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Protocols for C-Brick DNA Standard Assembly Using Cpf1
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Structural Basis for the Canonical and Non-canonical PAM Recognition by CRISPR-Cpf1
Takashi Yamano1, Bernd Zetsche2, Ryuichiro Ishitani1
1Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
Molecular Cell
|August 8, 2017
Summary
Cpf1 nucleases, used in genome editing, show relaxed recognition of DNA targets. Structural studies reveal how LbCpf1 adapts to varied protospacer adjacent motif (PAM) sequences for DNA cleavage.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Cpf1 (Cas12a) is an RNA-guided nuclease essential for CRISPR-Cas genome editing.
- Cpf1 recognizes specific protospacer adjacent motif (PAM) sequences on target DNA.
- While typically recognizing TTTV PAMs, Cpf1 also binds suboptimal C-containing PAMs.
Purpose of the Study:
- To elucidate the structural basis of Cpf1's relaxed PAM recognition.
- To understand how LbCpf1 accommodates diverse PAM sequences during DNA binding and cleavage.
Main Methods:
- X-ray crystallography was used to determine the structures of LbCpf1.
- Complexes of LbCpf1 with crRNA and target DNA containing TTTA, TCTA, TCCA, or CCCA PAMs were analyzed.
Main Results:
- Four crystal structures revealed LbCpf1 in complex with crRNA and various PAM-containing DNA targets.
- LbCpf1 undergoes distinct conformational changes in response to different PAM sequences.
- These conformational alterations mediate varied interactions with the PAM-DNA duplex.
Conclusions:
- The study provides atomic-level insights into Cpf1's PAM-dependent DNA recognition mechanism.
- Understanding these structural dynamics enhances knowledge of Cpf1 family nucleases for genome editing applications.
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