Complete Disruption of UlK1, FAT10 and CtIP Genes by Homology-Independent Multiallelic Knock-in Yielded Distinct Functional Outcomes
Refereed conference paper presented and published in conference proceedings



摘要Human cell lines maintained under culture conditions are pivotal models for direct analysis of human gene functions. Since most cultured cells possess diploid or hyperploid genomes, meaning that a single gene is often presented as two or more copies in the genome, knockout or targeted disruption to introduce complete loss-of-function of a selected gene has been technically challenging in these cells. Recently, studies have exploited the NHEJ repair mechanism to knock-in DNA at CRISPR-induced DSBs in both zebrafish and mammalian cells. By targeting constitutively expressed house-keeping gene GAPDH at 3’-UTR using promoterless fluorescence reporters, we directly compared frequencies of knock-in mediated by CRISPR-induced NHEJ and HDR repair mechanisms. We found that knock-in via CRISPR/Cas9-induced NHEJ is superior to the commonly used HDR-based method in all human cell lines examined. Here, exemplified by hyperploid human cell line LO2, we demonstrated that simultaneous knock-in of dual reporters through CRISPR/Cas9-induced homology-independent DNA repair, permits one-step generation of cells carrying complete gene disruption of multiple alleles. Through knocking-in large DNA fragments at a coding exon, we generated stable single-cell clones carrying complete disruption of all four copies of ULK1 gene, lacking all three copies of intact FAT10 gene, or devoid of intact CtIP gene at both alleles. Importantly, we fully confirmed the depletion of ULK1 and FAT10 transcripts as well as corresponding proteins; and in subsequent functional analysis of the ULK1−/− and FAT10−/− cell clones, we observed defect in mitophagy and cytokine-induced cell death, respectively; which are consistent with previous reports. In conclusion, Multiallelic gene disruption could be readily introduced through CRISPR/Cas9-induced homology-independent knock-in of dual fluorescence reporters followed by direct tracing and cell isolation. Robust cellular mechanisms exist to spare essential genes from loss-of-function modifications, by generating partial functional transcripts through diverse DNA and RNA processing mechanisms.
著者Chenzi ZHANG, Xiangjun HE, Yvonne K KWOK, Feng WANG, Junyi XUE, Hui ZHAO, Kin Wah SUEN, Chi Chiu WANG, Jianwei REN, George G. CHEN, Paul B. S. LAI, Jiangchao LI, Yin XIA, Andrew M CHAN, Wai-Yee CHAN, Bo FENG
會議名稱The 22nd Annual Meeting of the American Society of Gene and Cell Therapy (ASGCT)
會議地點Washington D.C.
期次4 (suppl 1)
頁次355 - 355

上次更新時間 2021-21-09 於 00:29