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Boosting activity of high-fidelity CRISPR/Cas9 variants using a tRNAGln-processing system in human cells.


ABSTRACT: CRISPR/Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. High-fidelity Cas9 variants have been identified; however, they often have reduced activity, constraining their utility, which presents a major challenge for their use in research applications and therapeutics. Here we developed a tRNAGln-processing system to restore the activity of multiple high-fidelity Cas9 variants in human cells, including SpCas9-HF1, eSpCas9, and xCas9. Specifically, acting on previous observations that small guide RNAs (sgRNAs) harboring an extra A or G (A/G) in the first 5' nucleotide greatly affect the activity of high-fidelity Cas9 variants and that tRNA-sgRNA fusions improve Cas9 activity, we investigated whether a GN20 sgRNA fused to different tRNAs (G-tRNA-N20) could restore the activity of SpCas9 variants in human cells. Using flow cytometry, a T7E1 assay, deep sequencing-based DNA cleavage activity assays, and HEK-293 cells, we observed that a tRNAGln-sgRNA fusion system enhanced the activity of Cas9 variants, which could be harnessed for efficient correction of a pathogenic mutation in the retinoschisin 1 (RS1) gene, resulting in 6- to 8-fold improved Cas9 activity. We propose that the tRNA-processing system developed here specifically for human cells could facilitate high-fidelity Cas9-mediated human genome-editing applications.

SUBMITTER: He X 

PROVIDER: S-EPMC6556588 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Boosting activity of high-fidelity CRISPR/Cas9 variants using a tRNA<sup>Gln</sup>-processing system in human cells.

He Xiubin X   Wang Yufei Y   Yang Fayu F   Wang Bang B   Xie Haihua H   Gu Lingkai L   Zhao Tianyuan T   Liu Xiexie X   Zhang Dingbo D   Ren Qianwen Q   Liu Xiaoyu X   Liu Yong Y   Gao Caixia C   Gu Feng F  

The Journal of biological chemistry 20190422 23


CRISPR/Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. High-fidelity Cas9 variants have been identified; however, they often have reduced activity, constraining their utility, which presents a major challenge for their use in research applications and therapeutics. Here we developed a tRNA<sup>Gln</sup>-processing system to restore the activity of multiple high-fidelity Cas9 variants in human cells, including SpCas9-HF1, eSpCas9, and xCas9. Specifi  ...[more]

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