Genome Editing with CRISPR-Cas9: Can It Get Any Better?

Maximilian Haeussler; Jean-Paul Concordet

doi:10.1016/j.jgg.2016.04.008

Volume 43 Issue 5

May 2016

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Article Navigation > Journal of Genetics and Genomics > 2016 > 43(5): 239-250

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Genome Editing with CRISPR-Cas9: Can It Get Any Better?

doi: 10.1016/j.jgg.2016.04.008

Maximilian Haeussler^a,
Jean-Paul Concordet^b

a
Santa Cruz Genomics Institute, MS CBSE, 1156 High Street, University of California, Santa Cruz, CA 95064, USA
b
Laboratoire Structure et Instabilité des Génomes, Inserm U1154, CNRS UMR7196, Muséum national d'Histoire naturelle, 43 rue Cuvier, 75005 Paris, France

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Corresponding author: E-mail address: jean-paul.concordet@mnhn.fr (Jean-Paul Concordet)
Received Date: 2016-02-12
Accepted Date: 2016-04-23
Rev Recd Date: 2016-04-13

Available Online: 2016-04-24

Publish Date: 2016-05-20

Abstract

Abstract

The CRISPR-Cas revolution is taking place in virtually all fields of life sciences. Harnessing DNA cleavage with the CRISPR-Cas9 system of Streptococcus pyogenes has proven to be extraordinarily simple and efficient, relying only on the design of a synthetic single guide RNA (sgRNA) and its co-expression with Cas9. Here, we review the progress in the design of sgRNA from the original dual RNA guide for S. pyogenes and Staphylococcus aureus Cas9 (SpCas9 and SaCas9). New assays for genome-wide identification of off-targets have provided important insights into the issue of cleavage specificity in vivo. At the same time, the on-target activity of thousands of guides has been determined. These data have led to numerous online tools that facilitate the selection of guide RNAs in target sequences. It appears that for most basic research applications, cleavage activity can be maximized and off-targets minimized by carefully choosing guide RNAs based on computational predictions. Moreover, recent studies of Cas proteins have further improved the flexibility and precision of the CRISPR-Cas toolkit for genome editing. Inspired by the crystal structure of the complex of sgRNA-SpCas9 bound to target DNA, several variants of SpCas9 have recently been engineered, either with novel protospacer adjacent motifs (PAMs) or with drastically reduced off-targets. Novel Cas9 and Cas9-like proteins called Cpf1 have also been characterized from other bacteria and will benefit from the insights obtained from SpCas9. Genome editing with CRISPR-Cas9 may also progress with better understanding and control of cellular DNA repair pathways activated after Cas9-induced DNA cleavage.
- Genome editing,
- CRISPR-Cas9 system,
- Guide RNA

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References

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