Studies On Editing Properties Of PE-Cas9 System In Chicken Cell Gene Editing

The outbreak of poultry infectious diseases is accompanied by high mortality of poultry,and specific highly virulent infectious strains may even pose a threat to public health and cause significant economic losses to the global poultry industry.Vaccines are the best way to prevent large-scale outbreaks of highly pathogenic avian influenza virus,but the vaccination strategy cannot have a lasting effect.Only relying on traditional breeding methods to breed chickens with disease-resistant effects has a long cycle and genetic resources are limited.With the emergence of new breeding technologies such as gene editing,the targeted modification of endogenous genes and the targeted integration of exogenous genes have become possible,and extensive research has enabled the poultry industry to make great progress.The PE-Cas9(prime editing-Cas9)-Based gene editing system(hereinafter referred to as PE-Cas9),which has emerged in recent years,can achieve large fragment deletion and small fragment insertion without the need for exogenous donors,which is of great value.In this study,chicken DF-1 cells were used as the experimental object.According to the editing window of PE-Cas9,design peg RNA(prime editing guide RNA)knockout DNA fragments on chicken disease resistance and reproductive-related genes ANP32 A and DMRT1 to verify editing activity;then analyze the editing characteristics of PE-Cas9,including the editing efficiency of PE-Cas9 in DF-1 cells,the characteristics of the interaction between PE-Cas9 and a single peg RNA,and compare the editing efficiency of different knockout lengths on the DMRT1 gene to find the optimal knockout length;finally,the exon knockout is realized on ANP32 A,DMRT1,and DAZL,which is applied to exon knockout editing and precise replacement,laying the foundation for related research on chicken disease resistance and reproduction.The specific research results are as follows:(1)Editing activity of PE-Cas9 in DF-1 cells: two pairs of peg RNAs were designed on the ANP32 A and DMRT1 genes,and the paired peg RNAs and PE-Cas9 were cotransfected into chicken DF-1 cells,and the positive cell sequencing results were collected,it shows that PE-Cas9 has successfully knocked out ANP32 A peg RNAF+ANP32A peg RNA-R sites and DMRT1 peg RNA-F+DMRT1 peg RNA-R sites,and under the guidance of a single peg RNA,PE-Cas9 can also achieve editing,It was shown that PE-Cas9 has editing activity on chicken cells.(2)Analysis of the editing characteristics of PE-Cas9: analyze the NGS(Nextgeneration sequencing)sequencing results through bioinformatics,and count and classify the editing types according to the base changes at the cleavage site when PE-Cas9 is edited.Cas9 achieved knockout while achieving 18 bp fragment insertion,ANP32 A gene locus(86.49%),DMRT1 gene locus(12.53%),and PE-Cas9 and Cas9 can achieve similar total knockout efficiency at the same site;in addition,under the guidance of a single peg RNA,when PE-Cas9 or Cas9 realizes editing,PE-Cas9 mainly edits the insertion of non-3bp(or integer multiples)bases,and Cas9 mainly edits the deletion of non-3bp(or integer multiples)bases;through Design 5 deletion lengths that increase in gradient on the DMRT1 gene,and detect the optimal knockout length of PE-Cas9 on the DMRT1 gene,19bp(11.35%),82bp(4.02%),160bp(16.98%),213bp(37.45%),551bp(12.53%);indicating that the distance between two paired peg RNAs on the DMRT1 gene is within545bp(including PAM(protospacer-adjacent motifs)),and the editing efficiency of PECas9 is the highest when the knockout length is 213 bp.(3)PE-Cas9 achieves exon knockout on ANP32 A,DMRT1,and DAZL genes: single cell sorting using flow cytometry to detect PE-Cas9 in paired peg RNA sites on different genes(ANP32A peg RNA-F+ANP32A peg RNA-R,DMRT1 peg RNA-F+DMRT1peg RNA-R,DAZL peg RNA-F+DAZL peg RNA-R)exon knockout efficiency,ANP32A57.44%,DMRT1 65.95%,DAZL 46.80%.In summary: In this study,the PE-Cas9 system was used to successfully achieve efficient,accurate and predictable fragment knockout of ANP32 A,DMRT1,and DAZL,three genes related to chicken disease resistance and reproduction,under the condition of pairing peg RNA targeting DNA complementary sequences.When the knockout length increases with the gradient,PE-Cas9 can still achieve accurate non-homology armdependent short-segment knock-in;exon knock-out can be achieved without foreign DNA and small-segment insertion editing can be performed to achieve precise replacement.This study analyzed the characteristics of PE-Cas9 gene editing in chicken cells,and achieved exon knockout and precise replacement,providing the basis for this system in chicken disease resistance,reproduction and wider research.