Effective isolation of targeted mutations generated by CRISPR/Cas9 requires not only reasonable editing efficiency, but also an easy method to screen for the mutations. Editing events generated by CRISPR/Cas9 are normally identified by restriction enzyme digestion of PCR fragments or by in vitro digestion using purified Cas9 protein. Both methods are time-consuming and laborious. Simplified screening methods are urgently needed.
Researchers at UC San Diego have developed an effective strategy to reliably isolate Cas9-free T2 plants that contain stably heritable mutations in Arabidopsis. The technology uses a cassette that enables the expression of a visual marker (e.g., luminescent or fluorescent marker) gene under the control of a strong promoter inserted into the CRISPR/Cas9 vector. The visual marker cassette allows one to visually select Cas9-free plants at T2 generation.
Crop improvement through genome editing. This invention allows a very fast and efficient identification of Cas9-free plants with the desired genome modifications. This is especially important for crop improvement using CRISPR/cas9 genome editing technology because of public concern over transgenes.
The current technology is much faster and less laborious compared to traditional PCR based methods. The method is suitable for use across a broad range of plant species.
Arabidopsis was used as a model to generate targeted mutations and demonstrate that the technology could successfully identify that the mutations were stably transmitted.
A provisional patent has been submitted and the technology is available for licensing.
|United States Of America||Published Application||0048646-A1||02/13/2020||2016-309|
|Patent Cooperation Treaty||Published Application||2018165249||09/13/2018||2016-309|
Cas9-mediated genome editing, CRISPR, mutagenesis, Arabidopsis, screening of mutations, fluorescent marker