Genome editing is the targeted modification of the genome of living cells
Genome editing is enabled by the development of tools to make precise, targeted changes to the genome of living cells.
Recently a new tool based on a bacterial CRISPR-associated protein-9 nuclease (Cas9) from Streptococcus pyogenes has generated considerable excitement.
Video: Genome Editing
CRISPR Cas9 Genome Editing
The simplicity of the CRISPR nuclease system, with only three components (Cas9 , crRNA and trRNA) makes this system amenable to adaptation for genome editing. By combining the crRNA and trRNA into a single synthetic guide RNA (sgRNA), a further simplified two component system can be used to introduce a targeted double stranded break. This break activates repair through error prone non-homologous end joining (NHEJ) or Homology directed Repair (HDR). In the presence of a donor template with homology to the targeted locus, the HDR pathway operates allowing for precise mutations to be made. In the absence of a template, NHEJ is activated resulting in insertions and/or deletions (indels) which disrupt the target locus.
Cas9 Nuclease Gene Editing
Transfecting cells with Cas9 RNPs (Ribonucleoprotein) complexes enables high efficiency gene editing
Different approaches are possible to deliver the Cas9 protein and the sgRNA into the cell during gene editing experiments:
- Transfection of sgRNA/Cas9 plasmids
- Transfection of sgRNA and Cas9 mRNA
- Co-Transfection of sgRNA and Cas9 protein (RNPs)
An HDR template plasmid can be added in all 3 scenarios.
Recent publications show that the use of sgRNA/Cas9 RNP complexes is currently regarded as the best method with the lowest off-target effects and highest efficiencies.
NEB’s scientists have shown that the new EnGen Cas9 NLS achieves the highest gene editing efficiencies in HEK293 cells.
Our Gene Editing 101 webinar will provide more information about the benefits of direct lipofection of Cas9 sgRNA RNPs.
Webinar: Gene Editing 101
Whatever you decide:
NEB offers a range of helpful and reliable reagents and kits to support your CRISPR Cas Genome Editing experiments.
Bacterial Adaptive Immunity with CRISPR/Cas9
CRISPR systems evolved as a means of bacterial adaptive immunity. Understanding their behavior in vivo is essential to harnessing their power in vitro.
Gene Editing Online Ressourcen
Selected products for CRISPR/Cas9 Workflow
As of: 22.04.2020
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