合物包含可检测的标记，例如荧光染料，例如FITC或DAPI。In certain embodiments, the conjugates of the invention comprise a detectable label, such as a fluorescent dye, such as FITC or DAPI.

在某些实施方案中，本发明的蛋白任选地通过接头与所述修饰部分偶联、缀合或融合。In certain embodiments, a protein of the invention is optionally coupled, conjugated or fused to the modified moiety by a linker.

在某些实施方案中，所述修饰部分直接连接至本发明的蛋白的N端或C端。In certain embodiments, the modified moiety is directly linked to the N-terminus or C-terminus of the protein of the invention.

在某些实施方案中，所述修饰部分通过接头连接至本发明的蛋白的N端或C端。这类接头是本领域熟知的，其实例包括但不限于包含一个或多个(例如，1个，2个，3个，4个或5个)氨基酸(如，Glu或Ser)或氨基酸衍生物(如，Ahx、β-Ala、GABA或Ava)的接头，或PEG等。In certain embodiments, the modified moiety is linked to the N-terminus or C-terminus of the protein of the invention by a linker. Such linkers are well known in the art, examples of which include, but are not limited to, one or more (eg, 1, 2, 3, 4 or 5) amino acids (eg, Glu or Ser) or amino acid derivatives. A linker (eg, Ahx, β-Ala, GABA, or Ava), or PEG, and the like.

融合蛋白Fusion protein

在第三方面，本发明提供了一种融合蛋白，其包含本发明的蛋白以及另外的蛋白或多肽。In a third aspect, the invention provides a fusion protein comprising a protein of the invention and an additional protein or polypeptide.

在某些实施方案中，本发明的融合蛋白包含一个或多个NLS序列，例如SV40病毒大T抗原的NLS。在某些实施方案中，所述NLS序列位于、靠近或接近本发明的蛋白的末端(例如，N端或C端)。在某些示例性实施方案中，所述NLS序列位于、靠近或接近本发明的蛋白的C端。In certain embodiments, a fusion protein of the invention comprises one or more NLS sequences, such as the NLS of the SV40 viral large T antigen. In certain embodiments, the NLS sequence is at, near, or near the end of the protein of the invention (eg, the N-terminus or the C-terminus). In certain exemplary embodiments, the NLS sequence is located at, near, or near the C-terminus of the protein of the invention.

在某些实施方案中，本发明的融合蛋白包含表位标签。In certain embodiments, a fusion protein of the invention comprises an epitope tag.

在某些实施方案中，本发明的融合蛋白包含报告基因序列。In certain embodiments, a fusion protein of the invention comprises a reporter gene sequence.

在某些实施方案中，本发明的融合蛋白包含能够与DNA分子或细胞内分子结合的结构域。In certain embodiments, a fusion protein of the invention comprises a domain capable of binding to a DNA molecule or an intracellular molecule.

在某些实施方案中，本发明的蛋白任选地通过接头与所述另外的蛋白或多肽融合。In certain embodiments, a protein of the invention is optionally fused to the additional protein or polypeptide via a linker.

在某些实施方案中，所述另外的蛋白或多肽直接连接至本发明的蛋白的N端或C端。In certain embodiments, the additional protein or polypeptide is directly linked to the N-terminus or C-terminus of the protein of the invention.

在某些实施方案中，所述另外的蛋白或多肽通过接头连接至本发明的蛋白的N端或C端。In certain embodiments, the additional protein or polypeptide is linked to the N-terminus or C-terminus of the protein of the invention by a linker.

在某些示例性实施方案中，本发明的融合蛋白具有选自下列的氨基酸序列：SEQ ID NOs:74-91。In certain exemplary embodiments, the fusion proteins of the invention have an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-91.

本发明的蛋白、本发明的缀合物或本发明的融合蛋白不受其产生方式的限定，例如，其可以通过基因工程方法(重组技术)产生，也可以通过化学合成方法产生。The protein of the present invention, the conjugate of the present invention or the fusion protein of the present invention is not limited by the manner in which it is produced, for example, it can be produced by a genetic engineering method (recombination technique) or can be produced by a chemical synthesis method.

同向重复序列Co-directional repeat

在第四方面，本发明提供了一种分离的核酸分子，其包含选自下列的序列，或由选自下列的序列组成：In a fourth aspect, the invention provides an isolated nucleic acid molecule comprising a sequence selected from the group consisting of: or consisting of:

(i)SEQ ID NOs：37-54任一项所示的序列；(i) the sequence set forth in any one of SEQ ID NOs: 37-54;

(ii)与SEQ ID NOs：37-54任一项所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；(ii) having one or more base substitutions, deletions or additions as compared to the sequence set forth in any one of SEQ ID NOs: 37-54 (eg, 1, 2, 3, 4, 5, a sequence of 6, 7, 8, or 10 base substitutions, deletions, or additions;

(iii)与SEQ ID NOs：37-54任一项所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；(iii) having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the sequence set forth in any one of SEQ ID NOs: 37-54 a sequence of at least 95% sequence identity;

(iv)在严格条件下与(i)-(iii)任一项中所述的序列杂交的序列；或(iv) a sequence that hybridizes under stringent conditions to the sequence set forth in any one of (i)-(iii); or

(v)(i)-(iii)任一项中所述的序列的互补序列；(v) the complement of the sequence set forth in any of (i)-(iii);

并且，(ii)-(v)中任一项所述的序列基本保留了其所源自的序列的生物学功能，所述序列的生物学功能是指，作为CRISPR-Cas***中的同向重复序列的活性。Furthermore, the sequence of any one of (ii)-(v) substantially retains the biological function of the sequence from which it is derived, the biological function of the sequence being referred to as the same direction in the CRISPR-Cas system Repeat the activity of the sequence.

在某些实施方案中，所述分离的核酸分子是CRISPR-Cas***中的同向重复序列。In certain embodiments, the isolated nucleic acid molecule is a direct repeat in a CRISPR-Cas system.

在某些实施方案中，所述核酸分子包含选自下列的序列，或由选自下列的序列组成：In certain embodiments, the nucleic acid molecule comprises a sequence selected from the group consisting of: or consists of a sequence selected from the group consisting of:

(a)SEQ ID NOs：37-54任一项所示的核苷酸序列；(a) the nucleotide sequence set forth in any one of SEQ ID NOs: 37-54;

(b)在严格条件下与(a)中所述的序列杂交的序列；或(b) a sequence that hybridizes under stringent conditions to the sequence described in (a); or

(c)(a)中所述的序列的互补序列。(c) the complement of the sequence described in (a).

在某些实施方案中，所述分离的核酸分子是RNA。In certain embodiments, the isolated nucleic acid molecule is RNA.

在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：In certain embodiments, the isolated nucleic acid molecule comprises a sequence selected from the group consisting of: or a sequence selected from the group consisting of:

(i)SEQ ID NO：37所示的序列；(i) the sequence shown as SEQ ID NO:37;

(ii)与SEQ ID NO：37所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；(ii) a substitution, deletion or addition of one or more bases compared to the sequence set forth in SEQ ID NO: 37 (

eg

1, 2, 3, 4, 5, 6, 7) a sequence of 8, 8 or 10 base substitutions, deletions or additions;

(iii)与SEQ ID NO：37所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；(iii) having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the sequence set forth in SEQ ID NO:37 Sequence of sequence identity;

(v)(i)-(iii)任一项中所述的序列的互补序列。(v) the complement of the sequence set forth in any of (i)-(iii).

在某些实施方案中，所述分离的核酸分子包含选自下列的序列，或由选自下列的序列组成：In certain embodiments, the isolated nucleic acid molecule comprises a sequence selected from the group consisting of: or consists of:

(a)SEQ ID NO：37所示的核苷酸序列；(a) the nucleotide sequence shown in SEQ ID NO: 37;

(c)SEQ ID NO：37所示的核苷酸序列的互补序列。(c) the complement of the nucleotide sequence shown by SEQ ID NO:37.

(i)SEQ ID NO：38所示的序列；(i) the sequence shown as SEQ ID NO:38;

(ii)与SEQ ID NO：38所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；(ii) having one or more base substitutions, deletions or additions compared to the sequence set forth in SEQ ID NO: 38 (

eg

(iii)与SEQ ID NO：38所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；(iii) having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the sequence set forth in SEQ ID NO:38 Sequence of sequence identity;

(a)SEQ ID NO：38所示的核苷酸序列；(a) the nucleotide sequence shown in SEQ ID NO: 38;

(c)SEQ ID NO：38所示的核苷酸序列的互补序列。(c) the complement of the nucleotide sequence shown as SEQ ID NO:38.

(i)SEQ ID NO：39所示的序列；(i) the sequence shown as SEQ ID NO:39;

(ii)与SEQ ID NO：39所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；(ii) a substitution, deletion or addition of one or more bases compared to the sequence set forth in SEQ ID NO: 39 (

eg

(iii)与SEQ ID NO：39所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；(iii) having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the sequence set forth in SEQ ID NO:39 Sequence of sequence identity;

(a)SEQ ID NO：39所示的核苷酸序列；(a) the nucleotide sequence shown in SEQ ID NO: 39;

(b)在严格条件下与(a)中所述的序列杂交的序列；(b) a sequence that hybridizes under stringent conditions to the sequence described in (a);

(c)SEQ ID NO：39所示的核苷酸序列的互补序列。(c) the complement of the nucleotide sequence shown by SEQ ID NO:39.

(i)SEQ ID NO：40所示的序列；(i) the sequence shown as SEQ ID NO:40;

(ii)与SEQ ID NO：40所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；(ii) a substitution, deletion or addition of one or more bases compared to the sequence set forth in SEQ ID NO: 40 (

eg

(iii)与SEQ ID NO：40所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；(iii) having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the sequence set forth in SEQ ID NO: Sequence of sequence identity;

(a)SEQ ID NO：40所示的核苷酸序列；(a) the nucleotide sequence shown in SEQ ID NO: 40;

(c)SEQ ID NO：40所示的核苷酸序列的互补序列。(c) the complement of the nucleotide sequence shown by SEQ ID NO:40.

(i)SEQ ID NO：41所示的序列；(i) the sequence shown as SEQ ID NO: 41;

(ii)与SEQ ID NO：41所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；(ii) having one or more base substitutions, deletions or additions compared to the sequence shown in SEQ ID NO: 41 (for example, 1, 2, 3, 4, 5, 6, 7) a sequence of 8, 8 or 10 base substitutions, deletions or additions;

(iii)与SEQ ID NO：41所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；(iii) having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the sequence set forth in SEQ ID NO:41 Sequence of sequence identity;

(a)SEQ ID NO：41所示的核苷酸序列；(a) the nucleotide sequence shown in SEQ ID NO: 41;

(c)SEQ ID NO：41所示的核苷酸序列的互补序列。(c) the complement of the nucleotide sequence shown in SEQ ID NO:41.

(i)SEQ ID NO：42所示的序列；(i) the sequence shown as SEQ ID NO:42;

(ii)与SEQ ID NO：42所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；(ii) a substitution, deletion or addition of one or more bases compared to the sequence set forth in SEQ ID NO: 42 (

eg

(iii)与SEQ ID NO：42所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；(iii) having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the sequence set forth in SEQ ID NO: Sequence of sequence identity;

(a)SEQ ID NO：42所示的核苷酸序列；(a) the nucleotide sequence shown in SEQ ID NO: 42;

(c)SEQ ID NO：42所示的核苷酸序列的互补序列。(c) the complement of the nucleotide sequence set forth in SEQ ID NO:42.

(i)SEQ ID NO：43所示的序列；(i) the sequence shown as SEQ ID NO:43;

(ii)与SEQ ID NO：43所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；(ii) having one or more base substitutions, deletions or additions compared to the sequence shown in SEQ ID NO: 43 (

eg

(iii)与SEQ ID NO：43所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；(iii) having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the sequence set forth in SEQ ID NO: Sequence of sequence identity;

(a)SEQ ID NO：43所示的核苷酸序列；(a) the nucleotide sequence shown in SEQ ID NO: 43;

(c)SEQ ID NO：43所示的核苷酸序列的互补序列。(c) the complement of the nucleotide sequence shown in SEQ ID NO:43.

(i)SEQ ID NO：44所示的序列；(i) the sequence shown as SEQ ID NO: 44;

(ii)与SEQ ID NO：44所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；(ii) a substitution, deletion or addition of one or more bases compared to the sequence set forth in SEQ ID NO: 44 (

eg

(iii)与SEQ ID NO：44所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；(iii) having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the sequence set forth in SEQ ID NO: 44 Sequence of sequence identity;

(a)SEQ ID NO：44所示的核苷酸序列；(a) the nucleotide sequence shown in SEQ ID NO: 44;

(c)SEQ ID NO：44所示的核苷酸序列的互补序列。(c) the complement of the nucleotide sequence shown by SEQ ID NO:44.

(i)SEQ ID NO：45所示的序列；(i) the sequence shown as SEQ ID NO:45;

(ii)与SEQ ID NO：45所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；(ii) a substitution, deletion or addition of one or more bases compared to the sequence shown in SEQ ID NO: 45 (

eg

(iii)与SEQ ID NO：45所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；(iii) having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the sequence set forth in SEQ ID NO:45 Sequence of sequence identity;

(a)SEQ ID NO：45所示的核苷酸序列；(a) the nucleotide sequence shown in SEQ ID NO: 45;

(c)SEQ ID NO：45所示的核苷酸序列的互补序列。(c) the complement of the nucleotide sequence shown by SEQ ID NO:45.

(i)SEQ ID NO：46所示的序列；(i) the sequence shown as SEQ ID NO:46;

(ii)与SEQ ID NO：46所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；(ii) having one or more base substitutions, deletions or additions compared to the sequence set forth in SEQ ID NO: 46 (

eg

(iii)与SEQ ID NO：46所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；(iii) having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the sequence set forth in SEQ ID NO:46 Sequence of sequence identity;

(a)SEQ ID NO：46所示的核苷酸序列；(a) the nucleotide sequence shown as SEQ ID NO: 46;

(c)SEQ ID NO：46所示的核苷酸序列的互补序列。(c) the complement of the nucleotide sequence shown by SEQ ID NO:46.

(i)SEQ ID NO：47所示的序列；(i) the sequence shown as SEQ ID NO:47;

(ii)与SEQ ID NO：47所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；(ii) a substitution, deletion or addition of one or more bases compared to the sequence set forth in SEQ ID NO: 47 (

eg

(iii)与SEQ ID NO：47所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；(iii) having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the sequence set forth in SEQ ID NO:47 Sequence of sequence identity;

(a)SEQ ID NO：47所示的核苷酸序列；(a) the nucleotide sequence shown in SEQ ID NO: 47;

(c)SEQ ID NO：47所示的核苷酸序列的互补序列。(c) the complement of the nucleotide sequence shown by SEQ ID NO:47.

(i)SEQ ID NO：48所示的序列；(i) the sequence shown as SEQ ID NO:48;

(ii)与SEQ ID NO：48所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；(ii) having one or more base substitutions, deletions or additions compared to the sequence set forth in SEQ ID NO: 48 (

eg

(iii)与SEQ ID NO：48所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；(iii) having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the sequence set forth in SEQ ID NO:48 Sequence of sequence identity;

(a)SEQ ID NO：48所示的核苷酸序列；(a) the nucleotide sequence shown in SEQ ID NO:48;

(c)SEQ ID NO：48所示的核苷酸序列的互补序列。(c) the complement of the nucleotide sequence set forth in SEQ ID NO:48.

(i)SEQ ID NO：49所示的序列；(i) the sequence shown as SEQ ID NO:49;

(ii)与SEQ ID NO：49所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；(ii) a substitution, deletion or addition of one or more bases compared to the sequence set forth in SEQ ID NO: 49 (

eg

(iii)与SEQ ID NO：49所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；(iii) having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the sequence set forth in SEQ ID NO:49 Sequence of sequence identity;

(a)SEQ ID NO：49所示的核苷酸序列；(a) the nucleotide sequence shown in SEQ ID NO: 49;

(c)SEQ ID NO：49所示的核苷酸序列的互补序列。(c) the complement of the nucleotide sequence shown in SEQ ID NO:49.

(i)SEQ ID NO：50所示的序列；(i) the sequence shown as SEQ ID NO:50;

(ii)与SEQ ID NO：50所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；(ii) a substitution, deletion or addition of one or more bases compared to the sequence set forth in SEQ ID NO: 50 (

eg

(iii)与SEQ ID NO：50所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；(iii) having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the sequence set forth in SEQ ID NO: Sequence of sequence identity;

(a)SEQ ID NO：50所示的核苷酸序列；(a) the nucleotide sequence shown as SEQ ID NO: 50;

(c)SEQ ID NO：50所示的核苷酸序列的互补序列。(c) the complement of the nucleotide sequence shown by SEQ ID NO:50.

(i)SEQ ID NO：51所示的序列；(i) the sequence shown as SEQ ID NO: 51;

(ii)与SEQ ID NO：51所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；(ii) having one or more base substitutions, deletions or additions compared to the sequence shown in SEQ ID NO: 51 (for example, 1, 2, 3, 4, 5, 6, 7) a sequence of 8, 8 or 10 base substitutions, deletions or additions;

(iii)与SEQ ID NO：51所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；(iii) having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the sequence set forth in SEQ ID NO:51 Sequence of sequence identity;

(a)SEQ ID NO：51所示的核苷酸序列；(a) the nucleotide sequence shown in SEQ ID NO: 51;

(c)SEQ ID NO：51所示的核苷酸序列的互补序列。(c) the complement of the nucleotide sequence shown by SEQ ID NO:51.

(i)SEQ ID NO：52所示的序列；(i) the sequence shown as SEQ ID NO:52;

(ii)与SEQ ID NO：52所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；(ii) a substitution, deletion or addition of one or more bases compared to the sequence set forth in SEQ ID NO: 52 (

eg

(iii)与SEQ ID NO：52所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；(iii) having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the sequence set forth in SEQ ID NO:52 Sequence of sequence identity;

(a)SEQ ID NO：52所示的核苷酸序列；(a) the nucleotide sequence shown in SEQ ID NO: 52;

(c)SEQ ID NO：52所示的核苷酸序列的互补序列。(c) the complement of the nucleotide sequence shown by SEQ ID NO:52.

(i)SEQ ID NO：53所示的序列；(i) the sequence shown as SEQ ID NO:53;

(ii)与SEQ ID NO：53所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；(ii) a substitution, deletion or addition of one or more bases compared to the sequence shown in SEQ ID NO: 53 (

eg

(iii)与SEQ ID NO：53所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；(iii) having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the sequence set forth in SEQ ID NO: Sequence of sequence identity;

(a)SEQ ID NO：53所示的核苷酸序列；(a) the nucleotide sequence shown in SEQ ID NO: 53;

(c)SEQ ID NO：53所示的核苷酸序列的互补序列。(c) the complement of the nucleotide sequence shown as SEQ ID NO:53.

(i)SEQ ID NO：54所示的序列；(i) the sequence set forth in SEQ ID NO:54;

(ii)与SEQ ID NO：54所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；(ii) a substitution, deletion or addition of one or more bases compared to the sequence set forth in SEQ ID NO: 54 (

eg

(iii)与SEQ ID NO：54所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；(iii) having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the sequence set forth in SEQ ID NO:54 Sequence of sequence identity;

(a)SEQ ID NO：54所示的核苷酸序列；(a) the nucleotide sequence shown in SEQ ID NO: 54;

(c)SEQ ID NO：54所示的核苷酸序列的互补序列。(c) the complement of the nucleotide sequence shown in SEQ ID NO:54.

CRISPR/Cas复合物CRISPR/Cas Complex

在第五方面，本发明提供了一种复合物，其包含：In a fifth aspect, the invention provides a composite comprising:

(i)蛋白组分，其选自：本发明的蛋白、缀合物或融合蛋白，及其任意组合；和(i) a protein component selected from the group consisting of a protein, conjugate or fusion protein of the invention, and any combination thereof;

(ii)核酸组分，其从5’至3’方向包含如第四方面所述的分离的核酸分子和能够与靶序列杂交的导向序列，(ii) a nucleic acid component comprising, in the 5' to 3' direction, the isolated nucleic acid molecule of the fourth aspect and a targeting sequence capable of hybridizing to the target sequence,

其中，所述蛋白组分与核酸组分相互结合形成复合物。Wherein the protein component and the nucleic acid component are combined with each other to form a complex.

在某些实施方案中，所述导向序列连接于所述核酸分子的3’端。In certain embodiments, the targeting sequence is linked to the 3' end of the nucleic acid molecule.

在某些实施方案中，所述导向序列包含所述靶序列的互补序列。In certain embodiments, the targeting sequence comprises the complement of the target sequence.

在某些实施方案中，所述核酸组分是CRISPR-Cas***中的导向RNA。In certain embodiments, the nucleic acid component is a targeting RNA in a CRISPR-Cas system.

在某些实施方案中，所述核酸分子是RNA。In certain embodiments, the nucleic acid molecule is RNA.

在某些实施方案中，所述复合物不包含反式作用crRNA(tracrRNA)。In certain embodiments, the complex does not comprise a trans-acting crRNA (tracrRNA).

在某些实施方案中，所述导向序列在长度上为至少5个、至少10个、在某些实施方案中，所述导向序列在长度上为10-30个、或15-25个、或15-22个、或19-25个或19-22个核苷酸。In certain embodiments, the targeting sequence is at least 5, at least 10 in length, and in certain embodiments, the targeting sequence is 10-30, or 15-25 in length, or 15-22, or 19-25 or 19-22 nucleotides.

在某些实施方案中，所述分离的核酸分子在长度上为55-70个核苷酸，例如55-65个核苷酸，例如60-65个核苷酸，例如62-65个核苷酸，例如63-64个核苷酸。在某些实施方案中，所述分离的核酸分子在长度上为15-30个核苷酸，例如15-25个核苷酸，例如20-25个核苷酸，例如22-24个核苷酸，例如23个核苷酸。In certain embodiments, the isolated nucleic acid molecule is 55-70 nucleotides in length, such as 55-65 nucleotides, such as 60-65 nucleotides, such as 62-65 nucleosides. Acid, for example 63-64 nucleotides. In certain embodiments, the isolated nucleic acid molecule is 15-30 nucleotides in length, such as 15-25 nucleotides, such as 20-25 nucleotides, such as 22-24 nucleosides. Acid, for example 23 nucleotides.

编码核酸、载体及宿主细胞Encoding nucleic acids, vectors and host cells

在第六方面，本发明提供了一种分离的核酸分子，其包含：In a sixth aspect, the invention provides an isolated nucleic acid molecule comprising:

(i)编码本发明的蛋白或融合蛋白的核苷酸序列；(i) a nucleotide sequence encoding a protein or fusion protein of the invention;

(ii)编码如第四方面所述的分离的核酸分子；或(ii) an isolated nucleic acid molecule encoding a fourth aspect; or

(iii)包含(i)和(ii)的核苷酸序列。(iii) a nucleotide sequence comprising (i) and (ii).

在某些实施方案中，(i)-(iii)任一项中所述的核苷酸序列经密码子优化用于在原核细胞中进行表达。在某些实施方案中，(i)-(iii)任一项中所述的核苷酸序列经密码子优化用于在真核细胞中进行表达。In certain embodiments, the nucleotide sequence set forth in any of (i)-(iii) is codon optimized for expression in a prokaryotic cell. In certain embodiments, the nucleotide sequence set forth in any of (i)-(iii) is codon optimized for expression in eukaryotic cells.

在第七方面，本发明还提供了一种载体，其包含如第六方面所述的分离的核酸分子。本发明的载体可以是克隆载体，也可以是表达载体。在某些实施方案中，本发明的载体是例如质粒，粘粒，噬菌体，柯斯质粒等等。在某些选实施方案中，所述载体能够在受试者(例如哺乳动物，例如人)体内表达本发明的蛋白、融合蛋白、如第四方面所述的分离的核酸分子或如第五方面所述的复合物。In a seventh aspect, the invention provides a vector comprising the isolated nucleic acid molecule of the sixth aspect. The vector of the present invention may be a cloning vector or an expression vector. In certain embodiments, vectors of the invention are, for example, plasmids, cosmids, phage, cosmid, and the like. In certain selected embodiments, the vector is capable of expressing a protein of the invention, a fusion protein, an isolated nucleic acid molecule of the fourth aspect, or a fifth aspect, in a subject (eg, a mammal, eg, a human) Said complex.

在第八方面，本发明还提供了包含如上所述的分离的核酸分子或载体的宿主细胞。此类宿主细胞包括但不限于，原核细胞例如大肠杆菌细胞，以及真核细胞例如酵母细胞，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。本发明的细胞还可以是细胞系，例如293T细胞。In an eighth aspect, the invention also provides a host cell comprising an isolated nucleic acid molecule or vector as described above. Such host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (eg, mammalian cells, such as mouse cells, human cells, etc.). The cells of the invention may also be cell lines, such as 293T cells.

组合物及载体组合物Composition and carrier composition

在第九方面，本发明还提供了一种组合物，其包含：In a ninth aspect, the present invention also provides a composition comprising:

(i)第一组分，其选自：本发明的蛋白、缀合物、融合蛋白、编码所述蛋白或融合蛋白的核苷酸序列，以及其任意组合；和(i) a first component selected from the group consisting of a protein, a conjugate, a fusion protein, a nucleotide sequence encoding the protein or fusion protein, and any combination thereof;

(ii)第二组分，其为包含导向RNA的核苷酸序列，或者编码所述包含导向RNA的核苷酸序列的核苷酸序列；(ii) a second component which is a nucleotide sequence comprising a targeting RNA or a nucleotide sequence encoding the nucleotide sequence comprising the targeting RNA;

其中，所述导向RNA从5’至3’方向包含同向重复序列和导向序列，所述导向序列能够与靶序列杂交；Wherein the targeting RNA comprises a homologous repeat sequence and a targeting sequence from the 5' to 3' direction, the targeting sequence being capable of hybridizing to the target sequence;

所述导向RNA能够与(i)中所述的蛋白、缀合物或融合蛋白形成复合物。The targeting RNA is capable of forming a complex with the protein, conjugate or fusion protein described in (i).

在某些实施方案中，所述同向重复序列是如第四方面所定义的分离的核酸分子。In certain embodiments, the isotropic repeat is an isolated nucleic acid molecule as defined in the fourth aspect.

在某些实施方案中，所述导向序列连接至所述同向重复序列的3’端。在某些实施方案中，所述导向序列包含所述靶序列的互补序列。In certain embodiments, the targeting sequence is ligated to the 3' end of the isotropic repeat. In certain embodiments, the targeting sequence comprises the complement of the target sequence.

在某些实施方案中，所述组合物不包含tracrRNA。In certain embodiments, the composition does not comprise tracrRNA.

在某些实施方案中，所述组合物是非天然存在的或经修饰的。在某些实施方案中，所述组合物中的至少一个组分是非天然存在的或经修饰的。在某些实施方案中，所述第一组分是非天然存在的或经修饰的；和/或，所述第二组分是非天然存在的或经修饰的。In certain embodiments, the composition is non-naturally occurring or modified. In certain embodiments, at least one component of the composition is non-naturally occurring or modified. In certain embodiments, the first component is non-naturally occurring or modified; and/or the second component is non-naturally occurring or modified.

在某些实施方案中，当所述靶序列为DNA时，所述靶序列位于原间隔序列临近基序(PAM)的3’端，并且所述PAM具有5’-NTN或5’-TNN所示的序列，其中，N选自A、G、T、C。In certain embodiments, when the target sequence is DNA, the target sequence is located at the 3' end of the proximate spacer adjacent motif (PAM) and the PAM has a 5'-NTN or 5'-TNN The sequence shown, wherein N is selected from the group consisting of A, G, T, and C.

在某些实施方案中，当所述靶序列为RNA时，所述靶序列不具有PAM结构域限制。In certain embodiments, when the target sequence is RNA, the target sequence does not have a PAM domain restriction.

在某些实施方案中，所述靶序列是来自原核细胞或真核细胞的DNA或RNA序列。在某些实施方案中，所述靶序列是非天然存在的DNA或RNA序列。In certain embodiments, the target sequence is a DNA or RNA sequence derived from a prokaryotic or eukaryotic cell. In certain embodiments, the target sequence is a non-naturally occurring DNA or RNA sequence.

在某些实施方案中，所述靶序列存在于细胞内。在某些实施方案中，所述靶序列存在于细胞核内或细胞质(例如，细胞器)内。在某些实施方案中，所述细胞是真核细胞。在某些实施方案中，所述细胞是原核细胞。In certain embodiments, the target sequence is present in a cell. In certain embodiments, the target sequence is present within the nucleus or within the cytoplasm (eg, an organelle). In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the cell is a prokaryotic cell.

在某些实施方案中，所述蛋白连接有一个或多个NLS序列。在某些实施方案中，所述缀合物或融合蛋白包含一个或多个NLS序列。在某些实施方案中，所述NLS序列连接至所述蛋白的N端或C端。在某些实施方案中，所述NLS序列融合至所述蛋白的N端或C端。In certain embodiments, the protein is linked to one or more NLS sequences. In certain embodiments, the conjugate or fusion protein comprises one or more NLS sequences. In certain embodiments, the NLS sequence is linked to the N-terminus or C-terminus of the protein. In certain embodiments, the NLS sequence is fused to the N-terminus or C-terminus of the protein.

在第十方面，本发明还提供了一种组合物，其包含一种或多种载体，所述一种或多种载体包含：In a tenth aspect, the present invention also provides a composition comprising one or more carriers, the one or more carriers comprising:

(i)第一核酸，其为编码本发明的蛋白或融合蛋白的核苷酸序列；任选地所述第一核酸可操作地连接至第一调节元件；以及(i) a first nucleic acid which is a nucleotide sequence encoding a protein or fusion protein of the invention; optionally the first nucleic acid is operably linked to a first regulatory element;

(ii)第二核酸，其编码包含导向RNA的核苷酸序列；任选地所述第二核酸可操作地连接至第二调节元件；(ii) a second nucleic acid encoding a nucleotide sequence comprising a targeting RNA; optionally the second nucleic acid is operably linked to a second regulatory element;

其中：among them:

所述第一核酸与第二核酸存在于相同或不同的载体上；The first nucleic acid and the second nucleic acid are present on the same or different carrier;

所述导向RNA从5’至3’方向包含同向重复序列和导向序列，所述导向序列能够与靶序列杂交；The targeting RNA comprises a homologous repeat sequence and a targeting sequence from the 5' to 3' direction, the targeting sequence being capable of hybridizing to the target sequence;

所述导向RNA能够与(i)中所述的效应蛋白或融合蛋白形成复合物。The targeting RNA is capable of forming a complex with the effector protein or fusion protein described in (i).

在某些实施方案中，所述组合物是非天然存在的或经修饰的。在某些实施方案中，所述组合物中的至少一个组分是非天然存在的或经修饰的。In certain embodiments, the composition is non-naturally occurring or modified. In certain embodiments, at least one component of the composition is non-naturally occurring or modified.

在某些实施方案中，所述第一调节元件是启动子，例如诱导型启动子。In certain embodiments, the first regulatory element is a promoter, such as an inducible promoter.

在某些实施方案中，所述第二调节元件是启动子，例如诱导型启动子。In certain embodiments, the second regulatory element is a promoter, such as an inducible promoter.

在某些实施方案中，一种类型的载体是质粒，其是指其中可以例如通过标准分子克隆技术***另外的DNA片段的环状双链DNA环。另一种类型的载体是病毒载体，其中病毒衍生的DNA或RNA序列存在于用于包装病毒(例如，逆转录病毒、复制缺陷型逆转录病毒、腺病毒、复制缺陷型腺病毒、以及腺相关病毒)的载体中。病毒载体还包含由用于转染到一种宿主细胞中的病毒携带的多核苷酸。某些载体(例如，具有细菌复制起点的细菌载体和附加型哺乳动物载体)能够在它们被导入的宿主细胞中自主复制。其他载体(例如，非附加型哺乳动物载体)在引入宿主细胞后整合到该宿主细胞的基因组中，并且由此与该宿主基因组一起复制。而且，某些载体能够指导它们可操作连接的基因的表达。这样的载体在此被称为“表达载体”。在重组DNA技术中使用的普通表达栽体通常是质粒形式。In certain embodiments, one type of vector is a plasmid, which refers to a circular double stranded DNA loop in which additional DNA fragments can be inserted, for example, by standard molecular cloning techniques. Another type of vector is a viral vector in which a virus-derived DNA or RNA sequence is present for packaging a virus (eg, retrovirus, replication-defective retrovirus, adenovirus, replication-defective adenovirus, and adeno-associated In the vector of the virus). The viral vector also comprises a polynucleotide carried by a virus for transfection into a host cell. Certain vectors (e.g., bacterial vectors having bacterial origins of replication and episomal mammalian vectors) are capable of autonomous replication in the host cell into which they are introduced. Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell upon introduction into the host cell and thereby replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors." Common expression vectors used in recombinant DNA techniques are typically in the form of plasmids.

重组表达载体可包含处于适合于在宿主细胞中的核酸表达的形式的本发明的核酸分子，这意味着这些重组表达载体包含基于待用于表达的宿主细胞而选择的一种或多种调节元件，所述调节元件可操作地连接至待表达的核酸序列。The recombinant expression vector can comprise a nucleic acid molecule of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vector comprises one or more regulatory elements selected based on the host cell to be used for expression. The regulatory element is operably linked to the nucleic acid sequence to be expressed.

递送及递送组合物Delivery and delivery composition

本发明的蛋白、缀合物、融合蛋白、如第四方面所述的分离的核酸分子、本发明的复合物、如第六方面所述的分离的核酸分子、如第七方面所述的载体、如第九方面及第十方面所述的组合物，可以通过本领域已知的任何方法进行递送。此类方法包括但不限于，电穿孔、脂转染、核转染、显微注射、声孔效应、基因枪、磷酸钙介导的转染、阳离子转染、脂质体转染、树枝状转染、热激转染、核转染、磁转染、脂转染、穿刺转染、光学转染、试剂增强性核酸摄取、以及经由脂质体、免疫脂质体、病毒颗粒、人工病毒体等的递送。The protein, the conjugate, the fusion protein of the invention, the isolated nucleic acid molecule of the fourth aspect, the complex of the invention, the isolated nucleic acid molecule of the sixth aspect, the vector of the seventh aspect The compositions of the ninth and tenth aspects can be delivered by any method known in the art. Such methods include, but are not limited to, electroporation, lipofection, nuclear transfection, microinjection, sonoporation, gene gun, calcium phosphate mediated transfection, cation transfection, lipofection, dendritic Transfection, heat shock transfection, nuclear transfection, magnetic transfection, lipofection, puncture transfection, optical transfection, reagent-enhanced nucleic acid uptake, and via liposomes, immunoliposomes, viral particles, artificial viruses Delivery of body, etc.

因此，在另一个方面，本发明提供了一种递送组合物，其包含递送载体，以及选自下列的一种或多种：本发明的蛋白、缀合物、融合蛋白、如第四方面所述的分离的核酸分子、本发明的复合物、如第六方面所述的分离的核酸分子、如第七方面所述的载体、如第九方面及第十方面所述的组合物。Accordingly, in another aspect, the present invention provides a delivery composition comprising a delivery vehicle, and one or more selected from the group consisting of a protein, a conjugate, a fusion protein of the invention, as in the fourth aspect The isolated nucleic acid molecule, the complex of the invention, the isolated nucleic acid molecule of the sixth aspect, the carrier of the seventh aspect, the composition of the ninth and tenth aspects.

在某些实施方案中，所述递送载体是粒子。In certain embodiments, the delivery vehicle is a particle.

在某些实施方案中，所述递送载体选自脂质颗粒、糖颗粒、金属颗粒、蛋白颗粒、脂质体、外泌体、微泡、基因枪或病毒载体(例如，复制缺陷型逆转录病毒、慢病毒、腺病毒或腺相关病毒)。In certain embodiments, the delivery vehicle is selected from the group consisting of a lipid particle, a sugar particle, a metal particle, a protein particle, a liposome, an exosome, a microvesicle, a gene gun, or a viral vector (eg, replication defective reverse transcription) Virus, lentivirus, adenovirus or adeno-associated virus).

试剂盒Kit

在另一个方面，本发明提供了一种试剂盒，其包含如上所述的组分中的一种或多种。在某些实施方案中，所述试剂盒包含一种或多种选自下列的组分：本发明的蛋白、缀合物、融合蛋白、如第四方面所述的分离的核酸分子、本发明的复合物、如第六方面所述的分离的核酸分子、如第七方面所述的载体、如第九方面及第十方面所述的组合物。In another aspect, the invention provides a kit comprising one or more of the components described above. In certain embodiments, the kit comprises one or more components selected from the group consisting of a protein, a conjugate, a fusion protein of the invention, an isolated nucleic acid molecule of the fourth aspect, the invention The complex, the isolated nucleic acid molecule of the sixth aspect, the carrier of the seventh aspect, the composition of the ninth and tenth aspects.

在某些实施方案中，本发明的试剂盒包含如第九方面所述的组合物。在某些实施方案中，所述试剂盒还包含使用所述组合物的说明书。In certain embodiments, the kit of the invention comprises the composition of the ninth aspect. In certain embodiments, the kit further comprises instructions for using the composition.

在某些实施方案中，本发明的试剂盒包含如第十方面所述的组合物。在某些实施方案中，所述试剂盒还包含使用所述组合物的说明书。In certain embodiments, the kit of the invention comprises the composition of the tenth aspect. In certain embodiments, the kit further comprises instructions for using the composition.

在某些实施方案中，本发明的试剂盒中包含的组分可以被提供于任何适合的容器中。In certain embodiments, the components contained in the kits of the invention can be provided in any suitable container.

在某些实施方案中，所述试剂盒还包含一种或多种缓冲液。缓冲液可以是任何缓冲液，包括但不限于碳酸钠缓冲液、碳酸氢钠缓冲液、硼酸盐缓冲液、Tris缓冲液、MOPS缓冲液、HEPES缓冲液及其组合。在某些实施方案中，该缓冲液是碱性的。在某些实施方案中，该缓冲液具有从约7至约10的pH。In certain embodiments, the kit further comprises one or more buffers. The buffer can be any buffer including, but not limited to, sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer, and combinations thereof. In certain embodiments, the buffer is basic. In certain embodiments, the buffer has a pH of from about 7 to about 10.

在某些实施方案中，该试剂盒还包括一个或多个寡核苷酸，该一个或多个寡核苷酸对应于一个用于***进载体中的导向序列，以便可操作地连接该导向序列和调节元件。在某些实施方案中，该试剂盒包括用于同源重组的编辑模板。In certain embodiments, the kit further comprises one or more oligonucleotides corresponding to a targeting sequence for insertion into a vector for operably linking the guide Sequence and adjustment elements. In certain embodiments, the kit includes an editing template for homologous recombination.

方法及用途Method and use

在另一个方面，本发明提供了一种修饰靶基因的方法，其包括：将如第五方面所述的复合物、如第九方面所述的组合物或如第十方面所述的组合物与所述靶基因接触，或者递送至包含所述靶基因的细胞中；所述靶序列存在于所述靶基因中。In another aspect, the invention provides a method of modifying a target gene, comprising: the complex of the fifth aspect, the composition of the ninth aspect, or the composition of the tenth aspect Contacting the target gene or delivering to a cell comprising the target gene; the target sequence is present in the target gene.

在某些实施方案中，所述方法用于体外(in vitro)或离体(ex vivo)修饰靶基因。在某些实施方案中，所述方法不是通过疗法来治疗人或动物的方法。在某些实施方案中，所述方法不包括修饰人类种系遗传特性的步骤。In certain embodiments, the methods are used to modify a target gene in vitro or ex vivo. In certain embodiments, the method is not a method of treating a human or animal by therapy. In certain embodiments, the method does not include the step of modifying the genetic characteristics of the human germline.

在某些实施方案中，所述靶基因存在于细胞内。在某些实施方案中，所述细胞是原核细胞。在某些实施方案中，所述细胞是真核细胞。在某些实施方案中，所述细胞是哺乳动物细胞。在某些实施方案中，所述细胞是人类细胞。在某些实施方案中，所述细胞选自非人灵长类动物、牛、猪或啮齿类动物细胞。在某些实施方案中，所述细胞是非哺乳动物真核细胞，例如家禽或鱼等。在某些实施方案中，所述细胞是植物细胞，例如栽培植物(如木薯、玉米、高粱、小麦或水稻)、藻类、树或蔬菜具有的细胞。In certain embodiments, the target gene is present in a cell. In certain embodiments, the cell is a prokaryotic cell. In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a human cell. In certain embodiments, the cell is selected from a non-human primate, bovine, porcine or rodent cell. In certain embodiments, the cell is a non-mammalian eukaryotic cell, such as a poultry or fish. In certain embodiments, the cell is a plant cell, such as a cell of a cultivated plant (such as cassava, corn, sorghum, wheat, or rice), algae, tree, or vegetable.

在某些实施方案中，所述靶基因存在于体外的核酸分子(例如，质粒)中。在某些实施方案中，所述靶基因存在于质粒中。In certain embodiments, the target gene is present in a nucleic acid molecule (eg, a plasmid) in vitro. In certain embodiments, the target gene is present in a plasmid.

在某些实施方案中，所述修饰是指所述靶序列的断裂，如DNA的双链断裂或RNA的单链断裂。In certain embodiments, the modification refers to cleavage of the target sequence, such as double-strand breaks in DNA or single-strand breaks in RNA.

在某些实施方案中，所述断裂导致靶基因的转录降低。In certain embodiments, the cleavage results in a decrease in transcription of the target gene.

在某些实施方案中，所述方法还包括：将编辑模板与所述靶基因接触，或者递送至包含所述靶基因的细胞中。在此类实施方案中，所述方法通过与所述编辑模板同源重组修复所述断裂的靶基因，其中所述修复导致一种突变，包括所述靶基因的一个或多个核苷酸的***、缺失、或取代。在某些实施方案中，所述突变导致在从包含该靶序列的基因表达的蛋白质中的一个或多个氨基酸改变。In certain embodiments, the method further comprises contacting the editing template with the target gene or delivering to a cell comprising the target gene. In such embodiments, the method repairs the disrupted target gene by homologous recombination with the editing template, wherein the repair results in a mutation comprising one or more nucleotides of the target gene Insert, delete, or replace. In certain embodiments, the mutation results in a change in one or more amino acids in a protein expressed from a gene comprising the target sequence.

因此，在某些实施方案中，所述修饰还包括将编辑模板(例如外源核酸)***所述断裂中。Thus, in certain embodiments, the modification further comprises inserting an editing template (eg, an exogenous nucleic acid) into the fragmentation.

在某些实施方案中，所述的蛋白、缀合物、融合蛋白、分离的核酸分子、复合物、载体或组合物包含于递送载体中。In certain embodiments, the protein, conjugate, fusion protein, isolated nucleic acid molecule, complex, vector or composition is included in a delivery vehicle.

在某些实施方案中，所述递送载体选自脂质颗粒、糖颗粒、金属颗粒、蛋白颗粒、脂质体、外泌体、病毒载体(如复制缺陷型逆转录病毒、慢病毒、腺病毒或腺相关病毒)。In certain embodiments, the delivery vehicle is selected from the group consisting of a lipid particle, a sugar particle, a metal particle, a protein particle, a liposome, an exosome, a viral vector (eg, a replication defective retrovirus, a lentivirus, an adenovirus) Or adeno-associated virus).

在某些实施方案中，所述方法其用于改变靶基因或编码靶基因产物的核酸分子中的一个或多个靶序列来修饰细胞、细胞系或生物体。In certain embodiments, the methods are used to modify a target gene or one or more target sequences in a nucleic acid molecule encoding a target gene product to modify a cell, cell line or organism.

在另一个方面，本发明提供了一种改变基因产物的表达的方法，其包括：将如第五方面所述的复合物、如第九方面所述的组合物或如第十方面所述的组合物与编码所述基因产物的核酸分子接触，或者递送至包含所述核酸分子的细胞中，所述靶序列存在于所述核酸分子中。In another aspect, the invention provides a method of altering the expression of a gene product, comprising: the complex of the fifth aspect, the composition of the ninth aspect, or the The composition is contacted with a nucleic acid molecule encoding the gene product or delivered to a cell comprising the nucleic acid molecule, the target sequence being present in the nucleic acid molecule.

在某些实施方案中，所述核酸分子存在于细胞内。在某些实施方案中，所述细胞是原核细胞。在某些实施方案中，所述细胞是真核细胞。在某些实施方案中，所述细胞是哺乳动物细胞。在某些实施方案中，所述细胞是人类细胞。在某些实施方案中，所述细胞选自非人灵长类动物、牛、猪或啮齿类动物细胞。在某些实施方案中，所述细胞是非哺乳动物真核细胞，例如家禽或鱼等。在某些实施方案中，所述细胞是植物细胞，例如栽培植物(如木薯、玉米、高粱、小麦或水稻)、藻类、树或蔬菜具有的细胞。In certain embodiments, the nucleic acid molecule is present in a cell. In certain embodiments, the cell is a prokaryotic cell. In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a human cell. In certain embodiments, the cell is selected from a non-human primate, bovine, porcine or rodent cell. In certain embodiments, the cell is a non-mammalian eukaryotic cell, such as a poultry or fish. In certain embodiments, the cell is a plant cell, such as a cell of a cultivated plant (such as cassava, corn, sorghum, wheat, or rice), algae, tree, or vegetable.

在某些实施方案中，所述核酸分子存在于体外的核酸分子(例如，质粒)中。在某些实施方案中，所述核酸分子存在于质粒中。In certain embodiments, the nucleic acid molecule is present in a nucleic acid molecule (eg, a plasmid) in vitro. In certain embodiments, the nucleic acid molecule is present in a plasmid.

在某些实施方案中，所述基因产物的表达被改变(例如，增强或降低)。在某些实施方案中，所述基因产物的表达被增强。在某些实施方案中，所述基因产物的表达被降低。In certain embodiments, the expression of the gene product is altered (eg, increased or decreased). In certain embodiments, the expression of the gene product is enhanced. In certain embodiments, the expression of the gene product is reduced.

在某些实施方案中，所述基因产物是蛋白。In certain embodiments, the gene product is a protein.

在另一个方面，本发明涉及如第一方面所述的蛋白、如第二方面所述的缀合物、如第三方面所述的融合蛋白、如第四方面所述的分离的核酸分子、如第五方面所述的复合物、如第六方面所述的分离的核酸分子、如第七方面所述的载体、如第九方面所述的组合物、如第十方面所述的组合物、本发明的试剂盒或递送组合物，用于核酸编辑(例如，体外或离体核酸编辑)的用途，或者在制备制剂中的用途，所述制剂用于核酸编辑。In another aspect, the invention relates to the protein of the first aspect, the conjugate of the second aspect, the fusion protein of the third aspect, the isolated nucleic acid molecule of the fourth aspect, The complex of claim 5, the isolated nucleic acid molecule of the sixth aspect, the carrier of the seventh aspect, the composition of the ninth aspect, the composition of the tenth aspect A kit or delivery composition of the invention for use in nucleic acid editing (eg, in vitro or ex vivo nucleic acid editing), or in the preparation of a formulation for nucleic acid editing.

在某些实施方案中，待被编辑的核酸存在于细胞内。在某些实施方案中，所述细胞是原核细胞或真核细胞。在某些实施方案中，待被编辑的核酸存在于体外的核酸分子(例如，质粒)中。In certain embodiments, the nucleic acid to be edited is present within the cell. In certain embodiments, the cell is a prokaryotic cell or a eukaryotic cell. In certain embodiments, the nucleic acid to be edited is present in a nucleic acid molecule (eg, a plasmid) in vitro.

在某些实施方案中，所述核酸编辑包括基因或基因组编辑，例如修饰基因、敲除基因、改变基因产物的表达、修复突变、和/或***多核苷酸。在某些实施方案中，所述基因或基因组编辑不包括修饰人类种系遗传特性的步骤。在某些实施方案中，所述用途不是通过疗法来治疗人或动物的方法。In certain embodiments, the nucleic acid editing comprises genetic or genomic editing, such as modifying a gene, knocking out a gene, altering the expression of a gene product, repairing a mutation, and/or inserting a polynucleotide. In certain embodiments, the gene or genome editing does not include the step of modifying the genetic characteristics of the human germline. In certain embodiments, the use is not a method of treating a human or animal by therapy.

在某些实施方案中，所述用途还包括通过与外源模板多核苷酸的同源重组来修复被编辑的靶序列，其中所述修复可以产生该靶序列的突变，包括一个或多个核苷酸的***、缺失或取代。In certain embodiments, the use further comprises repairing the edited target sequence by homologous recombination with an exogenous template polynucleotide, wherein the repair can produce a mutation in the target sequence, including one or more nuclei Insertion, deletion or substitution of a nucleotide.

在另一个方面，本发明涉及如第一方面所述的蛋白、如第二方面所述的缀合物、如第三方面所述的融合蛋白、如第四方面所述的分离的核酸分子、如第五方面所述的复合物、如第六方面所述的分离的核酸分子、如第七方面所述的载体、如第九方面所述的组合物、如第十方面所述的组合物、本发明的试剂盒或递送组合物，在制备制剂中的用途，所述制剂用于：In another aspect, the invention relates to the protein of the first aspect, the conjugate of the second aspect, the fusion protein of the third aspect, the isolated nucleic acid molecule of the fourth aspect, The complex of claim 5, the isolated nucleic acid molecule of the sixth aspect, the carrier of the seventh aspect, the composition of the ninth aspect, the composition of the tenth aspect Use of a kit or delivery composition of the invention in the preparation of a formulation for:

(i)体外或离体单链DNA的检测(例如原核细胞中的单链DNA的检测)；(i) detection of in vitro or ex vivo single-stranded DNA (eg, detection of single-stranded DNA in prokaryotic cells);

(ii)编辑靶基因座中的靶序列来修饰生物或非人类生物(例如，原核生物)。(ii) Editing target sequences in the target locus to modify biological or non-human organisms (eg, prokaryotes).

细胞及细胞子代Cell and cell progeny

在某些情况下，由本发明的方法引入到细胞的修饰可以使得细胞和其子代被改变以改进其生物产物(如抗体、淀粉、乙醇或其他期望的细胞输出物)的产生。在某些情况下，由本发明的方法引入到细胞的修饰可以使得细胞和其子代包括使所生产生物产物发生变化的改变。In some cases, modifications introduced to cells by the methods of the invention can cause the cells and their progeny to be altered to improve the production of their biological products, such as antibodies, starch, ethanol, or other desired cellular output. In some cases, modifications introduced into the cell by the methods of the invention can cause the cell and its progeny to include changes that result in a change in the produced biological product.

因此，在另一方面，本发明还涉及如上所述的方法获得的细胞或其子代，其中所述细胞含有在其野生型中不存在的修饰。Thus, in another aspect, the invention relates to a cell or a progeny thereof obtained by the method as described above, wherein said cell contains a modification which is not found in its wild type.

本发明还涉及如上所述的细胞或其子代的细胞产物。The invention also relates to a cell product of a cell or a progeny thereof as described above.

本发明还涉及一种体外的、离体的或体内的细胞或细胞系或它们的子代，所述细胞或细胞系或它们的子代包含：如第一方面所述的蛋白、如第二方面所述的缀合物、如第三方面所述的融合蛋白、如第四方面所述的分离的核酸分子、如第五方面所述的复合物、如第六方面所述的分离的核酸分子、如第七方面所述的载体、如第九方面所述的组合物、如第十方面所述的组合物、本发明的试剂盒或递送组合物。The invention also relates to an in vitro, ex vivo or in vivo cell or cell line or a progeny thereof, the cell or cell line or a progeny thereof comprising: the protein of the first aspect, such as the second The conjugate of the aspect, the fusion protein of the third aspect, the isolated nucleic acid molecule of the fourth aspect, the complex of the fifth aspect, the isolated nucleic acid of the sixth aspect A molecule, a carrier according to the seventh aspect, a composition according to the ninth aspect, a composition according to the tenth aspect, a kit of the invention or a delivery composition.

在某些实施方案中，所述细胞是原核细胞。In certain embodiments, the cell is a prokaryotic cell.

在某些实施方案中，所述细胞是真核细胞。在某些实施方案中，所述细胞是哺乳动物细胞。在某些实施方案中，所述细胞是人类细胞。某些实施方案中，所述细胞是非人哺乳动物细胞，例如非人灵长类动物、牛、羊、猪、犬、猴、兔、啮齿类(如大鼠或小鼠)的细胞。在某些实施方案中，所述细胞是非哺乳动物真核细胞，例如家禽鸟类(如鸡)、鱼类或甲壳动物(如蛤蜊、虾)的细胞。在某些实施方案中，所述细胞是植物细胞，例如单子叶植物或双子叶植物具有的细胞或栽培植物或粮食作物如木薯、玉米、高粱、大豆、小麦、燕麦或水稻具有的细胞，例如藻类、树或生产植物、果实或蔬菜(例如，树类如柑橘树、坚果树；茄属植物、棉花、烟草、番茄、葡萄、咖啡、可可等)。In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a human cell. In certain embodiments, the cell is a non-human mammalian cell, such as a cell of a non-human primate, cow, sheep, pig, dog, monkey, rabbit, rodent (eg, rat or mouse). In certain embodiments, the cell is a non-mammalian eukaryotic cell, such as a poultry bird (eg, chicken), a fish or a crustacean (eg, scorpion, shrimp) cells. In certain embodiments, the cell is a plant cell, such as a cell possessed by a monocot or a dicot or a cultivated plant or a cell of a food crop such as cassava, corn, sorghum, soybean, wheat, oat or rice, for example Algae, tree or production of plants, fruits or vegetables (for example, trees such as citrus, nut trees; nightshade, cotton, tobacco, tomatoes, grapes, coffee, cocoa, etc.).

在某些实施方案中，所述细胞是干细胞或干细胞系。In certain embodiments, the cell is a stem cell or stem cell line.

术语定义Definition of Terms

在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的分子遗传学、核酸化学、化学、分子生物学、生物化学、细胞培养、微生物学、细胞生物学、基因组学和重组DNA等操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。In the present invention, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art, unless otherwise stated. Furthermore, the procedures of molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA used herein are routine steps widely used in the corresponding fields. . Also, for a better understanding of the present invention, definitions and explanations of related terms are provided below.

在本发明中，表述“Cas12g”是指，本发明人首次发现并鉴定的一种Cas效应蛋白，其具有选自下列的氨基酸序列：In the present invention, the expression "Cas12g" means a Cas effector protein first discovered and identified by the present inventors having an amino acid sequence selected from the group consisting of:

eg

本发明的Cas12g是一种在导向RNA引导下与靶序列特定位点结合并切割的核酸内切酶，同时具有DNA和RNA内切酶活性。The Cas12g of the present invention is an endonuclease which binds to a specific site of a target sequence and cleaves under the guidance of a guide RNA, and has both DNA and RNA endonuclease activity.

在本发明中，表述“Cas12h”是指，本发明人首次发现并鉴定的一种Cas效应蛋白，其具有选自下列的氨基酸序列：In the present invention, the expression "Cas12h" refers to a Cas effector protein first discovered and identified by the present inventors having an amino acid sequence selected from the group consisting of:

(i)SEQ ID NO：3或4所示的序列；(i) the sequence of SEQ ID NO: 3 or 4;

eg

本发明的Cas12h是一种在导向RNA引导下与靶序列特定位点结合并切割的核酸内切酶，同时具有DNA和RNA内切酶活性。The Cas12h of the present invention is an endonuclease which binds to a specific site of a target sequence and cleaves under the guidance of a guide RNA, and has both DNA and RNA endonuclease activity.

在本发明中，表述“Cas12w”是指，本发明人首次发现并鉴定的一种Cas效应蛋白，其具有选自下列的氨基酸序列：In the present invention, the expression "Cas12w" refers to a Cas effector protein first discovered and identified by the present inventors having an amino acid sequence selected from the group consisting of:

(i)SEQ ID NO：5或6所示的序列；(i) the sequence of SEQ ID NO: 5 or 6;

eg

本发明的Cas12w是一种在导向RNA引导下与靶序列特定位点结合并切割的核酸内切酶，同时具有DNA和RNA内切酶活性。The Cas12w of the present invention is an endonuclease which binds to a specific site of a target sequence and cleaves under the guidance of a guide RNA, and has both DNA and RNA endonuclease activity.

在本发明中，表述“Cas12j”是指，本发明人首次发现并鉴定的一种Cas效应蛋白，其具有选自下列的氨基酸序列：In the present invention, the expression "Cas12j" refers to a Cas effector protein first discovered and identified by the present inventors having an amino acid sequence selected from the group consisting of:

eg

本发明的Cas12j是一种在导向RNA引导下与靶序列特定位点结合并切割的核酸内切酶，同时具有DNA和RNA内切酶活性。The Cas12j of the present invention is an endonuclease which binds to a specific site of a target sequence and cleaves under the guidance of a guide RNA, and has both DNA and RNA endonuclease activity.

如本文中所使用的，术语“规律成簇的间隔短回文重复(CRISPR)-CRISPR-相关(Cas)(CRISPR-Cas)***”或“CRISPR***”可互换地使用并且具有本领域技术人员通常理解的含义，其通常包含与CRISPR相关(“Cas”)基因的表达有关的转录产物或其他元件，或者能够指导所述Cas基因活性的转录产物或其他元件。此类转录产物或其他元件可以包含编码Cas效应蛋白的序列和包含CRISPR RNA(crRNA)的导向RNA，以及在CRISPR-Cas9***中所含有的反式作用crRNA(tracrRNA)序列，或来自CRISPR基因座的其他序列或转录产物。As used herein, the term "regular clustered interspersed short palindrome repeat (CRISPR)-CRISPR-related (Cas) (CRISPR-Cas) system" or "CRISPR system" is used interchangeably and has skill in the art. A person generally understands the meaning, which typically includes a transcription product or other element associated with the expression of a CRISPR-associated ("Cas") gene, or a transcription product or other element capable of directing the activity of the Cas gene. Such transcription products or other elements may comprise a sequence encoding a Cas effector protein and a targeting RNA comprising CRISPR RNA (crRNA), and a trans-acting crRNA (tracrRNA) sequence contained in the CRISPR-Cas9 system, or from a CRISPR locus Other sequences or transcripts.

如本文中所使用的，术语“Cas效应蛋白”、“Cas效应酶”可互换地使用并且是指，C RISPR-Cas***中呈现的任一种大于长度900个氨基酸的蛋白质。在某些情况下，这类蛋白是指从Cas基因座中鉴定的蛋白。As used herein, the terms "Cas effector protein", "Cas effector enzyme" are used interchangeably and refer to any of the proteins presented in the CRISPR-Cas system that are greater than 900 amino acids in length. In some cases, such proteins refer to proteins identified from the Cas locus.

如本文中所使用的，术语“导向RNA(guide RNA)”、“成熟crRNA”可互换地使用并且具有本领域技术人员通常理解的含义。一般而言，导向RNA可以包含同向(direct)重复序列和导向序列(guide sequence)，或者基本上由或由同向重复序列和导向序列(在内源性CRISPR***背景下也称为间隔序列(spacer))组成。在某些情况下，导向序列是与靶序列具有足够互补性从而与所述靶序列杂交并引导CRISPR/Cas复合物与所述靶序列的特异性结合的任何多核苷酸序列。在某些实施方案中，当最佳比对时，导向序列与其相应靶序列之间的互补程度为至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、或至少99％。确定最佳比对在本领域的普通技术人员的能力范围内。例如，存在公开和可商购的比对算法和程序，诸如但不限于ClustalW、matlab中的史密斯-沃特曼算法(Smith-Waterman)、Bowtie、Geneious、Biopython以及SeqMan。As used herein, the terms "guide RNA", "mature crRNA" are used interchangeably and have the meaning as commonly understood by one of ordinary skill in the art. In general, the targeting RNA may comprise a direct repeat sequence and a guide sequence, or consist essentially of or consist of a homologous repeat sequence and a guide sequence (also referred to as a spacer sequence in the context of an endogenous CRISPR system). (spacer)) composition. In some cases, the targeting sequence is any polynucleotide sequence that is sufficiently complementary to the target sequence to hybridize to the target sequence and direct the specific binding of the CRISPR/Cas complex to the target sequence. In certain embodiments, when optimally aligned, the degree of complementarity between the targeting sequence and its corresponding target sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, Or at least 99%. Determining the optimal alignment is within the abilities of one of ordinary skill in the art. For example, there are publicly available and commercially available alignment algorithms and programs such as, but not limited to, ClustalW, Smith-Waterman in Matlab, Bowtie, Geneious, Biopython, and SeqMan.

在某些情况下，所述导向序列在长度上为至少5个、至少10个、至少15个、至少16个、至少17个、至少18个、至少19个、至少20个、至少21个、至少22个、至少 23个、至少24个、至少25个、至少26个、至少27个、至少28个、至少29个、至少30个、至少35个、至少40个、至少45个或至少50个核苷酸。在某些情况下，所述导向序列在长度上为不超过50个、45个、40个、35个、30个、25个、24个、23个、22个、21个、20个、15个、10个或更少个核苷酸。在某些实施方案中，所述导向序列在长度上为10-30个、或15-25个、或15-22个、或19-25个或19-22个核苷酸。In some cases, the targeting sequence is at least 5, at least 10, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 in length, At least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 35, at least 40, at least 45 or at least 50 Nucleotides. In some cases, the guide sequence is no more than 50, 45, 40, 35, 30, 25, 24, 23, 22, 21, 20, 15 in length. , 10 or fewer nucleotides. In certain embodiments, the targeting sequence is 10-30, or 15-25, or 15-22, or 19-25 or 19-22 nucleotides in length.

在某些情况下，所述同向重复序列在长度上为至少10个、至少15个、至少16个、至少17个、至少18个、至少19个、至少20个、至少21个、至少22个、至少23个、至少24个、至少25个、至少26个、至少27个、至少28个、至少29个、至少30个、至少35个、至少40个、至少45个、至少50个、至少55个、至少56个、至少57个、至少58个、至少59个、至少60个、至少61个、至少62个、至少63个、至少64个、至少65个或至少70个核苷酸。在某些情况下，所述同向重复序列在长度上为不超过70个、65个、64个、63个、62个、61个、60个、59个、58个、57个、56个、55个、50个、45个、40个、35个、30个、29个、28个、27个、26个、25个、24个、23个、22个、21个、20个、15个、10个或更少个核苷酸。在某些实施方案中，所述同向重复序列在长度上为55-70个核苷酸，例如55-65个核苷酸，例如60-65个核苷酸，例如62-65个核苷酸，例如63-64个核苷酸。在某些实施方案中，所述同向重复序列在长度上为15-30个核苷酸，例如15-25个核苷酸，例如20-25个核苷酸，例如22-24个核苷酸，例如23个核苷酸。In some cases, the isotropic repeats are at least 10, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22 in length. , at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 35, at least 40, at least 45, at least 50, At least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65 or at least 70 nucleotides . In some cases, the same direction repeat sequence is no more than 70, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56 in length. , 55, 50, 45, 40, 35, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 15 , 10 or fewer nucleotides. In certain embodiments, the isotropic repeat is 55-70 nucleotides in length, such as 55-65 nucleotides, such as 60-65 nucleotides, such as 62-65 nucleosides. Acid, for example 63-64 nucleotides. In certain embodiments, the isotropic repeat is 15-30 nucleotides in length, such as 15-25 nucleotides, such as 20-25 nucleotides, such as 22-24 nucleosides. Acid, for example 23 nucleotides.

如本文中所使用的，术语“CRISPR/Cas复合物”是指，导向RNA(guide RNA)或成熟crRNA与Cas蛋白结合所形成的核糖核蛋白复合体，其包含杂交到靶序列上并且与Cas蛋白结合的导向序列。该核糖核蛋白复合体能够识别并切割能与该导向RNA或成熟crRNA杂交的多核苷酸。As used herein, the term "CRISPR/Cas complex" refers to a ribonucleoprotein complex formed by the binding of a guide RNA or a mature crRNA to a Cas protein, which comprises hybridization to a target sequence and with Cas Protein-directed targeting sequences. The ribonucleoprotein complex is capable of recognizing and cleaving a polynucleotide that hybridizes to the targeting RNA or mature crRNA.

因此，在形成CRISPR/Cas复合物的情况下，“靶序列”是指被设计为具有靶向性的导向序列所靶向的多核苷酸，例如与该导向序列具有互补性的序列，其中靶序列与导向序列之间的杂交将促进CRISPR/Cas复合物的形成。完全互补性不是必需的，只要存在足够互补性以引起杂交并且促进一种CRISPR/Cas复合物的形成即可。靶序列可以包含任何多核苷酸，如DNA或RNA。在某些情况下，所述靶序列位于细胞的细胞核或细胞质中。在某些情况下，该靶序列可位于真核细胞的一个细胞器例如线粒体或叶绿体内。可被用于重组到包含该靶序列的靶基因座中的序列或模板被称为“编辑模板”或“编辑多核苷酸”或“编辑序列”。在某些实施方案中，所述编辑模板为外源核酸。在某些实施方案中，该重组是同源重组。Thus, in the context of the formation of a CRISPR/Cas complex, a "target sequence" refers to a polynucleotide that is designed to be targeted by a targeting sequence, such as a sequence that is complementary to the targeting sequence, wherein the target Hybridization between the sequence and the targeting sequence will promote the formation of the CRISPR/Cas complex. Complete complementarity is not required as long as sufficient complementarity exists to cause hybridization and promote the formation of a CRISPR/Cas complex. The target sequence can comprise any polynucleotide, such as DNA or RNA. In some cases, the target sequence is located in the nucleus or cytoplasm of the cell. In some cases, the target sequence can be located in an organelle of a eukaryotic cell, such as a mitochondria or chloroplast. Sequences or templates that can be used for recombination into a target locus comprising the target sequence are referred to as "editing templates" or "editing polynucleotides" or "editing sequences." In certain embodiments, the editing template is an exogenous nucleic acid. In certain embodiments, the recombination is homologous recombination.

在本发明中，表述“靶序列”或“靶多核苷酸”可以是对细胞(例如，真核细胞)而言任何内源或外源的多核苷酸。例如，该靶多核苷酸可以是一种存在于真核细胞的细胞核中的多核苷酸。该靶多核苷酸可以是一个编码基因产物(例如，蛋白质)的序列或一个非编码序列(例如，调节多核苷酸或无用DNA)。在某些情况下，据信该靶序列应该与原间隔序列临近基序(PAM)相关。对PAM的精确序列和长度要求取决于使用的Cas效应酶而不同，但是PAM典型地是临近原间隔序列(也即，靶序列)的2-5个碱基对序列。本领域技术人员能够鉴定与给定的Cas效应蛋白一起使用的PAM序列。In the present invention, the expression "target sequence" or "target polynucleotide" may be any endogenous or exogenous polynucleotide to a cell (eg, a eukaryotic cell). For example, the target polynucleotide can be a polynucleotide present in the nucleus of a eukaryotic cell. The target polynucleotide can be a sequence encoding a gene product (eg, a protein) or a non-coding sequence (eg, a regulatory polynucleotide or unwanted DNA). In some cases, it is believed that the target sequence should be associated with the original spacer sequence adjacent motif (PAM). The exact sequence and length requirements for PAM will vary depending on the Cas effector enzyme used, but PAM is typically a 2-5 base pair sequence adjacent to the original spacer sequence (ie, the target sequence). One skilled in the art will be able to identify PAM sequences for use with a given Cas effector protein.

在某些情况下，靶序列或靶多核苷酸可以包括多个疾病相关基因和多核苷酸以及信号传导生化途径相关基因和多核苷酸。此类靶序列或靶多核苷酸的非限制性实例，包括分别提交于2012年12月12日和2013年1月2日的美国临时专利申请61/736,527和61/748,427、提交于2013年12月12日的国际申请PCT/US2013/074667中所列举的那些，其全部通过引用并入本文。In some cases, the target sequence or target polynucleotide can include a plurality of disease associated genes and polynucleotides as well as signaling biochemical pathway related genes and polynucleotides. Non-limiting examples of such target sequences or target polynucleotides include U.S. Provisional Patent Application Nos. 61/736,527 and 61/748,427, filed on Dec. 12, 2012, and January 2, 2013, filed on Dec. Those listed in International Application No. PCT/US2013/074667, the entire contents of which is incorporated herein by reference.

在某些情况下，靶序列或靶多核苷酸的实例包括与信号传导生化途径相关的序列，例如信号传导生化途径相关基因或多核苷酸。靶多核苷酸的实例包括疾病相关基因或多核苷酸。“疾病相关”基因或多核苷酸是指与非疾病对照的组织或细胞相比，在来源于疾病影响的组织的细胞中以异常水平或以异常形式产生转录或翻译产物的任何基因或多核苷酸。在改变的表达与疾病的出现和/或进展相关的情况下，它可以是一个以异常高的水平被表达的基因；或者，它可以是一个以异常低的水平被表达的基因。疾病相关基因还指具有一个或多个突变或直接负责或与一个或多个负责疾病的病因学的基因连锁不平衡的遗传变异的基因。转录的或翻译的产物可以是已知的或未知的，并且可以处于正常或异常水平。In certain instances, examples of target sequences or target polynucleotides include sequences associated with a signaling biochemical pathway, such as a signaling biochemical pathway-related gene or polynucleotide. Examples of target polynucleotides include disease-related genes or polynucleotides. A "disease-associated" gene or polynucleotide refers to any gene or polynucleoside that produces a transcriptional or translational product at an abnormal level or in an abnormal form in a cell derived from a disease-affected tissue as compared to a non-disease-controlled tissue or cell. acid. Where the altered expression is associated with the appearance and/or progression of the disease, it may be a gene that is expressed at an abnormally high level; alternatively, it may be a gene that is expressed at an abnormally low level. A disease-associated gene also refers to a gene having one or more mutations or a genetic variation that is directly responsible for or incompatible with one or more genes responsible for the etiology of the disease. The transcribed or translated product may be known or unknown and may be at normal or abnormal levels.

如本文中所使用的，术语“野生型”具有本领域技术人员通常理解的含义，其表示生物、菌株、基因的典型形式或者当它在自然界存在时区别于突变体或变体形式的特征，其可从自然中的来源分离并且没有被人为有意地修饰。As used herein, the term "wild type" has the meaning commonly understood by those skilled in the art to mean a typical form of a organism, a strain, a gene, or a feature that distinguishes it from a mutant or variant when it exists in nature. It can be isolated from sources in nature and not intentionally modified.

如本文中所使用的，术语“非天然存在的”或“工程化的”可互换地使用并且表示人工的参与。当这些术语用于描述核酸分子或多肽时，其表示该核酸分子或多肽至少基本上从它们在自然界中或如发现于自然界中的与其结合的至少另一种组分游离出来。As used herein, the terms "non-naturally occurring" or "engineered" are used interchangeably and refer to artificial participation. When these terms are used to describe a nucleic acid molecule or polypeptide, it is meant that the nucleic acid molecule or polypeptide is at least substantially freed from at least one other component of its association in nature or as found in nature.

如本文中所使用的，术语“直系同源物(orthologue,ortholog)”具有本领域技术人员通常理解的含义。作为进一步指导，如本文中所述的蛋白质的“直系同源物”是指属于不同物种的蛋白质，该蛋白质执行与作为其直系同源物的蛋白相同或相似的功能。As used herein, the term "orthologue, ortholog" has the meaning as commonly understood by one of ordinary skill in the art. As a further guide, an "ortholog" of a protein as referred to herein refers to a protein belonging to a different species that performs the same or similar function as a protein as an ortholog thereof.

如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4:11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48:444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。As used herein, the term "identity" is used to mean the matching of sequences between two polypeptides or between two nucleic acids. When a position in the two sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a position in each of the two DNA molecules is occupied by adenine, or two Each position in each of the polypeptides is occupied by lysine, and then each molecule is identical at that position. The "percent identity" between the two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 of the 10 positions of the two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match). Typically, the comparison is made when the two sequences are aligned to produce maximum identity. Such alignment can be achieved by, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). It is also possible to use the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)) integrated into the ALIGN program (version 2.0), using the PAM 120 weight residue table. The gap length penalty of 12 and the gap penalty of 4 were used to determine the percent identity between the two amino acid sequences. In addition, the Needleman and Wunsch (J MoI Biol. 48: 444-453 (1970)) algorithms in the GAP program integrated into the GCG software package (available at www.gcg.com) can be used, using the Blossum 62 matrix or The PAM250 matrix and the gap weight of 16, 14, 12, 10, 8, 6 or 4 and the length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences .

如本文中所使用的，术语“载体”是指，可将多聚核苷酸***其中的一种核酸运载工具。当载体能使***的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、***瘤病毒、***多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。The term "vector," as used herein, refers to a nucleic acid vehicle into which a polynucleotide can be inserted. A vector is referred to as an expression vector when the vector enables expression of the protein encoded by the inserted polynucleotide. The vector can be introduced into the host cell by transformation, transduction or transfection, and the genetic material element carried thereby can be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1 derived artificial chromosomes (PAC). Phage such as lambda phage or M13 phage and animal virus. Animal viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, nipples Multi-tumor vacuolar virus (such as SV40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication.

如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as Escherichia coli or Bacillus subtilis, such as a fungal cell such as a yeast cell or an Aspergillus. For example, S2 Drosophila cells or insect cells such as Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

本领域技术人员将理解，表达载体的设计可取决于诸如待转化的宿主细胞的选择、所希望的表达水平等因素。一种载体可以被引入到宿主细胞中而由此产生转录物、蛋白质、或肽，包括由如本文所述的蛋白、融合蛋白、分离的核酸分子等(例如，CRISPR转录物，如核酸转录物、蛋白质、或酶)。One of skill in the art will appreciate that the design of the expression vector can depend on factors such as the choice of host cell to be transformed, the level of expression desired, and the like. A vector can be introduced into a host cell to thereby produce a transcript, protein, or peptide, including a protein, fusion protein, isolated nucleic acid molecule, etc. as described herein (eg, a CRISPR transcript, such as a nucleic acid transcript) , protein, or enzyme).

如本文中所使用的，术语“调节元件”旨在包括启动子、增强子、内部核糖体进入位点(IRES)、和其他表达控制元件(例如转录终止信号，如多聚腺苷酸化信号和多聚U序列)，其详细描述可参考戈德尔(Goeddel)，《基因表达技术：酶学方法》(GENE EXPRE SSION TECHNOLOGY:METHODS IN ENZYMOLOGY)185，学术出版社(Academic Press)，圣地亚哥(San Diego)，加利福尼亚州(1990)。在某些情况下，调节元件包括指导一个核苷酸序列在许多类型的宿主细胞中的组成型表达的那些序列以及指导该核苷酸序列只在某些宿主细胞中表达的那些序列(例如，组织特异型调节序列)。组织特异型启动子可主要指导在感兴趣的期望组织中的表达，所述组织例如肌肉、神经元、骨、皮肤、血液、特定的器官(例如肝脏、胰腺)、或特殊的细胞类型(例如淋巴细胞)。在某些情况下，调节元件还可以时序依赖性方式(如以细胞周期依赖性或发育阶段依赖性方式)指导表达，该方式可以是或者可以不是组织或细胞类型特异性的。在某些情况下，术语“调节元件”涵盖的是增强子元件，如WPRE；CMV增强子；在HTLV-I的LTR中的R-U5’片段((Mol.Cell.Biol.，第8(1)卷，第466-472页，1988)；SV40增强子；以及在兔β-珠蛋白的外显子2与3之间的内含子序列(Proc.Natl.Acad.Sci.USA.，第78(3)卷，第1527-31页，1981)。As used herein, the term "regulatory element" is intended to include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (eg, transcription termination signals, such as polyadenylation signals and Poly U sequence), a detailed description can be found in Goeddel, GENE EXPRE SSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego ), California (1990). In certain instances, regulatory elements include those sequences that direct constitutive expression of a nucleotide sequence in a plurality of types of host cells, as well as those sequences that direct expression of the nucleotide sequence only in certain host cells (eg, Tissue-specific regulatory sequence). Tissue-specific promoters can primarily direct expression in a desired tissue of interest, such as muscle, neurons, bone, skin, blood, specific organs (eg, liver, pancreas), or specific cell types (eg, Lymphocytes). In some cases, the regulatory elements may also direct expression in a time-dependent manner (eg, in a cell cycle dependent or developmental stage dependent manner), which may or may not be tissue or cell type specific. In some cases, the term "regulatory element" encompasses enhancer elements such as WPRE; CMV enhancer; R-U5' fragment in LTR of HTLV-I ((Mol. Cell. Biol., 8th ( 1) Vol., pp. 466-472, 1988); SV40 enhancer; and intron sequence between

exons

2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), pp. 1527-31, 1981).

如本文中所使用的，术语“启动子”具有本领域技术人员公知的含义，其是指一段位于基因的上游能启动下游基因表达的非编码核苷酸序列。组成型(constitutive)启动子是这样的核苷酸序列：当其与编码或者限定基因产物的多核苷酸可操作地相连时，在细胞的大多数或者所有生理条件下，其导致细胞中基因产物的产生。诱导型启动子是这样的核苷酸序列，当可操作地与编码或者限定基因产物的多核苷酸相连时，基本上只有当对应于所述启动子的诱导物在细胞中存在时，其导致所述基因产物在细胞内产生。组织特异性启动子是这样的核苷酸序列：当可操作地与编码或者限定基因产物的多核苷酸相连时，基本上只有当细胞是该启动子对应的组织类型的细胞时，其才导致在细胞中产生基因产物。As used herein, the term "promoter" has the meaning well-known to those skilled in the art and refers to a non-coding nucleotide sequence located upstream of the gene that initiates expression of the downstream gene. A constitutive promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in a gene product in the cell under most or all physiological conditions of the cell. The production. An inducible promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in substantially only when an inducer corresponding to the promoter is present in the cell The gene product is produced intracellularly. A tissue-specific promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, is substantially only caused when the cell is a cell of the tissue type corresponding to the promoter Gene products are produced in the cells.

如本文中所使用的，术语“可操作地连接”旨在表示感兴趣的核苷酸序列以一种允许该核苷酸序列的表达的方式被连接至该一种或多种调节元件(例如，处于一种体外转录/翻译***中或当该载体被引入到宿主细胞中时，处于该宿主细胞中)。As used herein, the term "operably linked" is intended to mean that a nucleotide sequence of interest is linked to the one or more regulatory elements in a manner that allows expression of the nucleotide sequence (eg, In an in vitro transcription/translation system or in the host cell when the vector is introduced into a host cell).

如本文中所使用的，术语“互补性”是指核酸与另一个核酸序列借助于传统的沃森-克里克或其他非传统类型形成一个或多个氢键的能力。互补百分比表示一个核酸分子中可与一个第二核酸序列形成氢键(例如，沃森-克里克碱基配对)的残基的百分比(例如，10个之中有5、6、7、8、9、10个即为50％、60％、70％、80％、90％、和100％互补)。“完全互补”表示一个核酸序列的所有连续残基与一个第二核酸序列中的相同数目的连续残基形成氢键。如本文使用的“基本上互补”是指在一个具有8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、30、35、40、45、50个或更多个核苷酸的区域上至少为60％、65％、70％、75％、80％、85％、90％、95％、97％、98％、99％、或100％的互补程度，或者是指在严格条件下杂交的两个核酸。As used herein, the term "complementarity" refers to the ability of a nucleic acid to form one or more hydrogen bonds with another nucleic acid sequence by means of conventional Watson-Crick or other non-traditional types. Percent complement indicates the percentage of residues in a nucleic acid molecule that can form a hydrogen bond (eg, Watson-Crick base pairing) with a second nucleic acid sequence (eg, 5, 6, 7, 8 out of 10) 9, 10, that is 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Completely complementary" means that all contiguous residues of one nucleic acid sequence form a hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. As used herein, "substantially complementary" means having 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, At least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98 in the region of 30, 35, 40, 45, 50 or more nucleotides %, 99%, or 100% complementarity, or two nucleic acids that hybridize under stringent conditions.

如本文中所使用的，对于杂交的“严格条件”是指与靶序列具有互补性的一个核酸主要地与该靶序列杂交并且基本上不杂交到非靶序列上的条件。严格条件通常是序列依赖性的，并且取决于许多因素而变化。一般而言，该序列越长，则该序列特异性地杂交到其靶序列上的温度就越高。严格条件的非限制性实例描述于蒂森(Tijssen)(1993)的《生物化学和分子生物学中的实验室技术-核酸探针杂交》(Laboratory Techniques In Biochemi stryAnd Molecular Biology-Hybridization With Nucleic Acid Probes)，第I部分，第二章，“杂交原理概述和核酸探针分析策略”(“Overview of principles of hybridization and the strategy of nucleic acid probe assay”)，爱思唯尔(Elsevier)，纽约。As used herein, "stringent conditions" for hybridization refers to conditions under which a nucleic acid that is complementary to a target sequence primarily hybridizes to the target sequence and does not substantially hybridize to a non-target sequence. Stringent conditions are usually sequence dependent and vary depending on many factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions are described in "Technology Techniques In Biochemi stry And Molecular Biology-Hybridization With Nucleic Acid Probes" by Tijssen (1993). ), Part I, Chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assay", Elsevier, New York.

如本文中所使用的，术语“杂交”是指其中一个或多个多核苷酸反应形成一种复合物的反应，该复合物经由这些核苷酸残基之间的碱基的氢键键合而稳定化。氢键键合可以借助于沃森-克里克碱基配对、Hoogstein结合或以任何其他序列特异性方式而发生。该复合物可包含形成一个双链体的两条链、形成多链复合物的三条或多条链、单个自我杂交链、或这些的任何组合。杂交反应可以构成一个更广泛的过程(如PCR的开始、或经由一种酶的多核苷酸的切割)中的一个步骤。能够与一个给定序列杂交的序列被称为该给定序列的“互补物”。As used herein, the term "hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that hydrogen bonds through the bases between these nucleotide residues. And stabilized. Hydrogen bonding can occur by means of Watson-Crick base pairing, Hoogstein binding or in any other sequence specific manner. The complex may comprise two chains forming one duplex, three or more chains forming a multi-strand complex, a single self-hybridizing strand, or any combination of these. The hybridization reaction can constitute a step in a broader process, such as the initiation of PCR, or the cleavage of a polynucleotide via an enzyme. A sequence that is capable of hybridizing to a given sequence is referred to as the "complement" of the given sequence.

如本文中所使用的，术语“表达”是指，藉此从DNA模板转录成多核苷酸(如转录成 mRNA或其他RNA转录物)的过程和/或转录的mRNA随后藉此翻译成肽、多肽或蛋白质的过程。转录物和编码的多肽可以总称为“基因产物”。如果多核苷酸来源于基因组DNA，表达可以包括真核细胞中mRNA的剪接。As used herein, the term "expression" refers to a process by which a DNA template is transcribed into a polynucleotide (eg, transcribed into mRNA or other RNA transcript) and/or transcribed mRNA, which is subsequently translated into a peptide, The process of a polypeptide or protein. The transcripts and encoded polypeptides may be collectively referred to as "gene products." If the polynucleotide is derived from genomic DNA, expression can include splicing of mRNA in eukaryotic cells.

如本文中所使用的，术语“接头”是指，由多个氨基酸残基通过肽键连接形成的线性多肽。本发明的接头可以为人工合成的氨基酸序列，或天然存在的多肽序列，例如具有铰链区功能的多肽。此类接头多肽是本领域众所周知的(参见例如，Holliger,P.等人(1993)Proc.Natl.Acad.Sci.USA 90:6444-6448；Poljak,R.J.等人(1994)Structure 2:1121-1123)。As used herein, the term "linker" refers to a linear polypeptide formed by the joining of multiple amino acid residues by peptide bonds. The linker of the invention may be a synthetic amino acid sequence, or a naturally occurring polypeptide sequence, such as a polypeptide having the function of a hinge region. Such linker polypeptides are well known in the art (see, for example, Holliger, P. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ et al. (1994) Structure 2: 1121- 1123).

如本文中所使用的，术语“治疗”是指，治疗或治愈病症，延缓病症的症状的发作，和/或延缓病症的发展。As used herein, the term "treating" refers to treating or curing a condition, delaying the onset of symptoms of the condition, and/or delaying the progression of the condition.

如本文中所使用的，术语“受试者”包括但不限于各种动物，例如哺乳动物，例如牛科动物、马科动物、羊科动物、猪科动物、犬科动物、猫科动物、兔科动物、啮齿类动物(例如，小鼠或大鼠)、非人灵长类动物(例如，猕猴或食蟹猴)或人。在某些实施方式中，所述受试者(例如人)患有病症(例如，疾病相关基因缺陷所导致的病症)。As used herein, the term "subject" includes, but is not limited to, various animals, such as mammals, such as bovine, equine, ovine, porcine, canine, feline, A rabbit, a rodent (eg, a mouse or rat), a non-human primate (eg, a macaque or a cynomolgus monkey) or a human. In certain embodiments, the subject (eg, a human) has a condition (eg, a condition caused by a disease-related gene defect).

发明的有益效果Advantageous effects of the invention

与现有技术相比，本发明的Cas蛋白及***具有显著的有利方面。例如，本发明的Cas效应蛋白的PAM结构域具有的5’-TG结构，可以与其他PAM为5’-TTN的***相互补充，扩大识别范围。例如，本发明的Cas效应蛋白能在真核生物体内进行DNA切割，在分子大小上比Cpf1和Cas9蛋白小约200-400个氨基酸，因此转染效率上明显优于Cpf1和Cas9。The Cas proteins and systems of the invention have significant advantages over the prior art. For example, the 5'-TG structure of the PAM domain of the Cas effector of the present invention can complement each other with a system in which the PAM is 5'-TTN, expanding the recognition range. For example, the Cas effector of the present invention is capable of DNA cleavage in eukaryotes and is about 200-400 amino acids smaller in size than Cpf1 and Cas9 proteins, and thus is significantly more efficient than Cpf1 and Cas9 in transfection efficiency.

附图说明DRAWINGS

图1为实施例3中Cas12h.1的PAM结构域分析结果。Figure 1 is a graph showing the results of PAM domain analysis of Cas12h.1 in Example 3.

序列信息Sequence information

本发明涉及的部分序列的信息提供于下面的表1中。Information on the partial sequences involved in the present invention is provided in Table 1 below.

表1：序列的描述Table 1: Description of the sequence

SEQ ID NO:SEQ ID NO:

描述description

11	Cas12g.1的氨基酸序列Amino acid sequence of Cas12g.1
22	Cas12g.2的氨基酸序列Amino acid sequence of Cas12g.2
33	Cas12h.1的氨基酸序列Amino acid sequence of Cas12h.1
44	Cas12h.2的氨基酸序列Amino acid sequence of Cas12h.2
55	Cas12w.1的氨基酸序列Amino acid sequence of Cas12w.1
66	Cas12w.2的氨基酸序列Amino acid sequence of Cas12w.2
77	Cas12j.1的氨基酸序列Amino acid sequence of Cas12j.1
88	Cas12j.2的氨基酸序列Amino acid sequence of Cas12j.2
99	Cas12j.3的氨基酸序列Amino acid sequence of Cas12j.3
1010	Cas12j.4的氨基酸序列Amino acid sequence of Cas12j.4
1111	Cas12j.5的氨基酸序列Amino acid sequence of Cas12j.5
1212	Cas12j.6的氨基酸序列Amino acid sequence of Cas12j.6
1313	Cas12j.7的氨基酸序列Amino acid sequence of Cas12j.7
1414	Cas12j.8的氨基酸序列Amino acid sequence of Cas12j.8
1515	Cas12j.9的氨基酸序列Amino acid sequence of Cas12j.9
1616	Cas12j.10的氨基酸序列Amino acid sequence of Cas12j.10
1717	Cas12j.11的氨基酸序列Amino acid sequence of Cas12j.11
1818	Cas12j.12的氨基酸序列Amino acid sequence of Cas12j.12
1919	Cas12g.1编码核酸序列Cas12g.1 encoding nucleic acid sequence
2020	Cas12g.2编码核酸序列Cas12g.2 encoding nucleic acid sequence
21twenty one	Cas12h.1编码核酸序列Cas12h.1 encoding nucleic acid sequence
22twenty two	Cas12h.2编码核酸序列Cas12h.2 encoding nucleic acid sequence
23twenty three	Cas12w.1编码核酸序列Cas12w.1 encoding nucleic acid sequence
24twenty four	Cas12w.2编码核酸序列Cas12w.2 encoding nucleic acid sequence
2525	Cas12j.1编码核酸序列Cas12j.1 encoding nucleic acid sequence
2626	Cas12j.2编码核酸序列Cas12j.2 encoding nucleic acid sequence
2727	Cas12j.3编码核酸序列Cas12j.3 encoding nucleic acid sequence
2828	Cas12j.4编码核酸序列Cas12j.4 encoding nucleic acid sequence
2929	Cas12j.5编码核酸序列Cas12j.5 encoding nucleic acid sequence
3030	Cas12j.6编码核酸序列Cas12j.6 encoding nucleic acid sequence
3131	Cas12j.7编码核酸序列Cas12j.7 encoding nucleic acid sequence
3232	Cas12j.8编码核酸序列Cas12j.8 encoding nucleic acid sequence
3333	Cas12j.9编码核酸序列Cas12j.9 encoding nucleic acid sequence
3434	Cas12j.10编码核酸序列Cas12j.10 encoding nucleic acid sequence
3535	Cas12j.11编码核酸序列Cas12j.11 encoding nucleic acid sequence
3636	Cas12j.12编码核酸序列Cas12j.12 encoding nucleic acid sequence
3737	Cas12g.1原型同向重复序列Cas12g.1 prototype isotropic repeat
3838	Cas12g.2原型同向重复序列Cas12g.2 prototype isotropic repeat
3939	Cas12h.1原型同向重复序列Cas12h.1 prototype isotropic repeat
4040	Cas12h.2原型同向重复序列Cas12h.2 prototype isotropic repeat
4141	Cas12w.1原型同向重复序列Cas12w.1 prototype isotropic repeat
4242	Cas12w.2原型同向重复序列Cas12w.2 prototype isotropic repeat

4343	Cas12j.1原型同向重复序列Cas12j.1 prototype isotropic repeat
4444	Cas12j.2原型同向重复序列Cas12j.2 prototype isotropic repeat
4545	Cas12j.3原型同向重复序列Cas12j.3 prototype isotropic repeat
4646	Cas12j.4原型同向重复序列Cas12j.4 prototype isotropic repeat
4747	Cas12j.5原型同向重复序列Cas12j.5 prototype isotropic repeat
4848	Cas12j.6原型同向重复序列Cas12j.6 prototype isotropic repeat
4949	Cas12j.7原型同向重复序列Cas12j.7 prototype isotropic repeat
5050	Cas12j.8原型同向重复序列Cas12j.8 prototype isotropic repeat
5151	Cas12j.9原型同向重复序列Cas12j.9 prototype isotropic repeat
5252	Cas12j.10原型同向重复序列Cas12j.10 prototype isotropic repeat
5353	Cas12j.11原型同向重复序列Cas12j.11 prototype isotropic repeat
5454	Cas12j.12原型同向重复序列Cas12j.12 prototype isotropic repeat
5555	Cas12g.1原型同向重复序列的编码核酸序列Encoding nucleic acid sequence of Cas12g.1 prototype homologous repeat
5656	Cas12g.2原型同向重复序列的编码核酸序列Encoding nucleic acid sequence of Cas12g.2 prototype homologous repeat
5757	Cas12h.1原型同向重复序列的编码核酸序列Encoding nucleic acid sequence of Cas12h.1 prototype homologous repeat
5858	Cas12h.2原型同向重复序列的编码核酸序列Encoding nucleic acid sequence of Cas12h.2 prototype homologous repeat
5959	Cas12w.1原型同向重复序列的编码核酸序列Encoding nucleic acid sequence of Cas12w.1 prototype homologous repeat
6060	Cas12w.2原型同向重复序列的编码核酸序列Encoding nucleic acid sequence of Cas12w.2 prototype homologous repeat
6161	Cas12j.1原型同向重复序列的编码核酸序列Encoding nucleic acid sequence of Cas12j.1 prototype homologous repeat
6262	Cas12j.2原型同向重复序列的编码核酸序列Encoding nucleic acid sequence of Cas12j.2 prototype homologous repeat
6363	Cas12j.3原型同向重复序列的编码核酸序列Encoding nucleic acid sequence of Cas12j.3 prototype homologous repeat
6464	Cas12j.4原型同向重复序列的编码核酸序列Encoding nucleic acid sequence of Cas12j.4 prototype homologous repeat
6565	Cas12j.5原型同向重复序列的编码核酸序列Encoding nucleic acid sequence of Cas12j.5 prototype homologous repeat
6666	Cas12j.6原型同向重复序列的编码核酸序列Encoding nucleic acid sequence of Cas12j.6 prototype homologous repeat
6767	Cas12j.7原型同向重复序列的编码核酸序列Encoding nucleic acid sequence of Cas12j.7 prototype homologous repeat
6868	Cas12j.8原型同向重复序列的编码核酸序列Encoding nucleic acid sequence of Cas12j.8 prototype homologous repeat
6969	Cas12j.9原型同向重复序列的编码核酸序列Encoding nucleic acid sequence of Cas12j.9 prototype homologous repeat
7070	Cas12j.10原型同向重复序列的编码核酸序列Encoding nucleic acid sequence of Cas12j.10 prototype homologous repeat
7171	Cas12j.11原型同向重复序列的编码核酸序列Encoding nucleic acid sequence of Cas12j.11 prototype homologous repeat
7272	Cas12j.12原型同向重复序列的编码核酸序列Encoding nucleic acid sequence of Cas12j.12 prototype homologous repeat
7373	NLS序列NLS sequence
7474	Cas12g.1-NLS融合蛋白的氨基酸序列Amino acid sequence of Cas12g.1-NLS fusion protein
7575	Cas12g.2-NLS融合蛋白的氨基酸序列Amino acid sequence of Cas12g.2-NLS fusion protein
7676	Cas12h.1-NLS融合蛋白的氨基酸序列Amino acid sequence of Cas12h.1-NLS fusion protein
7777	Cas12h.2-NLS融合蛋白的氨基酸序列Amino acid sequence of Cas12h.2-NLS fusion protein
7878	Cas12w.1-NLS融合蛋白的氨基酸序列Amino acid sequence of Cas12w.1-NLS fusion protein
7979	Cas12w.2-NLS融合蛋白的氨基酸序列Amino acid sequence of Cas12w.2-NLS fusion protein
8080	Cas12j.1-NLS融合蛋白的氨基酸序列Amino acid sequence of Cas12j.1-NLS fusion protein
8181	Cas12j.2-NLS融合蛋白的氨基酸序列Amino acid sequence of Cas12j.2-NLS fusion protein
8282	Cas12j.3-NLS融合蛋白的氨基酸序列Amino acid sequence of Cas12j.3-NLS fusion protein
8383	Cas12j.4-NLS融合蛋白的氨基酸序列Amino acid sequence of Cas12j.4-NLS fusion protein

8484	Cas12j.5-NLS融合蛋白的氨基酸序列Amino acid sequence of Cas12j.5-NLS fusion protein
8585	Cas12j.6-NLS融合蛋白的氨基酸序列Amino acid sequence of Cas12j.6-NLS fusion protein
8686	Cas12j.7-NLS融合蛋白的氨基酸序列Amino acid sequence of Cas12j.7-NLS fusion protein
8787	Cas12j.8-NLS融合蛋白的氨基酸序列Amino acid sequence of Cas12j.8-NLS fusion protein
8888	Cas12j.9-NLS融合蛋白的氨基酸序列Amino acid sequence of Cas12j.9-NLS fusion protein
8989	Cas12j.10-NLS融合蛋白的氨基酸序列Amino acid sequence of Cas12j.10-NLS fusion protein
9090	Cas12j.11-NLS融合蛋白的氨基酸序列Amino acid sequence of Cas12j.11-NLS fusion protein
9191	Cas12j.12-NLS融合蛋白的氨基酸序列Amino acid sequence of Cas12j.12-NLS fusion protein
9292	Cas12g.1***表达盒的核苷酸序列Nucleotide sequence of the Cas12g.1 system expression cassette
9393	PAM文库序列PAM library sequence
9494	Cas12h.1***表达盒的核苷酸序列Nucleotide sequence of Cas12h.1 system expression cassette
95-11295-112	SEQ ID NOs:74-91所示融合蛋白的编码核酸序列Encoding nucleic acid sequence of the fusion protein shown in SEQ ID NOs: 74-91

具体实施方式detailed description

现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。The invention is described with reference to the following examples which are intended to illustrate, but not limit the invention.

除非特别指明，否则基本上按照本领域内熟知的以及在各种参考文献中描述的常规方法进行实施例中描述的实验和方法。例如，本发明中所使用的免疫学、生物化学、化学、分子生物学、微生物学、细胞生物学、基因组学和重组DNA等常规技术，可参见参见萨姆布鲁克(Sambrook)、弗里奇(Fritsch)和马尼亚蒂斯(Maniatis)，《分子克隆：实验室手册》(MOLECULAR CLONING:A LABORATORY MANUAL)，第2次编辑(1989)；《当代分子生物学实验手册》(CURRENT PROTOCOLS IN MOLECULAR BIOLOGY)(F.M.奥苏贝尔(F.M.Ausubel)等人编辑，(1987))；《酶学方法》(METHODS IN ENZYMOLOGY)系列(学术出版公司)：《PCR 2：实用方法》(PCR 2:A PRACTICAL APPROACH)(M.J.麦克弗森(M.J.MacPherson)、B.D.黑姆斯(B.D.Hames)和G.R.泰勒(G.R.Taylor)编辑(1995))、哈洛(Harlow)和拉内(Lane)编辑(1988)《抗体：实验室手册》(ANTIBODIES,A LABORATORY MANUAL)，以及《动物细胞培养》(ANIMAL CELL CULTURE)(R.I.弗雷谢尼(R.I.Freshney)编辑(1987))。The experiments and methods described in the Examples are carried out essentially according to conventional methods well known in the art and described in various references, unless otherwise indicated. For example, conventional techniques such as immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA used in the present invention can be found in Sambrook, Fry ( Fritsch) and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY) (FMAubel et al., (1987)); METHOS IN ENZYMOLOGY series (academic publishing company): "PCR 2: Practical Methods" (PCR 2: A PRACTICAL) APPROACH) (MJ MacPherson, BD Hames, and GR Taylor (1995)), Harlow, and Lane Editor (1988) : ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (RI Freshney, eds. (1987)).

另外，实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。本文中提及的全部公开案和其他参考资料以其全文通过引用合并入本文。In addition, those which do not specify the specific conditions in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained commercially. The invention is described by way of example, and is not intended to limit the scope of the invention. All publications and other references mentioned herein are incorporated by reference in their entirety.

以下实施例涉及的部分试剂的来源如下：The sources of some of the reagents involved in the following examples are as follows:

LB液体培养基：10g胰蛋白胨(Tryptone)，5g酵母提取物(Yeast Extract)，10g NaCl，定容至1L，灭菌。若需加抗生素，则待培养基冷却后加，50μg/ml的终浓度。LB liquid medium: 10 g tryptone, 5 g yeast extract (Yeast Extract), 10 g NaCl, made up to 1 L, sterilized. If antibiotics are required, add the final concentration of 50 μg/ml after the medium is cooled.

氯仿/异戊醇：240ml的氯仿加10ml的异戊醇，混匀。Chloroform / isoamyl alcohol: 240 ml of chloroform plus 10 ml of isoamyl alcohol, and mix.

RNP缓冲液：100mM氯化钠,50mM Tris-HCl,10mM MgCl ₂,100μg/ml BSA,pH 7.9。 RNP buffer: 100 mM sodium chloride, 50 mM Tris-HCl, 10 mM MgCl ₂ , 100 μg/ml BSA, pH 7.9.

原核表达载体pACYC-Duet-1和pUC19购自北京全式金生物技术有限公司。The prokaryotic expression vectors pACYC-Duet-1 and pUC19 were purchased from Beijing Quanjin Biotechnology Co., Ltd.

大肠杆菌感受态EC100购自Epicentre公司。E. coli competent EC100 was purchased from Epicentre.

除非特别指明，以下实施例中涉及的序列合成均由南京金斯瑞生物科技有限公司完成，涉及的测序均由上海英骏生物技术有限公司完成。Unless otherwise specified, the sequence synthesis involved in the following examples was completed by Nanjing Kingsray Biotechnology Co., Ltd., and the sequencing involved was completed by Shanghai Yingjun Biotechnology Co., Ltd.

实施例1.Cas12g基因和Cas12g导向RNA的获得Example 1. Acquisition of Cas12g gene and Cas12g targeting RNA

1、CRISPR和基因的注释：使用Prodigal对将NCBI和JGI数据库的微生物基因组和宏基因组数据进行基因注释得到所有蛋白，同时用Piler-CR进行CRISPR座的注释，参数均为默认参数。1. CRISPR and gene annotation: Prodigal was used to genetically annotate the microbial genome and metagenomic data of the NCBI and JGI databases to obtain all proteins, and the Pyrer-CR was used to annotate the CRISPR block. The parameters are default parameters.

2、蛋白质的过滤：通过序列一致性对注释蛋白去冗余，去除序列完全一致的蛋白，同时将长度大于800个氨基酸的蛋白划分为大分子蛋白。由于目前发现的所有第二类CRISPR/Cas***的效应蛋白长度多大于900个氨基酸，所以为了降低计算复杂度，我们在挖掘CRISPR效应蛋白的时候只对大分子蛋白进行考虑。2. Protein filtration: The sequence protein is de-redundant by sequence identity, and the proteins with completely identical sequences are removed, and proteins with a length of more than 800 amino acids are classified into macromolecular proteins. Since all second-generation CRISPR/Cas systems have effector proteins longer than 900 amino acids, in order to reduce computational complexity, we only consider macromolecular proteins when mining CRISPR effector proteins.

3、CRISPR相关大分子蛋白的获得：将每一个CRISPR座上下游延伸10Kb，将对CRISPR邻近区间内的非冗余大分子蛋白进行鉴定。3. Acquisition of CRISPR-related macromolecular proteins: Each of the CRISPR blocks is extended 10Kb upstream and downstream, and non-redundant macromolecular proteins in the proximity region of CRISPR will be identified.

4、CRISPR相关大分子蛋白质的聚类：使用BLASTP对非冗余大分子CRISPR相关蛋白进行内部的两两比对，输出Evalue<1E-10的比对结果。使用MCL对BLASTP的输出结果进行聚类分析，CRISPR相关蛋白质家族。4. Clustering of CRISPR-related macromolecular proteins: BLASTP was used to perform internal pairwise alignment of non-redundant macromolecular CRISPR-related proteins, and the results of the alignment of Evalue<1E-10 were output. The MCL was used to cluster the output of BLASTP, a family of CRISPR-related proteins.

5、CRISPR富集大分子蛋白质家族的鉴定：使用BLASTP对CRISPR相关蛋白质家族的蛋白比对到去除去CRISPR相关蛋白的非冗余大分子蛋白数据库，输出Evalue<1E-10的比对结果。如果一个非CRISPR相关蛋白数据库发现的同源蛋白小于100％，那么则说明这个家族的蛋白在CRISPR区域是富集的，通过这种方法我们对CRISPR富集大分子蛋白质家族进行鉴定。5. Identification of CRISPR-enriched macromolecular protein families: BLASTP was used to compare the proteins of the CRISPR-related protein family to a database of non-redundant macromolecular proteins from which the CRISPR-related proteins were removed, and the results of the alignment of Evalue<1E-10 were output. If a non-CRISPR-related protein database finds less than 100% homologous protein, then this family of proteins is enriched in the CRISPR region, and we have identified the CRISPR-rich macromolecular protein family.

6、蛋白功能和结构域的注释：利用Pfam数据库，NR数据库以及从NCBI收集的Cas蛋白对CRISPR富集大分子蛋白质家族进行注释，得到新的CRISPR/Cas蛋白质家族。利用Mafft对每个CRISPR/Cas家族蛋白进行多重序列比对，然后用JPred和HHpred进行保守结构域分析，鉴定含有RuvC结构域的蛋白质家族。6. Notes on protein function and domain: The CRISPR/Cas protein family was obtained by annotating the CRISPR-rich macromolecular protein family using the Pfam database, the NR database, and the Cas protein collected from NCBI. Multiple sequence alignments of each CRISPR/Cas family protein were performed using Mafft, followed by conserved domain analysis with JPred and HHpred to identify a family of proteins containing the RuvC domain.

在此基础上，本发明人获得了一种全新的Cas效应蛋白，即Cas12g，其包括2种活性同源物序列，分别命名为Cas12g.1(SEQ ID NO:1)、Cas12g.2(SEQ ID NO:2)，两种同源物的编码DNA分别如SEQ ID NOs:19、20所示。Cas12g.1、Cas12g.2所对应的原型同向重复序列(pre-crRNA中所含有的repeat序列)分别如SEQ ID NOs:37、38所示。On this basis, the inventors obtained a novel Cas effector protein, Cas12g, which includes two active homolog sequences, named Cas12g.1 (SEQ ID NO: 1) and Cas12g. 2 (SEQ. ID NO: 2), the coding DNAs of the two homologs are shown in SEQ ID NOs: 19, 20, respectively. The prototype homologous repeat sequences (repeat sequences contained in the pre-crRNA) corresponding to Cas12g.1 and Cas12g.2 are shown in SEQ ID NOs: 37 and 38, respectively.

实施例2.Cas12h基因和Cas12h导向RNA的获得Example 2. Acquisition of Cas12h gene and Cas12h targeting RNA

6、蛋白功能和结构域的注释：利用Pfam数据库，NR数据库以及从NCBI收集的Cas蛋白对CRISPR富集大分子蛋白质家族进行注释，得到新的CRISPR/Cas蛋白质家族。利用Mafft对每个CRISPR/Cas家族蛋白进行多重序列比对，然后用JPred和HHpred进行保守结构域分析，鉴定含有RuvC结构域的蛋白质家族。6. Notes on protein function and domain: The CRISPR/Cas protein family was obtained by annotating the CRISPR-enriched macromolecular protein family using the Pfam database, the NR database, and the Cas protein collected from NCBI. Multiple sequence alignments of each CRISPR/Cas family protein were performed using Mafft, followed by conserved domain analysis with JPred and HHpred to identify a family of proteins containing the RuvC domain.

在此基础上，本发明人获得了一种全新的Cas效应蛋白，即Cas12h，其包括2种活性同源物序列，分别命名为Cas12h.1(SEQ ID NO:3)、Cas12h.2(SEQ ID NO:4)，两种同源物的编码DNA分别如SEQ ID NOs:21、22所示。Cas12h.1、Cas12h.2所对应的原型同向重复序列(pre-crRNA中所含有的repeat序列)分别如SEQ ID NOs:39、40所示。On this basis, the inventors obtained a novel Cas effector protein, Cas12h, which includes two active homolog sequences, named Cas12h.1 (SEQ ID NO: 3) and Cas12h.2 (SEQ. ID NO: 4), the coding DNAs of the two homologs are shown in SEQ ID NOs: 21, 22, respectively. The prototype homologous repeat sequences (repeat sequences contained in the pre-crRNA) corresponding to Cas12h.1 and Cas12h.2 are shown in SEQ ID NOs: 39 and 40, respectively.

实施例3.Cas12h.1蛋白的PAM结构域鉴定Example 3. Identification of PAM domain of Cas12h.1 protein

1.构建重组质粒pACYC-Duet-1+CRISPR/Cas12h.1并测序。根据测序结果，对重组质粒pACYC-Duet-1+CRISPR/Cas12h.1进行结构描述如下：将载体pACYC-Duet-1的限制性内切酶Pml I和Kpn I识别序列间的小片段替换为SEQ ID NO:21所示的序列中自5’末端起第1至2664位所示的双链DNA分子。重组质粒pACYC-Duet-1+CRISPR/Cas12h表达SEQ ID NO:3所示的Cas12h.1蛋白和SEQ ID NO:39所示的Cas12h.1导向RNA。1. Construction of recombinant plasmid pACYC-Duet-1+CRISPR/Cas12h.1 and sequencing. Based on the sequencing results, the recombinant plasmid pACYC-Duet-1+CRISPR/Cas12h.1 was structurally described as follows: a small fragment between the restriction endonuclease Pml I and Kpn I recognition sequences of the vector pACYC-Duet-1 was replaced with SEQ ID NO: The double-stranded DNA molecule shown in positions 1 to 2664 from the 5' end in the sequence shown by 21. The recombinant plasmid pACYC-Duet-1+CRISPR/Cas12h expresses the Cas12h.1 protein shown in SEQ ID NO: 3 and the Cas12h.1 targeting RNA shown in SEQ ID NO:39.

2.重组质粒pACYC-Duet-1+CRISPR/Cas12h.1中含有表达盒，该表达盒的核苷酸序列如SEQ ID NO:94所示。SEQ ID NO:94所示的序列中，自5’末端起第1至44位为pLacZ启动子的核苷酸序列，第45至2708位为Cas12h.1基因的核苷酸序列，第2709至2794位为终止子的核苷酸序列(用于终止转录)。自5’末端起第2795至2834位为J23119启动子的核苷酸序列，第2835至3007位为CRISPR阵列的核苷酸序列，第3008至3093位为rrnB-T1终止子的核苷酸序列(用于终止转录)。2. The recombinant plasmid pACYC-Duet-1+CRISPR/Cas12h.1 contains an expression cassette, and the nucleotide sequence of the expression cassette is shown in SEQ ID NO:94. In the sequence shown in SEQ ID NO: 94, the nucleotide sequence of the pLacZ promoter from positions 1 to 44 from the 5' end, and the nucleotide sequence of the Cas12h.1 gene from positions 45 to 2708, from page 2709 to The 2794 position is the nucleotide sequence of the terminator (used to terminate transcription). From the 5' end, positions 2795 to 2834 are the nucleotide sequence of the J23119 promoter, positions 2835 to 3007 are the nucleotide sequence of the CRISPR array, and positions 3008 to 3093 are the nucleotide sequence of the rrnB-T1 terminator. (used to terminate transcription).

3.重组大肠杆菌的获得：将重组质粒pACYC-Duet-1+CRISPR/Cas12h.1导入大肠杆菌EC100中，得到重组大肠杆菌，命名为EC100/pACYC-Duet-1+CRISPR/Cas12h.1。将重组质粒pACYC-Duet-1导入大肠杆菌EC100中，得到重组农杆菌，命名为EC100/pACYC-Duet-1。3. Recombination of recombinant E. coli: The recombinant plasmid pACYC-Duet-1+CRISPR/Cas12h.1 was introduced into Escherichia coli EC100 to obtain recombinant Escherichia coli, named EC100/pACYC-Duet-1+CRISPR/Cas12h.1. The recombinant plasmid pACYC-Duet-1 was introduced into Escherichia coli EC100 to obtain recombinant Agrobacterium, and was named EC100/pACYC-Duet-1.

4.PAM文库的构建：人工合成SEQ ID NO:93所示的序列，并连接到pUC19载体，其中SEQ ID NO:93所示的序列包括5’端八个随机碱基和靶序列。对PAM文库的靶标序列5’端前面设计了8个随机碱基构建质粒文库。将质粒分别转入到含有Cas12h.1基因座的大肠杆菌中和不含有Cas.12h.1基因座的大肠杆菌中。在37℃下处理1小时后，我们对质粒进行提取，并对PAM区域序列进行PCR扩增和测序。4. Construction of a PAM library: The sequence shown in SEQ ID NO: 93 was artificially synthesized and ligated into the pUC19 vector, wherein the sequence set forth in SEQ ID NO: 93 includes eight random bases and a target sequence at the 5' end. Eight random base construction plasmid libraries were designed in front of the 5' end of the target sequence of the PAM library. The plasmids were separately transferred into E. coli containing the Cas12h.1 locus and E. coli containing no Cas.12h.1 locus. After 1 hour of treatment at 37 ° C, we extracted the plasmid and PCR amplification and sequencing of the PAM region sequence.

5.PAM文库结构域的获得：分别统计实验组和对照组中65,536种组合的PAM序列出现次数，并用各自组所有的PAM序列数目进行标准化。对于任意一条PAM序列，当 log2(对照组标准化值/实验组标准化值)大于2时，认为这条PAM被显著消耗。用Weblogo对显著消耗的PAM序列进行预测，结果如图1所示，Cas12h.1的PAM结构域为5’-TG结构，这种PAM可以与其他PAM为5’-TTN的***相互补充，扩大识别范围。5. Acquisition of PAM library domain: The number of occurrences of 65,536 combinations of PAM sequences in the experimental and control groups were separately counted and normalized by the number of all PAM sequences in each group. For any one PAM sequence, when log2 (control group normalized value / experimental group normalized value) is greater than 2, this PAM is considered to be significantly consumed. The significantly consumed PAM sequence was predicted by Weblogo. The results are shown in Figure 1. The PAM domain of Cas12h.1 is a 5'-TG structure. This PAM can complement other systems with PAM 5'-TTN. Recognition range.

尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。While the invention has been described in detail, it will be understood by the . The invention is generally divided into the appended claims and any equivalents thereof.

Claims

一种蛋白，其具有SEQ ID NOs:1-18任一项所示的氨基酸序列或其直系同源物(ortholog)、同源物、变体或功能性片段；其中，所述直系同源物、同源物、变体或功能性片段基本保留了其所源自的序列的生物学功能；A protein having the amino acid sequence set forth in any one of SEQ ID NOs: 1-18, or an ortholog, homolog, variant or functional fragment thereof; wherein the ortholog a homologue, variant or functional fragment substantially retains the biological function of the sequence from which it is derived;

例如，所述直系同源物、同源物、变体与其所源自的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性，并且基本保留了其所源自的序列的生物学功能；For example, the ortholog, homolog, variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94 compared to the sequence from which it is derived. %, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, and substantially retains the biological function of the sequence from which it is derived;

例如，所述直系同源物、同源物、变体与SEQ ID NOs：1-18任一项所示的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性，并且基本保留了其所源自的序列的生物学功能；For example, the ortholog, homolog, variant has at least 80%, at least 85%, at least 90%, at least 91%, at least compared to the sequence set forth in any one of SEQ ID NOs: 1-18. 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity and substantially retains the biological function of the sequence from which it is derived ;

例如，所述蛋白具有Cas效应蛋白活性；For example, the protein has Cas effector activity;

例如，所述蛋白是CRISPR/Cas***中的效应蛋白。For example, the protein is an effector protein in the CRISPR/Cas system.
权利要求1所述的蛋白，其来自选自下列的物种：Sulfuricurvum sp、Omnitrophica WOR_2、Smithella sp和Agrobacterium sp；The protein of claim 1 which is derived from a species selected from the group consisting of Sulfuricurvum sp, Omnitrophica WOR 2, Smithella sp and Agrobacterium sp;

例如，所述蛋白为包含于选自下列的物种的CRISPR基因座(CRISPR locus)中的Cas效应蛋白：Sulfuricurvum sp、Omnitrophica WOR_2、Smithella sp和Agrobacterium sp；For example, the protein is a Cas effector protein contained in a CRISPR locus selected from the following species: Sulfuricurvum sp, Omnitrophica WOR_2, Smithella sp and Agrobacterium sp;

例如，所述蛋白具有SEQ ID NOs：5-18任一项所示的氨基酸序列或其直系同源物、同源物、变体或功能性片段；其中，所述直系同源物、同源物、变体或功能性片段基本保留了其所源自的序列的Cas效应蛋白活性；For example, the protein has the amino acid sequence set forth in any one of SEQ ID NOs: 5-18 or an ortholog, homolog, variant or functional fragment thereof; wherein the ortholog, homolog a variant, or a functional fragment substantially retains the Cas effector activity of the sequence from which it is derived;

例如，所述直系同源物、同源物、变体与SEQ ID NOs：5-18任一项所示的氨基酸序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性，并且基本保留了其所源自的序列的Cas效应蛋白活性。For example, the ortholog, homologue, variant has at least 80%, at least 85%, at least 90%, at least 91%, compared to the amino acid sequence set forth in any one of SEQ ID NOs: 5-18. At least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, and substantially retains the Cas effect of the sequence from which it is derived Protein activity.
权利要求1所述的蛋白，其包含选自下列的序列，或由选自下列的序列组成：The protein of claim 1 comprising a sequence selected from the group consisting of: or consisting of a sequence selected from the group consisting of:

(i)SEQ ID NOs：1-18任一项所示的序列；(i) the sequence set forth in any one of SEQ ID NOs: 1-18;

(ii)与SEQ ID NOs：1-18任一项所示的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)的序列；或(ii) a substitution, deletion or addition of one or more amino acids compared to the sequence set forth in any one of SEQ ID NOs: 1-18 (eg 1, 2, 3, 4, 5, 6 a sequence of 7, 7 or 9 amino acid substitutions, deletions or additions; or

(iii)与SEQ ID NOs：1-18任一项所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性的序列；(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% of the sequence set forth in any one of SEQ ID NOs: 1-18 a sequence of at least 96%, at least 97%, at least 98%, or at least 99% sequence identity;

例如，所述蛋白具有SEQ ID NOs：1-18任一项所示的氨基酸序列。For example, the protein has the amino acid sequence set forth in any one of SEQ ID NOs: 1-18.
权利要求1所述的蛋白，其包含选自下列的序列，或由选自下列的序列组成：The protein of claim 1 comprising a sequence selected from the group consisting of: or consisting of a sequence selected from the group consisting of:

(i)SEQ ID NO：3所示的序列；(i) the sequence shown as SEQ ID NO:3;

(ii)与SEQ ID NO：3所示的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加)的序列；或(ii) having one or more amino acid substitutions, deletions or additions compared to the sequence set forth in SEQ ID NO: 3 (eg 1, 2, 3, 4, 5, 6, 7) a sequence of 8, 9 or 10 amino acid substitutions, deletions or additions; or

(iii)与SEQ ID NO：3所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的序列同一性的序列；(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, and the sequence set forth in SEQ ID NO: a sequence of at least 97%, at least 98%, or at least 99% sequence identity;

例如，所述蛋白具有SEQ ID NO：3所示的氨基酸序列。For example, the protein has the amino acid sequence set forth in SEQ ID NO:3.
一种缀合物，其包含权利要求1-4任一项所述的蛋白以及修饰部分；A conjugate comprising the protein of any one of claims 1 to 4 and a modified moiety;

例如，所述修饰部分选自另外的蛋白或多肽、可检测的标记，及其任意组合；For example, the modified moiety is selected from additional proteins or polypeptides, detectable labels, and any combination thereof;

例如，所述修饰部分任选地通过接头连接至所述蛋白的N端或C端；For example, the modified moiety is optionally linked to the N-terminus or C-terminus of the protein via a linker;

例如，所述修饰部分融合至所述蛋白的N端或C端；For example, the modified moiety is fused to the N-terminus or C-terminus of the protein;

例如，所述另外的蛋白或多肽选自表位标签、报告基因序列、核定位信号(NLS)序列、靶向部分、转录激活结构域(例如，VP64)、转录抑制结构域(例如，KRAB结构域或SID结构域)、核酸酶结构域(例如，Fok1)，具有选自下列的活性的结构域：甲基化酶活性,去甲基化酶,转录激活活性,转录抑制活性,转录释放因子活性,组蛋白修饰活性,核酸酶活性,单链RNA切割活性,双链RNA切割活性,单链DNA切割活性,双链DNA切割活性和核酸结合活性；以及其任意组合；For example, the additional protein or polypeptide is selected from the group consisting of an epitope tag, a reporter gene sequence, a nuclear localization signal (NLS) sequence, a targeting moiety, a transcriptional activation domain (eg, VP64), a transcriptional repression domain (eg, a KRAB structure) a domain or SID domain), a nuclease domain (eg, Fok1), having a domain selected from the group consisting of methylase activity, demethylase, transcriptional activation activity, transcriptional repression activity, transcriptional release factor Activity, histone modification activity, nuclease activity, single-strand RNA cleavage activity, double-stranded RNA cleavage activity, single-strand DNA cleavage activity, double-strand DNA cleavage activity and nucleic acid binding activity; and any combination thereof;

例如，所述缀合物包含表位标签；For example, the conjugate comprises an epitope tag;

例如，所述缀合物包含NLS序列；For example, the conjugate comprises an NLS sequence;

例如，所述NLS序列如SEQ ID NO:73所示；For example, the NLS sequence is set forth in SEQ ID NO:73;

例如，所述NLS序列位于、靠近或接近所述蛋白的末端(例如，N端或C端)。For example, the NLS sequence is located at, near, or near the end of the protein (eg, the N-terminus or the C-terminus).
一种融合蛋白，其包含权利要求1-4任一项所述的蛋白以及另外的蛋白或多肽；A fusion protein comprising the protein of any of claims 1-4 and an additional protein or polypeptide;

例如，所述另外的蛋白或多肽任选地通过接头连接至所述蛋白的N端或C端；For example, the additional protein or polypeptide is optionally linked to the N-terminus or C-terminus of the protein via a linker;

例如，所述另外的蛋白或多肽选自表位标签、报告基因序列、核定位信号(NLS)序列、靶向部分、转录激活结构域(例如，VP64)、转录抑制结构域(例如，KRAB结构域或SID结构域)、核酸酶结构域(例如，Fok1)，具有选自下列的活性的结构域：甲基化酶活性,去甲基化酶,转录激活活性,转录抑制活性,转录释放因子活性,组蛋白修饰活性,核酸酶活性,单链RNA切割活性,双链RNA切割活性,单链DNA切割活性,双链DNA切割活性和核酸结合活性；以及其任意组合；For example, the additional protein or polypeptide is selected from the group consisting of an epitope tag, a reporter gene sequence, a nuclear localization signal (NLS) sequence, a targeting moiety, a transcriptional activation domain (eg, VP64), a transcriptional repression domain (eg, a KRAB structure) a domain or SID domain), a nuclease domain (eg, Fok1), having a domain selected from the group consisting of methylase activity, demethylase, transcriptional activation activity, transcriptional repression activity, transcriptional release factor Activity, histone modification activity, nuclease activity, single-strand RNA cleavage activity, double-stranded RNA cleavage activity, single-strand DNA cleavage activity, double-strand DNA cleavage activity and nucleic acid binding activity; and any combination thereof;

例如，所述融合蛋白包含表位标签；For example, the fusion protein comprises an epitope tag;

例如，所述融合蛋白包含NLS序列；For example, the fusion protein comprises an NLS sequence;

例如，所述NLS序列如SEQ ID NO:73所示；For example, the NLS sequence is set forth in SEQ ID NO:73;

例如，所述NLS序列位于、靠近或接近所述蛋白的末端(例如，N端或C端)；For example, the NLS sequence is located at, near, or near the end of the protein (eg, the N-terminus or the C-terminus);

例如，所述融合蛋白具有选自下列的氨基酸序列：SEQ ID NOs:74-91。For example, the fusion protein has an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-91.
一种分离的核酸分子，其包含选自下列的序列，或由选自下列的序列组成：An isolated nucleic acid molecule comprising a sequence selected from the group consisting of: or consisting of a sequence selected from the group consisting of:

(i)SEQ ID NOs：37-54任一项所示的序列；(i) the sequence set forth in any one of SEQ ID NOs: 37-54;

(ii)与SEQ ID NOs：37-54任一项所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；(ii) having one or more base substitutions, deletions or additions as compared to the sequence set forth in any one of SEQ ID NOs: 37-54 (eg, 1, 2, 3, 4, 5, a sequence of 6, 7, 8, or 10 base substitutions, deletions, or additions;

(iii)与SEQ ID NOs：37-54任一项所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；(iii) having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the sequence set forth in any one of SEQ ID NOs: 37-54 a sequence of at least 95% sequence identity;

(iv)在严格条件下与(i)-(iii)任一项中所述的序列杂交的序列；或(iv) a sequence that hybridizes under stringent conditions to the sequence set forth in any one of (i)-(iii); or

(v)(i)-(iii)任一项中所述的序列的互补序列；(v) the complement of the sequence set forth in any of (i)-(iii);

并且，(ii)-(v)中任一项所述的序列基本保留了其所源自的序列的生物学功能；Furthermore, the sequence of any one of (ii)-(v) substantially retains the biological function of the sequence from which it is derived;

例如，所述核酸分子包含一个或多个茎环或优化的二级结构；For example, the nucleic acid molecule comprises one or more stem loops or an optimized secondary structure;

例如，(ii)-(v)中任一项所述的序列保留了其所源自的序列的二级结构；For example, the sequence of any of (ii)-(v) retains the secondary structure of the sequence from which it is derived;

例如，所述核酸分子包含选自下列的序列，或由选自下列的序列组成：For example, the nucleic acid molecule comprises a sequence selected from the group consisting of: or consists of a sequence selected from the group consisting of:

(a)SEQ ID NOs：37-54任一项所示的核苷酸序列；(a) the nucleotide sequence set forth in any one of SEQ ID NOs: 37-54;

(b)在严格条件下与(a)中所述的序列杂交的序列；或(b) a sequence that hybridizes under stringent conditions to the sequence described in (a); or

(c)(a)中所述的序列的互补序列；(c) the complement of the sequence described in (a);

例如，所述分离的核酸分子是RNA；For example, the isolated nucleic acid molecule is RNA;

例如，所述分离的核酸分子是CRISPR/Cas***中的同向重复序列。For example, the isolated nucleic acid molecule is a homologous repeat in the CRISPR/Cas system.
权利要求7所述的分离的核酸分子，其包含选自下列的序列，或由选自下列的序列组成：The isolated nucleic acid molecule of claim 7, comprising a sequence selected from the group consisting of: or consisting of a sequence selected from the group consisting of:

(i)SEQ ID NO：39所示的序列；(i) the sequence shown as SEQ ID NO:39;

(ii)与SEQ ID NO：39所示的序列相比具有一个或多个碱基的置换、缺失或添加(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个碱基的置换、缺失或添加)的序列；(ii) a substitution, deletion or addition of one or more bases compared to the sequence set forth in SEQ ID NO: 39 (eg 1, 2, 3, 4, 5, 6, 7) a sequence of 8, 8 or 10 base substitutions, deletions or additions;

(iii)与SEQ ID NO：39所示的序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％的序列同一性的序列；(iii) having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the sequence set forth in SEQ ID NO:39 Sequence of sequence identity;

(iv)在严格条件下与(i)-(iii)任一项中所述的序列杂交的序列；或(iv) a sequence that hybridizes under stringent conditions to the sequence set forth in any one of (i)-(iii); or

(v)(i)-(iii)任一项中所述的序列的互补序列；(v) the complement of the sequence set forth in any of (i)-(iii);

例如，所述核酸分子包含选自下列的序列，或由选自下列的序列组成：For example, the nucleic acid molecule comprises a sequence selected from the group consisting of: or consists of a sequence selected from the group consisting of:

(a)SEQ ID NO：39所示的核苷酸序列；(a) the nucleotide sequence shown in SEQ ID NO: 39;

(b)在严格条件下与(a)中所述的序列杂交的序列；或(b) a sequence that hybridizes under stringent conditions to the sequence described in (a); or

(c)SEQ ID NO：39所示的核苷酸序列的互补序列。(c) the complement of the nucleotide sequence shown by SEQ ID NO:39.
一种复合物，其包含：A composite comprising:

(i)蛋白组分，其选自：权利要求1-4任一项所述的蛋白、权利要求5所述的缀合物、权利要求6所述的融合蛋白，及其任意组合；和(i) a protein component selected from the group consisting of the protein of any one of claims 1 to 4, the conjugate of claim 5, the fusion protein of claim 6, and any combination thereof;

(ii)核酸组分，其从5’至3’方向包含权利要求7或8所述的分离的核酸分子和能够与靶序列杂交的导向序列，(ii) a nucleic acid component comprising the isolated nucleic acid molecule of claim 7 or 8 and a targeting sequence capable of hybridizing to the target sequence from the 5' to 3' direction,

其中，所述蛋白组分与核酸组分相互结合形成复合物；Wherein the protein component and the nucleic acid component are combined with each other to form a complex;

例如，所述导向序列连接于所述核酸分子的3’端；For example, the targeting sequence is ligated to the 3' end of the nucleic acid molecule;

例如，所述导向序列包含所述靶序列的互补序列；For example, the targeting sequence comprises the complement of the target sequence;

例如，所述核酸组分是CRISPR/Cas***中的导向RNA；For example, the nucleic acid component is a targeting RNA in a CRISPR/Cas system;

例如，所述核酸分子是RNA；For example, the nucleic acid molecule is RNA;

例如，所述复合物不包含反式作用crRNA(tracrRNA)。For example, the complex does not comprise a trans-acting crRNA (tracrRNA).
权利要求9所述的复合物，其包含：The composite of claim 9 comprising:

(i)蛋白组分，其选自：权利要求4所述的蛋白、包含所述蛋白的缀合物或融合蛋白；以及(i) a protein component selected from the group consisting of the protein of claim 4, a conjugate comprising the protein, or a fusion protein;

(ii)核酸组分，其包含权利要求8所述的分离的核酸分子以及所述导向序列。(ii) a nucleic acid component comprising the isolated nucleic acid molecule of claim 8 and the targeting sequence.
一种分离的核酸分子，其包含：An isolated nucleic acid molecule comprising:

(i)编码权利要求1-4任一项所述的蛋白、或权利要求6所述的融合蛋白的核苷酸序列；(i) a nucleotide sequence encoding the protein of any one of claims 1 to 4, or the fusion protein of claim 6;

(ii)编码权利要求7或8所述的分离的核酸分子的核苷酸序列；和/或，(ii) a nucleotide sequence encoding the isolated nucleic acid molecule of claim 7 or 8; and/or,

(iii)包含(i)和(ii)的核苷酸序列；(iii) a nucleotide sequence comprising (i) and (ii);

例如，(i)-(iii)任一项中所述的核苷酸序列经密码子优化用于在原核细胞或真核细胞中进行表达。For example, the nucleotide sequence set forth in any of (i)-(iii) is codon optimized for expression in prokaryotic or eukaryotic cells.
权利要求11的分离的核酸分子，其包含：The isolated nucleic acid molecule of claim 11 comprising:

(i)编码权利要求4所述的蛋白、或包含所述蛋白的融合蛋白的核苷酸序列；(i) a nucleotide sequence encoding the protein of claim 4, or a fusion protein comprising the protein;

(ii)编码权利要求8所述的分离的核酸分子的核苷酸序列；和/或，(ii) a nucleotide sequence encoding the isolated nucleic acid molecule of claim 8; and/or,

(iii)包含(i)和(ii)的核苷酸序列。(iii) a nucleotide sequence comprising (i) and (ii).
一种载体，其包含权利要求11或12所述的分离的核酸分子。A vector comprising the isolated nucleic acid molecule of claim 11 or 12.
一种宿主细胞，其包含权利要求11或12所述的分离的核酸分子或权利要求13所述的载体。A host cell comprising the isolated nucleic acid molecule of claim 11 or 12 or the vector of claim 13.
一种组合物，其包含：A composition comprising:

(i)第一组分，其选自：权利要求1-4任一项所述的蛋白、权利要求5所述的缀合物、权利要求6所述的融合蛋白、编码所述蛋白或融合蛋白的核苷酸序列，以及其任意组合；和(i) a first component selected from the group consisting of the protein of any one of claims 1 to 4, the conjugate of claim 5, the fusion protein of claim 6, the protein or fusion a nucleotide sequence of a protein, and any combination thereof; and

(ii)第二组分，其为包含导向RNA的核苷酸序列，或者编码所述包含导向RNA的核苷酸序列的核苷酸序列；(ii) a second component which is a nucleotide sequence comprising a targeting RNA or a nucleotide sequence encoding the nucleotide sequence comprising the targeting RNA;

其中，所述导向RNA从5’至3’方向包含同向重复序列和导向序列，所述导向序列能够与靶序列杂交；Wherein the targeting RNA comprises a homologous repeat sequence and a targeting sequence from the 5' to 3' direction, the targeting sequence being capable of hybridizing to the target sequence;

所述导向RNA能够与(i)中所述的蛋白、缀合物或融合蛋白形成复合物；The targeting RNA is capable of forming a complex with the protein, conjugate or fusion protein described in (i);

例如，所述同向重复序列是权利要求7或8中所定义的分离的核酸分子；For example, the isotropic repeat is an isolated nucleic acid molecule as defined in claim 7 or 8;

例如，所述导向序列连接至所述同向重复序列的3’端；For example, the targeting sequence is ligated to the 3' end of the isotropic repeat;

例如，所述导向序列包含所述靶序列的互补序列；For example, the targeting sequence comprises the complement of the target sequence;

例如，所述组合物不包含反式作用crRNA(tracrRNA)；For example, the composition does not comprise a trans-acting crRNA (tracrRNA);

例如，所述组合物是非天然存在的或经修饰的；For example, the composition is non-naturally occurring or modified;

例如，所述组合物中的至少一个组分是非天然存在的或经修饰的；For example, at least one component of the composition is non-naturally occurring or modified;

例如，所述第一组分是非天然存在的或经修饰的；和/或，所述第二组分是非天然存在的或经修饰的。For example, the first component is non-naturally occurring or modified; and/or the second component is non-naturally occurring or modified.
权利要求15所述的组合物，其中：The composition of claim 15 wherein:

所述第一组分选自：权利要求4所述的蛋白、或包含所述蛋白的缀合物或融合蛋白、或编码所述蛋白或融合蛋白的核苷酸序列，以及其任意组合；The first component is selected from the group consisting of: the protein of claim 4, or a conjugate or fusion protein comprising the protein, or a nucleotide sequence encoding the protein or fusion protein, and any combination thereof;

所述同向重复序列是权利要求8中所定义的分离的核酸分子。The isotropic repeat sequence is an isolated nucleic acid molecule as defined in claim 8.
一种组合物，其包含一种或多种载体，所述一种或多种载体包含：A composition comprising one or more carriers, the one or more carriers comprising:

(i)第一核酸，其为编码权利要求1-4任一项所述的蛋白或权利要求6所述的融合蛋白的核苷酸序列；任选地所述第一核酸可操作地连接至第一调节元件；以及(i) a first nucleic acid, which is a nucleotide sequence encoding the protein of any of claims 1-4 or the fusion protein of claim 6; optionally the first nucleic acid is operably linked to First adjustment element;

(ii)第二核酸，其编码包含导向RNA的核苷酸序列；任选地所述第二核酸可操作地连接至第二调节元件；(ii) a second nucleic acid encoding a nucleotide sequence comprising a targeting RNA; optionally the second nucleic acid is operably linked to a second regulatory element;

其中：among them:

所述第一核酸与第二核酸存在于相同或不同的载体上；The first nucleic acid and the second nucleic acid are present on the same or different carrier;

所述导向RNA从5’至3’方向包含同向重复序列和导向序列，所述导向序列能够与靶序列杂交；The targeting RNA comprises a homologous repeat sequence and a targeting sequence from the 5' to 3' direction, the targeting sequence being capable of hybridizing to the target sequence;

所述导向RNA能够与(i)中所述的效应蛋白或融合蛋白形成复合物；The targeting RNA is capable of forming a complex with the effector protein or fusion protein described in (i);

例如，所述同向重复序列是权利要求7或8中所定义的分离的核酸分子；For example, the isotropic repeat is an isolated nucleic acid molecule as defined in claim 7 or 8;

例如，所述导向序列连接至所述同向重复序列的3’端；For example, the targeting sequence is ligated to the 3' end of the isotropic repeat;

例如，所述导向序列包含所述靶序列的互补序列；For example, the targeting sequence comprises the complement of the target sequence;

例如，所述组合物不包含反式作用crRNA(tracrRNA)；For example, the composition does not comprise a trans-acting crRNA (tracrRNA);

例如，所述组合物是非天然存在的或经修饰的；For example, the composition is non-naturally occurring or modified;

例如，所述组合物中的至少一个组分是非天然存在的或经修饰的；For example, at least one component of the composition is non-naturally occurring or modified;

例如，所述第一调节元件是启动子，例如诱导型启动子；For example, the first regulatory element is a promoter, such as an inducible promoter;

例如，所述第二调节元件是启动子，例如诱导型启动子。For example, the second regulatory element is a promoter, such as an inducible promoter.
权利要求17所述的组合物，其中：The composition of claim 17 wherein:

所述第一核酸为编码权利要求4所述的蛋白或包含所述蛋白的融合蛋白的核苷酸序列；The first nucleic acid is a nucleotide sequence encoding the protein of claim 4 or a fusion protein comprising the protein;

所述同向重复序列是权利要求8中所定义的分离的核酸分子。The isotropic repeat sequence is an isolated nucleic acid molecule as defined in claim 8.
权利要求17或18所述的组合物，其中，当所述靶序列为DNA时，所述靶序列位于原间隔序列临近基序(PAM)的3’端，并且所述PAM具有5’-TG所示的序列；当所述靶序列为RNA时，所述靶序列不具有PAM结构域限制。The composition according to claim 17 or 18, wherein when the target sequence is DNA, the target sequence is located at the 3' end of the proto-sequence adjacent motif (PAM), and the PAM has 5'-TG The sequence shown; when the target sequence is RNA, the target sequence does not have a PAM domain restriction.
权利要求15-19任一项所述的组合物，其中，所述靶序列是来自原核细胞或真核细胞的DNA或RNA序列；或者，所述靶序列是非天然存在的DNA或RNA序列。The composition of any one of claims 15 to 19, wherein the target sequence is a DNA or RNA sequence derived from a prokaryotic cell or a eukaryotic cell; or the target sequence is a non-naturally occurring DNA or RNA sequence.
权利要求15-20任一项所述的组合物，其中，所述靶序列存在于细胞内；或者，所述靶序列存在于体外的核酸分子(例如，质粒)中；The composition of any one of claims 15 to 20, wherein the target sequence is present in a cell; or the target sequence is present in a nucleic acid molecule (eg, a plasmid) in vitro;

例如，所述靶序列存在于细胞核内或细胞质(例如，细胞器)内；For example, the target sequence is present in the nucleus or in the cytoplasm (eg, organelle);

例如，所述细胞是真核细胞；For example, the cell is a eukaryotic cell;

例如，所述细胞是原核细胞。For example, the cell is a prokaryotic cell.
权利要求15-21任一项所述的组合物，其中，所述蛋白连接有一个或多个NLS序列，或者，所述缀合物或融合蛋白包含一个或多个NLS序列；The composition of any one of claims 15 to 21, wherein the protein is linked to one or more NLS sequences, or the conjugate or fusion protein comprises one or more NLS sequences;

例如，所述NLS序列连接至所述蛋白的N端或C端；For example, the NLS sequence is linked to the N-terminus or C-terminus of the protein;

例如，所述NLS序列融合至所述蛋白的N端或C端。For example, the NLS sequence is fused to the N-terminus or C-terminus of the protein.
一种试剂盒，其包括一种或多种选自下列的组分：权利要求1-4任一项所述的蛋白、权利要求5所述的缀合物、权利要求6所述的融合蛋白、权利要求7或8所述的分离的核酸分子、权利要求9或10所述的复合物、权利要求11或12所述的分离的核酸分子、权利要求13所述的载体、权利要求15-22任一项所述的组合物；A kit comprising one or more components selected from the group consisting of the protein of any one of claims 1 to 4, the conjugate of claim 5, and the fusion protein of claim 6. The isolated nucleic acid molecule of claim 7 or 8, the complex of claim 9 or 10, the isolated nucleic acid molecule of claim 11 or 12, the vector of claim 13, and claim 15- The composition of any of 22;

例如，所述试剂盒包含权利要求15、16、19-22任一项所述的组合物，以及使用所述组合物的说明书；For example, the kit comprises the composition of any one of claims 15, 16, 19-22, and instructions for using the composition;

例如，所述试剂盒包含权利要求17、18、19-22任一项所述的组合物，以及使用所述组合物的说明书。For example, the kit comprises the composition of any one of claims 17, 18, 19-22, and instructions for using the composition.
一种递送组合物，其包含递送载体，以及选自下列的一种或多种：权利要求1-4任一项所述的蛋白、权利要求5所述的缀合物、权利要求6所述的融合蛋白、权利要求7或8所述的分离的核酸分子、权利要求9或10所述的复合物、权利要求11或12所述的分离的核酸分子、权利要求13所述的载体、权利要求15-22任一项所述的组合物；A delivery composition comprising a delivery vehicle, and one or more selected from the group consisting of the protein of any of claims 1-4, the conjugate of claim 5, and the method of claim 6. The fusion protein, the isolated nucleic acid molecule of claim 7 or 8, the complex of claim 9 or 10, the isolated nucleic acid molecule of claim 11 or 12, the vector of claim 13, the right The composition of any of 15-22;

例如，所述递送载体是粒子；For example, the delivery vehicle is a particle;

例如，所述递送载体选自脂质颗粒、糖颗粒、金属颗粒、蛋白颗粒、脂质体、外泌体、微泡、基因枪或病毒载体(例如，复制缺陷型逆转录病毒、慢病毒、腺病毒或腺相关病毒)。For example, the delivery vehicle is selected from the group consisting of a lipid particle, a sugar particle, a metal particle, a protein particle, a liposome, an exosome, a microvesicle, a gene gun, or a viral vector (eg, a replication-defective retrovirus, a lentivirus, Adenovirus or adeno-associated virus).
一种修饰靶基因的方法，其包括：将权利要求9或10所述的复合物或权利要求15-22任一项所述的组合物与所述靶基因接触，或者递送至包含所述靶基因的细胞中；所述靶序列存在于所述靶基因中；A method of modifying a target gene, comprising: contacting the complex of claim 9 or 10 or the composition of any of claims 15-22 with the target gene, or delivering to the target comprising In the cell of the gene; the target sequence is present in the target gene;

例如，所述靶基因存在于细胞内，或者，所述靶基因存在于体外的核酸分子(例如，质粒)中；For example, the target gene is present in a cell, or the target gene is present in a nucleic acid molecule (eg, a plasmid) in vitro;

例如，所述细胞是原核细胞；For example, the cell is a prokaryotic cell;

例如，所述细胞是真核细胞；For example, the cell is a eukaryotic cell;

例如，所述细胞选自(例如，哺乳动物细胞，例如人类细胞)、植物细胞；For example, the cell is selected from (eg, a mammalian cell, such as a human cell), a plant cell;

例如，所述修饰是指所述靶序列的断裂，如DNA的双链断裂或RNA的单链断裂；For example, the modification refers to cleavage of the target sequence, such as double-strand break of DNA or single-strand break of RNA;

例如，所述修饰还包括将外源核酸***所述断裂中。For example, the modification further comprises inserting an exogenous nucleic acid into the fragment.
权利要求25所述的方法，其包括将权利要求10所述的复合物、权利要求16所述的组合物或权利要求18所述的组合物与所述靶基因接触，或者递送至包含所述靶基因的细胞中。The method of claim 25, comprising contacting the complex of claim 10, the composition of claim 16, or the composition of claim 18 with the target gene, or delivering to the In the cells of the target gene.
一种改变基因产物的表达的方法，其包括：将权利要求9或10所述的复合物或权利要求15-22任一项所述的组合物与编码所述基因产物的核酸分子接触，或者递送至包含所述核酸分子的细胞中，所述靶序列存在于所述核酸分子中；A method of altering the expression of a gene product, comprising: contacting the complex of claim 9 or 10 or the composition of any of claims 15-22 with a nucleic acid molecule encoding the gene product, or Delivered into a cell comprising the nucleic acid molecule, the target sequence being present in the nucleic acid molecule;

例如，所述核酸分子存在于细胞内，或者所述核酸分子存在于体外的核酸分子(例如，质粒)中；For example, the nucleic acid molecule is present in a cell, or the nucleic acid molecule is present in a nucleic acid molecule (eg, a plasmid) in vitro;

例如，所述细胞是原核细胞；For example, the cell is a prokaryotic cell;

例如，所述细胞是真核细胞；For example, the cell is a eukaryotic cell;

例如，所述细胞选自(例如，哺乳动物细胞，例如人类细胞)、植物细胞；For example, the cell is selected from (eg, a mammalian cell, such as a human cell), a plant cell;

例如，所述基因产物的表达被改变(例如，增强或降低)；For example, the expression of the gene product is altered (eg, enhanced or decreased);

例如，所述基因产物是蛋白。For example, the gene product is a protein.
权利要求27所述的方法，其包括将权利要求10所述的复合物、权利要求16所述的组合物或权利要求18所述的组合物与编码所述基因产物的核酸分子接触，或者递送至包含所述核酸分子的细胞中。The method of claim 27, comprising contacting the complex of claim 10, the composition of claim 16, or the composition of claim 18 with a nucleic acid molecule encoding the gene product, or delivering To cells containing the nucleic acid molecule.
权利要求27或28所述的方法，其中所述的蛋白、缀合物、融合蛋白、分离的核酸分子、复合物、载体或组合物包含于递送载体中；The method of claim 27 or 28, wherein the protein, conjugate, fusion protein, isolated nucleic acid molecule, complex, vector or composition is included in a delivery vehicle;

例如，所述递送载体选自脂质颗粒、糖颗粒、金属颗粒、蛋白颗粒、脂质体、外泌体、病毒载体(如复制缺陷型逆转录病毒、慢病毒、腺病毒或腺相关病毒)。For example, the delivery vehicle is selected from the group consisting of lipid particles, sugar particles, metal particles, protein particles, liposomes, exosomes, viral vectors (eg, replication defective retroviruses, lentiviruses, adenoviruses, or adeno-associated viruses) .
权利要求27-29任一项所述的方法，其用于改变靶基因或编码靶基因产物的核酸分子中的一个或多个靶序列来修饰细胞、细胞系或生物体。The method of any one of claims 27-29 for modifying a target gene or one or more target sequences in a nucleic acid molecule encoding a target gene product to modify a cell, cell line or organism.
一种由权利要求27-30任一项所述的方法获得的细胞或其子代，其中所述细胞包含在其野生型中不存在的修饰。A cell obtained by the method of any one of claims 27-30, or a progeny thereof, wherein the cell comprises a modification which is not found in its wild type.
权利要求31所述的细胞或其子代的细胞产物。The cell product of the cell of claim 31 or a progeny thereof.
一种体外的、离体的或体内的细胞或细胞系或它们的子代，所述细胞或细胞系或它们的子代包含：权利要求1-4任一项所述的蛋白、权利要求5所述的缀合物、权利要求6所述的融合蛋白、权利要求7或8所述的分离的核酸分子、权利要求9或10所述的复合物、权利要求11或12所述的分离的核酸分子、权利要求13所述的载体、权利要求15-22任一项所述的组合物；An in vitro, ex vivo or in vivo cell or cell line or a progeny thereof, the cell or cell line or a progeny thereof comprising: the protein of any one of claims 1-4, claim 5 The conjugate, the fusion protein of claim 6, the isolated nucleic acid molecule of claim 7 or 8, the complex of claim 9 or 10, the isolated according to claim 11 or 12 a nucleic acid molecule, the vector of claim 13 or the composition of any one of claims 15-22;

例如，所述细胞是原核细胞或真核细胞。For example, the cell is a prokaryotic cell or a eukaryotic cell.
权利要求1-4任一项所述的蛋白、权利要求5所述的缀合物、权利要求6所述的融合蛋白、权利要求7或8所述的分离的核酸分子、权利要求9或10所述的复合物、权利要求11或12所述的分离的核酸分子、权利要求13所述的载体、权利要求15-22任一项所述的组合物或权利要求23所述的试剂盒，用于核酸编辑的用途，或者在制备制剂中的用途，所述制剂用于核酸编辑；A protein according to any one of claims 1 to 4, a conjugate according to claim 5, a fusion protein according to claim 6, an isolated nucleic acid molecule according to claim 7 or 8, claim 9 or 10 The complex, the isolated nucleic acid molecule of claim 11 or 12, the vector of claim 13, the composition of any one of claims 15 to 22, or the kit of claim 23. Use for nucleic acid editing, or in the preparation of a preparation for nucleic acid editing;

例如，核酸编辑包括基因或基因组编辑；For example, nucleic acid editing includes gene or genome editing;

例如，所述基因或基因组编辑包括修饰基因、敲除基因、改变基因产物的表达、修复突变、和/或***多核苷酸。For example, the gene or genome editing includes modifying the gene, knocking out the gene, altering the expression of the gene product, repairing the mutation, and/or inserting the polynucleotide.
权利要求1-4任一项所述的蛋白、权利要求5所述的缀合物、权利要求6所述的融合蛋白、权利要求7或8所述的分离的核酸分子、权利要求9或10所述的复合物、权利要求11或12所述的分离的核酸分子、权利要求13所述的载体、权利要求15-22任一项所述的组合物或权利要求23所述的试剂盒，在制备制剂中的用途，所述制剂用于：A protein according to any one of claims 1 to 4, a conjugate according to claim 5, a fusion protein according to claim 6, an isolated nucleic acid molecule according to claim 7 or 8, claim 9 or 10 The complex, the isolated nucleic acid molecule of claim 11 or 12, the vector of claim 13, the composition of any one of claims 15 to 22, or the kit of claim 23. Use in the preparation of a preparation for:

(i)体外或离体单链DNA的检测；(i) detection of in vitro or ex vivo single-stranded DNA;

(ii)编辑靶基因座中的靶序列来修饰生物或非人类生物。(ii) Editing target sequences in the target locus to modify biological or non-human organisms.