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Making CRISPR-Targeted Genome Editing Safer

The CRISPR-Cas9 technology is a powerful system for genome editing. It works by making targeted modifications to DNA. Guided by short RNAs known as CRISPR RNA or crRNA, the Cas9 protein is targeted to a specific sequence that is complementary to the crRNA sequence. Cas9 is highly efficient at cleaving at its target site. However, off-target cleavage, that is, binding to and cutting sites that are not fully complementary to the guide RNA, is a major drawback of the system.

Recently, researchers at the Broad Institute of MIT and Harvard and the McGovern Institute of Brain Research at MIT modified Cas9 to reduce off-target cleavage and hence editing errors. In a paper published in Science, the scientists report that changing three amino acids in the Cas9 enzyme are sufficient to reduce off-target cleavage to undetectable levels. Called enhanced S. pyogenes Cas9 or eSpCas9, this newly-engineered Cas9 addresses many genome-editing safety concerns.

Using their detailed knowledge of the structure of the Cas9 protein, the researchers were able to improve the specificity of Cas9 by replacing some positively charged amino acids with neutral ones. Using targeted deep sequencing and unbiased whole-genome off-target analysis, they were able to monitor Cas9-mediated DNA cleavage in human cells and show that eSpCas9 reduced off-target effects and maintained specific on-target cleavage.

Source: Broad Institute of MIT and Harvard

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