Gene editing tools are also being continuously improved and refined, which may help address the issue of off-target effects. Originally CRISPR/Cas9 edits a genomic locus by inducing DNA double-strand breaks using a single guide RNA-directed wild type Cas9 nuclease. The nickase version of Cas9 (D10A mutant) directed by paired guide RNAs or the engineered Cas9 nuclease variants with enhanced specificity (eSpCas9) is now being used increasingly for genome editing32–34, because both have been shown to reduce off-target effects substantially while retaining rigorous on-target cleavage34,35. Furthermore, catalytically dead Cas9 (dCas9) fused with transcriptional activator or suppressor has been used to modulate transcription of endogenous genes (so-called CRISPRi or CRISPRa) or image genomic loci by fusing with a fluorescent protein32–34,36. Modifications of the CRISPR/Cas9 system also enable explicit introduction of DNA sequence changes in a precise mono-allelic or bi-allelic manner with high efficiency37. A recent development in base editing takes advantage of the fusion of CRISPR/Cas9 and a cytidine deaminase enzyme to allow direct conversion of cytidine to uridine without the need of double strand DNA break38. This new approach enhances gene editing efficiency and will further facilitate gene editing in human ESCs and iPSCs.
