Figure 1. Tools to induce mutations in vivo.

(A) Upon recognition of a specific DNA sequence targeted through the guide RNA (gRNA) and nickase Cas9 (nCas9), AID to create C→T transitions in the genome. The DNA nick introduced by nCas9 increases the efficiency of mutagenesis. Uracil glycosylase inhibitor (UGI) inhibits uracil excision and recruitment of the DNA repair machinery such indels are avoided. (B) In the EvolvR system, enhancer-nickase Cas9 (enCas9) targeted to a locus makes the DNA accessible for an error prone DNA polymerase I from E. coli to transcribe the DNA. The DNA nick introduced by enCas9 is also required for mutagenesis. (C) Multiple gRNAs targeting an genomic region are expressed in a single RNA transcript flanked by tRNAs.