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Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair.

ABSTRACT: CRISPR/Cas9-induced site-specific DNA double-strand breaks (DSBs) can be repaired by homology-directed repair (HDR) or non-homologous end joining (NHEJ) pathways. Extensive efforts have been made to knock-in exogenous DNA to a selected genomic locus in human cells; which, however, has focused on HDR-based strategies and was proven inefficient. Here, we report that NHEJ pathway mediates efficient rejoining of genome and plasmids following CRISPR/Cas9-induced DNA DSBs, and promotes high-efficiency DNA integration in various human cell types. With this homology-independent knock-in strategy, integration of a 4.6 kb promoterless ires-eGFP fragment into the GAPDH locus yielded up to 20% GFP+ cells in somatic LO2 cells, and 1.70% GFP+ cells in human embryonic stem cells (ESCs). Quantitative comparison further demonstrated that the NHEJ-based knock-in is more efficient than HDR-mediated gene targeting in all human cell types examined. These data support that CRISPR/Cas9-induced NHEJ provides a valuable new path for efficient genome editing in human ESCs and somatic cells.

SUBMITTER: He X

PROVIDER: S-EPMC4872082 | biostudies-other | 2016 May

REPOSITORIES: biostudies-other

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Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair.

He Xiangjun X Tan Chunlai C Wang Feng F Wang Yaofeng Y Zhou Rui R Cui Dexuan D You Wenxing W Zhao Hui H Ren Jianwei J Feng Bo B

Nucleic acids research 20160204 9

CRISPR/Cas9-induced site-specific DNA double-strand breaks (DSBs) can be repaired by homology-directed repair (HDR) or non-homologous end joining (NHEJ) pathways. Extensive efforts have been made to knock-in exogenous DNA to a selected genomic locus in human cells; which, however, has focused on HDR-based strategies and was proven inefficient. Here, we report that NHEJ pathway mediates efficient rejoining of genome and plasmids following CRISPR/Cas9-induced DNA DSBs, and promotes high-efficiency ...[more]

PMID: 26850641

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Project description:BackgroundCultured human cells are pivotal models to study human gene functions, but introducing complete loss of function in diploid or aneuploid cells has been a challenge. The recently developed CRISPR/Cas9-mediated homology-independent knock-in approach permits targeted insertion of large DNA at high efficiency, providing a tool for insertional disruption of a selected gene. Pioneer studies have showed promising results, but the current methodology is still suboptimal and functional outcomes have not been well examined. Taking advantage of the promoterless fluorescence reporter systems established in our previous study, here, we further investigated potentials of this new insertional gene disruption approach and examined its functional outcomes.ResultsExemplified by using hyperploid LO2 cells, we demonstrated that simultaneous knock-in of dual fluorescence reporters through CRISPR/Cas9-induced homology-independent DNA repair permitted one-step generation of cells carrying complete disruption of target genes at multiple alleles. Through knocking-in at coding exons, we generated stable single-cell clones carrying complete disruption of ULK1 gene at all four alleles, lacking intact FAT10 in all three alleles, or devoid of intact CtIP at both alleles. We have confirmed the depletion of ULK1 and FAT10 transcripts as well as corresponding proteins in the obtained cell clones. Moreover, consistent with previous reports, we observed impaired mitophagy in ULK1-/- cells and attenuated cytokine-induced cell death in FAT10-/- clones. However, our analysis showed that single-cell clones carrying complete disruption of CtIP gene at both alleles preserved in-frame aberrant CtIP transcripts and produced proteins. Strikingly, the CtIP-disrupted clones raised through another two distinct targeting strategies also produced varied but in-frame aberrant CtIP transcripts. Sequencing analysis suggested that diverse DNA processing and alternative RNA splicing were involved in generating these in-frame aberrant CtIP transcripts, and some infrequent events were biasedly enriched among the CtIP-disrupted cell clones.ConclusionMultiallelic 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 partially functional transcripts through diverse DNA and RNA processing mechanisms.

Dataset Information

Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair.

Publications

Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair.

Similar Datasets

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