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.

Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single‐cell mutation detection to mutant plant regeneration

“…We evaluated the parameters of transfection and single‐cell analysis by targeting mutations in the tobacco PDS gene ( NtPDS ). Tobacco ( N. tabacum ) is an amphidiploid derived from N. sylvestris and N. tomentosiformis (Endo et al ., ; Sierro et al ., ). Specific primers were designed to amplify PDS genes from the N .…”

Transforming plant biology and breeding with CRISPR/Cas9, Cas12 and Cas13

“…In contrast to Cas9 which generates bluntended DSBs proximal to the PAM, Cas12a generates staggered DSBs distal from the PAM. In these studies it could be shown that via the CRISPR/Cas12a system targeted mutagenesis can be accomplished and that the appropriate mutations are heritable [72,73]. Similar to Cas9, only a subset of Cas12a orthologues shows robust activity in eukaryotic cells.…”

CRISPR‐Cas system: Toward a more efficient technology for genome editing and beyond

“…In the last few years, researchers purposed several CRISPR‐Cas effector proteins that may have differences in experimental use and targeting specificity (Table ) . Interestingly, the possibility of RNA transcripts editing is valuable for treating genetic disorders.…”