Guide-target mismatch effects on dCas9-sgRNA binding activity in living bacterial cells.

ABSTRACT: As an effective programmable DNA targeting tool, CRISPR-Cas9 system has been adopted in varieties of biotechnological applications. However, the off-target effects, derived from the tolerance towards guide-target mismatches, are regarded as the major problems in engineering CRISPR systems. To understand this, we constructed two sgRNA libraries carrying saturated single- and double-nucleotide mismatches in living bacteria cells, and profiled the comprehensive landscape of in vivo binding affinity of dCas9 toward DNA target guided by each individual sgRNA with particular mismatches. We observed a synergistic effect in seed, where combinatorial double mutations caused more severe activity loss compared with the two corresponding single mutations. Moreover, we found that a particular mismatch

SUBMITTER: Feng H 

PROVIDER: S-EPMC7897480 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature