Targeted genome editing in human cells using CRISPR/Cas nucleases and truncated guide RNAs.
CRISPR RNA-guided nucleases have recently emerged as a robust genome-editing platform that functions in a wide range of organisms. To reduce off-target effects of these nucleases, we developed and validated a modified system that uses truncated guide RNAs (tru-gRNAs). The use of tru-gRNAs leads to decreases in off-target effects and does not generally compromise the on-target efficiencies of these genome-editing nucleases. In this chapter, we describe guidelines for identifying potential tru-gRNA target sites and protocols for measuring the on-target efficiencies of CRISPR RNA-guided nucleases in human cells.
Molecular Pathology Unit, Center for Computational and Integrative Biology, and Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA; Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA.
Yanfang
Y
Fu
Molecular Pathology Unit, Center for Computational and Integrative Biology, and Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA; Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA.
Deepak
D
Reyon
Molecular Pathology Unit, Center for Computational and Integrative Biology, and Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA; Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA. Electronic address: jjoung@mgh.harvard.edu.
J Keith
JK
Joung
D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000378:metabolism; D064113:CRISPR-Cas Systems; D002460:Cell Line; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004720:Endonucleases / Q000378:metabolism*; D018390:Gene Targeting; D015894:Genome, Human; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics* / Q000378:metabolism
D064130:CRISPR-Associated Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D004720:Endonucleases
CRISPR RNA-guided nucleases; CRISPR/Cas nucleases; Cas9; Genome editing; Off-target effects; Off-target mutations; Truncated guide RNAs