Method for preparing single mutant on basis of SaKKHn-pBE system

Abstract

The invention discloses a method for preparing a single mutant on the basis of a SaKKHn-pBE system. The preparation method for the single mutant comprises the following steps: introducing a SaKKHn-pBEbase editing system into an organism or a biological cell, and editing a genome target sequence through the SaKKHn-pBE base editing system so as to obtain the single mutant, wherein the single mutantis a biological mutant of which a single site C in a target sequence is mutated into T; and the SaKKHn-pBE base editing system comprises a SaKKHn protein, tRNA-sgRNA and a PmCDA1 protein. Results ofa test on 23 rice genome target sequences through the method disclosed by the invention show that almost all targets can obtain single mutants of a unit point C with highest mutation efficiency, and the proportions of the single mutants mostly exceed 50%.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing single mutants based on the SaKKHn-pBE system. 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 greatl...

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Problems solved by technology

At present, there are very limited reports on the types of mutations produced by CBE in plants. In the existing reports, not all CBEs can obtain single mutations for their editing targets (Wu, Y. et al. (2019) Increasing cytosine basedediting scope and efficiency with engineered Cas9-PmCDA1 fusions and the modified sgRNA in rice.Front.Genet.10,379.), or even if a single mutation is obtained for some targets, the proportion of single mutants is not high (Zhang,C.et al. (2019) Expanding the base editing scope to GA and relaxed NG PAM sites by improved xCas9 system. Plant Biotechnol. J. doi: 10.1111 / pbi.13259. [Epub ahead of print]; Zong, Y. (2018) Efficient C-to -T basedediting in plants using a fusion of nCas9 and humanAPOBEC3A.Nat.Biotechnol.36,950-953.)

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