In vitro Generation of CRISPR-Cas9 Complexes with Covalently Bound Repair Templates for Genome Editing in Mammalian Cells.
Bio Protoc
; 9(1)2019 Jan 05.
Article
em En
| MEDLINE
| ID: mdl-30675496
ABSTRACT
The CRISPR-Cas9 system is a powerful genome-editing tool that promises application for gene editing therapies. The Cas9 nuclease is directed to the DNA by a programmable single guide (sg)RNA, and introduces a site-specific double-stranded break (DSB). In mammalian cells, DSBs are either repaired by non-homologous end joining (NHEJ), generating small insertion/deletion (indel) mutations, or by homology-directed repair (HDR). If ectopic donor templates are provided, the latter mechanism allows editing with single-nucleotide precision. The preference of mammalian cells to repair DSBs by NHEJ rather than HDR, however, limits the potential of CRISPR-Cas9 for applications where precise editing is needed. To enhance the efficiency of DSB repair by HDR from donor templates, we recently engineered a CRISPR-Cas9 system where the template DNA is bound to the Cas9 enzyme. In short, single-stranded oligonucleotides were labeled with O6-benzylguanine (BG), and covalently linked to a Cas9-SNAP-tag fusion protein to form a ribonucleoprotein-DNA (RNPD) complex consisting of the Cas9 nuclease, the sgRNA, and the repair template. Here, we provide a detailed protocol how to generate O6-benzylguanine (BG)-linked DNA repair templates, produce recombinant Cas9-SNAP-tag fusion proteins, in vitro transcribe single guide RNAs, and transfect RNPDs into various mammalian cells.
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1
Coleções:
01-internacional
Base de dados:
MEDLINE
Tipo de estudo:
Guideline
Idioma:
En
Revista:
Bio Protoc
Ano de publicação:
2019
Tipo de documento:
Article