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Review
. 2021 Jul:189:114492.
doi: 10.1016/j.bcp.2021.114492. Epub 2021 Feb 27.

Small nucleic acids and the path to the clinic for anti-CRISPR

Christopher L Barkau  1 , Daniel O'Reilly  2 , Seth B Eddington  1 , Masad J Damha  3 , Keith T Gagnon  4
Affiliations
Review

Small nucleic acids and the path to the clinic for anti-CRISPR

Christopher L Barkau et al. Biochem Pharmacol. 2021 Jul.

Abstract

CRISPR-based therapeutics have entered clinical trials but no methods to inhibit Cas enzymes have been demonstrated in a clinical setting. The ability to inhibit CRISPR-based gene editing or gene targeting drugs should be considered a critical step in establishing safety standards for many CRISPR-Cas therapeutics. Inhibitors can act as a failsafe or as an adjuvant to reduce off-target effects in patients. In this review we discuss the need for clinical inhibition of CRISPR-Cas systems and three existing inhibitor technologies: anti-CRISPR (Acr) proteins, small molecule Cas inhibitors, and small nucleic acid-based CRISPR inhibitors, CRISPR SNuBs. Due to their unique properties and the recent successes of other nucleic acid-based therapeutics, CRISPR SNuBs appear poised for clinical application in the near-term.

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Figures

Figure 1.
Figure 1.
Comparison of anti-CRISPR technologies. Characteristics noted are based on published or implied properties and predicted delivery methods. Kd value for CRISPR SNuB [Anti1_PAM-tracr(FL), reported in Barkau et al., 2019] is unpublished.
Figure 2.
Figure 2.
Mechanism of Three Anti-CRISPR Technologies. An Acr protein, AcrIIA4, and small molecule, BRD0539, bind the PI domain of SpCas9, preventing PAM recognition and target binding. Different Acr proteins exhibit a diversity of mechanisms, but each usually has only one target in the Cas9 RNP. A CRISPR SNuB (Anti-PAM_tracr FL) exhibits a dual mechanism. The SNuB binds a sgRNA, obstructing proper assembly of the Cas9 RNP, and provides a DNA hairpin containing an NGG motif in close proximity to the PI domain as a substrate analog.
Figure 3.
Figure 3.
Methods of Anti-CRISPR Delivery. Small molecules can be taken orally and are cell permeable. Acr proteins must be encoded in an AAV vector or packaged in lipid nanoparticles (LNPs) and delivered by injection. Oligonucleotides (SNuBs) can be injected directly or via LNP and exhibit uptake and distribution patterns modulated by chemical modifications and conjugates.
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