Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage.

ABSTRACT:

Background

The propensity for off-target activity of Streptococcus pyogenes Cas9 (SpCas9) has been considerably decreased by rationally engineered variants with increased fidelity (eSpCas9; SpCas9-HF1). However, a subset of targets still generate considerable off-target effects. To deal specifically with these targets, we generated new "Highly enhanced Fidelity" nuclease variants (HeFSpCas9s) containing mutations from both eSpCas9 and SpCas9-HF1 and examined these improved nuclease variants side by side to decipher the factors that affect their specificities and to determine the optimal nuclease for applications sensitive to off-target effects.

Results

These three increased-fidelity nucleases can routinely be used only with perfectly matching 20-nucleotide-long spacers, a ma

SUBMITTER: Kulcsar PI 

PROVIDER: S-EPMC6389135 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature