The Establishment And Preliminary Application Of HeLa Cell Genome-wide Knockout Library Based On CRISPR/Cas9 Gene Editing Technology

Forward genetic screens can reveal the genetic genes corresponding to specific phenotypes,and directly link biological phenomena with their corresponding genetic cause.In mammalian genome-wide screening,CRISPR/Cas9 gene editing system is a powerful and unbiased method for conducting forward genetic screens.The genome-wide single-guide RNA?sgRNA?library directing Cas9 endonuclease enables the generation of mutant cell libraries and this is a perfect platform for phenotypic highthroughput screening.Based on this genome editing technique,all kinds of cell libraries have been widely used in the screening of various functional genes,including drug targeted genes,tumor proliferation genes and genes associated with viral infection.In this research,we have successfully established a genomewide knockout HeLa cell library by using CRISPR/Cas9 gene editing technology,and preliminarily applied it to the screen of HIV host-dependent factors in the early stage of replication,which also provides an effective high-throughput screening platform for the following studies.In this study,the two-vector system was used to construct knockout cell pool.HeLa cells were transduced with lentiCRISPRv2-Hyg virus.HeLa cells expressed Cas9 protein with high-efficiency cleavage activity to ensure lower off-target efficiency.Then,a CRISPR sgRNA library was introduced into HeLa-Cas9 cells.This study was divided into four parts: 1.Establishment of He La-Cas9 cell lines.HeLa cell lines expressing Cas9 protein were constructed through lentivirus infection.We selected nine monoclonal cell lines expressing Cas9 by flow cytometry.HeLa-Cas9 cell lines were transduced with pXPR-011 which contains the GFP sequence and the specific sgRNA-targeting GFP and active Cas9-expressing lines will result in a reduction in GFP.The two cell lines with high KO efficiency was 79% and 81%,respectively.After the cell activity was detected by CCK-8,we chose HeLa-Cas9₈ cells to sort single cell through flow cytometry.Furthermore,we got HeLa-Cas9₈-6 cell as a candidate.2.Amplified a single guide RNA library.In this study,the commercialized genome-scale CRISPR knock-out library were amplified by means of electrical transformation,and a number of colonies per sgRNA was greater than 100 copies.After NGS sequencing was finished,the sgRNA distribution was 100 % and the homogeneity was about 5.2?general standard ?15?.3.Construct knockout cell pool.The GeCKO library?A library?lentivirus titer was detected and the viruses infected the candidate cell line with a low titer.Cells were cultured in medium containing appropriate concentration of puromycin antibiotic for one or two weeks to get the collection of pooled HeLa knockout cells.Then we extracted the genome and amplified the sgRNA for NGS.In addition,the abundances of sgRNA in HeLa KO pools were analyzed by next generation sequencing and bioinformatics.The coverage rate of sgRNA was 97.80%,98.38% and 94.37%,respectively.4.Preliminary application of HeLa knockout library cells.The HIV-GFP?VSV-G?virus infected HeLa-GeCKO cell lines with viruses at a multiplicity of infection?MOI?of approximately 5 10.Flow cytometry showed significant differences in infection,revealing the existence of early host dependence factors related to HIV-1 replication.It is necessary to increase the number of cells to obtain more gDNA and further verify it by deep sequencing.Based on the above results,this study successfully screened a candidate cell line HeLa-Cas9₈-6 with high knockout efficiency and no significant difference in cell activity compared with the wild type.Under the GeCKO library with high coverage and good uniformity,the genome-wide knockout library of human HeLa cells CRISPR/Cas9?HeLa-GeCKO?was established.As a screen platform for various specific phenotypes,more unknown functions of known genes can be discovered,lying a foundation for the treatment of genetic diseases and the screening of drug targets.