Construction Of An Adenovirus-mediated SpCas9 Efficient Knockout System For Chicken AMH

Anti-Müllerian Hormone(AMH),also known as Müllerian inhibiting substance,is one of the most important hormones controlling gonadal differentiation during embryonic development.AMH is mainly responsible for the degeneration of oviductal gonadal precursors in early embryo of male animals and the degeneration of the right oviduct in early embryo of female poultry.However,the molecular mechanism of AMH for gonadal differentiation and oviduct precursor degradation in early stage of embryonic development is still unclear.In recent years,the Precise Genome Editing technology has provided a good technical support for researching on the function of AMH.In particular,CRISPR/Cas9 makes it possible to influence the AMH expression endogenously.CRISPR/Cas9 is a RNA-guided nuclease system with cutting enzyme activity which is largely dependent on the targeting efficiency of sgRNA and the transduction efficiency of exogenous nucleotides.Therefore,this experiment optimized the CRISPR/Cas9 knockout system of targeted chicken AMH gene from two aspects of improving the targeting efficiency of sgRNA and transduction efficiency of SpCas9 to improve the editing efficiency of CRISPR/Cas9 on chicken AMH gene.1.To improve the targeting efficiency of sgRNA on chicken AMH gene,we constructed paired sgRNA knockout vectors pX330-U6-AMHsg1_dBsaI-CBh-SpCas9 and pX330-U6-AMHsg2_dBsaI-CBh-SpCas9 that targeting the first exon of chicken AMH gene.We also constructed a corresponding SSA-RPG dual fluorescence reporter vector pB-CMV-DsRedCAG-AMH12.200 bp repeat.Puro-T2A-GFP which is based on the SSA cell repair mechanism.We transfected those plasmids to chicken fibroblast cell line(DF-1 cells)with Lipofectamine 3000 transfection reagent.The efficiency of the CRISPR/Cas9 system was initially assessed by the fluorescence of SSA-RPG reporter.Positive cells were enriched by Puromycin at a concentration of 3 ?g/mL for 8 days.TA-clone sequencing showed that the targeting efficiency of the chicken AMH gene by the CRISPR/Cas9 system designed by paired sgRNA(83%)was significantly improved compared with the traditional single sgRNA design(40%).2.To improve the transduction efficiency of CRISPR/Cas9 in cells,the Ad-Easy adenovirus packaging system was used in this study.We constructed the SpCas9 coding sequence into the adenovirus shuttle vector pAdTrack_CMV through the MCS of the backbone plasmid.pAdEasy-SpCas9,a recombinant adenovirus vector carrying the SpCas9 sequence,was obtained by homologous recombination of the BJ5183 bacterial cells.pAdEasy-SpCas9 was linearized by enzyme digestion and transfected into 293 A packaging cells,then we generated recombinant adenovirus reAdv-SpCas9 which carrying SpCas9 expression cassette.In addition,the paired sgRNA expression vector msgRNA-AMH12 and the SSA-RFP single red fluorescence reportor pB-puro-RFP.SSA-AMH_target12 based on single strand annealing were constructed in this study.The constructed plasmids(msgRNA-AMH12 and pB-puro-RFP.SSA-AMH_target12)were co-transferred into 293 T cells together with the recombinant virus.The SpCas9 protein expressed by recombinant adenovirus reAdv-SpCas9 and knocked out the chicken AMH sequence of reporter pB-puro-RFP.SSA-AMH_target12 successfully under the guidance of paired sgRNA expressed by plasmid msgRNA-AMH12.In summary,adenovirus-mediated SpCas9 coupled with paired sgRNA design can significantly improve CRISPR/Cas9 transduction efficiency and chicken AMH gene targeting efficiency.This experiment provided SpCas9 expression vector with high infection activity for the study of gene function in vivo.The paired structure of sgRNA design provides a new idea for the efficient knock out of target genes.