Viral Packaging and Cell Culture for CRISPR-Based Screens
- 1Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139;
- 2Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142;
- 3Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142;
- 4David H. Koch Institute for Integrative Cancer Research at MIT, Cambridge, Massachusetts 02139;
- 5Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139;
- 6Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115
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↵7 These authors contributed equally to this work
Abstract
This protocol describes how to perform the tissue culture and high-throughput sequencing library preparation for a CRISPR-based screen. First, pantropic lentivirus is prepared from a single guide RNA (sgRNA) plasmid pool and applied to the target cells. Following antibiotic selection and a harvest of the initial population, cells are then cultured under the desired screening condition(s) for 14 population doublings. The sgRNA barcode sequences integrated in the genomic DNA of each cell population are amplified and subject to high-throughput sequencing. Guidelines for downstream analysis of the sequencing data are also provided.
Footnotes
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↵8 Correspondence: lander{at}broadinstitute.org; sabatini{at}wi.mit.edu
- © 2016 Cold Spring Harbor Laboratory Press
