Challenges Of CRISPR-Cas Gene-Editing Tool

As there are ten-times the number of phages compared to bacteria, there is constant selective pressure on bacterium to develop a formidable arsenal of mechanisms to resist infection. These mechanisms in-turn exert selective pressure on the phages, forcing them to counter-evolve new ways to fight off against the bacteria. This is the ‘arms-race’ between phage and bacteria as they continuously coevolve in attempts to outcompete each other. Studies of this ongoing arms-race led to the discovery of the bacterial defense system: CRISPR-Cas9, which recognizes and targets phage DNA/RNA. Scientists have adapted this natural defense mechanism to become a powerful gene-editing tool. One of the problems associated with the CRISPR-Cas system was that after the intended cut was made, the system would look for similar sequences and shred up the genome due to off-target effects.

Scientists sought to use the natural phage response to fix this problem, which was to use anti-CRISPRs (Acrs). Acrs are protein inhibitors encoded in the phage genome that allow them to evade the CRISPR-Cas defence system1 In this issue of Nature Microbiology, Hynes et al. identify Acr that inhibits the activity of Streptococcus thermophilus Cas9 (stCas9) and Streptococcus pyogenes Cas9 (spCas9). This Acr is now known as: acrII5A, and is the second discovered Acr for spCas9, a popular Cas9 protein for gene editing. 1 This discovery is brought into light because of the arms-race between lactic acid bacteria and phage in cheese factories as companies are concerned with how it may drastically affect dairy product production.

Hynes et al. , as well as scientists from DuPont sought to discover new Acrs by screening phages for their ability to evade S. thermophilus DGCC7854’s Cas9. S. thermophilus DGCC7854 is one of the many strains of lactic acid bacterium used in dairy production that Dupont genetically modifies using CRISPR to create phage-resistant bacterium.

This screening process involved incubating the phages with S. thermophilus DGCC7854 to test if the phage were capable of surviving and led to the observation that one phage, D4276, survived and was thereby immune to S. thermophilus DGCC7854’s Cas9. 1 (Figure 1A) This resistance indicated that there was a property of the D4276 phage that modulated immunity against the CRISPR-Cas system and was thereby a candidate to harbour an Acr. To test this hypothesis, D4276 genes were investigated and expressed in the CRISPR-sensitive phage D5842, which meant that it was capable of being targeted and degraded by CRISPR-Cas9. This allowed Hynes et al. to determine that the gene: acrIIA5, encoded for the Acr that provided CRISPR immunity. When acrIIA5 was expressed, D5842 was able to survive against stCas9, strengthening the hypothesis of a newly discovered Acr as acrIIA5 was likely the cause of newly acquired immunity. Structural analysis of acrIIA5 revealed that it was 140 amino acids long, the typical length of Acrs being 50-150 amino acids, and was predicted to contain a distinctive coiled-coil motif that played a role in nucleic acid binding, much like other motifs in other Acrs.

Hynes et al. were also prompted to test acrIIA5’s ability to function against a variety of CRISPR-Cas systems as a broad-functioning Acr as acrIIA5 was a successful in inhibiting and restoring phage sensitivity S. thermophilus DGCC7710’s CRISPR system and S. thermophilus DGCC7854’s CRISPR system, whose Cas9 amino acid sequences differed by approximately 75%. Hynes et al. tested acrIIA5’s potential to function as a genome-editing off-switch against Streptococcus pyogenes’ CRISPR-Cas system (spCas9), a very popular Cas protein for gene editing to investigate whether this newly discovered Acr has potential for vast biotechnological purposes.

A spCas9 was adjusted for use in Lactococcus lactis, another lactic acid bacterium used by dairy companies. Using CRISPR-sensitive virulent phage p2 in the presence of acrIIA5, phage resistance was observed against spCas9 – proving that this newly discovered Acr can also inhibit spCas9. Hynes et al. ’s discovery is undoubtably not the last Acr that will be discovered as scientists continue to practice and advance gene therapy. The next step in the field of CRISPR technology would be to screen for more Acr proteins and determine the optimal Acr-CRISPR-Cas9 pairings to promote maximum efficacy in genome editing procedures.

As demonstrated by Hynes et al. when they unsuccessfully attempted to express acrIIA5 in E. coli, there are limitations to which Acr protein and CRISPR-Cas9 pairings that are capable of being expressed at the same time, hence the need to investigate the relationship between Acrs and CRISPR-Cas9 systems. Acrs have the potential to become the last component of the genome-editing process to function as the off-switch that stops CRISPR-Cas9 from functioning. This would provide scientists more control over the CRISPR-Cas9 system as Acrs greatly decrease the likelihood of off-target effects, improving CRISPR’s capabilities as a genome editing tool.