Abstract
CRISPR/Cas9 systems are nowadays applied extensively to effect genome editing in various organisms including plants. CRISPR from Prevotella and Francisella 1 (Cpf1) is a newly characterized RNA-guided endonuclease that has two distinct features as compared to Cas9. First, Cpf1 utilizes a thymidine-rich protospacer adjacent motif (PAM) while Cas9 prefers a guanidine-rich PAM. Cpf1 could be used as a sequence-specific nuclease to target AT-rich regions of a genome that Cas9 had difficulty accessing. Second, Cpf1 generates DNA ends with a 5′ overhang, whereas Cas9 creates blunt DNA ends after cleavage. “Sticky” DNA ends should increase the efficiency of insertion of a desired DNA fragment into the Cpf1-cleaved site using complementary DNA ends. Therefore, Cpf1 could be a potent tool for precise genome engineering. To evaluate whether Cpf1 can be applied to plant genome editing, we selected Cpf1 from Francisella novicida (FnCpf1), which recognizes a shorter PAM (TTN) within known Cpf1 proteins, and applied it to targeted mutagenesis in tobacco and rice. Our results show that targeted mutagenesis had occurred in transgenic plants expressing FnCpf1 with crRNA. Deletions of the targeted region were the most frequently observed mutations. Our results demonstrate that FnCpf1 can be applied successfully to genome engineering in plants.
Highlights
Targeted mutagenesis and gene targeting using sequence specific nucleases (SSNs) are powerful strategies used to accelerate molecular breeding of crops
Since DSBs with compatible overhangs can be repaired via precise end joining[14], the sticky DNA ends generated by Cpf[1] are thought to be ideally suited to precise genome editing such as knock-in or replacement of a desired DNA fragment using compatible DNA ends
To express CRISPR RNA (crRNA) of FnCpf[1], Arabidopsis U6-26 and rice U6-2 small nuclear RNA gene promoters were used in tobacco and rice, respectively (Fig. 1a,b)[17,19]
Summary
Targeted mutagenesis and gene targeting using sequence specific nucleases (SSNs) are powerful strategies used to accelerate molecular breeding of crops. Since DSBs with compatible overhangs can be repaired via precise end joining[14], the sticky DNA ends generated by Cpf[1] are thought to be ideally suited to precise genome editing such as knock-in or replacement of a desired DNA fragment using compatible DNA ends These specific features of Cpf[1] can broaden the spectrum of genome editing that is possible using SSNs. To apply Cpf[1] to plant genome engineering, we selected FnCpf[1] for the following reasons. The shorter PAM of FnCpf[1] is a practical and favorable feature for targeted mutagenesis, the genome editing activity of FnCpf[1] in human cells is reported to be lower than that of AsCpf[1] and LbCpf[113]. We first engineered a binary vector to optimize the expression of FnCpf[1] in plants, designed a targeted mutagenesis experiment in tobacco and rice
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