CRISPR-Cas9 has become even more accurate
Researchers at the University of California at Berkeley and Massachusetts General Hospital have identified a key region of the Cas9 protein that regulates the accuracy of the CRISPR-Cas9 system's interaction with the target DNA sequence. This allowed them to create an exceptionally accurate gene editor that provides the lowest level of erroneous DNA cuts to date.
The Cas9 protein (gray) is an RNA-guided nuclease that can be programmed to bind and cut any target DNA sequence (dark blue double helix), which makes it a powerful tool for genome editing. After binding to the target, the Cas9 protein domains undergo conformational rearrangements (the movements of individual amino acids are represented by colored stripes), providing activation of the Cas9-sgRNA complex for targeted DNA cutting. The REC3 domain (blue) is responsible for target recognition, which is a signal for the REC2 domain (lilac) to rotate outward, opening the passage to the HNH nuclease domain (yellow). This active Cas9 conformation has the ability to trigger the coordinated cutting of two target DNA strands.
The CRISPR-Cas9 system is currently used for targeted cutting of DNA molecules and their subsequent editing. Researchers are constantly working to improve the accuracy of this approach. One of the strategies of this search is to create mutations in REC3 – the main domain of the Cas9 protein – and evaluate their positive impact on the accuracy of the method without reducing the effectiveness with respect to the number of inaccurate incisions. Experiments have shown that even minor changes in the REC3 domain affect the quantitative ratio between accurate and inaccurate sections.
In their latest work, the authors used a technique known as single-molecular resonance fluorescence energy transfer to accurately study how different domains of the Cas9-RNA protein complex move when it binds to DNA.
This allowed them to establish that the REC3 domain is responsible for determining the accuracy of binding to the target, which is a signal for the REC2 domain to rotate outward, opening the passage to the HNH nuclease domain, activating the "scissors". This active Cas9 conformation has the ability to trigger the coordinated cutting of two target DNA strands.
After that, the researchers showed that by mutating different REC3 regions, it is possible to change the specificity of the Cas9 protein in such a way that the HNH nuclease is activated only when the guide RNA and a specific fragment of the target DNA are in close proximity. They managed to create an improved hyper-accurate Cas9, called HypaCas9. In human cells, the new version of the enzyme distinguishes the target regions of DNA from non-target regions somewhat better while maintaining the effectiveness of the effect on the target fragments.
The authors hope that studying the relationship between the structure, functions and dynamics of Cas9 will allow them to create even more sensitive and effective variations of the enzyme for introducing various modifications into DNA.
Article by Janice S. Chen et al. Enhanced proofreading governments CRISPR–Cas9 targeting accuracy http://www.nature.com/nature/journal/vaap/ncurrent/full/nature24268.html published in the journal Nature.
Evgeniya Ryabtseva
Portal "Eternal youth" http://vechnayamolodost.ru based on the materials of the University of California, Berkeley: Discovery helps engineer more accurate Cas9s for CRISPR editing.
27.09.2017