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Characterization of Cas12a nucleases reveals diverse PAM profiles between closely-related orthologs

Thomas Jacobsen1, Fani Ttofali1, Chunyu Liao2

  • 1Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27695, USA.

Nucleic Acids Research
|April 25, 2020
PubMed

Insights

Exploring CRISPR-Cas12a nucleases reveals significant functional diversity among related subtypes. This diversity, particularly in PAM recognition, expands the potential of CRISPR gene editing technologies.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • CRISPR-Cas systems are prokaryotic adaptive immune mechanisms.
  • RNA-guided nucleases from CRISPR systems are vital for biotechnological applications.
  • Expanding the known CRISPR-Cas subtypes is crucial for discovering novel nucleases.

Purpose of the Study:

  • To characterize the functional diversity of six Cas12a nucleases from the V-A subtype.
  • To investigate the PAM recognition profiles and targeting activities of these Cas12a nucleases.
  • To explore the potential for expanding the CRISPR toolbox through ortholog mining and PAM engineering.

Main Methods:

  • Utilized cell-free transcription-translation systems and human cells for nuclease characterization.
  • Assessed guide RNA utilization, PAM profiles, and targeting activities.
  • Performed mutational analyses to engineer Cas12a nucleases with altered PAM specificities.

Main Results:

  • Cas12a nucleases from the V-A subtype exhibited distinct PAM profiles and targeting activities, irrespective of phylogenetic relatedness.
  • Two highly similar Prevotella Cas12a nucleases (PiCas12a and PdCas12a) displayed different PAM recognition.
  • Mutational analysis yielded novel Cas12a variants with distinct PAM profiles, enabling more flexible gene editing in human cells.

Conclusions:

  • Cas12a nucleases can display significant functional variation even among closely related orthologs.
  • Selective pressures likely drive the diversification of PAM recognition in Cas12a nucleases.
  • Ortholog mining and PAM engineering are promising strategies for expanding the CRISPR gene editing toolbox.

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