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Published on: August 30, 2021
Protocol for Discovery and Characterization of Miniature Cas12 Systems
Zhipeng Wang1, Yujue Wang1, Quanjiang Ji1,2,3
1School of Physical Science and Technology & State Key Laboratory of Advanced Medical Materials and Devices, ShanghaiTech University, Shanghai 201210, China.
None:
Efficient delivery remains a major challenge for therapeutic genome editing because many widely used CRISPR nucleases are large and leave limited space for regulatory elements or additional payloads in a single adeno-associated virus (AAV) vector. Miniature Cas12 nucleases are particularly appealing, as their reduced size alleviates packaging constraints while preserving RNA-guided DNA cleavage. Here, we outline a workflow that links large-scale sequence mining with phylogenetic and structural filtering, followed by PAM profiling, in vitro cleavage, bacterial genome interference, and genome-editing assays in human cells to confirm activity. This protocol is intended to distill broad sequence collections into a small set of compact Cas12 nucleases with demonstrated functions that can serve as starting points for further engineering in delivery-limited settings.
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