Targeted Mutagenesis Using FnCpf1 in Tobacco

Various CRISPR/Cas9 systems have been extensively applied for targeted mutagenesis to generate mutants that impaired in genes of interest. Clustered regularly interspersed short palindromic repeats (CRISPR) from Prevotella and Francisella 1 (Cpf1) is new RNA-directed endonuclease possessing some differences as compared to Cas9. Several papers have shown that Cpf1 could be a versatile tool in plant genome engineering. Cfp1 from Francisella novicida (FnCpf1) recognizes TTN as its protospacer adjacent motif (PAM). TTN is a shortest PAM among other known Cpf1s such as AsCpf1 or LbCpf1, which use TTTN as PAM. The length of PAM can be the restriction of the number of target sequences. Cpf1 generates cohesive DNA end after the digestion of target sequences. Sticky DNA end is thought to appropriate for in vivo ligation rather than blunt DNA end created by Cas9. Therefore, FnCpf1 is practical for targeted mutagenesis experiments. The application of FnCpf1-mediated targeted mutagenesis to the plant genome engineering could accelerate molecular breeding of crops. Here, we describe procedures for targeted mutagenesis in tobacco using FnCpf1.