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Synthetic CRISPR Inhibitors Suppress DNA Breaks Induced by Cas12a

NEW YORK (GenomeWeb) – Ohio State University researchers have developed synthetic oligonucleotides that inhibit the ability of the CRISPR-Cas12a enzyme to induce double-strand breaks (DSBs) in DNA in a time-dependent and sequence-independent manner.

Researchers have published studies in the past on the discoveries of anti-CRISPR (Acr) proteins — bacteria harboring Cas9 or Cas3 adaptive immune systems sometimes acquire genetic elements that encode Cas9, Cas3, or Cascade complex Acrs. In September, researchers from Jennifer Doudna's lab at the University of California, Berkeley also found Acrs for Cas12a for the first time.

Rather than discovering a naturally occurring Acr for Cas12a, however, the OSU team constructed CRISPR RNA (crRNA) variants consisting of chemically modified DNA-crRNA and RNA-crRNA duplexes and found that phosphorothioate (PS)-modified DNA-crRNA duplex completely blocked the function of Cas12a. As they wrote in Cell Reports yesterday, the researchers also found that these PS-modified DNA oligonucleotides were able to suppress DNA double-strand breaks induced by the Cas12a orthologs Acidaminococcus sp. Cas12a (AsCas12a) and Lachnospiraceae bacterium (LbCas12a), without the need for prehybridization.

The researchers — who referred to Cas12a in their study by its older name, Cpf1 — synthesized oligonucleotides complementary to different regions of the crRNA of Cas12a, which is composed of a 5' handle, seed region, and 3' end. They then generated their corresponding crDuplexes by annealing an equimolar oligonucleotide with the crRNA.

Experiments with the crDuplexes showed that the genome editing activity of Cas12a could be affected by factors such as different chemical modifications in the oligonucleotides or position of hybridization. Among all the oligonucleotides tested, both 2'-fluoro RNA and PS-modified DNA exhibited apparent inhibitory effects. Given how challenging it is to synthesize relatively long 2'-fluoro-modified oligonucleotides, the researchers then selected two PS-modified DNA-crRNA oligonucleotides — dubbed crDuplex11 and crDuplex13 — to study further. These two crDuplexes exhibited the most potent inhibition against Cas12a expression, they noted.

Further analysis found that crDuplex13 exhibited some detectable genome editing activity, whereas crDuplex11 was capable of completely disabling Cas12a function. When they compared the structures of crDuplex11 and crDuplex13, the researchers found that a PS-DNA oligonucleotide with the same length of crRNA as Cas12a may be the key to fully inhibiting the enzyme's editing activity.

The researchers also tested the inhibitory effects of PS DNA on CRISPR-Cas9. They synthesized a 100-nucleotide PS DNA oligonucleotide complementary to the full-length synthetic SpCas9 single-guide RNA targeting the EMX1 gene. However, when they added this oligonucleotide to a human cell line, they found that the genome editing activity of SpCas9 was dramatically lowered but not totally inhibited.

"In general, unmodified DNA and RNA oligonucleotides showed moderate effects on Cpf1-mediated genome-editing activity regardless of the hybrid region and the length of oligonucleotides tested. 2'-Fluoro-modified oligonucleotides substantially interfered with Cpf1-mediated cleavage. 2'-O-methyl-modified oligo-nucleotides maintained the performance of the wild-type crRNA. PS-modified DNA oligonucleotides can act as potent inhibitors to switch off Cpf1 function," the authors wrote. "Most important, these PS-modified DNA oligonucleotides enabled us to inhibit genome editing activities of AsCpf1, LbCpf1, and SpCas9. Also, the inhibition was observed in three human genomic loci in a time- and dose-dependent manner."

These synthetic oligonucleotides, as well as naturally occurring Acr proteins, can serve as useful tools for further analysis of Cas12a, the researchers concluded. And in case of acute toxic effects of the CRISPR system in clinical use, a combination of Arcs and synthetic oligonucleotides could serve as an antidote, they said.