WO2023193748A1 - 基于crispr技术的靶基因捕获的方法 - Google Patents

基于crispr技术的靶基因捕获的方法 Download PDF

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WO2023193748A1
WO2023193748A1 PCT/CN2023/086511 CN2023086511W WO2023193748A1 WO 2023193748 A1 WO2023193748 A1 WO 2023193748A1 CN 2023086511 W CN2023086511 W CN 2023086511W WO 2023193748 A1 WO2023193748 A1 WO 2023193748A1
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sgrna
target gene
sequence
cas protein
crispr technology
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宋东亮
刘倩
刘娜
罗元廷
曹振
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翌圣生物科技(上海)股份有限公司
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Definitions

  • This application relates to the field of molecular biology, and specifically to a method for capturing target genes based on CRISPR technology.
  • next-generation sequencing NGS
  • whole-genome sequencing 2 branches of sequencing technology have emerged: 1) whole-genome sequencing; 2) targeted capture sequencing.
  • DNA targeted capture sequencing technology can enrich target genes, increase the proportion of target genes in the library, and greatly increase sequencing depth. For example, exons only account for 1% to 2% of the whole genome.
  • IDT Integrated DNA Technologies
  • the sequencing data of exons can be captured to more than 90%; the lowest detection limit can be to 0.1%.
  • the cost is reduced by about 100 times. Therefore, targeted capture sequencing has wide applications in fields such as genetic diseases, tumor detection, and site mutation research. High-throughput sequencing can directly obtain a large amount of relevant site variation information, which is also of great significance for precision medicine.
  • conventional methods based on target capture mainly include solid-phase carrier chips and solid-phase carrier magnetic beads.
  • the magnetic beads and probe are modified with avidin.
  • the probe specifically binds to the target sequence, and the magnetic beads specifically bind to the probe. It requires overnight hybridization, which takes a long time, about 20 hours; and the cost Too high, about 240 yuan per mg.
  • CRISPR technology research becomes more in-depth, its functions become more complete.
  • CRISPR technology is mostly used for insertion or site-specific silencing activation.
  • Research on targeted sequencing technology based on CRISPR technology is still in the basic research stage.
  • CPCS CRISPR-Protect Capture Sequence
  • This application provides a method for capturing target genes based on CRISPR technology.
  • the method includes the following steps:
  • the Cas protein includes but is not limited to Cas9, Cas12, Cas13 and Cas14.
  • the sgRNA library is designed based on the sequences at both ends of the target gene.
  • the distance between the sgRNAs at both ends of the target gene sequence does not exceed 10 kb, that is, the target gene region to be enriched should preferably not exceed 10 kb.
  • the binding conditions in step (1) are binding in 1 ⁇ Cas protein reaction solution at 20°C to 40°C.
  • the Cas protein reaction solution contains Tris-HCl, NaCl, MgCl2, bovine serum albumin (BSA) and polysorbate (Tween);
  • the concentration of Tris-HCl is 10mM ⁇ 200mM
  • the NaCl concentration is 10mM ⁇ 200mM
  • the MgCl2 concentration is 1mM ⁇ 50mM
  • the BSA concentration is 10ng/ ⁇ L ⁇ 50ng/ ⁇ L.
  • the Tween concentration is 0.01% to 2%.
  • the incubation temperature in step (2) is 25°C to 50°C.
  • the nucleic acid hydrolase described in step (3) is Escherichia coli exonuclease 1, Escherichia coli exonuclease 3, Escherichia coli exonuclease 7, phage exonuclease, T7 phage gene VI exonuclease or Bal 31 nuclease;
  • the usage amount of the nucleic acid hydrolase is 1 U/rxn to 1000 U/rxn.
  • the hydrolysis conditions described in step (3) are 25°C to 50°C.
  • the nucleic acid hydrolase described in step (3) is Escherichia coli exonuclease III and/or T7 phage gene VI exonuclease, and the termination of the nucleic acid hydrolase reaction is specifically inactivation at 70°C to 80°C.
  • the nucleic acid hydrolase is Escherichia coli exonuclease III and/or T7 phage gene VI exonuclease, and the termination of the nucleic acid hydrolase reaction is specifically inactivation at 70°C to 80°C.
  • the sgRNA library is prepared by the following steps:
  • T7 promoter-gRNA-Cas protein backbone DNA sequence as the upstream primer, the 3-terminal sequence of the universal Cas protein backbone DNA sequence as the downstream primer, add the Cas protein backbone DNA as a template, and amplify to obtain T7 promoter.
  • this application provides a lung cancer-related gene sequencing kit based on the target gene capture method of CRISPR technology, which includes sgRNA sequence, Cas protein and nucleolytic acid hydrolase;
  • the sgRNA sequence is selected from at least one of SEQ ID NO. 168-334.
  • the universal skeleton sequence of the Cas protein in the sequencing kit provided by this application is SEQ ID NO. 336.
  • the nucleic acid hydrolase in the sequencing kit provided by the present application is Escherichia coli exonuclease III and/or T7 phage gene VI exonuclease white.
  • the target gene capture method of the present application can be used not only for mRNA library construction, but also for direct gDNA library construction.
  • this technology does not require the addition of a large number of non-target fragments to participate in library construction, and can maximize the amplification of genes in the target region.
  • this technology does not require the use of a large number of modified probes and expensive modified solid-phase carriers, and can minimize the cost of capturing target genes.
  • this capture method can completely retain one or more target genes, and the integrity of the target genes can be guaranteed to the maximum extent.
  • this technology can complete the complete capture and library construction process in as little as 5 hours, which greatly improves detection efficiency and saves time and cost.
  • Figure 1 is a schematic diagram of the capture process based on CRISPR technology.
  • Figure 2 is a schematic diagram of the capture of this application.
  • Figure 3 shows the gRNA oligo sequence design diagram.
  • Figure 4 is a diagram showing the proportion of EGFR, KRAS, BRAF, HER2, ALK, ROS1, MET, and RET genes in sequencing samples 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 in Example 2.
  • Figure 5 is a graph showing the proportion of single sgRNA of the EGFR gene in the Target Reads of sequencing sample 1 in Example 2.
  • Figure 6 is a graph showing the proportion of a single sgRNA of the KRAS gene in the Target Reads of sequencing sample 2 in Example 2.
  • Figure 7 is a graph showing the proportion of a single sgRNA of the BRAF gene in the Target Reads of sequencing sample 3 in Example 2.
  • Figure 8 is a graph showing the proportion of a single sgRNA of the HER2 gene in the Target Reads of sequencing sample 4 in Example 2.
  • Figure 9 is a graph showing the proportion of a single sgRNA of the ALK gene in the Target Reads of sequencing sample 5 in Example 2.
  • Figure 10 is a graph showing the proportion of a single sgRNA of the ROS1 gene in the Target Reads of sequencing sample 6 in Example 2.
  • Figure 11 is a diagram showing the proportion of a single sgRNA of the MET gene of sequencing sample 7 in Target Reads in Example 2.
  • Figure 12 is a graph showing the proportion of a single sgRNA of the RET gene of sequencing sample 8 in Target Reads in Example 2.
  • Figure 13 is a comparison chart of the sequencing analysis of the actual site mutation rates of the sequencing samples and standards in Example 3.
  • Lung cancer is a disease that currently has more research, more detection methods, and more clinical data. Therefore, lung cancer-related genes (EGFR, KRAS, BRAF, HER2, ALK, ROS1, MET, RET) are used for explanation.
  • Example 1 gRNA design sequence and sgRNA library preparation
  • sgRNA was designed based on the sequences of lung cancer-related genes (EGFR, KRAS, BRAF, HER2, ALK, ROS1, MET, RET). All primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.
  • the gRNA design sequence is shown in Table 1:
  • the final synthesized gRNAoligo sequence is: 5'-TTCTAATACGACTCACTATA(SEQ ID NO.335)-gRNA-GTTTTAGAGCTAGA(SEQ ID NO.336)-3', as shown in Figure 3, it is also necessary to add between the T7 promoter sequence and gRNA G, the adding rules are: if the first two digits of gRNA are G, no need to add, if the first digit is G, add one G, if the first and second digits are not G, add two GG.
  • the upstream primer uses 5'-AAAAGCACCGACTCGGTGCC-3' (SEQ ID NO.337) as the downstream universal primer, and add a small part of the Cas protein backbone sequence as a template to amplify single or multiple sgRNAs.
  • the Cas9 backbone sequence is : AATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTTTTT (SEQ ID NO. 338) to obtain the DNA product.
  • the sgRNA-Cas protein backbone RNA was transcribed in vitro. The complete sgRNA sequence after transcription is shown in Table 2.
  • sgRNA amplification uses Precision sgRNA Synthesis Kit, YEASEN):
  • sgRNA library in vitro transcription adopts T7 High Yield RNA Synthesis Kit, YEASEN):
  • the sgRNAs designed according to the 8 genes of EGFR, KRAS, BRAF, HER2, ALK, ROS1, MET, and RET were mixed in equal concentrations into EGFR-sgRNA Mix, KRAS-sgRNA Mix, BRAF-sgRNA Mix, HER2-sgRNA Mix, ALK-sgRNA Mix, ROS1-sgRNA Mix, MET-sgRNA Mix, RET-sgRNA Mix.
  • KRAS-sgRNA Mix Take KRAS-sgRNA Mix as an example.
  • 4 gRNAs are designed in the KRAS gene. After the steps in Example 1, four KRAS sgRNA libraries with inconsistent concentrations are obtained, and then equal amounts are mixed to obtain KRAS-sgRNA Mix.
  • RNA capture kit Hieff Prepare mRNA using the mRNA Isolation Master Kit (mRNA, YEASEN): (1) Add 1 ⁇ g of RNA template (RNA sample that needs to be captured and sequenced); add water to 50 ⁇ L; add 50 ⁇ L of mRNA Capture Beads, mix by pipetting; (2) 65°C, 5 min; 25°C, 5min; 25°C, hold; (3) Leave at room temperature for 5min; (4) Remove the supernatant; (5) Use 200 ⁇ L Beads Wash Buffer to resuspend the magnetic beads and wash, transfer Remove the supernatant and repeat twice; (6) Add 50 ⁇ L Tris Buffer to resuspend the magnetic beads, mix well, and let stand for 5 minutes; (7) Remove the supernatant, use 200 ⁇ L Beads Wash Buffer to resuspend the magnetic beads, and remove the supernatant. ; (8) Resuspend the magnetic beads in 15 ⁇ L DEPC-H 2 O
  • RNA library construction kit Hieff DNA&RNA Library Co-Prep Kit for Illumina, YEASEN
  • 3 ⁇ L; 16°C 30min; 4 °Chold Add 1.2 ⁇ DNA Cleaner (Hieff DNA Selection Beads, YEASEN) purification; (5) 22 ⁇ L H 2 O elution; use Qubit to determine CDNA concentration.
  • EGFR-sgRNA Mix is used for sgRNA
  • KRAS-sgRNA Mix is used for sgRNA
  • BRAF-sgRNA Mix is used for sgRNA
  • HER2-sgRNA is used for sgRNA.
  • sgRNA uses ALK-sgRNA Mix
  • sgRNA uses ROS1-sgRNA Mix
  • sgRNA uses MET-sgRNA Mix
  • sgRNA uses RET-sgRNA Mix
  • sgRNA uses EGFR-sgRNA Mix, KRAS-sgRNA Mix, BRAF-sgRNA Mix, HER2-sgRNA Mix, ALK-sgRNA Mix, ROS1-sgRNA Mix, MET-sgRNA Mix, RET-sgRNA Mix and other concentration mixed Mix
  • Target gene capture is not performed;
  • Mapping (%) Map Reads/Reads*100%
  • Target Reads (%) Target Reads/Map Reads*100%. Since the presence of sgRNA results in a very small part of sgRNA data remaining in the final data, the Mapping rate is slightly lower than that of conventional library construction by about 1% to 2%; compared with the capture efficiency of traditional probe method capture, this method The capture efficiency is higher than that of traditional probe methods, which is due to the specific cleavage of Cas protein and the joint action of nucleolytic enzymes.
  • the sgRNA sequence can be adjusted; among the 4 sgRNAs of the KRAS gene, there is no significant difference in efficiency, and it is within the acceptable range, and no adjustment is needed; among the 16 sgRNAs of the BRAF gene, there is no significant difference in efficiency, and it is within the acceptable range.
  • the sgRNA sequence can be adjusted appropriately; among the 44 sgRNAs of the ROS1 gene, the proportion of ROS1-1 and ROS1-43 in the capture site analysis is lower than the average number (1 /44*100%), the efficiency is low, the sgRNA sequence can be adjusted appropriately; among the 25 sgRNAs of the MET gene, MET-4, MET-6, MET-13, MET-14, and MET-21 are included in the capture site analysis The proportion is lower than the average (1/25*100%), and the efficiency is low.
  • the sgRNA sequence can be adjusted appropriately; there is no large gap in efficiency among the 17 sgRNA
  • Target gene capture Pre-binding: (1) sgRNA Mix 1 ⁇ g; Cas protein 10pmol; 10 ⁇ Cas protein reaction solution 3 ⁇ L; add water to 29 ⁇ L; 25°C ⁇ 30°C for 30min (25°C is used in this example); target sequence Cutting: (2) Add 100ng of lung cancer standard (Jingliang, GW-OGTM800) template; 30°C ⁇ 40°C for 60min ⁇ 120min (37°C60min is used in this example); Non-target sequence digestion: (3) Add nucleic acid hydrolysis In this example, Escherichia coli exonuclease III and T7 phage gene VI exonuclease are used. Each enzyme is 10U/rxn.
  • This example has a total of 1 sequencing sample: (1) sgRNA uses EGFR-sgRNA Mix, KRAS-sgRNA Mix, BRAF-sgRNA Mix, HER2-sgRNA Mix, ALK-sgRNA Mix, ROS1-sgRNA Mix, MET-sgRNA Mix, RET -sgRNA Mix equal concentration mix;
  • the sequencing platform is illumina.
  • mapping (%) Map Reads/Reads*100%
  • Target Reads (%) Target Reads/Map Reads*100%. There is no abnormality in the mapping rate and targeting rate of actual mutation samples.

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Abstract

一种基于CRISPR技术的新型靶基因捕获的方法（CPCS），所述方法包括：使用sgRNACas蛋白复合体进行对靶基因的序列切割捕获，同时使用核酸水解酶消解非靶基因序列得到靶基因样本。所述方法使用CRISPR技术结合核酸水解酶进行靶基因捕获，利用CRISPR的进行精准定位并切割的特点进行靶基因序列与非靶基因序列分开，同时利用Cas蛋白切割后保留在切割位点的特点使用核酸水解酶对非靶基因进行水解从而获得靶基因。其具有低成本，高特异性，短时长捕获的特点，最短仅需5小时即可完成靶基因捕获建库流程。

Description

基于CRISPR技术的靶基因捕获的方法 技术领域
本申请涉及分子生物学领域，具体涉及一种基于CRISPR技术的靶基因捕获的方法。
背景技术
随着二代测序(Next-generation sequencing，NGS)技术高速发展，测序技术出现了两个分支：1)全基因组测序；2)靶向捕获测序。
由于全基因组测序可获得某一物种的个体或群体核DNA的全部遗传信息，与参考基因组对比可获得非常全面的遗传标记信息，同时利用二代测序的技术可获得未知生物的序列，进而在科研、临床感染、物种进化等领域上具有广泛的应用。但面临单个或多个基因的精准定位研究时，由于在庞大的遗传信息中所需要的仅占0.0001％～2％，占比极低，极大部分均为无效数据，尽管测序数据有极少量的所需信息，但由于数据量过少导致无法得到准确的判断；由于在单个或多个基因的精准定位研究中无效数据过多，导致单个样本测序成本过高，迫使研究人员开发另一种方法：靶向捕获测序。
将DNA靶向捕获测序技术应用到二代测序中，可将目标基因富集，提高目标基因在文库中的占比，以及大幅度提高测序深度。例如，外显子在全基因组中仅占1％～2％，使用IDT(Integrated DNA Technologies)的全外显子捕获时，可将外显子的测序数据捕获至90％以上；检测极限最低可至0.1％。相较于非捕获的全基因组测序，成本下降约100倍。因此，靶向捕获测序在遗传病、肿瘤检测、位点突变研究等领域上具有广泛的应用；高通量测序可直接获得大量相关位点变异信息，对于精准医疗也具有重大意义。但常规基于靶向捕获的方法主要有固相载体芯片与固相载体磁珠的技术。均需要对固相载体进行修饰后，才能进行特异性结合。例如M-270磁珠，对磁珠与探针进行亲和霉素修饰，探针特异性结合靶序列，磁珠特异性结合探针，需要过夜杂交，耗费时间长，约20小时；且成本过高，约240元每毫克。
随着CRISPR技术研究越发深入，其功能也越发完整。CRISPR技术大都用于插入或定点沉默激活等用途。基于CRISPR技术的靶向测序技术研究仍处于基础研究阶段。
发明内容
本申请提供了一种新型靶基因捕获的方法(CPCS)及试剂，命名为：CRISPR-Protect Capture Sequence(CPCS)。
本申请提供了一种基于CRISPR技术的靶基因捕获的方法，所述方法包括如下步骤：
(1)设计并制备靶基因的sgRNA库，将制备好的sgRNA与Cas蛋白结合，获得sgRNA/Cas复合体；
(2)将sgRNA/Cas复合体与待测样本混合孵育，获得被sgRNA/Cas复合体切割后的被处理样本；以及
(3)在被处理样本中加入核酸水解酶，水解非靶基因的序列，然后终止核酸水解酶反应，纯化获得靶基因样本。
优选地，所述Cas蛋白包括但不限于Cas9、Cas12、Cas13和Cas14。
优选地，所述sgRNA库根据靶基因两端的序列进行设计，靶基因序列两端sgRNA间隔不超过10kb，也即欲富集的靶基因区域最好不要超过10kb。
优选地，步骤(1)中所述结合的条件为在1×Cas蛋白反应液中以20℃～40℃条件下结合。
优选地，所述的Cas蛋白反应液中含有Tris-HCl、NaCl、MgCl2、牛血清白蛋白(BSA)和聚山梨酯(Tween)；
其中，所述的Tris-HCl浓度为10mM～200mM；
所述的NaCl浓度为10mM～200mM；
所述的MgCl2浓度为1mM～50mM；
所述的BSA浓度为10ng/μL～50ng/μL；以及
所述的Tween浓度为0.01％～2％。
优选地，步骤(2)中所述孵育的温度为25℃～50℃。
优选地，步骤(3)中所述的核酸水解酶为大肠杆菌外切酶1、大肠杆菌外切酶3、大肠杆菌外切酶7、噬菌体核酸外切酶、T7噬菌体基因VI核酸外切酶或Bal 31核酸酶；
优选地，所述核酸水解酶的使用量为1U/rxn～1000U/rxn。
优选地，步骤(3)中所述水解的条件为25℃～50℃。
优选地，步骤(3)所述的核酸水解酶为大肠杆菌外切酶III和/或T7噬菌体基因VI核酸外切酶，所述终止核酸水解酶反应具体为70℃～80℃条件下灭活所述核酸水解酶。
优选地，所述sgRNA库的制备通过以下步骤制得：
A.合成T7启动子-gRNA-Cas蛋白骨架的DNA序列；
B.使用T7启动子-gRNA-Cas蛋白骨架DNA序列的5端重叠区域为上游引物，通用Cas蛋白骨架DNA序列的3端序列作为下游引物，添加Cas蛋白骨架DNA作为模板，扩增得到T7启动子-gRNA-Cas蛋白骨架DNA；以及
C.使用T7体外转录获得sgRNA库。
优选地，本申请提供一种基于CRISPR技术的靶基因捕获的方法的肺癌相关基因的测序试剂盒，其包括sgRNA序列、Cas蛋白和核酸水解酶；
其中，所述sgRNA序列选自SEQ ID NO.168-334中的至少一种。
优选地，本申请提供的测序试剂盒中的Cas蛋白的通用骨架序列为SEQ ID NO.336。
优选地，本申请提供的测序试剂盒中的所述核酸水解酶为大肠杆菌外切酶III和/或T7噬菌体基因VI核酸外切酶白。
本申请的靶基因捕获方法不但可以用于mRNA建库，也可以用于gDNA直接建库。
与现有技术相比，本申请具有如下有益效果：
(1)与传统捕获的方法相比，本技术无需在建库时加入大量的非目标片段参与建库，能最大限度的放大目标区域的基因。
(2)与传统捕获的方法相比，本技术无需使用大量带修饰的探针和昂贵的带修饰的固相载体，能最大限度的降低目标基因的捕获成本。
(3)与传统捕获的方法相比，本捕获方法可完整保留一条或多条目标基因，其目标基因的完整度能得到最大限度的保障。
(4)与传统捕获的方法相比，本技术最短可在5小时完成捕获建库完整流程，极大程度上提升了检测效率，节约时间成本。
附图说明
图1为基于CRISPR技术的捕获流程示意图。
图2为本申请的捕获原理图。
图3为gRNA oligo序列设计图。
图4为实施例2中测序样品1、2、3、4、5、6、7、8、9、10中EGFR、KRAS、BRAF、HER2、ALK、ROS1、MET、RET基因的占比图。
图5为实施例2中的测序样品1的EGFR基因的单条sgRNA在Target Reads中的占比图。
图6为实施例2中的测序样品2的KRAS基因的单条sgRNA在Target Reads中的占比图。
图7为实施例2中的测序样品3的BRAF基因的单条sgRNA在Target Reads中的占比图。
图8为实施例2中的测序样品4的HER2基因的单条sgRNA在Target Reads中的占比图。
图9为实施例2中的测序样品5的ALK基因的单条sgRNA在Target Reads中的占比图。
图10为实施例2中的测序样品6的ROS1基因的单条sgRNA在Target Reads中的占比图。
图11为实施例2中的测序样品7的MET基因的单条sgRNA在Target Reads中的占比图。
图12为实施例2中的测序样品8的RET基因的单条sgRNA在Target Reads中的占比图。
图13为实施例3中的测序样品与标准品实际位点突变率的测序分析对比图。
具体实施方式
为进一步阐述本申请所采取的技术手段及其效果，以下结合实施例对本申请作进一步地说明。基于CRISPR技术的捕获流程如图1所示。肺癌是目前研究较多、检测方法较多、临床数据较多的疾病。故采用肺癌相关基因(EGFR、KRAS、BRAF、HER2、ALK、ROS1、MET、RET)进行阐述。
实施例1 gRNA设计序列及sgRNA库制备
根据肺癌相关基因(EGFR、KRAS、BRAF、HER2、ALK、ROS1、MET、RET)序列设计下述sgRNA，所有的引物均由生工生物工程(上海)股份有限公司代为合成。
gRNA设计序列如表1所示：
表1.gRNA设计序列





表2.转录后完整sgRNA序列












根据表1所设计的gRNA oligo在合成时，在gRNA oligo前端加上T7启动子序列：5’-TTCTAATACGACTCACTATA-3’(SEQ ID NO.335)；并在gRNA oligo后端加上Cas蛋白通用骨架序列：5’-GTTTTAGAGCTAGA-3’(SEQ ID NO.336)。
最终合成gRNAoligo序列为：5’-TTCTAATACGACTCACTATA(SEQ ID NO.335)-gRNA-GTTTTAGAGCTAGA(SEQ ID NO.336)-3’，如图3所示，还需要在T7启动子序列与gRNA之间添加G，添加规则为：如果gRNA前两位是G就不用添加，如果第一位是G就添加一个G，如果第一位和第二位不是G就添加两个GG。以该序列作为上游引物，以5’-AAAAGCACCGACTCGGTGCC-3’(SEQ ID NO.337)为下游通用引物，添加少部分Cas蛋白骨架序列作为模板进行单条或多条sgRNA的扩增，Cas9骨架序列为：AATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT(SEQ ID NO.338)，得到DNA产物，以DNA产物为模板，进行sgRNA-Cas蛋白骨架RNA体外转录，转录后完整sgRNA序列如表2所示。
sgRNA库制备具体实施流程如下(sgRNA扩增采用Precision sgRNA Synthesis Kit，YEASEN)：
1)加入Scaffold模板1.25μL；gRNA oligo(10μM)；下游通用引物(10μM)；2×Canace Enzyme Mix 12.5μL；补水至25μL；
2)98℃10S；68℃10S；30cycles；
3)取5μL进行电泳，验证条带是否单一；
sgRNA库体外转录具体实施流程如下(sgRNA库体外转录采用T7 High Yield RNA Synthesis Kit，YEASEN)：
1)10×Transcription Buffer 2μL；CTP/GTP/ATP/UTP(100mM)各2μL；T7 RNA Polymerase Mix 2μL；sgRNA库1μg；补水至20μL；
2)37℃120min；
3)加入DNase I 2μL；
4)37℃15min；
5)2.0×RNA Cleaner(HieffRNA Cleaner，YEASEN)磁珠纯化；
6)取5μL进行电泳，验证条带是否单一；
实施例2 sgRNA测试
1.根据EGFR、KRAS、BRAF、HER2、ALK、ROS1、MET、RET 8个基因所设计的sgRNA分别等浓度混合为EGFR-sgRNA Mix、KRAS-sgRNA Mix、BRAF-sgRNA Mix、HER2-sgRNA Mix、ALK-sgRNA Mix、ROS1-sgRNA Mix、MET-sgRNA Mix、RET-sgRNA Mix。以KRAS-sgRNA Mix为例，比如KRAS基因中设计有4条gRNA，经过实施例1步骤后分别获得4条浓度不一致的KRAS的sgRNA文库，随后进行等量混合，即获得KRAS-sgRNA Mix。
2.实验方法：
1)使用mRNA捕获试剂盒(HieffmRNA Isolation Master Kit mRNA，YEASEN)制备mRNA：(1)RNA模板(需要进行捕获测序的RNA样本)投入1μg；补水至50μL；加入mRNA Capture Beads 50μL，吹打混匀；(2)65℃，5min；25℃，5min；25℃，hold；(3)室温静置5min；(4)去除上清；(5)使用200μL Beads Wash Buffer重悬磁珠洗涤，移 除上清，重复两次；(6)加入50μL Tris Buffer重悬磁珠，混匀，静置5min；(7)移除上清，使用200μL Beads Wash Buffer重悬磁珠洗涤，移除上清；(8)15μL DEPC-H2O重悬磁珠；80℃2min；立即转移至新PCR管。
2)使用RNA建库试剂盒(HieffDNA&RNA Library Co-Prep Kit for Illumina，YEASEN)制备二链CDNA：(1)Dt23VN 1μL；步骤1制得的mRNA 14μL；70℃5min；立即置于冰上；(2)1st Reaction Buffer 8μL；1st Strand Enzyme Mix 2μL；加热变性的RNA 15μL；25℃5min；42℃30min；85℃5min；4℃hold；(3)1st Strand cDNA 25μL；2nd Reaction Buffer 7μL；2nd Strand Enzyme Mix 3μL；16℃30min；4℃hold；(4)加入1.2×DNA Cleaner(HieffDNA Selection Beads，YEASEN)纯化；(5)22μL H2O洗脱；使用Qubit测定CDNA浓度。
3)靶基因捕获：预结合：(1)sgRNA Mix 1μg；Cas蛋白10pmol；10×Cas蛋白反应液3μL；补水至29μL；25℃～30℃30min(本实施例中使用25℃)；靶序列切割：(2)加入步骤2)制得的二链CDNA模板100ng；30℃～40℃60min～120min(本实施例中使用37℃60min)；非靶序列消化：(3)加入核酸水解酶本实施例中使用大肠杆菌外切酶III与T7噬菌体基因VI核酸外切酶混合，每种酶10U/rxn 37℃120min；75℃20min；纯化：(4)加入1.0～1.8×DNA Cleaner(HieffDNA Selection Beads，YEASEN)纯化；(5)22μL H2O洗脱。10μL用于电泳；10μL用于建库。
4)使用DNA建库试剂盒(HieffOnePot Pro DNA Library Prep Kit for Illumina，YEASEN)进行靶序列建库：(1)Buffer 10μL；Enzyme 5μL；靶基因捕获DNA 10μL；补水至60μL；4℃1min；30℃20min；65℃20min；4℃hold；(2)Ligation Enhancer 30μL；Novel T4 DNA Ligase 5μL；DNA Adapter 5μL；dA-tailed DNA(1)产物)60μL；20℃15min；4℃hold；(3)0.6×DNA Cleaner(HieffDNA Selection Beads，YEASEN)纯化；(4)22μL H2O洗脱；(5)Pro Amplifecation Mix 25μL；纯化产物20μL；I5/I7 5μL；98℃1min；(98℃10S；60℃30S；72℃30S)×10；72℃1min；4℃hold；(6)0.9×DNA Cleaner(HieffDNA Selection Beads，YEASEN)纯化；(7)22μL H2O洗脱；(8)使用Qubit测定浓度，并进行电泳与测序；
3.实验设计：
本实施例共10个测序样品，分别是：(1)sgRNA采用EGFR-sgRNA Mix；(2)sgRNA采用KRAS-sgRNA Mix；(3)sgRNA采用BRAF-sgRNA Mix；(4)sgRNA采用HER2-sgRNA Mix；(5)sgRNA采用ALK-sgRNA Mix；(6)sgRNA采用ROS1-sgRNA Mix；(7)sgRNA采用MET-sgRNA Mix；(8)sgRNA采用RET-sgRNA Mix；(9)sgRNA采用EGFR-sgRNA Mix、KRAS-sgRNA Mix、BRAF-sgRNA Mix、HER2-sgRNA Mix、ALK-sgRNA Mix、ROS1-sgRNA Mix、MET-sgRNA Mix、RET-sgRNA Mix等浓度混合Mix；(10)不进行靶基因捕获；
4.结果分析：
1)靶向富集原理：Cas蛋白切割后蛋白保留在gRNA结合位点，具有空间位置的占据效果，核酸水解酶在水解过程中无法通过被Cas蛋白占据的位点，及Cas蛋白对该靶位点具有保护作用；靶序列上下游分别设计gRNA对靶序列进行切割保护(图2)。切割完成后使用核酸水解酶对未被保护的序列区域进行水解；随后进行失活、纯化、建库。分析结果如图4-12所示。
表3.各样本测序数据
                                               *测序平台为illumina。
2)Mapping(％)及Target Reads(％)分析：在测序数据分析中，Mapping(％)＝Map Reads/Reads*100％；Target Reads(％)＝Target Reads/Map Reads*100％。由于sgRNA的存在导致最终数据中有极小部分的sgRNA数据残留，故Mapping率相较于常规建库略低约1％～2％；相较于传统的探针法捕获的捕获效率，本方法在的捕获效率高于传统的探针法，这得益于Cas蛋白的特异性切割与核酸水解酶的共同作用。
表4.各基因在样本中的占比如下(％)
3)基因占比(％)分析：各样本中的EGFR、KRAS、BRAF、HER2、ALK、ROS1、MET、RET 8个基因占比数据拆分进行分析。单个基因具有良好的特异性富集率，在面对多个基因的复合Mix时同样具有良好的特异性富集率，其靶向率并没有受靶序列的增多而减少。在多个基因的复合Mix中，单个基因的富集率受其sgRNA效率所影响；如HER2基因所设计的sgRNA具有良好的特异性及工作效率，故HER2基因无论在单基因富集还是在多个基因富集时相较于其他基因具有最高的靶向率。
表5.Target Reads中各sgRNA所占reads数




4)单条sgRNA在单个基因样本(％)的占比分析：根据EGFR、KRAS、BRAF、HER2、ALK、ROS1、MET、RET 8个基因中共134条sgRNA序列进行逐条序列的所在位点进行分析，分析单条sgRNA在单基因中占比，对其评定工作效率。在EGFR基因的22条sgRNA中：EGFR-4、EGFR-5、EGFR-7、EGFR-9、EGFR-10在捕获位点分析中占比远低于平均数(1/21*100％)，效率过低，可调整sgRNA序列；在KRAS基因的4条sgRNA中效率无大幅度差距，在可接受范围内，无需调整；在BRAF基因的16条sgRNA中效率无大幅度差距，在可接受范围内，无需调整；在HER2基因的10条sgRNA中效率无大幅度差距，在可接受范围内，无需调整；在ALK基因的29条sgRNA中：ALK-13在捕获位点分析中占比远于平均数(1/29*100％)，效率偏低，可适当调整sgRNA序列；在ROS1基因的44条sgRNA中ROS1-1、ROS1-43在捕获位点分析中占比低于平均数(1/44*100％)，效率偏低，可适当调整sgRNA序列；在MET基因的25条sgRNA中MET-4、MET-6、MET-13、MET-14、MET-21在捕获位点分析中占比低于平均数(1/25*100％)，效率偏低，可适当调整sgRNA序列；在RET基因的17条sgRNA中效率无大幅度差距，在可接受范围内，无需调整。
实施例3肺癌标准品捕获建库
1.根据EGFR、KRAS、BRAF、HER2、ALK、ROS1、MET、RET 8个基因所设计的sgRNA等浓度混合为EGFR-sgRNA Mix、KRAS-sgRNA Mix、BRAF-sgRNA Mix、HER2-sgRNA Mix、ALK-sgRNA Mix、ROS1-sgRNA Mix、MET-sgRNA Mix、RET-sgRNA Mix。
2.实验方法：
1)靶基因捕获：预结合：(1)sgRNA Mix 1μg；Cas蛋白10pmol；10×Cas蛋白反应液3μL；补水至29μL；25℃～30℃30min(本实施例中使用25℃)；靶序列切割：(2)加入肺癌标准品(菁良，GW-OGTM800)模板100ng；30℃～40℃60min～120min(本实施例中使用37℃60min)；非靶序列消化：(3)加入核酸水解酶本实施例中使用大肠杆菌外切酶III与T7噬菌体基因VI核酸外切酶混合，每种酶10U/rxn 37℃120min；75℃20min，纯化； (4)加入1.0～1.8×DNA Cleaner(HieffDNA Selection Beads，YEASEN)纯化；(5)22μL H2O洗脱。10μL用于电泳；10μL用于建库。
2)使用DNA建库试剂盒(HieffOnePot Pro DNA Library Prep Kit for Illumina，YEASEN)进行靶序列建库：(1)Buffer 10μL；Enzyme 5μL；靶基因捕获DNA 10μL；补水至60μL；4℃1min；30℃20min；65℃20min；4℃hold；(2)Ligation Enhancer 30μL；Novel T4 DNA Ligase 5μL；DNA Adapter 5μL；dA-tailed DNA(1)产物)60μL；20℃15min；4℃hold；(3)0.6×DNA Cleaner(HieffDNA Selection Beads，YEASEN)纯化；(4)22μL H2O洗脱；(5)Pro Amplification Mix 25μL；纯化产物20μL；I5/I7 5μL；98℃1min；(98℃10S；60℃30S；72℃30S)×10；72℃1min；4℃hold；(6)0.9×DNA Cleaner(HieffDNA Selection Beads，YEASEN)纯化；(7)22μL H2O洗脱；(8)使用Qubit测定浓度，并进行电泳与测序；结果如图13所示。
3.实验设计：
本实施例共1个测序样品：(1)sgRNA采用EGFR-sgRNA Mix、KRAS-sgRNA Mix、BRAF-sgRNA Mix、HER2-sgRNA Mix、ALK-sgRNA Mix、ROS1-sgRNA Mix、MET-sgRNA Mix、RET-sgRNA Mix等浓度混合Mix；
4.结果分析：
表6.测序数据
                                      *测序平台为illumina。
1)Mapping(％)及Target Reads(％)分析：在测序数据分析中，Mapping(％)＝Map Reads/Reads*100％；Target Reads(％)＝Target Reads/Map Reads*100％。实际突变样本可Map率与靶向率无异常。
表7.肺癌标准品(菁良，GW-OGTM800)突变频率与实际测序突变频率
                                                *测序平台为illumina。
2)肺癌标准品(菁良，GW-OGTM800)突变频率与实际测序突变频率分析：肺癌标准品(菁良，GW-OGTM800)的预期突变频率与本方法测试出的突变频率差距在可控范围以内。

Claims (11)

  1. 一种基于CRISPR技术的靶基因捕获的方法，其包括：
    (1)设计并制备靶基因的sgRNA库，将制备好的sgRNA与Cas蛋白结合，获得sgRNA/Cas复合体；
    (2)将sgRNA/Cas复合体与待测样本混合孵育，sgRNA/Cas复合体结合到靶序列的对应位置，获得sgRNA/Cas/靶序列三元复合物；以及
    (3)在被处理样本中加入核酸水解酶，水解非靶基因的序列，然后终止核酸水解酶反应，纯化获得靶基因样本。
  2. 根据权利要求1所述的基于CRISPR技术的靶基因捕获的方法，其中，所述Cas蛋白包括但不限于Cas9、Cas12、Cas13和Cas14。
  3. 根据权利要求1所述的基于CRISPR技术的靶基因捕获的方法，其中，所述sgRNA库根据靶基因两端的序列进行设计，靶基因序列两端sgRNA间隔不超过10kb。
  4. 根据权利要求1所述的基于CRISPR技术的靶基因捕获的方法，其中，步骤(1)中所述结合的条件为在1×Cas蛋白反应液中以20℃～40℃条件下结合。
  5. 根据权利要求4所述的基于CRISPR技术的靶基因捕获的方法，其中，所述的Cas蛋白反应液中含有Tris-HCl、NaCl、MgCl2、牛血清白蛋白和聚山梨酯；
    所述的Tris-HCl浓度为10mM～200mM；
    所述的NaCl浓度为10mM～200mM；
    所述的MgCl2浓度为1mM～50mM；
    所述的牛血清白蛋白浓度为10ng/μL～50ng/μL；以及
    所述的聚山梨酯浓度为0.01％～2％体积比。
  6. 根据权利要求1所述的基于CRISPR技术的靶基因捕获的方法，其中，步骤(2)中所述孵育的温度为25℃～50℃。
  7. 根据权利要求1所述的基于CRISPR技术的靶基因捕获的方法，其中，步骤(3)中所述的核酸水解酶为大肠杆菌外切酶1、大肠杆菌外切酶3、大肠杆菌外切酶7、噬菌体核酸外切酶、T7噬菌体基因VI核酸外切酶或Bal 31核酸酶。
  8. 根据权利要求7所述的基于CRISPR技术的靶基因捕获的方法，其中，所述的核酸水解酶的使用量为1U/rxn～1000U/rxn。
  9. 根据权利要求1所述的基于CRISPR技术的靶基因捕获的方法，其中，步骤(3)中所述水解的条件为25℃～50℃。
  10. 根据权利要求1所述的基于CRISPR技术的靶基因捕获的方法，其中，所述sgRNA库的制备通过以下步骤制得：
    A.合成T7启动子-gRNA-Cas蛋白骨架的DNA序列；
    B.使用T7启动子-gRNA-Cas蛋白骨架DNA序列的5端重叠区域为上游引物，通用Cas蛋白骨架DNA序列的3端序列作为下游引物，添加Cas蛋白骨架DNA作为模板，扩增得到T7启动子-gRNA-Cas蛋白骨架DNA；以及
    C.使用T7体外转录获得sgRNA库。
  11. 一种根据权利要求1-10任一项所述的基于CRISPR技术的靶基因捕获的方法的肺癌相关基因的测序试剂盒，其包括sgRNA序列、Cas蛋白和核酸水解酶；
    其中，所述sgRNA序列选自SEQ ID NO.168-334中的至少一种。
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