Efficient targeted mutagenesis of rice and tobacco genomes using Cpf1 from Francisella novicida

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.

Figure 1. T-DNA constructions for FnCpf1 expression in tobacco and rice.

(a) Construct for targeted mutagenesis in tobacco. FnCpf1 (At) was inserted downstream of the PcUbi promoter. The Athsp terminator was placed at the end of FnCpf1 ORF. The AtADH 5′-UTR was introduced between the PcUbi promoter and FnCpf1 (At) to enhance translation. the nuclear localization signal (NLS) from the SV40 large T-antigen was fused translationally to the C-terminus of FnCpf1. The crRNA is under the control of Arabidopsis U6-26 promoter. To isolate transformants with kanamycin resistance, an NPT II cassette was included in the construct. AtADH 5′-UTR: 5′ untranslated region of Arabidopsis thaliana ALCOHOLDEHYDROGENASE gene. Athsp ter: the terminator region of Arabidopsis thaliana HEAT SHOCK PROTEIN 18.2 gene. (b) Construct used for targeted mutagenesis in rice. The ZmUbi-1 promoter drives expression of FnCpf1 (Os). The OsADH 5′-UTR was introduced between the ZmUbi promoter and FnCpf1 (At) to enhance translation. An NLS was fused translationally to the C-terminus of FnCpf1 (Os). Pea3A and OsAct1 terminators were inserted tandemly downstream of FnCpf1 to terminate transcription. Expression of crRNA is driven by the rice U6-2 promoter. To screen transformants with hygromycin resistance, HPT cassettes were included in the construct. OsADH 5′-UTR: 5′ untranslated region of Oryza sativa ALCOHOLDEHYDROGENASE gene. Pea3A ter: the terminator region of Pisum sativum rbcS 3A gene. OsAct ter: the terminator region of Oryza sativa Actin gene.

Figure 2. Analyses of FnCpf1-induced mutations in tobacco.

(a) Heteroduplex mobility assay to detect mutation on crNtPDS-1 (upper two panels) and crNtPDS-2 (lower panel) loci. (S) and (T) indicate PCR products amplified from the loci including each target sequence on N. sylvestris and N. tomentosiformis genomes, respectively. (b) Patterns of mutations detected in crNtPDS-1 (top), crNtPDS-2 (middle) and crNtSTF-4 (bottom) loci. The target DNA sequences of each crRNA are shown as wild-type (WT) at the top with underlined. (S) and (T) indicate the target sequence on both S and T genomes. The PAM regions are shown by green. Mismatched nucleotide is indicated in red. Line numbers of transgenic plants were indicated as # at left side of each sequence. DNA deletions are presented as dashes. The length of indel and the number of clones are represented at the right side of each sequence (+, insertion; −, deletion; ×, number of clones). Mut. Freq. (%): Mutation frequency. (c) CAPS analysis of crNtSTF-4 locus in T0 generation. (d) CAPS analysis of crNtSTF-4 locus in T1 generation of line #7. −: Non-digested PCR products, +: EcoRV-digested PCR products. Arrow head indicated the position of undigested PCR products. An undigested band indicates mutation at the crNtSTF-4 locus.

Figure 3. Analyses of FnCpf1-induced mutations in rice.

(a) CAPS analysis of crOsDL-1~2 and crOsALS-1~2 loci. −: Non-digested PCR products, +: PstI or AseI-digested PCR products. Arrow head indicated the position of undigested PCR products. An undigested band indicates mutation in the target loci. (b) Patterns of mutations detected in crOsDL-1~2 and crOsALS-1~2 loci. The target DNA sequences of each crRNA are shown as wild-type (WT) at the top with underlined. (S) and (T) indicate the target sequence on both S and T genomes. The PAM regions are shown by green. Mismatched nucleotide is indicated in red. Line numbers of transgenic plants were indicated as # at left side of each sequence. DNA deletions are presented as dashes. The length of indel and the number of clones are represented at the right side of each sequence (+, insertion; −, deletion; ×, number of clones). Mut. Freq. (%): Mutation frequency.