Research highlight

Gene therapy: CRISPR/Cas9 editing of single letters of the genetic code


April 21, 2016

An improved method for targeting and modifying single letters of the genetic code, without introducing random insertions and deletions into the genome, is reported in a paper published in Nature. The new ‘base editing’ method, which uses a modified CRISPR/Cas9 protein engineered to work with two other proteins, is more efficient at correcting single base mutations than existing methods, and has been used in cultured cells to successfully reverse single base mutations that are associated with diseases, including late-onset Alzheimer’s and breast cancer.

Most genetic diseases arise from mutations to a single nucleotide (point mutations). The CRISPR/Cas9 method currently widely used for genome editing involves cutting both strands of DNA, forming a double-stranded break in the target DNA sequence. However, when used to correct a single nucleotide, the standard CRISPR/Cas9 method is usually inefficient, and frequently introduces random insertions or deletions (collectively known as indels) at the target location, mainly as a result of the cellular response to a double-stranded DNA break.

To increase the efficiency of correcting point mutations while minimizing the frequency of indels, David Liu and colleagues modified the Cas9 protein so that it no longer cuts both strands of DNA, but can still bind to a target DNA sequence. By attaching a base modifying enzyme (APOBEC1) to Cas9, the authors were able to directly convert cytosine (C) to uracil (U), which forms base pairs as thymine (T). To cause the edited base pair to become permanent in cells, the authors used a third protein to manipulate normal cellular DNA repair processes, resulting in the conversion of a target C:G base pair to a T:A base pair. The authors show that their base editing system can be used to efficiently correct a variety of point mutations relevant to human disease in mouse and human cell lines, with minimal indel formation.

Two other studies published in Nature provide new information on the mechanism and structure of the Cpf1 enzyme, which can be used as an alternative to Cas9 in CRISPR-mediated genome editing. Emmanuelle Charpentier and colleagues show that Cpf1, unlike Cas9, can perform both the RNA processing and DNA cleavage activities required for targeted genome editing, possibly opening up new avenues for sequence-specific genome engineering and silencing. In a separate paper, Zhiwei Huang and colleagues report the crystal structure of Cpf1 protein bound to RNA, and describe how Cpf1 shape changes when bound.

doi: 10.1038/nature17946

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