The CRISPR Cas9 system was originally identified in bacteria as an adaptive immune response to defend itself against bacteriophage. The system was re-appropriated for use as gene-editing tool only a few years ago, and has had demonstrated success in bacteria, zebrafish embryos and mammalian cells.
CRISPR technology consists of the Cas9 RNA-guided nuclease along with the crRNA and a tracrRNA, which can be linked together to serve as the guideRNA (gRNA). The largest advantage of the CRISPR/Cas9 system over the TALENs and ZFNs is its ease in design. Unlike TALENs and ZFNs which rely on protein-DNA interactions for targeting, CRISPR/Cas9 relies on gRNA-DNA interactions for targeting. As such, the gRNA sequence can be easily manipulated in vitro to allow for rapid design to target a sequence of interest. The only limitation for gRNA design is that it must bind next to the protospacer
adjacent motif (PAM), which is typically a 5’-NGG, which can reduce the number of potential target sites.
Due to its ease of use and lower cost, the CRISPR/Cas9 system is rapidly becoming the prevalent system for genetic manipulation of cell and animal models. Numerous studies have demonstrated its efficacy in creating knockouts, deletions and mutations. Several studies have also utilized CRISPR/Cas9 technology with the piggyBac™ transposon system in a seamless manner. Overall, the CRISPR gene editing system has proven to be powerful.
Perhaps the largest drawback of the CRISPR/Cas9 system is the high “off-target” mutation rate. Numerous design algorithms have been developed to circumvent this obstacle, but without full-genome sequencing for numerous clones, the identification of a modified line with little to no non-specific mutations is largely unavoidable. This obstacle, however, has been largely overcome by advances in the CRISPR/Cas9 system and specifically the development of dimeric CRISPR RNA-guided FokI nucleases (RFNs, marketed as NextGEN™ CRISPR). In this system the dimerization-dependent FokI endonucleases are fused to an inactive Cas9 (dCas9), and two gRNAs are needed facilitate the binding of each dCas9-FokI monomer to their respective half sites. Upon binding of each monomer a catalytically-active dimer forms that creates the double-stranded break. Since two gRNAs are needed for the binding of their respective monomers, this greatly increases the specificity and reduces the “off-target” mutation rate. Therefore, this system provides all the advantages of the CRISPR/Cas9 and TALEN systems, and none of their drawbacks.
Singh et al. (2015) Gene Editing in Human Pluripotent Stem Cells: Choosing the Correct Path. J Stem Cell Regen Biol.
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