title: CRISPR/Cpf1-mediated DNA-free plant genome editing.
abstract: Cpf1, a type V CRISPR effector, recognizes a thymidine-rich protospacer-adjacent motif and induces cohesive double-stranded breaks at the target site guided by a single CRISPR RNA (crRNA). Here we show that Cpf1 can be used as a tool for DNA-free editing of plant genomes. We describe the delivery of recombinant Cpf1 proteins with in vitro transcribed or chemically synthesized target-specific crRNAs into protoplasts isolated from soybean and wild tobacco. Designed crRNAs are unique and do not have similar sequences (≤3 mismatches) in the entire soybean reference genome. Targeted deep sequencing analyses show that mutations are successfully induced in FAD2 paralogues in soybean and AOC in wild tobacco. Unlike SpCas9, Cpf1 mainly induces various nucleotide deletions at target sites. No significant mutations are detected at potential off-target sites in the soybean genome. These results demonstrate that Cpf1-crRNA complex is an effective DNA-free genome-editing tool for plant genome editing.
authors.affiliation: Center for Genome Engineering, Institute for Basic Science, 70, Yuseong-daero 1689-gil, Yuseong-gu, Daejeon 34047, South Korea.
authors.forename: Hyeran
authors.initials: H
authors.lastname: Kim
authors.affiliation: Center for Genome Engineering, Institute for Basic Science, 70, Yuseong-daero 1689-gil, Yuseong-gu, Daejeon 34047, South Korea.
authors.forename: Sang-Tae
authors.initials: ST
authors.lastname: Kim
authors.affiliation: Center for Genome Engineering, Institute for Basic Science, 70, Yuseong-daero 1689-gil, Yuseong-gu, Daejeon 34047, South Korea.
authors.forename: Jahee
authors.initials: J
authors.lastname: Ryu
authors.affiliation: Center for Genome Engineering, Institute for Basic Science, 70, Yuseong-daero 1689-gil, Yuseong-gu, Daejeon 34047, South Korea.
authors.forename: Beum-Chang
authors.initials: BC
authors.lastname: Kang
authors.affiliation: Center for Genome Engineering, Institute for Basic Science, 70, Yuseong-daero 1689-gil, Yuseong-gu, Daejeon 34047, South Korea.|Department of Chemistry, Seoul National University, Seoul 08826, South Korea.
authors.forename: Jin-Soo
authors.initials: JS
authors.lastname: Kim
authors.affiliation: Center for Genome Engineering, Institute for Basic Science, 70, Yuseong-daero 1689-gil, Yuseong-gu, Daejeon 34047, South Korea.
authors.forename: Sang-Gyu
authors.initials: SG
authors.lastname: Kim
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D064130:CRISPR-Associated Proteins / Q000235:genetics / Q000378:metabolism; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D018744:DNA, Plant / Q000378:metabolism*; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism; D044943:Fatty Acid Desaturases / Q000235:genetics; D017353:Gene Deletion; D000072669:Gene Editing / Q000379:methods*; D018745:Genome, Plant; D009154:Mutation; D011523:Protoplasts / Q000378:metabolism; D012313:RNA / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins; D017421:Sequence Analysis; D013025:Glycine max / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism; D004706:Endodeoxyribonucleases
chemical_list: D001426:Bacterial Proteins; D064130:CRISPR-Associated Proteins; D018744:DNA, Plant; D004720:Endonucleases; D044943:Fatty Acid Desaturases; D012313:RNA; D011994:Recombinant Proteins; C523310:FAD2 protein, Arabidopsis; C000656745:Cas12a protein; D004706:Endodeoxyribonucleases