Deciphering relationship between microhomology and in-frame mutation occurrence in human CRISPR-based gene knockout.

Abstract

To the Editor: CRISPR-based gene editing is widely implemented in various cell types and has great potential for molecular therapy.1 The CRISPR-Cas9 system creates sequence-specific double-strand DNA breaks that are repaired by a dominant error-prone nonhomologous end-joining (NHEJ) pathway, often resulting in gene inactivation by generating frameshift alleles.1,2,3,4,5,6,7 CRISPR-based gene knockout (KO) often produces in-frame variants that retain functionality, however, which reduces KO efficiency. Recently, Sangsu Bae et al pioneered studies to use microhomology prediction to improve CRISPR-based KO efficiency in cell lines, by in-silico selection of target sites to reduce in-frame mutations.2 They presented that the preference of the in-frame mutations at a given target site can be predicted by the microhomology profile, and an alternative NHEJ pathway, i.e., the microhomology-mediated end joining (MMEJ) occurs.2,3 A score was defined to predict the microhomology-based out-of-frame mutation preferences.2,4