Methods and compositions for generating CRISPR/Cas guide RNAs
US11279926B2 · kind B2 · utility
Assignee
Inventors
Key dates
| Filing date | Jun 2, 2016 |
| Grant date | Mar 22, 2022 |
| Priority date | — |
| Expiry date | Jan 16, 2037 |
Classification
- Technology area (CPC C)Chemistry; Metallurgy
- CPC primaryC12Q2525/191
- WIPO fieldBiotechnology
- WIPO sectorChemistry
Abstract
The present disclosure provides methods, kits, and compositions for generating DNA molecules encoding CRISPR/Cas guide RNAs (e.g., Cas9 single guide RNAs or Cas9 targeter RNAs). A library of such DNA molecules can be generated from any DNA source. The methods include a step of contacting target DNA with one or more DNA endonucleases that specifically bind to and cleave within a recognition sequence that includes a PAM sequence, to generate a plurality of cleavage fragments, to which a DNA adapter can be attached. A distal-cleaving DNA endonuclease can be used that specifically binds to a recognition sequence in the DNA adapter and cleaves at a site within the attached DNA cleavage fragments to generate a library of CRISPR/Cas guide sequences. After removal of all or a portion of the DNA adapter, a constant region of a guide RNA can be attached to generate DNA molecules encoding CRISPR/Cas guide RNAs.
Source: USPTO / EPO open patent data. Objective bibliographic and citation counts.