Complete Gene Disruption by CRISPR-Based Multiallelic Knock-In Engages Alternative DNA Cleavage and RNA Splicing to Spare Essential Proteins
Refereed conference paper presented and published in conference proceedings



摘要Targeted gene disruption in diploid or aneuploid somatic cells has been a challenge hindering in-depth study on gene functions. 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. Among six genes attempted, we easily 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. While we fully confirmed the depletion of ULK1 and FAT10 transcripts as well as corresponding proteins, our study revealed robust flexibility of cellular mechanisms in generating in-frame aberrant CtIP transcripts after both CtIP alleles were disrupted. Through targeting different regions of CtIP gene with distinct donor designs, we consistently observed various chimeric CtIP transcripts that were in-frame and produced aberrant CtIP proteins. Sequence alignment showed that various alternative splicing at cryptic splice sites as well as alternative DNA cleavage at 4th nucleotide by CRISPR/Cas9 were engaged for preserving CtIP proteins. Altogether, our study demonstrates a universal dual-reporter knock-in approach for trackable gene disruption, which could be utilized in a wide range of applications. Meanwhile, these data suggest that more caution is needed when disrupting essential genes, where aberrant functional transcripts might be generated via flexible RNA splicing and alternative DNA cleavage.
著者Chenzi ZHANG, Xiangjun HE, Yvonne K KWOK, Hui ZHAO, Kin Wah SUEN, Chi Chiu WANG, Yin XIA, Jianwei REN, Andrew M CHAN, Wai-Yee CHAN, Bo FENG
會議名稱21st Annual Meeting of the American Society of Gene and Cell Therapy (ASGCT)
期次5 (Supplement 1)

上次更新時間 2021-17-01 於 01:57