The type I CRISPR protein Cas3 works like Pac-Man, chomping away at a continuous stream of nucleotides with intrinsic activity for introducing targeted large deletions from a few hundred base pairs to as large as 200 kb. However, without the molecular equivalent to the four colored ghosts who chase and capture Pac-Man, the broad and unidirectional genome editing activity of Cas3 is essentially unregulated.

Yan Zhang, PhD, assistant professor in the department of biological chemistry at the University of Michigan Medical School, and her collaborators at Cornell University identified two anti-CRISPR proteins that can “turn off” Cas3, paving the way toward safer and better-controlled CRISPR applications.

The research article, “Exploiting activation and inactivation mechanisms in type I-C CRISPR-Cas3 for genome-editing applications,” was published in Molecular Cell.

Anti-CRISPR proteins in biotechnological applications

Certain phages have evolved small inhibitor proteins known as anti-CRISPRs to inactivate bacteria’s use of CRISPR effectors for immune defense.

Researchers have used anti-CRISPR (Acrs) proteins that inactivate Cas9, a Type II CRISPR system, to change the timing, location, or tissue-specific spread of gene editing activity or lower the number of unwanted off-target events.

Acrs against the Cas9 nuclease have provided genetically encodable brakes to control gene editing in numerous application contexts. For instance, delayed introduction of AcrIIA4 after Cas9 RNP delivery can reduce off-target editing in human cells. Tissue-specific control of Acr expression reinforced safe in vivo Cas9 editing only in the target organs of mice. Acrs have also been exploited as safety controls for gene drives, enablers for biosensors and synthetic gene circuits, facilitators for phage therapies, and are used to capture ligands to quantify Cas9’s presence in vitro.

Though CRISPR-Cas9 can induce large genomic deletions by using two targeting RNAs, called single guide RNAs (sgRNAs), that flank the region to be deleted, Cas9 with two sgRNAs may also increase the chance of undesirable off-target mutations as well as unintended reverse mutations between the cutting sites.

Regulating Cas3-mediated genomic deletions

CRISPR-Cas3, a Type I CRISPR system, relies on a multi-subunit ribonucleoprotein (RNP) complex called the CRISPR-associated complex for anti-viral defense (Cascade) to facilitate DNA target searching and activation upon forming a complex with target DNA. Despite the growing list of type I CRISPR-enabled eukaryotic applications, no Acr off-switches have been developed to control these technologies.

By comparing and analyzing cryoelectron microscopy “snapshots” of several Cascade-Cas3 complexes, Zhang and colleagues revealed the binding and cleavage mechanisms in high resolution. With this structural evidence, they identified two highly efficient anti-CRISPR proteins, AcrIC8 and AcrlC9, which work through two slightly different mechanisms to block Cascade from engaging DNA targets. Importantly, in human genome editing experiments, both Acrs were shown to inhibit Cascade-Cas3-induced DNA deletion and gene activation mediated by Cascade fused with the P65 transcription activator domain.

Acrl8 and Acrl8 represent the first set of Acrs harnessed as off-switches for multi-subunit CRISPR gene editors, paving the way toward safer and better-controlled type I CRISPR applications. This sets the stage to improve type I editing outcomes through temporal, spatial, tissue-specific, and light- or drug-controlled Acr regulation. The dose-dependent response observed with AcrIC8 and AcrIC9 may allow tunable control beyond a simple on-off switch.

While Acr proteins carry great promises as off-switches to mitigate undesired editing by Cas effectors, Zhang and colleagues say that it is unlikely to find Acrs that are highly effective against a broad spectrum of CRISPR-Cas systems, as structural variations among Cas homologs may weaken the high-affinity interactions from Acr. Narrow-spectrum Acrs may allow control of one CRISPR variant without interfering with the others, whereas broad-spectrum Acrs can shield the entire organism from any leaky activity of all editing agents. It is, therefore, important to understand the cross-reactivity of each Acr against different CRISPR systems.