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Cold Spring Harb Protoc. 2016 Mar 1;2016(3):pdb.prot090803. doi: 10.1101/pdb.prot090803.

Single Guide RNA Library Design and Construction.

Author information

1
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;
2
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.

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.

PMID:
26933249
PMCID:
PMC4804709
DOI:
10.1101/pdb.prot090803
[Indexed for MEDLINE]
Free PMC Article

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