Last update: Sep 2020
One technique increasing the specificity of genome editing is using paired nickases. Currently, SpCas9 mutants are used for this purpose. Cas9 nickases have one of their nuclease domains inactivated, and thus can cleave only one DNA strand. When we use a pair of such nickases, we can reduce off-target editing.
For example, we can use D10A SpCas9 mutant, whose RuvC domain is inactivated, thus making the nickase cleave the target strand only. We can also use an H840A mutant, whose HNH domain is inactivated, and which can only cleave the non-target (PAM) strand. Using paired Cas9 nickases to make DSBs results in staggered cuts, as opposed to blunt cuts made by WT Cas9. For this application, we have to design 2 separate gRNAs targeting opposite DNA strands, which should be relatively close to each other. Using 2 gRNAs to target a region means that the sequence recognition requirement is doubled, which is a great strength of this technique.
When designing an experiment using Cas9 nickases, we must take the distance between the nicking sites and orientation of PAM sequences into consideration. PAM sequences can be located either flanking the target region (PAM-out) or in the middle of it (PAM-in). Researchers at IDT decided to investigate which orientation and distance between nicking sites yield better editing efficiency for knocking out a target gene. They used Human Embryonic Kidney cells (HEK293) in their experiments, generating indels using D10A and H840A with varying nick distances.
According to their experimental results (Fig.2), editing efficiency in HEK293 is way higher for the PAM-out configuration. Also, D10A has higher efficiency than H840A. For Cas9 D10A nickase, the optimum efficiency is achieved when the 2 nicking sites are 40–70 bp apart, while for Cas9 H840A, a distance of 50–70 bp yields better results. Another finding is the differing indel profiles of the two nickases- while D10A usually creates small deletions, H840A tends to produce large insertions.
Since D10A has higher editing efficiency than H840A, it is very common to use a pair of Cas9 D10A nickase mutants. For example, using SpyCas9 D10A is the preferred option in human cells. If the template is provided, they can be used to make insertion‐type genome edits too.
Since Cas12a possesses only RuvC domain that cleaves both strands, unlike Cas9 that has both RuvC and HNH domains that can be respectively inactivated to make single-strand breaks, Cas12a cannot be modified to create Cas12a nickase. In other words, so far, no Cas12a mutant that can make single-strand breaks was created. However, it is possible to deactivate Cas12a nuclease completely, making dCas12a, which can be used in CRISPRa and CRISPRi, as discussed in the next section.
Figure 1. Mary Gearing, (2018), Cleavage capabilities of Cas9 nickases https://blog.addgene.org/crispr-101-cas9-nickase-design-and-homology-directed-repair [Accessed 31 May 2020].
Figure 2. Mary Gearing, (2018), Optimizing nickase experimental design https://blog.addgene.org/crispr-101-cas9-nickase-design-and-homology-directed-repair [Accessed 31 May 2020].