title: [Establishment and validation of human cancer cell lines with stable Cas9 expression].
abstract: Objective:To establish human cancer cell strains with stable Cas9 expression, and to validate the gene editing activity of Cas9 for simple gene editing in future study.Methods:Fifteen cancer cell lines of different tissue origins were infected with pLv-EF1α-Cas9-Flag-Neo or pLv-EF1α-Cas9-Flag-Puro by lentivirus and clone selection was employed to screen Cas9 stably expressed cancer cell lines. Afterward designed guide RNA vectors targeting TSC22 gene were transiently transfected into 3 of cell lines, and subsequently the gene editing activity of Cas9 was evaluated by genomic PCR, sequencing and Western blot.Results:Sixty-nine human cancer cell strains with stable Cas9 expression from different cancers were established, and by transient transfection with designed guide RNA, long fragment deletion was detected in TSC22 gene.Conclusions:Sixty-nine human cancer cell strains are successfully established with stable expression of Cas9 protein and gene editing activity. These cell strains may be employed in large-scale drug screening, screening of new drug targets and gene function investigation.
authors.affiliation: Department of Pathology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & School of Basic Medicine, Peking Union Medical College, Beijing 100005, China.
authors.forename: X C
authors.initials: XC
authors.lastname: Bian
authors.forename: Z L
authors.initials: ZL
authors.lastname: Yang
authors.forename: H L
authors.initials: HL
authors.lastname: Feng
authors.forename: X M
authors.initials: XM
authors.lastname: Zhao
authors.forename: B
authors.initials: B
authors.lastname: Gu
authors.forename: J
authors.initials: J
authors.lastname: Li
authors.forename: H
authors.initials: H
authors.lastname: Sun
authors.forename: Y Q
authors.initials: YQ
authors.lastname: Liu
mesh_terms: D064130:CRISPR-Associated Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor / Q000378:metabolism*; D000072669:Gene Editing; D006801:Humans; D009363:Neoplasm Proteins / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics; D014162:Transfection; D000094704:RNA, Guide, CRISPR-Cas Systems
chemical_list: D064130:CRISPR-Associated Proteins; D009363:Neoplasm Proteins; D012097:Repressor Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems