2 min readA Crisper CRISPR: Safer Genome Modification

Hinxton, UK – Researchers from the Wellcome Trust Sanger Institute and the Model Animal Research Center of Nanjing University, China, have developed a safer way of genome editing. This method can be used on any organism and dramatically minimizes the problem caused by unplanned damage to other regions of the genome.

CRISPR is a molecular tool that edits the genomes of cells and organisms. The technology uses the DNA-cutting enzyme Cas9, with the help of a guide RNA sequence, to find and modify genetic targets.

Previously, researchers used a Cas9 endonuclease, an enzyme that cuts through both strands of DNA. Broken DNA strands can lead to new genetic mutations at the desired site but also at other related sites. In this study, the team found that by using a Cas9 enzyme that nicks a single strand of DNA, known as nickase, they can reduce unplanned, off-target DNA cuts elsewhere in the genome.

“The initial excitement around CRISPR technology has been tempered by recent studies showing significant damage at related sites of the genome,” says Dr. Bill Skarnes, senior author from the Wellcome Trust Sanger Institute. “We’ve solved this problem by using a form of Cas9 enzyme that doesn’t break DNA, except at the intended target. The improvement in specificity is remarkable.”

To determine the safest and most efficient option for DNA modification in mouse models, the team compared results with the Cas9 endonuclease, inducing double strand breaks on DNA, and the Cas9 nickase, inducing single strand nicks.

“The Cas9 endonuclease causes double strand breaks at related sites of the genome and these cannot be repaired – this is a real problem for CRISPR technology,” says Dr. Wensheng Zhang, co-first author from the Wellcome Trust Sanger Institute. “Because the Cas9 nickase breaks DNA only at target sites that are close together opposite strands of DNA, off-target nicks at related sequences are easily repaired.”

The team used the Cas9 nickase to make a genetic deletion on three genes in mice. Because they targeted two genes very close together – Rag1 and Rag2 – they were also able to successfully create a large deletion. The nickase successfully created cuts on both strands of DNA with no evidence of off-target damage elsewhere.

“This system greatly reduces the risk of off-target mutations in the genomes of mouse models and doesn’t seem to compromise the efficiency of the previous system,” says Dr. Xingxu Huang, senior author from Model Animal Research Center of Nanjing University, China. “It’s amazing to think that this strategy could potentially be applied to alter the genome of any organism.”

The team have also developed a freely available online resource that matches the correct genetic sequence with the gene you wish to modify through CRISPR technology.

Publication: Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Bin Shen, Wensheng Zhang, Jun Zhang, Jiankui Zhou, Jianying Wang, Li Chen, Wang, Alex Hodgkins, Vivek Iyer, Xingxu Huang, William C. Skarnes. Nature Methods (2014): http://www.nature.com/nmeth/journal/vaop/ncurrent/full/nmeth.2857.html

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