Engineering of efficiency-enhanced Cas9 and base editors with improved gene therapy efficacies.

ABSTRACT: Editing efficiency is pivotal for the efficacies of CRISPR-based gene therapies. We found that fusing an HMG-D domain to the N terminus of SpCas9 (named efficiency-enhanced Cas9 [eeCas9]) significantly increased editing efficiency by 1.4-fold on average. The HMG-D domain also enhanced the activities of non-NGG PAM Cas9 variants, high-fidelity Cas9 variants, smaller Cas9 orthologs, Cas9-based epigenetic regulators, and base editors in cell lines. Furthermore, we discovered that eeCas9 exhibits comparable off-targeting effects with Cas9, and its specificity could be increased through ribonucleoprotein delivery or using hairpin single-guide RNAs and high-fidelity Cas9s. The entire eeCas9 could be packaged into an adeno-associated virus vector and exhibited a 1.7- to 2.6-fold increase in editi

SUBMITTER: Yin S 

PROVIDER: S-EPMC10014233 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature