Fine-tuning CRISPR to Create Popular Mouse Models
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CRISPR has built a tremendous amount of excitement in the scientific community since 2013. Though it can be used to create simple gene-disrupted animal models, it is extremely challenging to use it to insert foreign cassettes into genomes to create knock-ins or more complex models such as conditional knockouts.
A team headed by Dr. Channabasavaiah B Gurumurthy (Guru) at the University of Nebraska Medical Center, Omaha, U.S.A., in collaboration with Dr. Masato Ohtsuka, Tokai University, Japan have developed a method they call Easi-CRISPR.
This new technique revolutionizes the speed at which, much-needed, mutant mouse models are created for biomedical research. This work was published in Genome Biology journal on May 17, 2017.
The Easi-CRISPR method employs long single stranded DNAs as donor cassettes for gene editing via CRISPR, unlike the typically very inefficient double stranded DNA donors commonly used by the scientific community. In addition, the ssDNA donors are combined with newer platforms of CRISPR guide RNAs (that constitute separated crRNA and tracrRNA) and Cas9 protein, together called ctRNP.
During the previous 4 years, many scientists have tried to use CRISPR to create knock-in models, that relied on homology-directed repair (HDR), but many were unsuccessful as their methods were not able to shift the balance from NHEJ to HDR for it to work efficiently. A recent Science Magazine news article captured the frustration of the research community about the limitations of the previously used CRISPR methods.
Guru’s and Masato’s labs first observed the robustness of ssDNA donors for HDR, in their Easi-CRISPR platform, in the summer of 2016. They posted their preliminary results on the preprint server biorXiv, started presenting their data at several conferences so that their method can immediately be available to the scientific community, before their manuscript was peer-reviewed and published in a journal.
Guru said “Several independent labs have already been able to use Easi-CRISPR for other genes, thanks to its early online posting on bioRxiv”.He added, “Hundreds of labs are interested in using the technology — we posted another bioRxiv article on this work today that describes detailed step-by-step protocols of Easi-CRISPR, which should help the community further.”