title: Viral Packaging and Cell Culture for CRISPR-Based Screens.
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.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142; Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142; David H. Koch Institute for Integrative Cancer Research at MIT, Cambridge, Massachusetts 02139; Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139;
authors.forename: Tim
authors.initials: T
authors.lastname: Wang
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142; Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Eric S
authors.initials: ES
authors.lastname: Lander
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142; Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142; David H. Koch Institute for Integrative Cancer Research at MIT, Cambridge, Massachusetts 02139; Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139;
authors.forename: David M
authors.initials: DM
authors.lastname: Sabatini
mesh_terms: D064113:CRISPR-Cas Systems; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D055786:Gene Knockout Techniques / Q000379:methods; D018390:Gene Targeting / Q000379:methods; D005820:Genetic Testing / Q000379:methods; D006801:Humans; D016086:Lentivirus / Q000235:genetics / Q000502:physiology*; D019065:Virus Assembly