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Chemical engineering of CRISPR–Cas systems for therapeutic application

Nature Reviews Drug Discovery volume 24, pages 209–230 (2025)Cite this article

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Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) technology has transformed molecular biology and the future of gene-targeted therapeutics. CRISPR systems comprise a CRISPR-associated (Cas) endonuclease and a guide RNA (gRNA) that can be programmed to guide sequence-specific binding, cleavage, or modification of complementary DNA or RNA. However, the application of CRISPR-based therapeutics is challenged by factors such as molecular size, prokaryotic or phage origins, and an essential gRNA cofactor requirement, which impact efficacy, delivery and safety. This Review focuses on chemical modification and engineering approaches for gRNAs to enhance or enable CRISPR-based therapeutics, emphasizing Cas9 and Cas12a as therapeutic paradigms. Issues that chemically modified gRNAs seek to address, including drug delivery, physiological stability, editing efficiency and off-target effects, as well as challenges that remain, are discussed.

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Fig. 1: Structural organization of the natural CRISPR–Cas9 and CRISPR–Cas12a complexes.
Fig. 2: Representative 2′-OH contacts between guide RNA and Cas proteins.
Fig. 3: General mechanism of double-stranded DNA cleavage and repair by natural CRISPR–Cas9 and CRISPR–Cas12a systems.
Fig. 4: Chemical modifications that link the crRNA and tracrRNA together to form a sgRNA in the CRISPR–Cas9 system.
Fig. 5: Chemical modifications used in CRISPR–Cas systems.
Fig. 6: Delivery methods for CRISPR–Cas systems.
Fig. 7: Engineered CRISPR–Cas-based editing without double-strand breaks.

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Acknowledgements

We acknowledge funding from the National Institutes of Health (1R01GM135646-01) to K.T.G. and the National Science and Engineering Council of Canada (NSERC) Discovery Grant #2022-03372 to M.J.D. Additionally, H.M.B. and M.J.D. are members of the Centre de recherche en biologie structurale, funded by Fonds de Recherche du Québec (Health Sector) Research Centres Grant #288558 and H.M.B. received graduate funding support from Fonds de Recherche du Québec - Nature et Technologies doctoral scholarship #332295 and the NSERC CREATE Programmed Molecules for Therapeutics, Sensing, and Diagnostics (PROMOTE) training program.

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Author notes

  1. These authors contributed equally: Halle M. Barber, Adrian A. Pater.

Authors and Affiliations

  1. Department of Chemistry, McGill University, Montreal, Quebec, Canada

    Halle M. Barber & Masad J. Damha

  2. Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, NC, USA

    Adrian A. Pater & Keith T. Gagnon

  3. Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX, USA

    Daniel O’Reilly

  4. Sealy Institute for Drug Discovery, University of Texas Medical Branch, Galveston, TX, USA

    Daniel O’Reilly

Authors
  1. Halle M. Barber
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  2. Adrian A. Pater
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  3. Keith T. Gagnon
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  4. Masad J. Damha
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All authors contributed to researching the literature, discussing the article’s content, preparing figures, and writing and/or substantial editing.

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Correspondence to Keith T. Gagnon, Masad J. Damha or Daniel O’Reilly.

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Glossary

2′-OH contacts

Hydrogen bonding interactions between the 2′-hydroxyl (2′-OH) groups of the gRNA nucleotides and the Cas amino acids, as well as between nucleotides.

Base editors

(BEs). Systems consisting of a dCas9 fused to either a cytidine deaminase, for cytosine base editors, or an adenosine deaminase, for adenine base editors, which allow for the precise conversion of specific DNA bases (such as, C to T or A to G) without double-strand breaks.

Chemical modification

Either a naturally occurring or synthetic modification to the phosphate, sugar or nucleobase of nucleotides.

Click chemistry

A biocompatible chemical reaction that allows for efficient and specific joining of two biomolecules.

CRISPR RNA

(crRNA). An RNA molecule composed of a spacer-derived guide region and a repeat-derived trans-activating CRISPR RNA-pairing or 5′ handle region.

dCas9

A Cas9 protein with mutations in both nuclease domains (RuvC and HNH), resulting in a catalytically inactive enzyme still capable of binding to DNA via guide RNA.

Double-strand breaks

(DSBs). DNA damage types in which both strands of the DNA double helix are cleaved, forming either a blunt or staggered break.

Ex vivo gene editing

A therapeutic approach in which patient cells are isolated, edited outside of the body and subsequently reintroduced back into the patient.

Guide RNA

(gRNA). A generalized term for an RNA molecule that directs the CRISPR–Cas effector enzyme to a specific DNA sequence through complementary base pairing.

Homology-directed repair

(HDR) A precise DNA repair pathway guided by a homologous DNA template to repair double-strand breaks.

In vivo gene editing

A therapeutic approach in which the gene editing components are delivered directly into a patient via local or systemic delivery.

Lipid nanoparticle

(LNP). A vesicle composed of lipid moieties used to encapsulate nucleic acids or proteins and deliver these therapeutic agents into cells.

nCas9

A Cas9 protein with either the RuvC or HNH catalytic domain mutated, resulting in a Cas9 that can introduce a single-strand break (that is, a nick) into DNA by cleaving only one DNA strand.

Non-homologous end joining

(NHEJ). A DNA repair pathway in which double-strand breaks are repaired by directly joining the ends, often resulting in insertions or deletions at the site.

Non-target strand

The DNA strand opposite the target strand that contains the protospacer adjacent motif and remains unbound during CRISPR–Cas-mediated cleavage.

Off-target effects

Unintended cleavages or modifications of DNA at sites other than the intended target sequence.

Prime editing guide RNA

(pegRNA). An RNA molecule used in prime editing that combines the properties of a gRNA, a reverse transcriptase template and a template sequence encoding the desired edit to direct the fusion protein to the target DNA site.

Prime editors

Systems consisting of an nCas9 fused to a reverse transcriptase enzyme and guided by a pegRNA to enable precise edits without double-strand breaks.

Protospacer adjacent motif

(PAM). A short, specific DNA sequence adjacent to the target site that is essential for the recognition and binding of the Cas enzyme.

Ribonucleoprotein (RNP) complex

A complex composed of both RNA and protein.

R-loop

A nucleic acid structure that forms when an RNA strand binds to one strand of a DNA double helix, creating an RNA:DNA hybrid duplex and displacing the other DNA strand.

Seed region

The first 5–10 nucleotides directly adjacent to the PAM that initiate base pairing between the gRNA and the target strand.

Single-guide RNA

(sgRNA). An RNA molecule created by fusing the Cas9 crRNA and trans-activating CRISPR RNA into a single construct.

Sugar pucker conformations

Conformations of the ribose ring in a nucleotide, with C2′-endo corresponding to DNA-like sugars with B-form helical structures and C3′-endo corresponding to RNA-like sugars with A-form helical structures.

Target strand

The DNA strand that is complementary to the gRNA.

Trans-activating CRISPR RNA

(tracrRNA). An RNA molecule that hybridizes to the crRNA and anchors the gRNA construct to Cas9 through its stem loop structures.

Transcriptional activation

(CRISPRa). A technique using dCas9 fused to transcriptional activation domains and directed by a gRNA to specific genomic loci to upregulate gene expression.

Transcriptional interference

(CRISPRi). A method using dCas9 fused to repressor domains to repress gene expression when guided to specific genomic loci by a gRNA.

Viral vectors

Modified viruses used to deliver nucleic acids into cells for therapeutic purposes.

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Barber, H.M., Pater, A.A., Gagnon, K.T. et al. Chemical engineering of CRISPR–Cas systems for therapeutic application. Nat Rev Drug Discov 24, 209–230 (2025). https://doi.org/10.1038/s41573-024-01086-0

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