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Structural Basis for the Altered PAM Recognition by Engineered CRISPR-Cpf1.

Hiroshi Nishimasu1, Takashi Yamano2, Linyi Gao3

  • 1Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan; JST, PRESTO, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.

Molecular Cell
|June 10, 2017
PubMed
Summary

Engineered Cpf1 variants (AsCpf1) with altered protospacer adjacent motif (PAM) recognition were studied. Crystal structures reveal how substituted residues in RVR and RR variants enable recognition of new PAM sequences for genome editing.

Keywords:
CRISPR-Cas systemCas12aCpf1crystal structureprotospacer adjacent motif

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Area of Science:

  • Molecular Biology
  • Structural Biology
  • Genomics

Background:

  • CRISPR-Cpf1 nucleases are RNA-guided enzymes for DNA cleavage.
  • Cpf1-mediated genome editing relies on recognizing specific protospacer adjacent motifs (PAMs).
  • Wild-type Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) recognizes TTTV PAMs.

Purpose of the Study:

  • To elucidate the mechanism of altered PAM recognition by engineered AsCpf1 variants.
  • To provide structural insights into the RVR and RR AsCpf1 variants.
  • To guide further engineering of Cpf1 nucleases for expanded genome editing applications.

Main Methods:

  • X-ray crystallography
  • Determination of crystal structures at 2.0 Å resolution
  • Structural analysis of RVR and RR AsCpf1 variants bound to crRNA and target DNA

Main Results:

  • High-resolution crystal structures of RVR and RR AsCpf1 variants were obtained.
  • The structures reveal that substituted residues in the variants are key for recognizing altered PAM sequences (TATV and TYCV).
  • The study delineates the specific molecular interactions responsible for the expanded PAM specificity.

Conclusions:

  • The structural data clarifies the altered PAM recognition mechanisms of AsCpf1 variants.
  • This understanding facilitates the rational design of novel Cpf1 nucleases with tailored PAM specificities.
  • The findings support the advancement of Cpf1-based genome editing technologies.