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

May 24, 2016Journal of genetics and genomics = Yi chuan xue bao

Can CRISPR-Cas9 Genome Editing Be Improved?

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Abstract

The CRISPR-Cas9 system has been shown to enable efficient DNA cleavage with well-designed synthetic guide RNAs.

The design of synthetic single guide RNAs (sgRNAs) has evolved from the original dual RNA guide systems.
Genome-wide assays have identified off-target effects, providing insights into the specificity of CRISPR-Cas9 cleavage.
On-target activity of thousands of guide RNAs has been assessed, leading to tools that aid in selecting effective guides.
Careful selection of guide RNAs based on computational predictions may maximize cleavage activity and minimize off-target effects.
Recent advancements in engineering Cas proteins have enhanced the precision and flexibility of CRISPR-Cas genome editing.
Newly characterized Cas9-like proteins from other bacteria could expand the applications of CRISPR technology.

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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.

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

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