Figure 1. One-step inactivation of chromosomal gene(s) by CRISPR-Cas9 assisted non-homologous end-joining (CA-NHEJ).

Cas9 and NHEJ-related proteins (Mt-Ku and Mt-LigD) are expressed in host cells, which are then transformed with a single-guide RNA (sgRNA) donor plasmid to generate double-stranded breaks (DSBs) and trigger indel mutations. Mutagenesis is attributed to the RNA-directed Cas9 cleavage system and the error-prone NHEJ repair system. First, site-specific DSB is generated via sgRNA-directed Cas9 cleavage. The DNA ends are recognized and stabilized by the DNA end-binding protein Mt-Ku. Next, the ATP-dependent DNA ligase Mt-LigD is recruited to the DNA ends; the imprecise repair of DSB results in a frameshift mutation. Finally, only the DSB-repaired colonies lacking the Cas9 targeting site survive CRISPR-Cas9 screening. To further engineer the strain, the sgRNA donor plasmid is cured via an inducible sgRNA-mediated “suicide” strategy, and the temperature-sensitive plasmid pCas9 (Ts)-NHEJ by growing the cells at 42 °C.