CRISPR/Cpf1 system-mediated homologous recombination method using RNA transcript as repair template

Abstract

The invention discloses a CRISPR / Cpf1 system-mediated homologous recombination method using an RNA transcript as a repair template. The method adopts rice ALS genes as a research object, and constructs a homologous recombination vector. RCR1-RCR2-RDR fragments are transcribed in vitro, a RNP method is used, the RNA transcript is used as the repair template, and homologous recombination repair of target genes is achieved in rice callus. The vector is introduced into the rice callus by a gene gun method, and rice plants with ALS gene fixed-point modification are obtained. The results show that RNA used as the repair template can successfully mediate homologous recombination of the target genes, and the method provides a novel idea for crop breeding, and has strong application potential in agricultural breeding.

Description

technical field [0001] The invention relates to a CRISPR / Cpf1 system-mediated homologous recombination method using RNA transcripts as repair templates. Background technique [0002] CRISPR / Cpf1 has greatly expanded the scope of gene editing and has begun to be applied to the research of genetic improvement of crops. Gene knockout using CRISPR / Cas9-mediated genome editing technology has been applied in rice and other crops. However, due to the low frequency of homologous recombination in plants, the use of CRISPR / Cas9-mediated homologous recombination to achieve gene-directed replacement or site-directed integration in crops has rarely been reported. At present, the replacement of target gene fragments mediated by the CRISPR / Cpf1 system has not been reported. [0003] It has been hypothesized that RNA transcripts can serve as repair templates to participate in DNA homologous recombination repair (HDR) caused by DNA double-strand breaks (DSBs), and this hypothesis has been ...

AI Technical Summary

Problems solved by technology

However, this technique has not been widely used in yeast and human cells, mainly because DNA repair templates can efficiently enter cells in yeast and human cells by transformation methods such as electroporation, microinjection, or transfection, thereby mediating DNA recombination repair

However, in plant cells, due to the presence of cell walls, these transformation methods are not applicable, especially for some crop varieties such as monocotyledonous plants such as corn, wheat, and rice.

Therefore, it is very difficult to achieve homologous recombination repair of target genes through the CRISPR / Cas system in crops, mainly because: 1) In plant cells, DSBs are mainly formed by non-homologous end joining (NHEJ) However, the probability of homologous recombination-mediated repair (homology-directed repair, HDR) is extremely small; 2) the amount of repair templates transferred into plant cells is very limited, and there are currently two ways to increase the amount of repair templates , but the effect is still unsatisfactory. One way is to introduce the repair template fragment into the cell through the gene gun transformation method; the other method is to connect the repair template into the replican vector derived from the virus, and transform the vector into the cell, thereby increasing the repair template amount

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