FokI fused catalytically inactive dead Cas9 or fCas9 (Part 30- CRISPR in gene editing and beyond)
Welcome to the 30th part of the multi-part series on applications of CRISPR in gene editing and beyond.
The second variant of SpCas9 is FokI fused catalytically inactive Cas9. The first variant Cas9 nickase has been discussed in Part 29. We have already discussed previously that in the case of other gene-editing techniques, Zinc Finger Nucleases and TALENS, the DNA binding domain is fused to the nuclease domain of the FokI enzyme. Also, the nuclease domain of the FokI enzyme requires dimerization for endonuclease activity on the target DNA (Fig 1a, b). Thus a FokI monomer is fused to each of the two Zinc finger and TALE proteins. This facilitates the zinc finger nuclease and TALENS to effectively bind to the longer recognition site within the desired DNA sequence of the target genome. Then dimerized FokI nucleases can cleave the target DNA and produce a double-stranded break. On the other hand, Cas9 is a monomeric nuclease that acts when coupled to its guide RNA to produce a double-stranded break at its target site (Fig 1c).
The dimeric and monomeric nature of nucleases are responsible for the off-target activity. Studies have shown that the likelihood of off-target binding events is considerably smaller when the dimeric nuclease is used. Like in the case of ZFNs and TALENs, the FokI nuclease only becomes active after dimerization, which means that both monomers need to bind to the target site simultaneously in order to create a double-stranded break. This dimerization is less likely to occur at off-target sites, as the chances of both monomers binding at an unintended location are lower.
On the other hand, monomeric nucleases like Cas9 can bind to off-target sites more easily than dimeric nucleases because they only require a single unit to bind and cut DNA. This increases the risk of off-target effects. Therefore, the dimeric nature of nucleases like FokI can result in enhanced specificity in genome editing applications.
With these principles in mind, researchers thought to use the FokI enzyme in the CRISPR-Cas system in order to reduce the off-target effects. For this, first, the SpCas9 is rendered catalytically inert by mutating both its nuclease domains, HNH and RuvC. The resulting mutated SpCas9 nuclease is called dead-Cas9, abbreviated as dCas9.
Though dCas9 loses its ability to cleave DNA, it retains its ability to bind to DNA based on gRNA specificity. Now, this dCas9 is fused to the Fok1 nuclease domain to generate the fCas9 enzyme. Like ZFNs and TALENs, the FokI domain in fCas9 only becomes active after dimerization, requiring two monomers to bind to the target site simultaneously. Therefore a FokI monomer is fused to each of the two dCas9 enzymes. Thus two pairs of gRNAs are required to guide the FokI fused dCas9 to bind to the target site of the genome and then cleave it. The cleavage activity of fCas9 depends on the simultaneous binding of two gRNAs to their target DNA with a spacer distance of 15–25 base pairs.
This approach doubled the sequence length required for target DNA recognition, and thus the chance of cleavage of off-targets can be significantly reduced. Though one monomer might still bind an off-target site, it cannot introduce a double-stranded break in it. Studies have reported that the fCas9 enzyme has more than 140-fold higher specificity than wild-type SpCas9 and with an efficiency similar to that of paired Cas9 nickases.
If you liked this article and want to know more about applications of CRISPR in gene editing and beyond, click the below links:
