Fig. 1.

Effective methods to improve editing efficiency using gRNA engineering and Cas nuclease-related design and covalent binding. gRNA engineering involves three aspects: gRNA array processing, gRNA structural engineering, and gRNA chemical modification. Cas nuclease-related design and covalent binding include nuclear localization signals, amino acid substitutions, chemical modification of Cas mRNA, fusion expression of Cas protein and exonuclease, codon optimization, and covalent conjugation. gRNA:guide RNA; Cas: CRISPR-associated proteins.