CRISPR Guide RNA Cloning for Mammalian Systems
Name
document(1).pdf
Description
Published version
Size
252.96 KB
Format
Adobe PDF
Checksum (MD5)
9ca40d209e8899177f7061017537400a
Author(s)
Nageshwaran, Sathiji
Chavez, Alejandro
Cher Yeo, Nan
Guo, Xiaoge
Lance-Byrne, Alissa
Tung, Angela
Collins, James J.
Church, George M.
Date Issued
October 2018
Journal
Journal of Visualized Experiments
Publisher
MyJove Corporation
Citation
Nageshwaran, Sathiji et al. "CRISPR Guide RNA Cloning for Mammalian Systems." Journal of Visualized Experiments 140 (October 2018): e57998 © 2018 Journal of Visualized Experiments
Version
Final published version
Abstract
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.
MIT Department
Massachusetts Institute of Technology. Institute for Medical Engineering & Science
Massachusetts Institute of Technology. Department of Biological Engineering
Massachusetts Institute of Technology. Synthetic Biology Center
Terms of Use
Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use.
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DOI of Published Version
https://doi.org/10.3791/57998
