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  • Review Article
  • Published:

What rheumatologists need to know about CRISPR/Cas9

Nature Reviews Rheumatology volume 13, pages 205–216 (2017)Cite this article

Subjects

Key Points

  • Advances in CRISPR technology have provided the capacity to precisely identify and define the function of genes and noncoding regulatory elements associated with disease development and susceptibility

  • CRISPR technology has made the generation of mouse models of disease much quicker and less expensive than traditional approaches, and has facilitated the development of much-needed larger animal models of disease

  • CRISPR technology has enabled the generation of gene drives, whereby genetic changes propagate rapidly through a species, providing the potential to eliminate disease vectors and thus vector-borne diseases such as malaria

  • Successful treatment of mouse models of human diseases suggests that CRISPR technology can be applied to treat human diseases in the future

  • CRISPR technology has the ability to facilitate a breakthrough in our understanding of the more common and complex human diseases, including rheumatic diseases

  • The potential of CRISPR/Cas9 technology in the development of new treatment strategies is confidently expected to have a major effect on the practice of rheumatology

Abstract

CRISPR/Cas9 genome editing technology has taken the research world by storm since its use in eukaryotes was first proposed in 2012. Publications describing advances in technology and new applications have continued at an unrelenting pace since that time. In this Review, we discuss the application of CRISPR/Cas9 for creating gene mutations — the application that initiated the current avalanche of interest — and new developments that have largely answered initial concerns about its specificity and ability to introduce new gene sequences. We discuss the new, diverse and rapidly growing adaptations of the CRISPR/Cas9 technique that enable activation, repression, multiplexing and gene screening. These developments have enabled researchers to create sophisticated tools for dissecting the function and inter-relatedness of genes, as well as noncoding regions of the genome, and to identify gene networks and noncoding regions that promote disease or confer disease susceptibility. These approaches are beginning to be used to interrogate complex and multilayered biological systems and to produce complex animal models of disease. CRISPR/Cas9 technology has enabled the application of new therapeutic approaches to treating disease in animal models, some of which are beginning to be seen in the first human clinical trials. We discuss the direct application of these techniques to rheumatic diseases, which are currently limited but are sure to increase rapidly in the near future.

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Figure 1: Adaptive immune system of bacteria and archaea.
Figure 2: Cas9 targeting using crRNA (CRISPR RNA)–tracrRNA (trans activating crRNA) or a single guide RNA chimera.
Figure 3: Endogenous repair of double-strand DNA breaks by non-homologous end joining (NHEJ) or homology directed repair (HDR).
Figure 4: Engineering nuclease-deactivated Cas9 (dCas9) for gene activation and repression.

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Authors and Affiliations

  1. Henry Ford Hospital, Bone and Joint Center, 6135 Woodward Avenue, Detroit, 48202, Michigan, USA

    Gary J. Gibson & Maozhou Yang

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  1. Gary J. Gibson
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G.J.G. contributed to researching data for the article, writing, and reviewing and editing the manuscript before submission. M.Y. contributed to discussion of content and writing the article.

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Correspondence to Gary J. Gibson.

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PowerPoint slides

Glossary

CRISPR

Segments of prokaryotic DNA containing short repetitions of DNA sequences, which are interrupted by so-called spacer DNA derived from past invaders. CRISPR serves as the bacterial adaptive immune system that protects against invading genetic materials.

CRISPR/Cas9

RNA-guided gene editing platform based on the Cas9–gRNA ribonucleoprotein complex. The two-component complex can mediate gRNA-programmed recognition of specific DNA sequences and create a site-specific double-strand cleavage of the targeted DNA.

Cas9

An RNA-guided DNA endonuclease enzyme that is the universal component of the RNA-guided CRISPR/Cas9 gene editing machinery. Cas9 by itself is inactive; upon binding to the gRNA scaffold, Cas9 goes through conformational changes that initiate its target recognition, binding and cleavage activity.

Spacer

The spacer sequence refers to the 5′ end, ∼20 nucleotide variable sequence of the targeting gRNA construct. The spacer contains a targeting sequence that matches a region of DNA substrate and guides Cas9 nuclease activity.

Protospacer

The protospacer sequence refers to the targeted site on the DNA substrate. The nucleotide sequences of the spacer and the corresponding protospacer are identical.

Protospacer adjacent motif (PAM)

A three base pair DNA sequence immediately following the protospacer or the DNA sequence targeted by the Cas9/gRNA ribonuclease. The canonical PAM sequence for CRISPR/Cas9 gene editing machinery is 5′-NGG-3′.

Guide RNA (gRNA)

gRNA, also known as short guide RNA (sgRNA) is a short synthetic RNA sequence consisting of a scaffold structure and a programmable ∼20 nucleotide spacer at the 5′ end. The ∼80 nucelotide RNA scaffold structure is essential for mediating both Cas9 protein binding and activation. The unique spacer sequence dictates the DNA target site to be recognized and cleaved by Cas9 protein.

Non-homologous end joining (NHEJ)

A cellular pathway that repairs double-strand breaks in DNA. NHEJ is active throughout the cell cycle and requires no repair template. NHEJ is frequently imprecise and the repair process can generate an open reading frame shift with insertions, deletions or mutations at the site of double-strand breaks. The inaccurate nature of the NHEJ repair process forms the basis of the CRISPR/Cas knockout strategy.

Homology-directed repair (HDR)

The HDR pathway (also known as homologous recombination), involving a homologous template (either a sister chromatid or an exogenous DNA template), repairs double-strand DNA breaks accurately according to the template. The template or donor DNA consists of left and right arms identical to sequences flanking the double-strand break. Between the arms, any DNA sequence or marker can be inserted and HDR will force the additional genetic material to be knocked in to the particular locus. HDR is usually believed to be active only during S and G2 phases of the cell cycle.

CRISPR/Cas9 genomic screen

CRISPR/Cas9-mediated genome-wide knockout screen system. In this platform, a gRNA library targeting most genes in the genome with multiple sites per gene is cloned into lentiviral vectors and delivered as a pool into target cells that express Cas9. A low multiplicity of infection is used to ensure that each cell will receive no more than one gRNA or viral particle. By proper phenotype selection, gRNAs that are enriched or depleted in cells are determined and, correspondingly, genes that are required for that particular phenotype can be systematically identified.

Gene drives

Gene drives are genetic manipulations that enable a gene to force its inheritance to all, rather than half, of its offspring.

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Gibson, G., Yang, M. What rheumatologists need to know about CRISPR/Cas9. Nat Rev Rheumatol 13, 205–216 (2017). https://doi.org/10.1038/nrrheum.2017.6

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