FnCpf1-Mediated Targeted Mutagenesis in Plants.

ABSTRACT

Sequence-specific nucleases (SSNs) are nowadays fundamental tools to generate mutants that impaired in genes of interest. The bioactive molecules screened in the chemical genomics studies affect specific physiological process by disrupting the function of its target protein(s). Mutation analysis of the gene(s) of target protein(s) of the screened chemical is necessary to resolve how the chemical works in plants. Clustered regularly interspersed short palindromic repeats (CRISPR) from Prevotella and Francisella 1 (Cpf1) are newly characterized RNA-directed endonuclease. Several papers have shown clearly that Cpf1 could be a versatile SSN in plant genome engineering. Cfp1 from Francisella novicida (FnCpf1) recognizes TTN as its protospacer adjacent motif (PAM). FnCpf1 utilizes a shorter PAM compared to other known Cpf1s such as AsCpf1 or LbCpf1, which use TTTN as PAM. Since PAM length can be a limiting factor in target selection, this feature of FnCpf1 is practical for targeted mutagenesis experiments. The application of FnCpf1-mediated targeted mutagenesis to the chemical genomics could accelerate to figure out the mechanism of action of screened chemicals. Here, we describe procedures for targeted mutagenesis in rice and tobacco using FnCpf1.