Construction Of Whole Genome Knockout Cell Library Based On CRISPR-Cas9 And Screening Of SADS-CoV Interaction Proteins

Since the 21 st century,there have been three outbreaks of coronavirus caused by novel human coronavirus(CoVs),including the first outbreak of severe acute respiratory syndrome coronavirus(SARS-CoV)in China in 2003,the Middle East Respiratory Syndrome coronavirus(MERS-CoV)outbreak in the Middle East in 2012 and the global pandemic of SARS-CoV-2 since 2019.Swine acute diarrhea syndrome-coronavirus,SADS-CoV,the research object of this paper,is a new type of piglet diarrhea coronavirus found in some large-scale pig farms in Guangdong in 2017,which causes fatal diarrhea in piglets and causes serious economic losses to the domestic pig industry.There is no vaccine or specific drug for SADS-CoV,and the latest research shows that SADS-CoV can infect a variety of human cells,which may be at risk of cross-species transmission.So far,the specific receptors of SADS-CoV invading host cells are unknown,and there are few studies on replication-related proteins.At present,the whole genome knockout system based on CRISPR-Cas9 technology has become the most widely used new tool in the screening of virus receptors and replication-related proteins.Therefore,in this paper,the whole genome knockout system based on CRISPR-Cas9 technology is used to screen the specific cell receptors needed for SADS-CoV invasion into the host and the proteins that interact with the host in the process of infection,which is helpful for further understanding the mechanism of SADS-CoV infection.The following are the experimental results of this paper:1.In this paper,HEK293 A,HEK293T and L929 were identified as sensitive cell lines for screening SADS-CoV interacting proteins,and the whole genome knockout cell libraries of these three kinds of cells were successfully constructed;2.Through the four-round infection screening experiment of SADS-CoV,four stable HEK293 A cell clones were obtained(numbered as 7p27 ～ 30 and 32,respectively).The other two cell libraries were screened and no surviving cells were obtained.Further immunofluorescence and Western Blot experiments showed that the four stable cell clones showed no signs of weakening by SADS-CoV invasion or replication,while real-time fluorescence quantitative experiments showed that the four stable cell clones showed signs of weakening SADS-CoV replication;3.Finally,7,27,30 and 32 monoclonal cells were divided into two parts and sequenced by whole genome NGS sequencing and TA cloning,respectively,and the corresponding sequencing results were obtained.The analysis of NGS sequencing results showed that MRPL47,C5AR1,BET3 L,SYNPR,ZNF224,PPP2R1 B,TNFRSF11B and other genes might interact with SADS-CoV,and then the whole genome was extracted from another part of cell clones,and the target sg RNA information was determined by TA clone sequencing,which is still under sequencing.In summary,we successfully constructed the whole genome knockout cell library of HEK293 A stable transformation,completed the SADS-CoV interaction protein screening experiment,and finally obtained the NGS sequencing results of the cell clone.These results will lay an experimental foundation for further exploration of the infection mechanism of SADS-CoV.