Nature biotechnology, 2013-09, Vol.31 (9), p.827-832
2013
Details
- Autor(en) / Beteiligte
- Titel
- DNA targeting specificity of RNA-guided Cas9 nucleases
- Ist Teil von
- Nature biotechnology, 2013-09, Vol.31 (9), p.827-832
- Ort / Verlag
- United States: Nature Publishing Group
- Erscheinungsjahr
- 2013
- Link zum Volltext
- Quelle
- MEDLINE
- Beschreibungen/Notizen
- The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.
- Sprache
- Englisch
- Identifikatoren
- ISSN: 1087-0156eISSN: 1546-1696DOI: 10.1038/nbt.2647Titel-ID: cdi_pubmed_primary_23873081
- Format
- –
- Schlagworte
- Bacterial Proteins - genetics, Bacteriology, Base Pair Mismatch, Base Sequence, Deoxyribonucleases - genetics, Deoxyribonucleic acid, DNA, DNA - genetics, DNA binding proteins, Genetic aspects, Genetic engineering, Genetic Engineering - methods, Genomics, Molecular Sequence Data, Physiological aspects, Ribonucleic acid, RNA, RNA sequencing, RNA, Guide - genetics, Streptococcus, Streptococcus pyogenes, Streptococcus pyogenes - genetics, Transcription Factors - genetics