Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells.
Programmable clustered regularly interspaced short palindromic repeats (CRISPR) Cpf1 endonucleases are single-RNA-guided (crRNA) enzymes that recognize thymidine-rich protospacer-adjacent motif (PAM) sequences and produce cohesive double-stranded breaks (DSBs). Genome editing with CRISPR-Cpf1 endonucleases could provide an alternative to CRISPR-Cas9 endonucleases, but the determinants of targeting specificity are not well understood. Using mismatched crRNAs we found that Cpf1 could tolerate single or double mismatches in the 3' PAM-distal region, but not in the 5' PAM-proximal region. Genome-wide analysis of cleavage sites in vitro for eight Cpf1 nucleases using Digenome-seq revealed that there were 6 (LbCpf1) and 12 (AsCpf1) cleavage sites per crRNA in the human genome, fewer than are present for Cas9 nucleases (>90). Most Cpf1 off-target cleavage sites did not produce mutations in cells. We found mismatches in either the 3' PAM-distal region or in the PAM sequence of 12 off-target sites that were validated in vivo. Off-target effects were completely abrogated by using preassembled, recombinant Cpf1 ribonucleoproteins.
Center for Genome Engineering, Institute for Basic Science (IBS), Seoul, Republic of Korea.|Department of Chemistry, Seoul National University, Seoul, Republic of Korea.
Daesik
D
Kim
Center for Genome Engineering, Institute for Basic Science (IBS), Seoul, Republic of Korea.|Department of Chemistry, Seoul National University, Seoul, Republic of Korea.
Jungeun
J
Kim
Center for Genome Engineering, Institute for Basic Science (IBS), Seoul, Republic of Korea.
Junho K
0000-0003-3794-1149
JK
Hur
Center for Genome Engineering, Institute for Basic Science (IBS), Seoul, Republic of Korea.|Department of Chemistry, Seoul National University, Seoul, Republic of Korea.
Kyung Wook
KW
Been
Department of Chemistry, Seoul National University, Seoul, Republic of Korea.
Sun-Heui
SH
Yoon
Center for Genome Engineering, Institute for Basic Science (IBS), Seoul, Republic of Korea.|Department of Chemistry, Seoul National University, Seoul, Republic of Korea.
Jin-Soo
JS
Kim
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D020137:Base Pair Mismatch; D001665:Binding Sites; D064113:CRISPR-Cas Systems / Q000235:genetics*; D002874:Chromosome Mapping / Q000379:methods; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000502:physiology; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism*; D004789:Enzyme Activation; D015894:Genome, Human / Q000235:genetics*; D006801:Humans; D011485:Protein Binding; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004720:Endonucleases