Homology-directed repair with DharmaconTM Edit-RTM CRISPR-Cas9 reagents and single-stranded DNA oligos
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چکیده
CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) is a revolutionary tool that utilizes an RNA-guided nuclease for efficient site-directed genome engineering in various eukaryotic systems. The double-strand breaks (DSBs) created by CRISPR-Cas9 are repaired in the cell by two predominant mechanisms: imprecise non-homologous end joining (NHEJ) and precise homology-directed repair (HDR). The DharmaconTM Edit-RTM CRISPR-Cas9 gene engineering platform consists of both synthetic and expressed guide RNAs. In conjunction with Cas9 expression plasmids and lentiviral particles, it supports multiple experimental workflows. Here we demonstrate an innovative workflow to guide the insertion of 10-12 nucleotides into a gene of interest by HDR with the Edit-R CRISPR-Cas9 system, Dharmacon DharmaFECTTM Duo Transfection Reagent, and a single-stranded donor DNA oligo (ssDNA).
منابع مشابه
Fluorescent tagging of an endogenous gene by homology-directed repair using DharmaconTM Edit-RTM CRISPR-Cas9 reagents
CRISPR-Cas9 has increased the accessibility of genome engineering due to its ease of use and ability to cause double-strand breaks (DSBs) at almost any locus of interest. The DSB that is created by Cas9, when guided to a specific genomic locus by crRNA and tracrRNA, can be repaired by various pathways, including homology-directed repair (HDR). The choice of repair pathway depends on numerous fa...
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تاریخ انتشار 2015