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CRISPR-Cas12a Possesses Unconventional DNase Activity that Can Be Inactivated by Synthetic Oligonucleotides

Bin Li1, Jingyue Yan2, Youxi Zhang2

  • 1Division of Pharmaceutics and Pharmacology, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA; Department of Infectious Disease, Shenzhen People's Hospital, The Second Clinical Medical College of Jinan University, The First Affiliated Hospital of Southern University of Science and Technology, Shenzhen 518020, China.

Molecular Therapy. Nucleic Acids
|February 12, 2020
PubMed

Insights

CRISPR-Cas12a nucleases exhibit diverse DNA-cleavage activities, with specific metal ions influencing their preference for single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA). This DNase activity offers new insights for biological and therapeutic applications.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • CRISPR-Cas12a (CRISPR-Cpf1) systems are known for their DNA-cleavage capabilities.
  • The influence of metal ions on Cas12a's DNase activity and substrate specificity, particularly without CRISPR RNA (crRNA), remains less explored.

Purpose of the Study:

  • To investigate the DNase activity and substrate specificity of various Cas12a orthologs.
  • To determine the role of different divalent metal ions in modulating Cas12a's DNA cleavage.
  • To explore potential applications of Cas12a's ssDNA degradation activity.

Main Methods:

  • Assessing DNase activity of Cas12a orthologs using single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) substrates.
  • Testing activity in the presence of diverse divalent metal ions (e.g., magnesium (II), manganese (II)).
  • Evaluating the inhibitory effect of phosphorothioate-modified DNA oligonucleotides.

Main Results:

  • Cas12a orthologs demonstrated varying abilities to degrade ssDNA and/or dsDNA, contingent on the specific metal ions present.
  • Acidaminococcus sp. Cas12a (AsCas12a) showed a strong preference for ssDNA cleavage over dsDNA in the presence of magnesium (II) ions.
  • Both AsCas12a and Francisella novicida Cas12a (FnCas12a) exhibited significant dsDNA cleavage activity with manganese (II) ions.
  • DNase activities were successfully inhibited by synthetic DNA oligonucleotides with phosphorothioate modifications.

Conclusions:

  • Cas12a orthologs possess intrinsic ssDNase activity, representing a distinct DNA cleavage mechanism.
  • The metal ion-dependent substrate specificity of Cas12a offers versatility for various applications.
  • Understanding these DNase activities provides a foundation for novel biological and therapeutic strategies.
Keywords:
CRISPR-Cas12aDNase activityinhibitionmetal ionssynthetic oligonucleotide

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