CRISPR Guide RNA Cloning for Mammalian Systems
Author(s)Nageshwaran, Sathiji; Chavez, Alejandro; Cher Yeo, Nan; Guo, Xiaoge; Lance-Byrne, Alissa; Tung, Angela; Collins, James J.; Church, George M.; ... Show more Show less
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The outlined protocol describes streamlined methods for the efficient and cost-effective generation of Cas9-associated guide RNAs. Two alternative strategies for guide RNA (gRNA) cloning are outlined based on the usage of the Type IIS restriction enzyme BsmBI in combination with a set of compatible vectors. Outside of the access to Sanger sequencing services to validate the generated vectors, no special equipment or reagents are required aside from those that are standard to modern molecular biology laboratories. The outlined method is primarily intended for cloning one single gRNA or one paired gRNA-expressing vector at a time. This procedure does not scale well for the generation of libraries containing thousands of gRNAs. For those purposes, alternative sources of oligonucleotide synthesis such as oligo-chip synthesis are recommended. Finally, while this protocol focuses on a set of mammalian vectors, the general strategy is plastic and is applicable to any organism if the appropriate gRNA vector is available.
DepartmentMassachusetts Institute of Technology. Institute for Medical Engineering & Science; Massachusetts Institute of Technology. Department of Biological Engineering; Massachusetts Institute of Technology. Synthetic Biology Center
Journal of Visualized Experiments
Nageshwaran, Sathiji et al. "CRISPR Guide RNA Cloning for Mammalian Systems." Journal of Visualized Experiments 140 (October 2018): e57998 © 2018 Journal of Visualized Experiments
Final published version