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Advances in CRISPR therapeutics

Nature Reviews Nephrology volume 19, pages 9–22 (2023)Cite this article

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Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR) renaissance was catalysed by the discovery that RNA-guided prokaryotic CRISPR-associated (Cas) proteins can create targeted double-strand breaks in mammalian genomes. This finding led to the development of CRISPR systems that harness natural DNA repair mechanisms to repair deficient genes more easily and precisely than ever before. CRISPR has been used to knock out harmful mutant genes and to fix errors in coding sequences to rescue disease phenotypes in preclinical studies and in several clinical trials. However, most genetic disorders result from combinations of mutations, deletions and duplications in the coding and non-coding regions of the genome and therefore require sophisticated genome engineering strategies beyond simple gene knockout. To overcome this limitation, the toolbox of natural and engineered CRISPR–Cas systems has been dramatically expanded to include diverse tools that function in human cells for precise genome editing and epigenome engineering. The application of CRISPR technology to edit the non-coding genome, modulate gene regulation, make precise genetic changes and target infectious diseases has the potential to lead to curative therapies for many previously untreatable diseases.

Key points

  • CRISPR systems are RNA-guided ribonucleoproteins that function as both sequence-specific nucleic acid-targeting proteins and nucleases; these systems are being developed as therapies for simple Mendelian disorders.

  • Novel approaches using newly discovered CRISPR systems and Cas protein engineering have expanded the available genome engineering toolbox, enabling the development of potentially curative therapies for diseases with complex drivers.

  • Targeting and altering the non-coding genome with CRISPR could potentially ameliorate disease by changing the transcription or translation of target genes.

  • The use of CRISPR systems with nuclease-dead Cas proteins fused to transcriptional or epigenetic modulators enables targeted gene regulation without inducing DNA damage or altering the genetic code.

  • CRISPR base editors and prime editors can be used to create precise genome edits such as therapeutic mutations, insertions or deletions that are difficult to achieve using wild-type CRISPR–Cas nucleases.

  • In addition to genome engineering, the CRISPR toolbox could potentially be used to prevent and treat infectious diseases.

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Fig. 1: The CRISPR–Cas9 system.
Fig. 2: Natural Cas systems.
Fig. 3: Genome engineering using engineered Cas proteins.
Fig. 4: Editing non-coding regions of the genome.

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Acknowledgements

M.C. acknowledges support from the National Science Foundation Graduate Research Fellowship Program, the ARCS Foundation Scholarship, the Ruth L. Kirschstein National Research Service Awards for Individual Predoctoral Fellowship (F31AI164936), Siebel Scholar award, and Stanford Maternal and Child Health Research Institute (MCHRI). X.C. acknowledges support from the Stanford Bio-X SIGF Fellowship Program. P.B.F. acknowledges support from California Institute of Regenerative Medicine (CIRM). L.S.Q. acknowledges support from National Science Foundation CAREER award (Award #2046650), NIH (Grant # 1U01DK127405, 1R01CA266470, 1R21CA270609), Stanford MCHRI through the Uytengsu-Hamilton 22q11 Neuropsychiatry Research Award Program, and CIRM (DISC2-12669). L.S.Q. is a Chan Zuckerberg Biohub investigator.

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  1. These authors contributed equally: Michael Chavez, Xinyi Chen.

Authors and Affiliations

  1. Department of Bioengineering, Stanford University, Stanford, CA, USA

    Michael Chavez, Xinyi Chen, Paul B. Finn & Lei S. Qi

  2. Sarafan ChEM-H, Stanford University, Stanford, CA, USA

    Lei S. Qi

  3. Chan Zuckerberg Biohub, San Francisco, CA, USA

    Lei S. Qi

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L.S.Q., M.C. and X.C. researched the data for the article and wrote the text. All authors made substantial contributions to discussion of the content and reviewed and/or edited the manuscript before submission.

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Correspondence to Lei S. Qi.

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L.S.Q. is a founder and scientific adviser of Epic Bio, and a scientific adviser of Laboratory of Genomics Research (LGR). The other authors declare no competing interests.

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Nature Reviews Nephrology thanks Aron Geurts and Susanna Tomasoni, who co-reviewed with Piera Trionfini, for their contribution to the peer review of this work.

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Glossary

Trans-activating CRISPR RNA

(tracrRNA). RNA that partially base pairs with a CRISPR RNA (crRNA) to form a crRNA–tracrRNA hybrid that binds to and acts as a guide for Cas protein to cleave the targeted DNA sequence.

Non-homologous end joining

(NHEJ). A pathway that repairs DNA double-strand breaks by ligating the break ends without the need for a homologous template.

Homology-directed repair

(HDR). A pathway that repairs DNA double-strand breaks guided by a homologous DNA template.

Mendelian diseases

Genetic disorders caused by mutation in single genes.

Zinc finger nucleases

(ZFNs). Artificial endonucleases that can target and alter specific DNA sequences in the genome. They are generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain.

Transcription activator-like effector nucleases

(TALENs). Artificial endonucleases that can target and alter specific DNA sequences in the genome. They are generated by fusing a TAL effector DNA-binding domain to a DNA-cleavage domain.

Logic-gated gene regulation

A process used to control the timing and intensity of gene expression by performing a Boolean operation on multiple biological signals and executing a functional response when appropriate.

Off-target effects

Effects that can occur when CRISPR molecules bind to and alter genomic sites that they were not intended to target, usually as a result of the presence of a similar sequence to the desired target site.

Complementary DNA

(cDNA). DNA synthesized from a single-stranded RNA template that is usually used to express a protein in a cell that does not normally express the protein.

MicroRNAs

(miRNAs). A class of single-stranded non-coding RNAs that can silence RNA and regulate gene expression by base pairing with complementary sequences in mRNA molecules.

Long non-coding RNAs

(lncRNAs). A class of RNAs longer than 200 nucleotides that are not translated into protein and have roles in regulation of gene expression.

Inteins

Protein segments that are capable of excising themselves and joining the flanking protein portions together through a peptide bond during protein splicing.

Anti-CRISPRs

Proteins found in phages that inactivate CRISPR systems, enabling control over CRISPR tools.

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Chavez, M., Chen, X., Finn, P.B. et al. Advances in CRISPR therapeutics. Nat Rev Nephrol 19, 9–22 (2023). https://doi.org/10.1038/s41581-022-00636-2

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