Boosting activity of high-fidelity CRISPR/Cas9 variants using a tRNA^Gln-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 tRNA^Gln-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 diff

SUBMITTER: He X 

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

REPOSITORIES: biostudies-literature