CN108893529A - 一种基于CRISPR技术特异性检测人KRAS基因2号及3号外显子突变的crRNA - Google Patents
一种基于CRISPR技术特异性检测人KRAS基因2号及3号外显子突变的crRNA Download PDFInfo
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Abstract
本发明公开了一种基于CRISPR技术特异性检测人KRAS基因2号及3号外显子突变的crRNA，其特征在于，包括CRISPR‑Cas13a***，还包括可与CRISPR‑Cas13a***结合的crRNA，所述crRNA序列格式为：5`‑Cas13a蛋白锚定序列‑crRNA向导序列‑3`，本发明的优点在于，所述检测结果可通过荧光读数直观判断人KRAS基因是否发生突变，而无需高通量测序，具有成本低、可多次重复检测、检测速度快，结果可直观分析等优势，适用于大规模的临床样本检测，本发明公开的核酸检测技术与以往的基于PCR技术的检测技术不同，反应全程无需复杂的温度控制仪器或***；通过将三步反应全部加入在一个反应体系中，进一步简化操作流程，可以在两个小时内检测出具有特异序列的核酸片段。
Description
技术领域
本发明涉及一种生物材料的检测，具体地说，涉及一种基于CRISPR-Cas13a技术适用于快速检测生物样本中特异性核酸片段的方法。
背景技术
从基因水平上对疾病进行研究和诊断，是实现个体化精准医疗这一医学概念的关键，Kras 是一种鼠类肉瘤病毒癌基因，ras基因家族与人类肿瘤相关的基因有三种—H-ras、K-ras和N-ras，分别定位在11、12和1号染色体上。K-ras因编码21kD的ras蛋白又名p21基因。在ras基因中，K-Ras对人类癌症影响最大，它好像分子开关：当正常时能控制调控细胞生长的路径；发生异常时，则导致细胞持续生长，并阻止细胞自我毁灭。它参与细胞内的信号传递，当K-ras基因突变时，该基因永久活化，不能产生正常的ras蛋白，使细胞内信号传导紊乱，细胞增殖失控而癌变。
目前常规的基因检测通过PCR技术实现，但现有的PCR技术存在一些缺陷如灵敏度不高、特异性不强、目的片段常采用DNA等，而造成成本高、重复性不强、检测耗时久等问题。
发明内容
为了解决现有技术中存在的问题，本发明的目的是提供一种检测人KRAS基因2号及3号外显子突变的crRNA。
一种基于CRISPR技术特异性检测人KRAS基因2号及3号外显子突变的crRNA，包括CRISPR-Cas13a***，还包括可与CRISPR-Cas13a***结合的crRNA，所述crRNA序列格式为：5`-Cas13a蛋白锚定序列-crRNA向导序列-3`。
所述Cas13a蛋白为Lw Cas13a，所述crRNA序列中的锚定序列为
GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAAC，
所述crRNA序列分别为
KRAS-EXON2
GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACCUUGCCUACGCCACCAGCUCC AACUA
KRAS-EXON3
GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGUACUCCUCUUGACCUGCUG UGUCGA；
所述用于转录crRNA的脱氧核酸序列为
KRAS-EXON2
TAGTTGGAGCTGGTGGCGTAGGCAAGGTTTTAGTCCCCTTCGTTTTTGGGGTAGTCTAAATCCCCTATAGTGAGTCGTATTAATTTC
KRAS-EXON3
TCGACACAGCAGGTCAAGAGGAGTACGTTTTAGTCCCCTTCGTTTTTGGGGTAGTCTAAATCCCCTATAGTGAGTCGTATTAATTTC。
所述Cas13a蛋白为Lsh Cas13a，crRNA序列中的锚定序列为
CCACCCCAAUAUCGAAGGGGACUAAAAC，
所述crRNA序列分别为
KRAS-EXON2
CCACCCCAAUAUCGAAGGGGACUAAAACCUUGCCUACGCCACCAGCUCCAACUA KRAS-EXON3
CCACCCCAAUAUCGAAGGGGACUAAAACGUACUCCUCUUGACCUGCUGUGUCGA 用于转录crRNA的脱氧核酸序列为
KRAS-EXON2
TAGTTGGAGCTGGTGGCGTAGGCAAGGTTTTAGTCCCCTTCGATATTGGGGTGGCCCTATAGTGAGTCGTATTAATTTC
KRAS-EXON3
TCGACACAGCAGGTCAAGAGGAGTACGTTTTAGTCCCCTTCGATATTGGGGTGGCCCTATAGTGAGTCGTATTAATTTC。
所述的一种基于CRISPR技术特异性检测人KRAS基因2号及3号外显子突变的crRNA在人KRAS基因外显子2和外显子3突变检测中的应用。
本发明公开的一种基于CRIPSR-Cas13a的核酸检测技术，其最主要的机制是Cas13a蛋白可以在向导RNA的帮助下识别具有靶向序列的RNA片段，随后被激活的不受序列限制的RNA 酶活性，通过在反应体系中加入RNA链降解导致的信号报告分子，最终实现具有靶向序列的 RNA片段的信号识别。
本发明通过T7RNA聚合酶作用下合成的RNA，不仅使该检测技术的应用范围扩大到RNA 和DNA都可以检测，同时明显提高了检测的灵敏度，可以检测出阿摩尔级别的核酸。
由于Cas13a蛋白在识别靶向序列时，通常可以耐受一个碱基的错配，本发明通过提前在向导RNA的序列中加入人为错配碱基，使本检测技术的特异性达到可以识别具有单碱基差异的核酸序列。
相比之下本发明模板可以是DNA也可是RNA，检测结果可通过荧光读数直观判断人KRAS 基因是否发生突变，而无需高通量测序，具有成本低、可多次重复检测、检测速度快，结果可直观分析等优势，适用于大规模的临床样本检测。
本发明公开的核酸检测技术与以往的基于PCR技术的检测技术不同，反应全程无需复杂的温度控制仪器或***；通过将三步反应全部加入在一个反应体系中，进一步简化操作流程，可以在两个小时内检测出具有特异序列的核酸片段。
具体实施方式
为详细说明本发明之技术内容、构造特征、所达成目的及功效，以下兹例举实施例详予说明。
一种基于CRISPR技术特异性检测人KRAS基因2号及3号外显子突变的crRNA，包括CRISPR-Cas13a***，还包括可与CRISPR-Cas13a***结合的crRNA，所述crRNA序列格式为：5`-Cas13a蛋白锚定序列-crRNA向导序列-3`；所述Cas13a蛋白可以为Lw Cas13a，所述Cas13a蛋白亦可为Lsh Cas13a。
Cas13a蛋白为Lw Cas13a时
crRNA序列中的锚定序列为GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAAC 所述crRNA序列分别为
KRAS-EXON2
GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACCUUGCCUACGCCACCAGC UCCAACUA
KRAS-EXON3
GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGUACUCCUCUUGACCUG CUGUGUCGA；
所述用于转录crRNA的脱氧核酸序列为
KRAS-EXON2
TAGTTGGAGCTGGTGGCGTAGGCAAGGTTTTAGTCCCCTTCGTTTTTGGGGTAGTCTAAATCCCCTATAGTGAGTCGTATTAATTTC
KRAS-EXON3
TCGACACAGCAGGTCAAGAGGAGTACGTTTTAGTCCCCTTCGTTTTTGGGGTAGTCTAAATCCCCTATAGTGAGTCGTATTAATTTC。
所述Cas13a蛋白为Lsh Cas13a时
crRNA序列中的锚定序列为CCACCCCAAUAUCGAAGGGGACUAAAAC，
所述crRNA序列分别为
KRAS-EXON2
CCACCCCAAUAUCGAAGGGGACUAAAACCUUGCCUACGCCACCAGCUCCAACUA
KRAS-EXON3
CCACCCCAAUAUCGAAGGGGACUAAAACGUACUCCUCUUGACCUGCUGUGUCGA
用于转录crRNA的脱氧核酸序列为
KRAS-EXON2
TAGTTGGAGCTGGTGGCGTAGGCAAGGTTTTAGTCCCCTTCGATATTGGGGTGGCCCTATAGTGAGTCGTATTAATTTC
KRAS-EXON3
TCGACACAGCAGGTCAAGAGGAGTACGTTTTAGTCCCCTTCGATATTGGGGTGGCCCTATAGTGAGTCGTATTAATTTC。
靶向人KRAS基因的crRNA设计原则
由于CRISPR-Cas13a***是一种新型的靶向RNA基因编辑***。其中Cas13a是一种双核糖核酸酶，Cas13a与crRNA结合形成监测复合物，crRNA的向导区域用互补序列识别目标RNA，并且Cas13a降解目标RNA链。目前由于没有明确的crRNA设计原则，根据前期工作和经验，靶向人KRAS基因的crRNA设计要求如下：
crRNA包括蛋白锚定序列和向导序列，序列格式为：5`-与Cas13a蛋白结合的锚定序列 -crRNA向导序列-3`，蛋白锚定序列需要根据Cas13a蛋白来确定，使其能够与选定的Cas13a 蛋白匹配并结合；向导序列则与靶向DNA中的片段匹配。
crRNA向导RNA的选择
根据NCBI查找人KRAS基因序列，确定外显子2和外显子3发生突变的位点；crRNA靶点不能离起始密码子(ATG)太近；长度为21-28个核苷酸；最终，设计出分别靶向人KRAS基因外显子2和外显子3的crRNA向导序列，其针对的靶位点DNA序列如表1所示，其中大写部分为会发生突变的密码子。
人KRAS基因的突变位点靶标序列
与Cas13a蛋白结合的锚定序列的设计：
当Cas13a蛋白选用Lw Cas13a时，crRNA序列中的锚定序列为
GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAAC
当Cas13a蛋白选用Lsh Cas13a时，crRNA序列中的锚定序列为
CCACCCCAAUAUCGAAGGGGACUAAAAC
crRNA的合成
将化学合成的crRNA反转录成DNA并在5`添加T7启动子序列，所述的T7启动子序列为5`-TAATACGACTCACTATAGGG-3`；将上述带T7启动子DNA在T7RNA聚合酶作用下生成RNA，回收纯化，得到足量的crRNA，亦可根据设计的带T7启动子的crRNA转录模板 DNA在T7RNA聚合酶作用下生成RNA，回收纯化，得到足量的crRNA。
将合成的的靶向人KRAS基因的crRNA、样本DNA转录生成的RNA、相应的Cas13a蛋白、 RNA reporter(RNAse Alert V2，Thermo Scientfic)在核酸酶缓冲液中，37℃孵育1～3小时，通过荧光读数可以直观的分析KRAS基因外显子2和外显子3突变情况。所述的基于CRISPR技术特异性检测人KRAS基因2号及3号外显子突变的crRNA可以应用于人KRAS 基因外显子2和外显子3突变的检测。
具体实施中，选用KRAS野生型基因片段和KRAS突变型基因片段作为检测模型，按照发明公布的核酸检测技术检测了两段基因，可以检测出约50aM的KRAS野生型基因片段，同时通过向导RNA的设计，可以区分出一个碱基差异的DNA核酸序列，即KRAS野生型基因片段和 KRAS-G12D突变型基因片段。
综上所述，仅为本发明之较佳实施例，不以此限定本发明的保护范围，凡依本发明专利范围及说明书内容所作的等效变化与修饰，皆为本发明专利涵盖的范围之内。
Claims (4)
1.一种基于CRISPR技术特异性检测人KRAS基因2号及3号外显子突变的crRNA，其特征在于，包括CRISPR-Cas13a***，还包括可与CRISPR-Cas13a***结合的crRNA，所述crRNA序列格式为：5`-Cas13a蛋白锚定序列-crRNA向导序列-3`。
2.根据权利要求1所述的一种基于CRISPR技术特异性检测人KRAS基因2号及3号外显子突变的crRNA，其特征在于：所述Cas13a蛋白为LwCas13a，所述crRNA序列中的锚定序列为GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAAC，所述crRNA序列分别为
KRAS-EXON2
GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACCUUGCCUACGCCACCAGCUCCAACUA
KRAS-EXON3
GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGUACUCCUCUUGACCUGCUGUGUCGA；
所述用于转录crRNA的脱氧核酸序列为
KRAS-EXON2
TAGTTGGAGCTGGTGGCGTAGGCAAGGTTTTAGTCCCCTTCGTTTTTGGGGTAGTCTAAATCCCCTATAGTGAGTCGTATTAATTTC
KRAS-EXON3
TCGACACAGCAGGTCAAGAGGAGTACGTTTTAGTCCCCTTCGTTTTTGGGGTAGTCTAAATCCCCTATAGTGAGTCGTATTAATTTC。
3.根据权利要求1所述的一种基于CRISPR技术特异性检测人KRAS基因2号及3号外显子突变的crRNA，其特征在于：所述Cas13a蛋白为LshCas13a，crRNA序列中的锚定序列为CCACCCCAAUAUCGAAGGGGACUAAAAC，所述crRNA序列分别为
KRAS-EXON2
CCACCCCAAUAUCGAAGGGGACUAAAACCUUGCCUACGCCACCAGCUCCAACUA
KRAS-EXON3
CCACCCCAAUAUCGAAGGGGACUAAAACGUACUCCUCUUGACCUGCUGUGUCGA
用于转录crRNA的脱氧核酸序列为
KRAS-EXON2
TAGTTGGAGCTGGTGGCGTAGGCAAGGTTTTAGTCCCCTTCGATATTGGGGTGGCCCTATAGTGAGTCGTATTAATTTC
KRAS-EXON3
TCGACACAGCAGGTCAAGAGGAGTACGTTTTAGTCCCCTTCGATATTGGGGTGGCCCTATAGTGAGTCGTATTAATTTC。
4.权利要求1-3所述的一种基于CRISPR技术特异性检测人KRAS基因2号及3号外显子突变的crRNA在人KRAS基因外显子2和外显子3突变检测中的应用。
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Cited By (27)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109943567A (zh) * | 2019-04-16 | 2019-06-28 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | 一种CRISPR-Cas9介导的外显子定点突变技术在细胞系中应用 |
| US10465176B2 (en) | 2013-12-12 | 2019-11-05 | President And Fellows Of Harvard College | Cas variants for gene editing |
| US10508298B2 (en) | 2013-08-09 | 2019-12-17 | President And Fellows Of Harvard College | Methods for identifying a target site of a CAS9 nuclease |
| US10597679B2 (en) | 2013-09-06 | 2020-03-24 | President And Fellows Of Harvard College | Switchable Cas9 nucleases and uses thereof |
| CN110923314A (zh) * | 2019-12-30 | 2020-03-27 | 广州白云山拜迪生物医药有限公司 | 一组检测SNP位点rs9263726的引物、crRNA序列及其应用 |
| US10682410B2 (en) | 2013-09-06 | 2020-06-16 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
| US10704062B2 (en) | 2014-07-30 | 2020-07-07 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
| US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants and uses thereof |
| US10947530B2 (en) | 2016-08-03 | 2021-03-16 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US11046948B2 (en) | 2013-08-22 | 2021-06-29 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
| US11214780B2 (en) | 2015-10-23 | 2022-01-04 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
| US11268082B2 (en) | 2017-03-23 | 2022-03-08 | President And Fellows Of Harvard College | Nucleobase editors comprising nucleic acid programmable DNA binding proteins |
| US11306324B2 (en) | 2016-10-14 | 2022-04-19 | President And Fellows Of Harvard College | AAV delivery of nucleobase editors |
| US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| CN115404268A (zh) * | 2022-10-11 | 2022-11-29 | 芜湖森爱驰生物科技有限公司 | 一种sry基因检测探针和试剂盒 |
| US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
| US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
| US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
| US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins and uses thereof |
| US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
| US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
| US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
| US11912985B2 (en) | 2020-05-08 | 2024-02-27 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
| US12006520B2 (en) | 2011-07-22 | 2024-06-11 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
| US12031126B2 (en) | 2023-12-08 | 2024-07-09 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017218512A1 (en) * | 2016-06-13 | 2017-12-21 | Grail, Inc. | Enrichment of mutated cell free nucleic acids for cancer detection |
| CN107557455A (zh) * | 2017-09-15 | 2018-01-09 | 国家纳米科学中心 | 一种基于CRISPR‑Cas13a的特异性核酸片段的检测方法 |
| WO2018107129A1 (en) * | 2016-12-09 | 2018-06-14 | The Broad Institute, Inc. | Crispr effector system based diagnostics |
-
2018
- 2018-06-25 CN CN201810664661.2A patent/CN108893529A/zh active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017218512A1 (en) * | 2016-06-13 | 2017-12-21 | Grail, Inc. | Enrichment of mutated cell free nucleic acids for cancer detection |
| WO2018107129A1 (en) * | 2016-12-09 | 2018-06-14 | The Broad Institute, Inc. | Crispr effector system based diagnostics |
| CN107557455A (zh) * | 2017-09-15 | 2018-01-09 | 国家纳米科学中心 | 一种基于CRISPR‑Cas13a的特异性核酸片段的检测方法 |
Non-Patent Citations (2)
| Title |
|---|
| ZHAO XIAO等: "A CRISPR-Cas13a system for efficient and specific therapeutic targeting of mutant KRAS for pancreatic cancer treatment", 《CANCER LETTERS》 * |
| 王君等: "102 例肺腺癌常见基因突变与中医证型关系的研究", 《中国中医基础医学杂志》 * |
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