Efficient specific sgRNA recognition site guide sequence for pig gene editing and screening method thereof

Abstract

The invention discloses an efficient specific sgRNA recognition site guide sequence for pig gene editing and a screening method thereof. The screening method includes: screening functional genes, performing ORF analysis, predicting a functional gene sgRNA recognition site guide sequence, detecting whole-genome off-target sites, grading predicted target sites according to off-target information and target site positions, sequencing, screening and statistically counting results, optimizing algorithms and developing software. The efficient specific sgRNA recognition site guide sequence and the screening method thereof have the advantages that the pig specific sgRNA recognition site guide sequence is obtained through strict screening and inspection and includes the sgRNA recognition site guide sequences, for CRISPR-Cas9 gene editing, of all pig protein encoding genes; the authenticating, grading and inspecting algorithms for specific sgRNA recognition and software corresponding to the algorithms and used for predicting and evaluating the pig functional gene sgRNA recognition site guide sequence are widely applicable to the sgRNA specific site prediction of non-model species with whole-genome sequences.

Description

technical field [0001] The invention belongs to the technical field of genomics and bioinformatics, and in particular, the invention relates to an efficient and specific sgRNA recognition site guide sequence for pig gene editing and a screening method thereof. Background technique [0002] Genome editing technology mediated by CRISPR (Clustered regularly interspaced short palindromicrepeats) / Cas9 system is the third generation genome editing technology after zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) (Brouns et al. al., 2008). CRISPR-Cas9 is an adaptive immune system that exists in bacteria to bacteriophage genomes or horizontal transfer plasmids. The Cas9 protein with endonuclease activity specifically recognizes and cuts double-stranded DNA under the guidance of sgRNA. Therefore, CRISPR / Cas9 technology is also mainly composed of two parts: one is sgRNA that specifically binds to the genome through base complementary pairing; ...

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Problems solved by technology

[0009] 5) Combining the sgRNA site in the position of the protein-coding gene and the problem of alternative splicing: the editing of the protein-coding gene near the N-terminus is more efficient due to the higher probability of causing early stop codons, while for the protein-coding gene with multiple alternative splicing Genes need to consider mutations for each transcript, and many programs do not take the above into account

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