Single Guide RNA Library Design and Construction
Name
Lander_Single guide.pdf
Size
146.94 KB
Format
Adobe PDF
Checksum (MD5)
f2088f7d783236d74581cbb41b9c9a60
Author(s)
Wang, Tim
Lander, Eric Steven
Sabatini, David
Date Issued
December 9, 2016
Journal
Cold Spring Harbor Protocols
Publisher
Cold Spring Harbor Laboratory Press
Citation
Wang, Tim, Eric S. Lander, and David M. Sabatini. “Single Guide RNA Library Design and Construction.” Cold Spring Harb Protoc 2016, no. 3 (March 2016): pdb.prot090803.
Version
Author's final manuscript
Abstract
This protocol describes how to generate a single guide RNA (sgRNA) library for use in genetic screens. There are many online tools available for predicting sgRNA sequences with high target specificity and/or cleavage activity. Here, we refer the user to genome-wide sgRNA sequence predictions that we have developed for both the human and mouse and that are available from the Broad Institute website. Once a set of target genes and corresponding sgRNA sequences has been identified, customized oligonucleotide pools can be rapidly synthesized by a number of commercial vendors. Thereafter, as described here, the oligonucleotides can be efficiently cloned into an appropriate lentiviral expression vector backbone. The resulting plasmid pool can then be packaged into lentiviral particles and used to generate knockouts in any cell line of choice.
MIT Department
Massachusetts Institute of Technology. Department of Biology
Terms of Use
Creative Commons Attribution-Noncommercial-Share Alike
Persistent DSpace Link
DOI of Published Version
https://doi.org/10.1101/pdb.prot090803
