Enhancing the CRISPR-Cas tool box

Date: 11th February 2019

CRISPRs are a favourite tool amongst synthetic biologists for gene editing. Discovered first Cas9 has been the enzyme of choice however, the less familiar Cas12a may provide some advantages such as retention of recognition sequence after repair, and reports of higher precision. However, Cas12a has an extended essential targeting component (PAM) which to date has been a hindrance to its wide spread use.

Keith Joung and colleagues have recently engineered a Cas12a variant to increase its targeting range, making previously inaccessible PAM regions available for cleavage. Furthermore, within this now extended PAM a two-fold increase in gene editing efficiency was also observed. However, the team did not stop there and subsequent engineering has allowed further optimisation of the Cas12a variant and as such has improved the efficiency of multiplex gene editing, endogenous gene activation and C-to-T base editing. Lending to its appeal this high-fidelity version of Cas12a also has reduced off-target effects.

In essence, this is likely to widen the applications and use of Cas12a for gene editing within the broader community. Indeed, this will be a valuable tool for the synthetic biologist which may filter through to the clinic. As the use of CRISPR applications are rapidly progressing a more stringent, active version is likely to provoke interest. With other Cas enzymes recently discovered (Cas12b and CasX) having a catalogue of Cas proteins to choose from is advantageous from a researcher point of view. Although, as each version has its own identity deciphering which one is best suited to different functional applications will be an important step. From an immunogenic view will this variety reflect differing immunogenic responses which can be harnessed for therapeutic use?

 

Kleinstiver, B. P., A. A. Sousa, R. T. Walton, Y. E. Tak, J. Y. Hsu, K. Clement, M. M. Welch, J. E. Horng, J. Malagon-Lopez, I. Scarfò, M. V. Maus, L. Pinello, M. J. Aryee and J. K. Joung (2019). “Engineered CRISPR–Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing.” Nature Biotechnology 37(3): 276-282.

https://doi.org/10.1038/s41587-018-0011-0

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