Gene Editing And Off-target Analysis Of Streptomyces Virginia IBL-14 I-B-sviCas3 System In Saccharomyces Cerevisiae BY4741

CRISPR/Cas technology is one of the most cutting-edge and hot gene editing technologies in gene editing field.Its advantages of low cost,high efficiency and easy operation greatly promote the development of gene editing technology.The CRISPR/Cas system exists in both bacteria and archaea in nature and is an autoimmunity mechanism of bacteria and archaea in the face of the invasion of foreign DNA.Studies on CRISPR/Cas system have found that CRISPR-Cas9 and V-type Cpf1 have the function of gene editing,among which CRISPR-Cas9 is the most typical,but crispr-Cas9 also has the problem of incompatibility to some organisms and high miss rate.This study to our laboratory to separation and purification of Streptomyces Virginia IBL14 of whole genome sequencing,which found that the two CRISPR locus of chromosome exists between a Cas order Cas7 Cas5,Cas3,Cas4,Cas1,Cas2,protein group,they in the same operon,including Cas3 has the ability of DNA to edit.This paper named I-B-svi type CRISPR-Cas system,our laboratory studies have confirmed that the system in procaryote microbiology,eukaryotic organisms and human cells are capable of gene editing,I-b-svi has a lower molecular weight than CRISPR-Cas9 system,combined with its derived from gram-positive bacteria,and the characteristics of the low rate of miss,showed that the system has broad application prospects.Protein expression plasmids and gene editing plasmids were gradually transferred into the receptive cells of Saccharomyces cerevisiae by means of electrical transformation.By analyzing the experimental results,we found that:(1)The gene editing plasmid inserted with GFP gene can still conduct gene editing on target genes of yeast,which further confirms that this system can conduct effective gene editing in eukaryotic microorganisms.(2)The homologous sequences of randomly selected target genes were analyzed without miss,which laid a foundation for further accurate miss analysis.It can be preliminarily determined that the system has a low miss rate in gene editing of Saccharomyces cerevisiae.