Cas9 Inhibitory Proteins for Modulating Editing Activity

Possible mechanisms of action of three anti-CRISPR proteins, AcrF1, 2, and 3 that block Type IF CRISPR-Cas systems albeit in different ways. Image Credit: Karen Maxwell in Phages Fight Back: Inactivation of the CRISPR-Cas Bacterial Immune System by Anti-CRISPR Proteins

Pawluk et al (2106) describe anti-CRISPR Cas9 proteins that can be used to regulate the activity of type II Cas9s and show that they function in human cells.   These inhibitory proteins are likely to become valuable tools for modulating temporal and spatial aspects of gene editing.

Pawluk et al (2106) imagined that there would strong selection pressures that could shape the evolution of mobile genetic elements invading bacterial cells such as bacteriophages.  This group reported protein inhibitors of type 1E and 1F CRISPR-Cas9 systems recently and speculated that others would be found, including those against type II systems.

Anti- CRISPR genes (acr) were originally found upstream of their putative transcriptional regulators that are referred to as anti-CRISPR associated proteins (Aca).  Using these observations Pawluk et al (2106) examined the mobile genetic elements associated with Proteobacteria and identified aca genes and a few associated, putative, type II-C anti-CRISPR acrs – one from Brackiella oedipodis and two from Neisseria meningitidis.

All three acrs inhibited NmCas9 cleavage both in vitro and in vivo, including in human cells.  The acr proteins are small – less than 14 kDa – and consequently were able to diffuse into the nucleus through the nuclear pores and nuclear localization signals were not required. While the acrs inhibited NmCas9 they did not inhibit Streptococcus pyogenes Cas9 (type II-C CRISPR system), nor were they toxic to cells.

The Neisseria meningitidis CRISPR-Cas9 system is a perfectly good gene editing system and, in fact, has the advantage over the S. pyogenes system of having a Cas9 that is significantly smaller than SpCas9 by hundreds of amino acids.  An advantage if trying to include Cas9 in viral vector systems with limited capacity to accommodate transgenes.

Cas9 + sgRNA

Cas9 + sgRNA

One of the acrsAcrIIC3Nme – was able to prevent DNA binding of dNmeCas9 (d = ‘dead’ or ‘disabled’).

There are likely many anti-CRISPR genes that remain to be found and these will enable Cas9 activities to be more tightly controlled both temporally and spatially, facilitating the development of Cas9-based technologies that satisfy the reasonable concerns about controlling and managing these system in particular contexts – such as in gene drive  and gene therapy systems.


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