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Evolving CRISPR/Cas Tools through Engineering Strategies

The Ben Kleinstiver lab is leveraging protein engineering strategies to improve the properties and activities of CRISPR/Cas enzymes allowing greater editing access. Their recent work has focused on Cas12a and Cas9 nuclease engineering, which resulted in the development of Cas variants with expanded targeting capabilities. Because the recognition of Protospacer Adjacent Motifs or PAM sequences located near target sites is a constraint for CRISPR/Cas genome editing applications, Kleinstiver and colleagues have engineered specific Cas sequence changes that significantly expand PAM preference.

Engineering CRISPR/Cas12a to expand targeting

As part of the Keith Joung lab in 2019, Kleinstiver and colleagues developed several Cas12a nuclease variants with expanded targeting capabilities (Kleinstiver et al. 2019). Cas12a orthologs (earlier identified as Cpf1) are DNases that recognize regions enriched with T residues, specifically PAMs with "TTTV" sequences where "V" may be A, C, or G residues.  To broaden the targeting potential of the commonly used Acidaminococcus sp. AsCas12a, Kleinstiver used a structure-guided approach to modify specific residues that may impart broader PAM access. Out of ten AsCas12a variants developed, they were able to identify two variants with significantly expanded targeting in human cells, able to efficiently edit sequences upon recognition of non-canonical PAMs. Specifically, a variant carrying three single amino acid substitutions (E174R/S542R/K548R), referred to as "enhanced AsCas12a", showed the greatest improvement in targeting capabilities. Of interest, these mutations also lead to improved editing efficiency of sequences with canonical PAMs. Additionally, Kleinstiver and colleagues showed that their "enhanced AsCas12a" demonstrated improved capabilities in other applications, such as multiplex and epigenetic editing. Lastly, the team introduced an additional mutation to reduce off-target events resulting in a higher fidelity nuclease, "enhanced AsCas12a" or enAsCas12a-HF1.

Differences between Cas9 and Cas12a: CRISPR/Cas9 and Cas12a are multidomain effector nucleases classified under the class II system based on their reliance on a single nuclease protein. Some key differences between Cas9 and Cas12a include: dependence on two RNA molecules (i.e., tracr and crRNA) by Cas9 but only one crRNA by Cas12a; two nuclease domains in Cas9 (i.e., RuvC and HNH) but only one in Cas12a (i.e., RuvC); DNA blunt end cuts by Cas9 while Cas12a cuts result in staggered ends; Cas9 requires host RNAse III for pre-crRNA processing while Cas12a has intrinsic RNAse activity; Cas9 recognizes 3' G-rich PAM sequences while Cas12a recognizes 5' T-rich sequences (Bijoya and Montoya, 2019). Adapted from "Novel CRISPR–Cas Systems: An Updated Review of the Current Achievements, Applications, and Future Research Perspectives," Nidhi et al. 2021. Figure 5 was modified by removing Cas13a diagram. https://creativecommons.org/licenses/by/4.0/

Expanding CRISPR/Cas9 reach

Continuing CRISPR/Cas engineering work at his lab at the Center for Genomic Medicine, Massachusetts General Hospital, Harvard Medical School, in 2020 Dr. Kleinstiver focused on expanding Cas9's sequence access. Applying the same principles of structure-guided engineering, specific mutations to the Streptococcus pyogenes Cas9 (SpCas9) nuclease sequence enabled the development of two variants with expanded PAM preference (Walton et al. 2020).

Briefly, by working from a variant previously generated at the Joung lab, SpCas9-VRQR (D1135V/R1335Q/T1337R/G1218R), additional changes were introduced within the PAM interacting domain to develop SpG, a new SpCas9 variant with an expanded preference for NGN PAMs (Kleinstiver et al. 2015). Additionally, mutagenesis of critical sites in the SpG(L1111R/A1322R) variant (i.e., A61R, N1317R, and R1333P) allowed the team to identify a second variant, SpRY, with the ability to target NRN (R=A/G) PAMs. Besides improving access to NGN and NAN PAM sequences, the new SpCas9 variants also supported the targeting of NCN and NTN PAMs. Finally, the team demonstrated the utility of SpG and SpRY for the correction of human disease genetic variations through cytosine base editing.

Therefore, by expanding the access to a broad range of PAM sequences limiting Cas targeting, the Kleinstiver lab is developing tools to facilitate correcting human disease associated mutations in almost any genomic site.

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1. Kleinstiver, B. P. et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature (2016) doi:10.1038/nature16526.

2. Kleinstiver, B. P. et al. Engineered CRISPR–Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Nat. Biotechnol. (2019) doi:10.1038/s41587-018-0011-0.

3. Nidhi, S. et al. Novel crispr–cas systems: An updated review of the current achievements, applications, and future research perspectives. International Journal of Molecular Sciences (2021) doi:10.3390/ijms22073327.

4. Paul, B. & Montoya, G. CRISPR-Cas12a: Functional overview and applications. Biomedical Journal (2020) doi:10.1016/j.bj.2019.10.005.

5. Walton, R. T., Christie, K. A., Whittaker, M. N. & Kleinstiver, B. P. Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. Science (80-. ). (2020) doi:10.1126/science.aba8853.

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