Newly discovered ‘anti-CRISPR proteins’ can prevent CRISPR mistakes, new study says

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CRISPR/CAS9 has emerged as a flexible and effective approach for genome editing with a promise for the correction of disease-causing mutations. However, most studies have so far demonstrated CRISPR as an on/off system, with little temporal control of its activity or function. Now, new research from the University of Toronto described the discover of a number of proteins that allow to precisely do this: control the activity of CRISPR specifically and conditionally, and at defined time points.

Published in Cell, the study is titled “Naturally Occurring Off-Switches for CRISPR-Cas9” from the lab of Dr. Alan Davidson.

The authors identified naturally occurring protein inhibitors of a CRISPR-Cas9 system. A bioinformatics-based approach allowed the authors to discover three proteins that inhibit N. meningitidis type II-C CRISPR-Cas system, by interacting with NmeCas9 to function as off-switches for NmeCas9 genome editing activity.


Images from Pawluk et al. (2016). (A) Schematic representation of plasmids used for expression of dNmeCas9-(sfGFP)3, dSpyCas9-(mCherry)3, their respective telomeric sgRNAs, and anti-CRISPR protein. (B–F) Fluorescence images of U2OS cells transiently transfected with plasmids depicted in (A). The specific version of each plasmid set (with or without sgRNAs, with or without anti-CRISPRs) is given to the right of each row. First column: differential interference contrast (DIC) and mTagBFP2 imaging, merged. Second column: dNmeCas9-(sfGFP)3. Third column: dSpyCas9-(mCherry)3. Fourth column: dNmeCas9-(sfGFP)3 and dSpyCas9-(mCherry)3, merged. (G) Quantitation of dNmeCas9-(sfGFP)3 telomeric foci.


They further showed that members of all three anti-CRISPR families bind directly to the NmeCas9/sgRNA complex and inhibit in vitro DNA cleavage. Interestingly, they displayed unrelated sequences, leading the authors to postulate that they operate under different mechanisms as well.

Evolutionarily, Cas9-associating anti-CRISPRs were postulated to prevent the acquisition of new spacers (such as viral DNA) in response to external invasions, with CRISPR having a profound effect on horizontal gene transfer. This implication has profound effects on therapeutics being developed based on CRISPR/CAS9 technology.

Therapeutically, these proteins can prevent off-target effects and are thus exciting potential therapeutic targets.

The entire study can be read here.


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