PE-P2 prime editing system and application thereof in genome base editing

Abstract

The invention discloses a PE-P2 prime editing system and an application thereof in genome base editing. The PE-P2 prime editing system comprises fusion protein and pegRNA; and the fusion protein is composed of a Cas9 cutting enzyme or variants thereof, a reverse transcriptase, a self-cleavage oligopeptide and a screening marker protein in sequence, or consists of a screening marker protein, self-cleavage oligopeptide, a Cas9 cutting enzyme or variants thereof and a reverse transcriptase in sequence. By virtue of self-cleavage oligopeptide P2A, fusion expression of RT, Cas9n (H840A) and HPT ina PE-P1 system is realized, and an sgRNA framework is replaced with an esgRNA framework to obtain the PE-P2 prime editing system. Compared with the PE-P1 prime editing system, the PE-P2 prime editingsystem of the invention not only can improve the editing efficiency of an editing target, but also realizes the effective editing of a non-editable target.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a PE-P2 guided editing system and its application in genome base editing. Background technique [0002] CRISPR-Cas9 technology has become a powerful genome editing method and has been widely used in many tissues and cells. The CRISPR / Cas9 protein-RNA complex is positioned on the target by the guide RNA (guide RNA), cuts and generates a DNA double-strand break (dsDNA break, DSB), and then the organism will instinctively initiate a DNA repair mechanism to repair the DSB. There are generally two repair mechanisms, one is non-homologous end joining (NHEJ), and the other is homologous recombination (homology-directed repair, HDR). Usually NHEJ accounts for the majority, so the random indels (insertions or deletions) generated by the repair are much higher than the precise repair. For precise base substitution, the application of HDR to achieve precise base substitution is gr...

AI Technical Summary

Problems solved by technology

For base-accurate substitution, the application of HDR to achieve base-accurate substitution is greatly limited due to the low efficiency of HDR and the need for DNA templates

Cytosine base editors and adenine base editors reported successively in 2016 and 2017 can accurately realize cytosine (Cytosine, C) to thymine (Thymine, T) and adenine (Adenine, A) to The conversion of Guanine (G) does not generate DSB and does not introduce DNA templates, but the transversion between purine and pyrimidine cannot be realized, that is, the replacement of A to T, T to A, and C to G cannot be realized Replacement of G to C, A to C, T to G, C to A, G to T

[0004] At present, although guided editing technology can realize all types of base replacements, there are still problems that base editing efficiency is not high, or some sites cannot be edited

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