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RNA-Independent DNA Cleavage Activities of Cas9 and Cas12a

Ramya Sundaresan1, Hari Priya Parameshwaran1, S D Yogesha1

  • 1Department of Chemistry and Biochemistry, Stephenson Life Sciences Research Center, University of Oklahoma, 101 Stephenson Parkway, Norman, OK 73019, USA.

Cell Reports
|December 28, 2017
PubMed

Insights

CRISPR-Cas enzymes like Cas9 and Cas12a can now cut DNA without guide RNA, using manganese ions. Different Cas enzymes show varied substrate preferences in this RNA-independent DNA targeting, impacting gene editing.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • CRISPR-Cas systems offer sequence-specific defense in prokaryotes.
  • Cas9 and Cas12a proteins utilize guide RNA and divalent metal ions for DNA targeting and cleavage.
  • Protospacer adjacent motif (PAM) recognition is crucial for RNA-guided DNA cleavage by Cas9 and Cas12a.

Purpose of the Study:

  • To investigate the RNA-independent DNA cleavage activity of specific Cas9 and Cas12a orthologs.
  • To determine the substrate requirements for RNA-independent DNA cleavage by Francisella tularensis novicida (Fno) Cas12a, FnoCas9, and Streptococcus pyogenes Cas9 (SpyCas9).

Main Methods:

  • Assessing DNA cleavage by FnoCas12a, FnoCas9, and SpyCas9 in the presence of Mn2+ ions.
  • Evaluating cleavage activity on both double-stranded and single-stranded plasmid DNA substrates.
  • Comparing the substrate specificities of different Cas orthologs in an RNA-independent manner.

Main Results:

  • Francisella tularensis novicida Cas12a (FnoCas12a), FnoCas9, and Streptococcus pyogenes Cas9 (SpyCas9) exhibit DNA cleavage activity independent of guide RNA, in the presence of manganese ions.
  • RNA-independent activity varied among the tested enzymes: FnoCas9 nicked double-stranded DNA, SpyCas9 degraded single-stranded DNA, and FnoCas12a cleaved both types of substrates.
  • The observed RNA-independent DNA targeting highlights the influence of metal ion concentrations and Cas ortholog identity on enzyme function.

Conclusions:

  • The discovery of RNA-independent DNA cleavage by certain Cas enzymes broadens the understanding of CRISPR-Cas mechanisms.
  • Intracellular metal ion composition and the specific Cas9/Cas12a variant used are critical factors that can influence the outcomes of gene editing applications.
  • Further research into these RNA-independent activities could reveal novel applications or necessitate adjustments in current gene editing protocols.
Keywords:
CRISPRCas12aCas9 endonucleasesCpf1FnoCas12aFnoCas9Francisella tularensis novicidaMn(2+)-specific CRISPR activityRNA-independent DNA cleavageSpyCas9

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