WO2023116681A1 - 靶序列随机sgRNA全覆盖组的制备方法 - Google Patents

靶序列随机sgRNA全覆盖组的制备方法 Download PDF

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WO2023116681A1
WO2023116681A1 PCT/CN2022/140314 CN2022140314W WO2023116681A1 WO 2023116681 A1 WO2023116681 A1 WO 2023116681A1 CN 2022140314 W CN2022140314 W CN 2022140314W WO 2023116681 A1 WO2023116681 A1 WO 2023116681A1
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sgrna
library
dna
target sequence
sequence
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宋东亮
刘倩
黄成�
侯策
王嫚
孙睿
陈晶晶
曹振
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翌圣生物科技(上海)股份有限公司
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  • the application belongs to the field of biotechnology, and in particular relates to a method for preparing a target sequence random sgRNA full coverage group.
  • CRISPR gene editing technology Since its development, CRISPR gene editing technology has been widely used in various fields such as gene therapy, in vitro diagnosis, gene capture and target gene removal, and won the 2020 Nobel Prize in Physiology and Medicine. It is an efficient and practical technology.
  • the practical CRISPR system is mainly composed of two parts, one is the Cas protein with two endonuclease active sites, which is responsible for cutting the two strands of DNA at the target site; the other is the DNA pairing sequence with the target site
  • the guide RNA (sgRNA) that binds to the Cas protein sequence is responsible for recruiting the Cas protein and guiding the Cas protein to bind to the complementary paired target site.
  • the Cas protein first binds to the sgRNA to form a Cas-sgRNA complex, which is retrieved on the DNA.
  • the region complementary to the sgRNA protospacer, ProtoSpacer
  • the Cas protein unwinds the target site, making the unwound double-stranded DNA Entering the DNA cutting active domain of the Cas protein
  • the Cas protein cuts the double-stranded DNA, resulting in a double-strand DNA break.
  • the broken DNA is repaired by homologous recombination (HR) or non-homologous end joining (NHEJ) and other DNA damage repair methods to complete the editing of the target gene.
  • HR homologous recombination
  • NHEJ non-homologous end joining
  • Cas proteins currently used in commercial applications mainly include Cas9, Cas12, Cas13, and Cas14 and their variants. Different Cas proteins recognize PAM sequences and requirements, and the length of ProtoSpacer is also different. Therefore, different CRISPR systems have different application scenarios.
  • sgRNA In addition to the purification and preparation of Cas protein, the in vitro construction and synthesis of sgRNA is also an important part of the commercial application of CRISPR.
  • Conventional methods need to use primer synthesis to synthesize target sgRNA primers containing the T7 promoter, then use overlapping PCR to obtain the full-length sgRNA backbone template, and use in vitro transcription to obtain the required sgRNA.
  • This method is time-consuming, low-cost, and highly controllable. It has been commercialized on a large scale and has become the main form of sgRNA preparation in vitro, but it still has the problem of low throughput.
  • Gene capture or removal usually requires the capture or removal of a large region of the genome, covering a length of 1Mbp or even the entire complete genomic DNA. This requires the design and synthesis of tens of millions of sgRNAs. In the design and synthesis of sgRNA, both cost and technology are great challenges.
  • the present application provides a method for preparing a target sequence random sgRNA full coverage group, the steps of which include:
  • step (2) The 3' end of the double-stranded DNA obtained in step (1) is connected to the sgRNA backbone, wherein the sgRNA backbone has a side-cutting active restriction enzyme site;
  • step (3) Cutting the ligation product of step (2) with a side-cutting activity restriction enzyme to obtain the DNA of the protospacer region, and phosphorylate its 5' end;
  • amplification obtains the sgRNA library template containing T7 promoter
  • the restriction enzyme described in step (1) is a mixture of one or more of ScrFI, MspI, HpaII, BstNI, BfaI, DdeI.
  • Mung Bean Nuclease is used in step (1) to cut the ends flat.
  • the sgRNA backbone described in step (2) is a double-stranded DNA formed by complementary pairing of two single-stranded DNAs, wherein the forward sequence is /rApp/-CGGTTGGAGCTAGAAATAGCAAGTCAACCTAACGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT-/NH2C6/ (SEQ ID NO: 5), and the reverse The forward sequence is /NH2C6/-AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCGTTAGGTTGACTTGCTATTTCTAGCTCCAACC-/ddG/ (SEQ ID NO: 6), with MmeI restriction site; or the forward sequence is /rApp/-CTGCTGGAGCTAGAAATAGCAAGTCAGCATAACGCTAGTCCGTTATCAACTTGAAAAA GTGGCACCGAGTCGGTGCTTTT-/NH2C6/ (SEQ ID NO: 7), The reverse sequence is /NH2C6/-
  • T4 DNA ligase mutant K159L is used in step (2) to connect the sgRNA backbone with the double-stranded DNA.
  • the side cutting activity restriction enzyme described in step (3) is MmeI
  • the forward sequence of the T7 promoter described in step (4) is /NH2C6/-TTCTAATACGACTCACTATAGGNN (SEQ NO:9)
  • the reverse sequence is /ddC/-CTATAGTGAGTCGTATTAGAA-/NH2C6/ (SEQ NO: 10).
  • the side-cutting activity restriction enzyme described in step (3) is EcoP15I
  • the forward sequence of the T7 promoter described in step (4) is /NH2C6/-TTCTAATACGACTCACTATAGG (SEQ NO: 11)
  • the reverse sequence is /ddN/-NCTATAGTGAGTCGTATTAGAA-/NH2C6/ (SEQ NO: 12).
  • step (4) uses T4 DNA ligase to connect.
  • a primer pair whose forward sequence is TTCTAATACGACTCACTATAGG (SEQ NO: 13) and whose reverse sequence is AAAAGCACCGACTCGGTGCC (SEQ NO: 14) is used for library amplification.
  • T7 RNA polymerase is used for transcription in step (6), and the sgRNA library is recovered using RNA recovery magnetic beads.
  • the application provides a ribosomal RNA sgRNA library preparation method, the steps comprising:
  • the application provides a method for removing ribosomal RNA from an RNA library, the steps comprising:
  • the present application provides a method for removing the whole human genome from the host genome, the steps comprising:
  • This application provides a target sequence random sgRNA preparation method RPTS (Random sgRNA Preparation of Target Sequence), which can prepare all sgRNA groups randomly covering the target region at one time.
  • RPTS Random sgRNA Preparation of Target Sequence
  • the principle and process of RPTS are as follows: Use restriction enzymes to cut the PAM region of the target sequence; connect the sgRNA backbone; obtain the sequence targeting the protospacer region through the side cutting active restriction enzyme site on the sgRNA backbone; connect the T7 promoter; The sgRNA library with T7 promoter was obtained by amplification; the target sequence sgRNA library was obtained by in vitro transcription.
  • RPTS has the advantages of low cost, simple production, uniform coverage, small bias, no restriction on the length of the target sequence, and no need to design sgRNA in large quantities.
  • This application also provides the application process of RPTS in ribosomal RNA removal and host genome removal, breaking the application limitations of CRISPR technology in these fields.
  • Figure 1 is a map of normal sgRNA binding target sites.
  • Figure 2 shows the modified backbone of the sgRNA containing the MmeI restriction site.
  • Figure 3 shows the modified backbone of sgRNA containing EcoP15I restriction site.
  • Figure 4 is a schematic diagram of the principle and flow of RPTS.
  • Figure 5 is a summary of the restriction enzyme recognition sites used in the RPTS technique, where the shadows indicate that the restriction enzyme recognition sites contain PAM sequence types.
  • Figure 6 shows the distribution of recognition sites of restriction enzymes used in RPTS technology on the DNA of 18S rRNA.
  • Figure 7 shows the amplification results of 18S and 28S cDNA.
  • the left band is Marker, the middle band is 28S, and the right band is 18S.
  • Figure 8 shows the RPTS library amplification results of 18S/28S DNA and human whole genome DNA (right).
  • the left band is Marker
  • the middle band is 18S/28S DNA
  • the right band is human whole genome DNA.
  • Figure 9 shows the in vitro transcription results of 18S/28S DNA (left) and human whole genome DNA (right).
  • the left band is Marker
  • the middle band is 18S/28S DNA
  • the right band is human whole genome DNA.
  • Figure 10 shows the library distribution of the 18S/28S RPTS library in the application of CRISPR to remove rRNA.
  • the light color is without the RPTS library, and the dark color is the RPTS library.
  • Figure 11 shows the RNA-seq verification of the removal effect of the 18S/28S RPTS library in the application of CRISPR removal of rRNA.
  • Figure 12 is the library distribution of the human genome-wide RPTS library in the application of CRISPR to remove the host genome.
  • the light color is without the RPTS library, and the dark color is the RPTS library.
  • Figure 13 is the DNA-seq verification of the removal effect of the human genome-wide RPTS library in the application of CRISPR removal of the host genome.
  • Example 1 Design of sgRNA backbone and flow of RPTS
  • the backbone of the traditional sgRNA is modified, and the enzyme cutting site MmeI or EcoP15I is incorporated, but the structure of the sgRNA and the binding of Cas9 are not changed.
  • the modified sgRNA sequence and structure are shown in Figure 1- Figure 3.
  • Backbone annealing Dissolve sgM-F, sgM-R, sgE-F and sgE-R with 100mM NaCl solution to 100 ⁇ M, take 10 ⁇ L sgM-F and 10 ⁇ L sgM-R in a PCR tube, take 10 ⁇ L sgE-F and 10 ⁇ L sgE- R In another PCR tube, in the same reaction system, choose one of MmeI-sgRNA backbone and EcoP15I-sgRNA backbone. React at 95°C for 5 minutes, decreasing by 1°C per minute. After the reaction, the annealed matrix was diluted to 10 ⁇ M with water.
  • T7M-F, T7M-R, T7E-F and T7E-R Dissolve T7M-F, T7M-R, T7E-F and T7E-R with 100mM NaCl solution to 100 ⁇ M, take 10 ⁇ L T7M-F and 10 ⁇ L T7M-R in a PCR tube, take 10 ⁇ L T7E-F and 10 ⁇ L T7E- R In another PCR tube, select the appropriate T7 linker according to the type of sgRNA backbone above. React at 95°C for 5 minutes, decreasing by 1°C per minute. After the reaction, the annealed linker was diluted to 10 ⁇ M with water.
  • RNA beads After reacting at 37°C for 4h, add 10U DNase I (TAKARA), and react at 37°C for 1h. Add 50 ⁇ L Ampure RNA beads (Beckman) to recover RNA. The size of sgRNA was detected by agarose gel electrophoresis.
  • the restriction enzyme combination designed to recognize the PAM sequence contains 6 kinds of restriction enzymes, and these 6 kinds of restriction enzyme sites include two PAM sequences of the Cas9 protein. On DNA, there are such restriction enzyme sites about every 64bp, so the prepared sgRNA can randomly cover the whole genome.
  • Figure 6 shows the distribution of these 6 restriction enzyme sites on the 18S rRNA (shaded part).
  • Embodiment 2 The RPTS preparation of 18S rRNA or 28S rRNA.
  • RPTS was used to prepare a random sgRNA library covering 18S rRNA or 28S rRNA.
  • the specific implementation is as follows:
  • Preparation of sgRNA library by RPTS Prepare 18S sgRNA library or 28S sgRNA library according to the method in Example 1.
  • RNA library preparation was carried out using the dual-mode RNA library construction kit (12252) of Yisheng Biotechnology. After ligating DNA adapters, use 0.6 ⁇ Ampure DNA beads to recover the library.
  • the prepared library was sequenced and analyzed on the Illumina NovaSeq 6000 platform after Qsep100 quality inspection.
  • Example 3 RPTS preparation of the whole human genome.
  • RPTS was used to prepare a random sgRNA library covering the whole human genome.
  • the specific implementation method is as follows:
  • sgRNA library was prepared according to the method in Example 1, and the DNA used was human genomic DNA standard NA12878 (Coriell).
  • DNA library preparation the DNA used was a DNA standard mixture in which the human genome DNA standard NA12878 (Coriell) and the Escherichia coli genome were mixed at a ratio of 100:1.
  • the one-step library construction kit (12204) of Yisheng Biotech was used for DNA library construction. After ligating DNA adapters, use 0.6 ⁇ Ampure DNA beads to recover the library.
  • the prepared library was sequenced and analyzed on the Illumina NovaSeq 6000 platform after Qsep100 quality inspection.
  • the DNA library construction results and sequencing results are shown in Figure 11 and Figure 12.
  • the sgRNA library prepared by the RPTS method can effectively remove the human host genomic DNA during the DNA library construction process.
  • this application discloses a random sgRNA preparation method for target sequence RPTS (Random sgRNA Preparation of Target Sequence), which can prepare all sgRNA groups randomly covering the target region at one time.
  • RPTS Random sgRNA Preparation of Target Sequence
  • the principle and process of RPTS are as follows: Use restriction enzymes to cut the PAM region of the target sequence; connect the sgRNA backbone; obtain the sequence targeting the protospacer region through the side cutting active restriction enzyme site on the sgRNA backbone; connect the T7 promoter; The sgRNA library with T7 promoter was obtained by amplification; the target sequence sgRNA library was obtained by in vitro transcription.
  • RPTS has the advantages of low cost, simple production, uniform coverage, small bias, no restriction on the length of the target sequence, and no need to design sgRNA in large quantities.
  • This application also discloses the application process of RPTS in ribosomal RNA removal and host genome removal, breaking the application limitations of CRISPR technology in these fields.

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Abstract

提供了一种靶序列随机sgRNA全覆盖组的制备方法，使用限制酶对目标序列的PAM区域进行切割；连接上sgRNA骨架；通过sgRNA骨架上的旁切活性限制酶位点进行靶向原间隔区域的序列获取；连接T7启动子；扩增获得带T7启动子的sgRNA文库；体外转录获得靶序列sgRNA文库。还公开了核糖体RNA的sgRNA文库制备方法以及去除RNA文库中核糖体RNA的方法，和在宿主基因组去除人全基因组的方法。该方法具有成本低、制作简单、覆盖均一、偏好性小、不受靶序列长度限制和无需大批量设计sgRNA等优点。

Description

靶序列随机sgRNA全覆盖组的制备方法 技术领域
本申请属于生物技术领域，具体涉及一种靶序列随机sgRNA全覆盖组的制备方法。
背景技术
CRISPR基因编辑技术自从被开发出来就被广泛应用在基因治疗、体外诊断、基因捕获和靶基因去除等各个领域，并获得2020年诺贝尔生理医学奖，是一种高效且实用的技术。实用型CRISPR***主要有两个部分组成，一个是具有两个核酸内切酶活性位点的Cas蛋白，负责切割靶位点DNA的两条链；另一个是具有与靶位点处DNA配对序列和Cas蛋白结合序列的引导RNA(sgRNA)，负责募集Cas蛋白并引导Cas蛋白结合到互补配对的靶位点上。在CRISPR***中，Cas蛋白先与sgRNA结合形成Cas-sgRNA复合物，并在DNA上进行检索。当检索到与sgRNA互补配对的区域(原间隔区域，ProtoSpacer)，并且ProtoSpacer区域的3’端存在NGG序列(PAM序列)时，Cas蛋白将靶位点进行解旋，使得解开的双链DNA进入到Cas蛋白的DNA切割活性结构域，Cas蛋白对双链DNA进行切割，产生双链DNA断裂。断裂的DNA通过同源重组修复HR或者非同源末端连接NHEJ等DNA损伤修复方式完成靶基因的编辑。目前用于商业化应用的Cas蛋白主要有Cas9、Cas12、Cas13和Cas14及其变体，不同的Cas蛋白识别的PAM序列和要求，ProtoSpacer的长度也不同。因此，不同的CRISPR***的具有不同的应用场景。
除Cas蛋白的纯化和制备外，sgRNA的体外构建和合成也是CRISPR商业化应用的重要环节。常规的方法需要利用引物合成的方式合成含T7启动子的靶标sgRNA引物，再利用重叠PCR获得全长sgRNA骨架模板，并利用体外转录的方法获得需要的sgRNA。这种方法耗时短、成本低、可控性高，已经大规模商业化，成为sgRNA体外制备的主要形式，但仍存在通量低的问题。基因捕获或者去除通常需要对大区域的基因组进行捕获或者去除，需要覆盖长度达1Mbp甚至整个完整的基因组DNA。这需要设计和合成千万级别条数的sgRNA。在sgRNA的设计合成上，无论成本还是技术都是很大的挑战。
发明内容
第一方面，本申请提供了一种靶序列随机sgRNA全覆盖组的制备方法，其步骤包括：
(1)采用识别PAM序列的限制酶切割样本DNA，并将末端切平，所述切平是指去除双链DNA上的3’和5’单链悬垂，以产生钝端；
(2)步骤(1)获得的切平后的双链DNA 3’端连接sgRNA骨架，其中所述sgRNA骨架带有旁切活性限制酶位点；
(3)采用旁切活性限制酶切割步骤(2)的连接产物，获取原间隔区域DNA，并将其5’端磷酸化；
(4)在原间隔区域DNA的5’端连接T7启动子序列；
(5)扩增获得含T7启动子的sgRNA文库模板；和
(6)体外转录获得sgRNA文库。
优选地，步骤(1)中所述的限制酶为ScrFI、MspI、HpaII、BstNI、BfaI、DdeI中的一种或多种的混合物。
优选地，步骤(1)中采用Mung Bean Nuclease将末端切平。
优选地，步骤(2)中所述的sgRNA骨架为两条单链DNA互补配对形成的双链DNA，其中正向序列为/rApp/-CGGTTGGAGCTAGAAATAGCAAGTCAACCTAACGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT-/NH2C6/(SEQ ID NO:5)，反向序列为/NH2C6/-AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCGTTAGGTTGACTTGCTATTTCTAGCTCCAACC-/ddG/(SEQ ID NO:6)，设有MmeI酶切位点；或者正向序列为/rApp/-CTGCTGGAGCTAGAAATAGCAAGTCAGCATAACGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT-/NH2C6/(SEQ ID NO:7)，反向序列为/NH2C6/-AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCGTTATGCTGACTTGCTATTTCTAGCTCCAGCA-/ddG/(SEQ ID NO:8)，设有EcoP15I酶切位点。
优选地，步骤(2)中使用T4 DNA连接酶突变体K159L连接sgRNA骨架 与双链DNA。
优选地，步骤(3)中所述的旁切活性限制酶为MmeI，步骤(4)中所述的T7启动子正向序列为/NH2C6/-TTCTAATACGACTCACTATAGGNN(SEQ NO:9)，反向序列为/ddC/-CTATAGTGAGTCGTATTAGAA-/NH2C6/(SEQ NO:10)。
优选地，步骤(3)中所述的旁切活性限制酶为EcoP15I，步骤(4)中所述的T7启动子正向序列为/NH2C6/-TTCTAATACGACTCACTATAGG(SEQ NO:11)，反向序列为/ddN/-NCTATAGTGAGTCGTATTAGAA-/NH2C6/(SEQ NO:12)。
优选地，步骤(4)采用T4 DNA连接酶进行连接。
优选地，步骤(5)中使用正向序列为TTCTAATACGACTCACTATAGG(SEQ NO:13)，反向序列为AAAAGCACCGACTCGGTGCC(SEQ NO:14)的引物对进行文库扩增。
优选地，步骤(6)中采用T7 RNA聚合酶进行转录，使用RNA回收磁珠回收sgRNA文库。
第二方面，本申请提供了一种核糖体RNA的sgRNA文库制备方法，其步骤包括：
A)逆转录制备18S和28S全长cDNA；
B)PCR扩增获得18S和28S全长双链DNA；和
C)采用上述的方法，以18S和28S全长双链DNA为样本DNA，制得靶向覆盖18S和28S的sgRNA文库。
第三方面，本申请提供了一种去除RNA文库中核糖体RNA的方法，其步骤包括：
A)将上面制得的sgRNA文库与Cas9蛋白预组装；
B)采用预组装的Cas9-sgRNA切割RNA文库；和
C)RNA文库扩增并测序。
第四方面，本申请提供了一种在宿主基因组去除人全基因组的方法，其步骤包括：
A)采用上述的方法，以人全基因组DNA为样本DNA，制得靶向覆盖人全基因组DNA的sgRNA文库；
B)制得的sgRNA文库与Cas9蛋白预组装；
C)采用预组装的Cas9-sgRNA切割宿主基因组DNA文库；和
D)DNA文库扩增并测序。
本申请具有如下有益效果：
本申请提供了一种靶序列随机sgRNA制备方法RPTS(Random sgRNA Preparation of Target Sequence)，可以一次性制备随机覆盖目标区域的所有sgRNA组。RPTS的原理和流程如下：使用限制酶对目标序列的PAM区域进行切割；连接上sgRNA骨架；通过sgRNA骨架上的旁切活性限制酶位点进行靶向原间隔区域的序列获取；连接T7启动子；扩增获得带T7启动子的sgRNA文库；体外转录获得靶序列sgRNA文库。RPTS具有成本低、制作简单、覆盖均一、偏好性小、不受靶序列长度限制和无需大批量设计sgRNA等优点。本申请还提供了RPTS在核糖体RNA去除和宿主基因组去除上的应用流程，打破了CRISPR技术在这些领域上的应用限制。
附图说明
图1为正常sgRNA结合靶位点图。
图2为包含MmeI酶切位点的sgRNA改造骨架。
图3为包含EcoP15I酶切位点的sgRNA改造骨架。
图4为RPTS原理和流程示意图。
图5为RPTS技术中使用限制酶识别位点汇总，其中阴影表示该限制酶识别位点中包含PAM序列类型。
图6为18S rRNA的DNA上包含RPTS技术中使用限制酶的识别位点分布。
图7为18S和28S cDNA扩增结果，左侧条带为Marker，中间条带为28S，右边条带为18S。
图8为18S/28S DNA和人全基因组DNA(右)的RPTS文库扩增结果，左侧条带为Marker，中间条带为18S/28S DNA，右边条带为人全基因组DNA。
图9为18S/28S DNA(左)和人全基因组DNA(右)的体外转录结果，左侧条带为Marker，中间条带为18S/28S DNA，右边条带为人全基因组DNA。
图10为18S/28S RPTS库在CRISPR去除rRNA应用上的文库分布。浅色是未加RPTS库，深色是加入RPTS库。
图11为RNA-seq验证18S/28S RPTS库在CRISPR去除rRNA应用上去除 效果。
图12为人类全基因组RPTS库在CRISPR去除宿主基因组应用上的文库分布。浅色是未加RPTS库，深色是加入RPTS库。
图13为DNA-seq验证人全基因组RPTS库在CRISPR去除宿主基因组应用上去除效果。
具体实施方式
为了进一步描述使本申请的具体内容，以下结合实施例对本申请进行详细说明。实施例所涉及的操作方法及试剂为业内技术人员所熟知，应当理解，以下所描述的具体实施例仅仅用于对申请进行详细说明，但本申请的实施方式并不受下述实施例的限制。本实施例所使用的接头序列及修饰如表1所示，N为A、T、C、G中的任意碱基。
表1 接头序列及修饰
Figure PCTCN2022140314-appb-000001
Figure PCTCN2022140314-appb-000002
Figure PCTCN2022140314-appb-000003
实施例1：sgRNA骨架的设计和RPTS的流程
本实施例改造了传统sgRNA的骨架，融入了酶切位点MmeI或EcoP15I，但不改变sgRNA的结构和Cas9的结合。改造后的sgRNA序列和结构如图1-图3所示。
测试RPTS的流程，流程如图4所示：
(1)识别PAM序列的限制酶切割：
表2 限制酶酶切体系
组分 用量
PUC19 plasmid DNA 1μg
10×rCutSmart buffer 8μL
ScrFI(NEB) 5U
MspI(NEB) 5U
HpaII(NEB) 5U
BstNI(NEB) 5U
BfaI(NEB) 5U
DdeI(NEB) 5U
补水至 80μL
37℃反应过夜。反应结束后，加入160μL Ampure DNA beads(Beckman)回收片段化的DNA。45μL水洗脱。
表3 末端切平体系
组分 用量
上述回收的DNA 43μL
10×Mung Bean Nuclease Reaction Buffer 5μL
Mung Bean Nuclease(NEB) 20U
补水至 50μL
30℃反应2h。反应结束后，加入100μL Ampure DNA beads(Beckman)回收片段化的DNA。42μL水洗脱。
(2)含旁切活性限制酶酶切位点的sgRNA骨架连接。
骨架退火：sgM-F、sgM-R、sgE-F和sgE-R用100mM NaCl溶液溶解至100μM，取10μL sgM-F和10μL sgM-R于PCR管中，取10μL sgE-F和10μL sgE-R于另一PCR管中，在同一反应体系中，MmeI-sgRNA骨架和EcoP15I-sgRNA骨架二选一。95℃反应5min，每分钟降低1℃。反应结束后，用水将退火好的骨架稀释至10μM。
骨架连接：
表4 骨架连接体系
组分 用量
上述回收的DNA 40μL
10μM sgRNA骨架接头 5μL
10×T4 Ligase Reaction Buffer 5μL
T4 DNA Ligase(K159L) 2000U
补水至 50μL
20℃反应1h。反应结束后，加入22.5μL Ampure DNA beads回收DNA，去除掉未连接的接头。22μL水洗脱。
(3)MmeI或EcoP15I酶切
表5 MmeI酶切体系
组分 用量
上述回收的含MmeI-sgRNA骨架的DNA 20μL
10×rCutSmart buffer 3μL
MmeI 5U
补水至 30μL
表6 EcoP15I酶切体系
组分 用量
上述回收的含EcoP15I-sgRNA骨架的DNA 20μL
10×rCutSmart buffer 3μL
10×ATP 3μL
EcoP15I 20U
补水至 30μL
37℃反应2h。反应结束后，加入60μL Ampure DNA beads回收DNA。42μL水洗脱。
(4)T7启动子连接。
接头退火：T7M-F、T7M-R、T7E-F和T7E-R用100mM NaCl溶液溶解至100μM，取10μL T7M-F和10μL T7M-R于PCR管中，取10μL T7E-F和10μL T7E-R于另一PCR管中，根据上面的sgRNA骨架的类型选用合适的T7接头。95℃反应5min，每分钟降低1℃。反应结束后，用水将退火好的接头稀释至10μM。
接头连接：
表7 接头连接体系
组分 用量
上述回收的DNA 40μL
10μM对应的T7接头 5μL
10×T4 Ligase Reaction Buffer 5μL
T4 DNA Ligase 2000U
补水至 50μL
20℃反应1h。反应结束后，加入22.5μL Ampure DNA beads回收DNA，去除掉未连接的接头。22μL水洗脱。
(5)sgRNA库扩增
表8 sgRNA文库扩增体系
组分 用量
上述回收的DNA 20μL
10μM sgPCR-F/R 5μL
Phusion High-Fidelity PCR Master Mix 25μL
补水至 50μL
在98℃变性3min后，通过98℃10s变性、60℃20s退火和72℃10s延伸进行文库循环扩增。扩增产物使用70μL Ampure DNA beads进行回收，22μL DEPC水洗脱。
(6)sgRNA库体外转录
表9 sgRNA文库体外转录体系
组分 用量
上述回收的DNA 1μg
10×Transcription Buffer 2μL
CTP/GTP/ATP/UTP(100mM each) 2μL
T7 RNA Polymerase Mix(Yeasen) 2μL
补水至 20μL
37℃反应4h后，加入10U DNase I(TAKARA)，37℃反应1h。加入50μL Ampure RNA beads(Beckman)回收RNA。琼脂糖凝胶电泳检测sgRNA大小。
如图5所述，设计的识别PAM序列(NRG)的限制酶组合包含6种限制性内切酶，这6种限制酶位点包括了Cas9蛋白的两个PAM序列。在DNA上，约每64bp就存在这样的限制酶位点，因此制备的sgRNA可以随机覆盖到整个基 因组上。图6展示了18S rRNA上这6中限制酶位点的分布(阴影部分)。
实施例2：18S rRNA或28S rRNA的RPTS制备。
在本实施例中，利用RPTS制备了覆盖18S rRNA或28S rRNA的随机sgRNA库。具体实施方式如下：
(1)DNA片段的获取
表10 逆转录体系
组分 用量
293细胞RNA 1μg
10μM逆转录引物18S-R或者28S-R 1μL
10mM dNTPs 1
75℃反应5min  
5×FS Buffer 4μL
0.1M DTT 1μL
SuperScript IV(Thermo) 2μL
总体积 20μL
42℃ 15min，50℃ 15min，55℃ 15min，50℃ 15min，55℃ 15min，70℃ 15min。
表11 PCR扩增体系
组分 用量
上述逆转录产物 1μL
10μM 18S-F/R或者28S-F/R 5μL
Phusion High-Fidelity PCR Master Mix 25μL
加水至 50μL
在98℃变性3min后，通过98℃ 10s变性、60℃ 20s退火和72℃ 3min延伸进行文库循环扩增。扩增产物使用35μL Ampure DNA beads进行回收，22μL DEPC水洗脱。
RPTS制备sgRNA库：按照实施例1的方法制备18S sgRNA库或28S sgRNA库。
(3)RNA文库制备。使用翌圣生物的双模式RNA建库试剂盒(12252)进行RNA文库构建。连接DNA接头后，使用0.6×Ampure DNA beads回收文库。
(4)CRISPR去除18S和28S DNA。
表12 sgRNA库与Cas9蛋白预组装体系：
组分 用量
18SsgRNA库和28S sgRNA库 2-10μg
Cas9(NEB) 0.2-0.5μg
500mM氯化钠 1μL
总体积 5μL
37℃ 30min。
表13 CRISPR切割体系
组分 用量
Cas9-sgRNA库 5μL
RNA文库 1-100ng
10×NEB buffer 3.1 1μL
总体积 10μL
37℃ 30-90min，90℃ 10min。
(5)文库扩增
表14 文库扩增体系
组分 用量
上述反应体系 10μL
Index Primer F/R(Yeasen,12610) 5μL
2×Canase PCR mix 25μL
补水至 50μL
在98℃变性3min后，通过98℃ 10s变性、60℃ 30s退火和72℃ 30s延伸进行文库循环扩增。扩增产物使用45μL Ampure DNA beads进行回收，22μL DEPC水洗脱。
制备好的文库经过Qsep100质检后，在Illumina的NovaSeq 6000平台上进行测序并分析。
18S和28S RPTS结果如图7、图8和图9所示，RPTS技术能够成功构建18S或28S的sgRNA库。RNA建库结果和测序结果如图10和图11所示，RPTS方法制备的sgRNA文库能够有效去除18S和28S rRNA。
实施例3：人全基因组的RPTS制备。
在本实施例中，利用RPTS制备了覆盖人全基因组的随机sgRNA库，具体实施方式如下：
(1)RPTS制备sgRNA库：按照实施例1的方法制备sgRNA库，DNA使用人基因组DNA标准品NA12878(Coriell)。
(2)DNA文库制备：DNA使用人基因组DNA标准品NA12878(Coriell)和大肠杆菌基因组按照100:1比例混合的DNA标准品混合物。使用翌圣生物的一步法建库试剂盒(12204)进行DNA文库构建。连接DNA接头后，使用0.6×Ampure DNA beads回收文库。
(3)CRISPR去除人基因组DNA
表15 sgRNA库与Cas9蛋白预组装体系：
组分 用量
人基因组RPTS库 2-10μg
Cas9(NEB) 0.2-0.5μg
500mM氯化钠 1μL
总体积 5μL
37℃ 30min。
表16 CRISPR切割体系
组分 用量
Cas9-sgRNA库 5μL
RNA文库 1-100ng
10×NEB buffer 3.1 1μL
总体积 10μL
37℃ 30-90min，90℃ 10min。
(5)文库扩增
表17 文库扩增体系
组分 用量
上述反应体系 10μL
Index Primer F/R(Yeasen,12610) 5μL
2×Canace pro PCR mix 25μL
补水至 50μL
在98℃变性3min后，通过98℃ 10s变性、60℃ 30s退火和72℃ 30s延伸进行文库循环扩增。扩增产物使用45μL Ampure DNA beads进行回收，22μL DEPC水洗脱。
制备好的文库经过Qsep100质检后，在Illumina的NovaSeq 6000平台上进行测序并分析。
DNA建库结果和测序结果如图11和图12所示，RPTS方法制备的sgRNA文库能够有效去除DNA建库过程中的人类宿主基因组DNA。
综上，本申请公开了一种靶序列随机sgRNA制备方法RPTS(Random sgRNA Preparation of Target Sequence)，可以一次性制备随机覆盖目标区域的所有sgRNA组。RPTS的原理和流程如下：使用限制酶对目标序列的PAM区域进行切割；连接上sgRNA骨架；通过sgRNA骨架上的旁切活性限制酶位点进行靶向原间隔区域的序列获取；连接T7启动子；扩增获得带T7启动子的sgRNA文库；体外转录获得靶序列sgRNA文库。RPTS具有成本低、制作简单、覆盖均一、偏好性小、不受靶序列长度限制和无需大批量设计sgRNA等优点。本申请还公开了RPTS在核糖体RNA去除和宿主基因组去除上的应用流程，打破了CRISPR技术在这些领域上的应用限制。

Claims (13)

  1. 一种靶序列随机sgRNA全覆盖组的制备方法，其步骤包括：
    (1)采用识别PAM序列的限制酶切割样本DNA，并将末端切平；
    (2)步骤(1)获得的切平后的双链DNA3’端连接sgRNA骨架，其中sgRNA骨架带有旁切活性限制酶位点；
    (3)采用旁切活性限制酶切割步骤(2)的连接产物，获取原间隔区域DNA，并将其5’端磷酸化；
    (4)在原间隔区域DNA的5’端连接T7启动子序列；
    (5)扩增获得含T7启动子的sgRNA文库模板；和
    (6)体外转录获得sgRNA文库。
  2. 根据权利要求1所述的靶序列随机sgRNA全覆盖组的制备方法，其中，步骤(1)中所述的限制酶为ScrFI、MspI、HpaII、BstNI、BfaI、DdeI中的一种或数种的混合物。
  3. 根据权利要求1所述的靶序列随机sgRNA全覆盖组的制备方法，其中，步骤(1)中采用Mung Bean Nuclease将末端切平。
  4. 根据权利要求1所述的靶序列随机sgRNA全覆盖组的制备方法，其中，步骤(2)中所述的sgRNA骨架为两条单链DNA互补配对形成的双链DNA，其中正向序列为/rApp/-CGGTTGGAGCTAGAAATAGCAAGTCAACCTAACGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT-/NH2C6/(SEQ ID NO:5)，反向序列为/NH2C6/-AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCGTTAGGTTGACTTGCTATTTCTAGCTCCAACC-/ddG/(SEQ NO:6)，设有MmeI酶切位点；或者正向序列为/rApp/-CTGCTGGAGCTAGAAATAGCAAGTCAGCATAACGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT-/NH2C6/(SEQ NO:7)，反向序列为/NH2C6/-AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCGTTATGCTGACTTGCTATTTCTAGCTCCAGCA-/ddG/(SEQ NO:8)，设有EcoP15I酶切位点。
  5. 根据权利要求6所述的靶序列随机sgRNA全覆盖组的制备方法，其中， 步骤(2)中使用T4 DNA连接酶突变体K159L连接sgRNA骨架与双链DNA。
  6. 根据权利要求1所述的靶序列随机sgRNA全覆盖组的制备方法，其中，步骤(3)中所述的旁切活性限制酶为MmeI，步骤(4)中所述的T7启动子正向序列为/NH2C6/-TTCTAATACGACTCACTATAGGNN(SEQ NO:9)，反向序列为/ddC/-CTATAGTGAGTCGTATTAGAA-/NH2C6/(SEQ NO:10)。
  7. 根据权利要求1所述的靶序列随机sgRNA全覆盖组的制备方法，其中，步骤(3)中所述的旁切活性限制酶为EcoP15I，步骤(4)中所述的T7启动子正向序列为/NH2C6/-TTCTAATACGACTCACTATAGG(SEQ NO:11)，反向序列为/ddN/-NCTATAGTGAGTCGTATTAGAA-/NH2C6/(SEQ NO:12)。
  8. 根据权利要求1所述的靶序列随机sgRNA全覆盖组的制备方法，其中，步骤(4)采用T4 DNA连接酶进行连接。
  9. 根据权利要求1所述的靶序列随机sgRNA全覆盖组的制备方法，其中，步骤(5)中使用正向序列为TTCTAATACGACTCACTATAGG，反向序列为AAAAGCACCGACTCGGTGCC的引物对进行文库扩增。
  10. 根据权利要求1所述的靶序列随机sgRNA全覆盖组的制备方法，其中，步骤(6)中采用T7 RNA聚合酶进行转录，使用RNA回收磁珠回收sgRNA文库。
  11. 一种核糖体RNA的sgRNA文库制备方法，其步骤包括：
    A)逆转录制备18S和28S全长cDNA；
    B)PCR扩增获得18S和28S全长双链DNA；和
    C)采用权利要求1-10中任一项所述的方法，以18S和28S全长双链DNA为样本DNA，制得靶向覆盖18S和28S的sgRNA文库。
  12. 一种去除RNA文库中核糖体RNA的方法，其步骤包括：
    A)将权利要求11制得的sgRNA文库与Cas9蛋白预组装；
    B)采用预组装的Cas9-sgRNA切割RNA文库；和
    C)RNA文库扩增并测序。
  13. 一种在宿主基因组去除人全基因组的方法，其步骤包括：
    A)采用权利要求1-10中任一项所述的方法，以人全基因组DNA为样本DNA，制得靶向覆盖人全基因组DNA的sgRNA文库；
    B)制得的sgRNA文库与Cas9蛋白预组装；
    C)采用预组装的Cas9-sgRNA切割宿主基因组DNA文库；和
    D)DNA文库扩增并测序。
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