Structural Basis for the Canonical and Non-canonical PAM Recognition by CRISPR-Cpf1

The RNA-guided Cpf1 (also known as Cas12a) nuclease associates with a CRISPR RNA (crRNA) and cleaves the double-stranded DNA target complementary to the crRNA guide. The two Cpf1 orthologs from Acidaminococcus sp. (AsCpf1) and Lachnospiraceae bacterium (LbCpf1) have been harnessed for eukaryotic gen...

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Bibliographic Details
Published in:Molecular cell Vol. 67; no. 4; p. 633
Main Authors: Yamano, Takashi, Zetsche, Bernd, Ishitani, Ryuichiro, Zhang, Feng, Nishimasu, Hiroshi, Nureki, Osamu
Format: Journal Article
Language:English
Published: United States 17.08.2017
Subjects:
Acidaminococcus - enzymology
Acidaminococcus - genetics
Bacterial Proteins - chemistry
Bacterial Proteins - genetics
Bacterial Proteins - metabolism
Binding Sites
Clostridiales - enzymology
Clostridiales - genetics
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR-Associated Proteins - chemistry
CRISPR-Associated Proteins - genetics
CRISPR-Associated Proteins - metabolism
CRISPR-Cas Systems
Crystallography, X-Ray
DNA - chemistry
DNA - genetics
DNA - metabolism
Endonucleases - chemistry
Endonucleases - genetics
Endonucleases - metabolism
Escherichia coli - enzymology
Escherichia coli - genetics
HEK293 Cells
Humans
Models, Molecular
Nucleic Acid Conformation
Nucleic Acid Heteroduplexes - chemistry
Nucleic Acid Heteroduplexes - genetics
Nucleic Acid Heteroduplexes - metabolism
Protein Binding
Protein Conformation
RNA, Guide, CRISPR-Cas Systems - chemistry
RNA, Guide, CRISPR-Cas Systems - genetics
RNA, Guide, CRISPR-Cas Systems - metabolism
Structure-Activity Relationship
CRISPR-Cas
Cpf1
crystal structure
protospacer adjacent motif
Cas12a
ISSN:1097-4164, 1097-4164
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Summary:The RNA-guided Cpf1 (also known as Cas12a) nuclease associates with a CRISPR RNA (crRNA) and cleaves the double-stranded DNA target complementary to the crRNA guide. The two Cpf1 orthologs from Acidaminococcus sp. (AsCpf1) and Lachnospiraceae bacterium (LbCpf1) have been harnessed for eukaryotic genome editing. Cpf1 requires a specific nucleotide sequence, called a protospacer adjacent motif (PAM), for target recognition. Besides the canonical TTTV PAM, Cpf1 recognizes suboptimal C-containing PAMs. Here, we report four crystal structures of LbCpf1 in complex with the crRNA and its target DNA containing either TTTA, TCTA, TCCA, or CCCA as the PAM. These structures revealed that, depending on the PAM sequences, LbCpf1 undergoes conformational changes to form altered interactions with the PAM-containing DNA duplexes, thereby achieving the relaxed PAM recognition. Collectively, the present structures advance our mechanistic understanding of the PAM-dependent, crRNA-guided DNA cleavage by the Cpf1 family nucleases.
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ISSN:1097-4164
1097-4164
DOI:10.1016/j.molcel.2017.06.035