Efficient CRISPR/Cas (Clustered Regulatory Interspaced Short Palindromic Repeats/CRISPR associated) 9 gene editing system for Kluyveromyces optimization

Abstract

The invention relates to a special safe and efficient CRISPR / Cas (Clustered Regulatory Interspaced Short Palindromic Repeats / CRISPR associated) 9 gene editing system for Kluyveromyces optimization, and belongs to the field of biotechnology. In the prior art, pCAS plasmids used in saccharomyces cerevisiae have Cas9 gene sequences and gRNA (guide RNA) elements at the same time, and efficient genomemodification in the saccharomyces cerevisiae can be realized through one-time transformation, but stable copying and expression cannot be realized in Kluyveromyces. According to the system, Cas9 / gRNAfusion plasmids are transformed in Kluyveromyces cells, the plasmids are targetedly delivered to endogenous DNA sequences of the Kluyveromyces cells, and double strand breaks are generated; and donorDNA sequences are transformed in the Kluyveromyces cells, the sequences generate homologous recombination at the double strand breaks and target sites, and Tag sequences are inserted in the target sites. Through the modification, the new safe and efficient CRISPR / Cas9 gene editing system which is specially used for the Kluyveromyces and can perform stable copying and expression in the Kluyveromyces and perform gene modification is established.

Description

technical field [0001] The invention relates to a CRISPR / Cas9 high-efficiency gene editing system specially optimized for Kluyveromyces, belonging to the field of biotechnology. Background technique [0002] Microbial genome modification mainly relies on two biological mechanisms: endogenous homologous recombination repair (endogenous homology-directed repair, HDR) and non-homologous end joining (non-homologous end joining, NHEJ) [1]. The basic process of HDR is that after double-strand breaks (DSBs) occur in the genome, the donor DNA (sequence homologous to the sequences on both sides of the break site) undergoes homologous recombination with the damaged genome sequence at the break site , to achieve repair, this process is not easy to introduce insertion or deletion mutations [2]. The basic process of NHEJ is that the two ends of the broken DNA are directly joined without the aid of homologous recombination mediated by the donor DNA. This process is prone to insertion or ...

AI Technical Summary

Problems solved by technology

However, usually the plasmid of Saccharomyces cerevisiae is a 2µ plasmid (including pCAS), which cannot be stably replicated and expressed in Kluyveromyces[11]

In the Kluyveromyces CRISPR / Cas9 transformation system reported so far, the Cas9 gene is directly inserted into the yeast genome, resulting in continuous expression of the Cas9 protein, which poses a certain safety hazard to the industrial production of Kluyveromyces[12]. An efficient and safe CRISPR / Cas9 system capable of stably replicating and expressing in Kluyveromyces is necessary for the development of biotechnology and will be a great advancement in the field of biomedicine

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