An Arms Race Can Make the CRISPR Toolkit Even Better

Approximately half of all studied bacteria and 90% of archaea use CRISPR-Cas systems to defend themselves from phages by recognising and eliminating hostile mobile genetic elements. Although this defence is effective and can ensure bacterial survival, it is a complex piece of machinery involving many genes and proteins, and it comes with a substantial metabolic burden.

To reduce the costs, prokaryotes have evolved multiple mechanisms to control CRISPR-Cas gene expression so the defence can be shut down or activated according to needs. On the other hand, phages hit back and try to counteract the defence via a diverse set of anti-CRISPR proteins (Acrs) that inhibit CRISPR-Cas activity and thus help the phage to succeed in attacking bacteria.

»Mechanistically understanding how CRISPR is regulated can reveal the strengths and weaknesses of CRISPR-Cas systems. This could help us to precisely control the timing and location of CRISPR-Cas function and improve CRISPR as a genome-editing tool,« says Haridha Shivram.

CRISPR-Cas Systems Are Highly Regulated

Shivram earned his PhD in molecular biology and bioinformatics at The University of Texas at Austin before starting as a postdoc in Jennifer Doudna’s lab at the University of California, Berkeley in 2019. His current research focus is to discover novel regulators of CRISPR-Cas systems, and he is the first author of a recent review in Nature Chemical Biology that discusses the mechanisms of natural CRISPR–Cas regulatory biomolecules and the potential applications of these regulators.

CRISPR-Cas systems are divided into two classes, six types and 19 subtypes based on whether foreign nucleic acids are degraded by a single large Cas-protein or by a complex of multiple proteins, and on unique, signature proteins used almost exclusively in that subtype. For example, the well-known Cas9-system belongs to class II since it depends on a single, large nuclease. The Cas9-containing type II CRISPR-Cas system can be further divided into three subsystems (A-C) depending on their employment of the ring-shaped DNA-binding protein Csn2 or the endonuclease Cas4.

A number of transcription factors and RNA regulatory mechanisms confer transcriptional and post-transcriptional control over the CRISPR-Cas system in each of these types and subtypes. The presence or absence of viral infection can up or down regulate the defence mechanism, and bacteria can also adapt their expression of CRISPR genes to external stimuli. Through a cascade of regulatory proteins, limiting glucose and iron levels can promote CRISPR-Cas gene expression and thus adapt expression to metabolic stress induced by phage infection.

CRISPR Writes the History of a Cell Into Its DNA

»The activity and existence of CRISPR-Cas systems could vary based on the environment, nutrient availability and the likelihood of bacteriophage onslaught. In the absence of infection, it is likely that the CRISPR-Cas system may not be critical for survival and the host might shut off CRISPR activity,« says Shivram.

»Anti-CRISPR proteins are a major example of the adaptations and counter adaptations that affect the CRISPR war. Phages have evolved these proteins that specifically inhibit CRISPR-Cas systems from their host bacterial species. It is possible that in order to escape these anti-CRISPR proteins, bacteria, in turn, have evolved variants of CRISPR-Cas systems,« explains Shivram.

Acrs have been exploited experimentally to reduce off-target editing by reducing the time available for Cas nuclease activity and increase tissue-specific editing by controlling their presence in various tissues. Moreover, Acrs have been used to interrupt gene drives and thus to avoid unwanted ecological consequences.

Regulation Opens up for Countless Possibilities

Shivram believes that engineered regulation of these control mechanisms will be the best way to exploit them in the lab or clinic. He says:

»Domains fused to the effector proteins directly or to their transcriptional components would allow us to control the intensity of CRISPR activity and its biological location.«

This approach is already being exploited with dead Cas9 fused to base editors and transcriptional regulators, and similar methods are also used in natural systems. For example, as the review points out, an accessory protein, RspWYL1, is believed to bind to Cas13d and function as a sponge that attracts multiple ssRNAs for cleavage by the nuclease.