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CRISPR-Cas9 system sgRNA action target screening method and apparatus

A target and function technology, applied in the field of screening of sgRNA targets in the CRISPR-Cas9 system, can solve the problems of lack of mature methods for genome-wide targets

Active Publication Date: 2017-06-13
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, in the field of animal research, there are many Cas9 targets designed based on a single functional gene, but there is still a lack of a mature method for screening genome-wide targets

Method used

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  • CRISPR-Cas9 system sgRNA action target screening method and apparatus
  • CRISPR-Cas9 system sgRNA action target screening method and apparatus
  • CRISPR-Cas9 system sgRNA action target screening method and apparatus

Examples

Experimental program
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Effect test

Embodiment 1

[0059] Example 1 Screening method for the sgRNA target of the CRISPR-Cas9 system designed for chicken

[0060] In this example, chicken, a representative animal of poultry, is taken as an example to design a genome-wide Cas9 target library.

[0061] First, download the chicken reference genome (version number Galgal4, GCA_000002315.2) and its corresponding gene annotation file from the Ensembl database (http: / / www.ensembl.org / index.html). Use the whole genome sequence and gene annotation information to obtain the candidate target 5'-(N of all genes in the genome 20 ) NGG-3' sequence (N stands for A / T / C / G). According to statistics, a total of 380,459 candidate target sequences were obtained in chicken, covering 16,821 genes and 28,915 exons. Then the genome is broken into 23bp fragments and the sequences ending with NGG and without repeats on the genome are screened, compared with the candidate target sequences on the exons, and the corresponding sequences are compared accordi...

Embodiment 2

[0062] Example 2 Screening method for the sgRNA target of the CRISPR-Cas9 system designed for pigs

[0063] In this example, pigs, the representative animal of mammals, were taken as an example to design a genome-wide Cas9 target library.

[0064] First, download the pig reference genome (version number Sscrofa10.2, GCA_000003025.4) and its corresponding gene annotation file from the Ensembl database (http: / / www.ensembl.org / index.html). Use the whole genome sequence and gene annotation information to obtain the candidate target 5'-(N of all genes in the genome 20 ) NGG-3' sequence (N stands for A / T / C / G), statistics show that a total of 626,236 candidate target sequences in pigs were obtained, covering 24,734 genes and 43,049 exons. Then the genome is broken into 23bp fragments and the sequences ending with NGG and without repeats on the genome are screened, compared with the candidate target sequences on the exons, and the corresponding sequences are compared according to the...

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Abstract

The invention relates to a CRISPR-Cas9 system sgRNA action target screening method. The method comprises the steps of (1) obtaining segments with 5'-Nx-NGG-3' sequences (x is an integer ranging from 19 to 22 and N represents A / T / C / G) in genomes by utilizing whole genome sequences and gene annotation information of published species to serve as candidate targets of CRISPR-Cas9 system sgRNA; (2) fragmenting the genomes into fragments of 22-25bp and screening sequences ending with NGG and non-repeated on the genomes; and (3) comparing the sequences of the candidate targets in the step (1) with the screened sequences in the step (2), and performing screening and sorting on corresponding optimal sequences according to mismatch information and a selection formula to obtain an optimal genome sgRNA action target set. The invention furthermore provides an apparatus used for realizing the screening method. The method is suitable for all species with known genomes and gene annotation information, and a universal sgRNA sequence set of whole genome level of the species is quickly and efficiently obtained to build a gene knockout mutant library or a gene knockout animal model.

Description

technical field [0001] The invention relates to the fields of bioinformatics, proteomics, transcriptomics and genetic engineering, in particular to a screening method and device for sgRNA targets of the CRISPR-Cas9 system. Background technique [0002] With the development of DNA sequencing technology, the genome sequence information of many model organisms has been published, and then researchers turned their research focus to the mining of gene function information. Gene knockout animal models have always been an important tool for studying gene function and finding suitable drug targets in living animals. However, the traditional gene knockout method needs to go through a series of steps such as complex targeting vector construction, embryonic stem cell (ES cell) screening, and chimera breeding. It takes a long time, and the success rate is also affected by many factors. Even for laboratories with relatively mature technology, it generally takes a long time to construct...

Claims

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Application Information

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IPC IPC(8): G06F19/22C40B40/08
Inventor 赵毅强高菲王宇哲许文杰胥春龙吴森胡晓湘
Owner CHINA AGRI UNIV
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