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CN-121992073-A - Method for batch detection of gene editing event and related sequencing library construction method thereof

CN121992073ACN 121992073 ACN121992073 ACN 121992073ACN-121992073-A

Abstract

The invention discloses a method for detecting gene editing events in batches and a related sequencing library construction method thereof. According to the invention, different sample PCR reaction systems are packaged into micro-droplets and mixed in advance, then the combination characteristics of LNA modified primers at different temperatures are utilized to finish amplification of a target detection sequence and embedding of sample specific barcodes in one reaction tube, finally a sequencing library obtained after mixing PCR products of all samples is subjected to high-throughput sequencing to obtain mixed sequencing data, and the mixed sequencing data is subjected to efficient splitting of the samples and analysis based on the barcodes, so that low-cost high-throughput detection of gene editing events and gene editing efficiency can be realized, meanwhile, the specificity and accuracy of detection are improved, the experimental period is obviously shortened, and the risks of human operation errors and cross contamination among the samples are reduced.

Inventors

  • XIE CHUANXIAO
  • XU XIAOJIE
  • QI XIANTAO
  • MAO WENBO
  • Shi Dianshi
  • Su yulu
  • GAO ZHAOXU
  • ZHU JINJIE
  • LIU CHANGLIN
  • HUANG JING

Assignees

  • 中国农业科学院作物科学研究所

Dates

Publication Date
20260508
Application Date
20260106

Claims (10)

  1. 1. A method for constructing a sequencing library for detecting gene editing events, which is characterized by comprising the following steps: A1 Preparing an n-fold independent PCR reaction system, wherein the PCR reaction system comprises a nucleic acid sequence template to be detected containing a target sequence, a specific binding primer pair 1 of the target sequence with a bridging sequence 1 and a primer pair 2 with m lock nucleic acid modifications and bar codes, wherein the primer pair 2 contains the bridging sequence 2, and the bridging sequence 1 and the bridging sequence 2 are complementarily paired; A2 Carrying out PCR amplification reaction on the n-fold independent PCR reaction system under the same reaction program to obtain n-fold PCR products, mixing the n-fold PCR products to obtain the sequencing library, wherein the reaction program comprises a program 1 and a program 2 temperature control reaction program according to time sequence, the annealing temperature of the program 1 is 50-62 ℃, and the annealing temperature of the program 2 is 65-75 ℃; And the natural number of n is more than or equal to 2, and the natural number of m is more than or equal to 1.
  2. 2. The method of claim 1, wherein the independent PCR reaction system is a droplet encapsulated by using droplet generation oil, the PCR reaction system further comprises a PCR buffer solution capable of maintaining the stability of the droplet, and the A1) further comprises the step of mixing n weight of the droplet to obtain a mixed droplet library.
  3. 3. The method according to claim 1 or 2, wherein n is a natural number of 8 or more and 56 or less.
  4. 4. The method according to claim 1 to 3, wherein m is a natural number of 1 to 5.
  5. 5. The method according to claim 1 to 4, wherein the annealing temperature of the process 1 is 58℃and the annealing temperature of the process 2 is 70 ℃.
  6. 6. The method according to claim 1 to 5, wherein the number of amplification cycles of the procedure 1 is 15 to 25 and the number of amplification cycles of the procedure 2 is 20 to 25.
  7. 7. A method for detecting a gene editing event or/and determining the efficiency of gene editing, which is characterized in that the method comprises the steps of constructing a sequencing library of a nucleic acid sequence to be detected by using the method of any one of claims 1-6, sequencing the sequencing library by using a sequencing platform to obtain sequencing data, and detecting the gene editing in batches based on the analysis result of the sequencing data.
  8. 8. A composition for constructing a sequencing library for batch detection of gene editing events, characterized in that the composition comprises a primer pair 2 as set forth in claim 1, a droplet-generating oil as set forth in claim 2, and a PCR buffer capable of maintaining the droplet stability.
  9. 9. The composition of claim 8, wherein the composition further comprises a PCR reaction component.
  10. 10. A system for detecting gene editing event or/and monitoring gene editing efficiency, characterized in that the system comprises the composition, a microfluidic device and a PCR instrument as claimed in claim 8 or 9.

Description

Method for batch detection of gene editing event and related sequencing library construction method thereof Technical Field The invention belongs to the technical field of biology, and particularly relates to a method for detecting gene editing events in batches and a related sequencing library construction method thereof. Background In recent years, gene editing techniques represented by CRISPR/Cas9 systems have been developed at an unprecedented rate, and have been widely used in fields such as functional genomics, disease model construction, and accurate medical treatment. Along with the continuous expansion of the experimental scale of gene editing, how to efficiently, accurately and inexpensively detect editing events in massive samples has become one of the bottlenecks that restrict the technology from moving to large-scale application. The traditional gene editing detection method, such as T7E1 enzyme digestion method and survivin method, has the inherent defects of low sensitivity (generally > 1-5%), incapability of providing specific mutation sequence information, easiness in interference by background polymorphism and the like. Sanger sequencing, while capable of accurately reading sequences, is extremely low in throughput, costly, and difficult to resolve complex chimeric mutations resulting from gene editing. These methods all have difficulty meeting the need for rapid screening of thousands of samples. Second generation sequencing (NGS) technology has become a powerful tool for analyzing gene editing results due to its advantages of high throughput, high sensitivity, and low cost. To analyze NGS data, a variety of bioinformatics tools have been developed, such as: BATCH-GE (Sci. Rep., 2016) and CRISPResso (Nat. Biotechnol., 2016) these tools enable BATCH analysis of NGS data, quantitative assessment of insertion/deletion (Indel) efficiency and Homology Directed Repair (HDR) efficiency. However, they require separate PCR amplification and library building for each sample prior to analysis, resulting in cumbersome sample processing steps, high reagent consumption, long cycle times, and risk of cross-contamination between samples. Hi-TOM (Sci. CHINA LIFE Sci., 2019, 2024) the platform allows for high throughput sequencing of multiple samples, multiple targets, by two rounds of PCR (target amplification and barcode labelling) and provides a convenient on-line analysis tool. However, the experimental flow still depends on the step-by-step PCR reaction, the extreme compression of the reaction system is not realized, and the bioinformatics flow is easy to use but has limited customization and flexibility. Cas-analyzers (bioengineers, 2017) and CRISPR-GA (bioengineers, 2014) that these online or local tools simplify data analysis, but typically support analysis of only a single or small number of samples, and are difficult to handle for large-scale screening projects. In addition, they only solve the data analysis problem at the "dry test" end, and no innovation is made in the sample pretreatment flow at the "wet test" end. In summary, the prior art has a common core problem that the pretreatment of the samples of the wet experiment and the data analysis of the dry experiment are mutually split processes. Current high-throughput detection strategies either do a large number of independent reactions at the experimental end, resulting in bottlenecks in throughput, cost, and efficiency, or lack intelligent and flexible solutions at the data analysis end that match the high-throughput experimental design. Particularly, how to extremely compress and mix the reaction systems of thousands of samples without introducing high pollution and develop an automatic bioinformatics flow which can accurately split and analyze the mixed data in a matched way is a technical problem which is not effectively solved at present. Therefore, there is an urgent need in the art to develop an integrated solution integrating revolutionary wet experimental procedures with intelligent dry analytical procedures that can fundamentally simplify the operation steps, greatly increase the detection throughput, significantly reduce the unit sample cost, and ensure the accuracy of the detection results. Disclosure of Invention The invention aims to solve the technical problems of accurately detecting high-flux gene edits and/or accurately detecting batch gene edits or mutations at low cost with high flux. In order to solve the above technical problems, the present invention firstly provides a method for constructing a sequencing library for (batch) detection of gene editing events, which may comprise the steps of: A1 Preparing an n-fold independent PCR reaction system, wherein the PCR reaction system comprises a nucleic acid sequence template to be detected containing a target sequence, a specific binding primer pair 1 of the target sequence with a bridging sequence 1 and a primer pair 2 with m lock nucleic acid modifications and bar codes,