Small Nucleic Acid Based Inhibitors of SpCas9 (SNuBs)

Jackson Champer, Ph.D. is a Postdoctoral Fellow at the Cornell University in Ithaca, NY where he is studying gene drive systems in Drosophila and mosquitoes using molecular biology, genetics and computational biology approaches. MORE ABOUT THE AUTHOR

Barkau et. al. 2019 report the development of small nucleic acid-based inhibitors (SNuBs) of SpCas9.  Due to considerations such as off-target activity, it is desirable that we have a high degree of control over CRISPR/Cas9 cleavage rates. However, while several mechanisms have been developed to activate Cas9 activity, little work has been done to develop ways of rapidly shutting down Cas9. This could be particularly important in human therapeutic applications where unintentional side effects of a treatment should be minimized. While protein inhibitors of Cas9 are available, they may be too large or costly for some applications, particularly in vivo.

Barkau et al. developed SNuBs of  two basic types, each of which bound to critical components, interfering with their function in Cas9-based cleavage. The first type was designed to bind the tracrRNA by direct homology, and the second type mimicked part of the tracrRNA to bind the protospacer adjacent motif (PAM)-binding domain of Cas9. However, the binding affinity of these initial designs was deemed insufficient to substantially inhibit Cas9 activity.

Inhibitors were modified oligonucleotides that bound the CRISPR RNA guide sequence (anti-guide) or repeat sequence (anti-tracr), or DNA oligonucleotides that bound the protospacer adjacent motif (PAM)-interaction domain (anti-PAM) of SpCas9.

To increase binding affinity, the authors combined both types of nucleic acid inhibitors with a flexible polyethylene glycol linker. This produced a high affinity for the Cas9/RNA target. To directly measure inhibition, the authors then measured cleavage rates of EGFP in vitro and when transfected into HEK 293T cells. The combined anti-PAM-tracr SNuB successfully produced substantial inhibition of Cas9 enzymatic activity, though results were more modest in the cell line. Versions of the construct with modified nucleotides had even greater binding affinity and inhibitory activity.

Image result for off target effectsThough not capable of completely silencing Cas9 in vivo, SNuBs were still effective in substantially reducing Cas9 cleavage rates. Further possible modifications of the nucleotide constructs may substantially increase inhibitory efficiency. They could thus potentially be used in therapeutic applications where fine control of Cas9 activity is necessary for successful treatment or to minimize adverse side effects. The authors also suggest that SNuBs could find uses in sensitizing bacteria for purposes of bacteriophage therapy, or even in the inhibition of gene drives that caused an undesired effect. Indeed, SNuBs designed for in vivo expression could potentially also increase the efficiency of a gene drive by limiting Cas9 activity during time windows when resistance alleles are liable to form. Overall, this study represents a significant step forward in our ability to fine-tune Cas9 activity in a flexible manner.

Barkau, C. L., O’Reilly, D., Rohilla, K. J., Damha, M. J., & Gagnon, K. T. (2019). Rationally Designed Anti-CRISPR Nucleic Acid Inhibitors of CRISPR-Cas9. Nucleic Acid Therapeutics, 29(3), 136–147. doi:10.1089/nat.2018.0758

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