RT Journal Article
SR Electronic
T1 Protocol: Genome-scale CRISPR-Cas9 Knockout and Transcriptional Activation Screening
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 059626
DO 10.1101/059626
A1 Julia Joung
A1 Silvana Konermann
A1 Jonathan S. Gootenberg
A1 Omar O. Abudayyeh
A1 Randall J. Platt
A1 Mark D. Brigham
A1 Neville E. Sanjana
A1 Feng Zhang
YR 2016
UL http://biorxiv.org/content/early/2016/06/18/059626.abstract
AB Forward genetic screens are powerful tools for the unbiased discovery and functional characterization of specific genetic elements associated with a phenotype of interest. Recently, the RNA-guided endonuclease Cas9 from the microbial immune system CRISPR (clustered regularly interspaced short palindromic repeats) has been adapted for genome-scale screening by combining Cas9 with guide RNA libraries. Here we describe a protocol for genome-scale knockout and transcriptional activation screening using the CRISPR-Cas9 system. Custom-or ready-made guide RNA libraries are constructed and packaged into lentivirus for delivery into cells for screening. As each screen is unique, we provide guidelines for determining screening parameters and maintaining sufficient coverage. To validate candidate genes identified from the screen, we further describe strategies for confirming the screening phenotype as well as genetic perturbation through analysis of indel rate and transcriptional activation. Beginning with library design, a genome-scale screen can be completed in 6-10 weeks followed by 3-4 weeks of validation.