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.
Downloads
Gene Editing Online Ressourcen
Plasmid Repositories:
http://www.addgene.org
CRISPR-gRNA Design Tools:
http://crispr.mit.edu
http://zifit.partners.org/ZiFiT/
http://www.e-crisp.org/E-CRISP/designcrispr.html
https://chopchop.rc.fas.harvard.edu/
Online Forums:
https://groups.google.com/forum/crispr
Organism-specific Resources:
http://wormcas9hr.weebly.com
http://www.flyrnai.org
Selected products for CRISPR/Cas9 Workflow
| CRISPR/Cas9 Application | PRODUCT | Prod.Nr.: | SIZE | INFO | |
|---|---|---|---|---|---|
| Zentrale Komponente des CRISPR/Cas9-Workflows | EnGen® Spy Cas9 NLS | M0646T | 400 pmol |  |
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| M0646M | 2000 pmol | ||||
| EnGen® Spy Cas9 HF1 | M0667T | 500 pmol | |
|
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| M0667M | 2500 pmol | ||||
| Zentrale Komponente des CRISPR/Cas9-Workflows, ohne NLS | Cas9 Nuclease, S. pyogenes |
M0386S | 70 pmol | |
|
| M0386T | 400 pmol | ||||
| M0386M | 2000 pmol | ||||
|
EnGen® Lba Cas12a (Cpf1) | M0653S | 70 pmol | |
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| M0653T | 2000 pmol | ||||
|
EnGen® Sau Cas9 | M0654S | 70 pmol | |
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| M0654T | 400 pmol | ||||
| Einfügen versetzter Doppelstrangbrüche an genomischen Loci durch zweifachen Einzelstrangbruch (Nick) | EnGen® Spy Cas9 Nickase | M0650S | 70 pmol | |
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| M0650T | 400 pmol | ||||
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EnGen®Spy dCas9 (SNAP-tag®) | M0652S | 70 pmol | |
|
| M0652T | 400 pmol | ||||
| Synthesis of sgRNA | EnGen® sgRNA Synthesis Kit,S. pyogenes | E3322S | 20 rxns | |
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| E3322V | 10 rxns | |
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| Überprüfung der Genome Editing-Effizienz | EnGen® Mutation Detection Kit | E3321S | 25 rxns | |
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Weitere ausgewählte NEB Produkte für den Einsatz in CRISPR Workflows: |
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| Synthese von sgRNA | HiScribe T7 High Yield RNA Synthesis Kit |
E2040S | 50 rxns | |
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| HiScribe T7 Quick High Yield RNA Synthesis Kit | E2050S | 50 rxns | |
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| Synthese von capped Cas9 mRNA | HiScribe T7 ARCA mRNA Kit (mit Tailing) | E2060S | 20 rxns | |
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| HiScribe T7 ARCA mRNA Kit | E2065S | 20 rxns | |
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| Insertion der Zielsequenz in den sgRNA Vektor | Q5 Site-directed Mutagenesis Kit (mit oder ohne comp. E. coli) |
E0554S | 10 rxns (+ komp. E.coli) |
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| E0552S | 10 rxns | |
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| Ultragenaue Amplifikation Ihrer Konstrukte im CRISPR Workflow | Q5 High-Fidelity DNA Polymerase |
M0491S | 100 u | |
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| M0491L | 500 u | ||||
| Q5 Hot Start High-Fidelity DNA Polymerase | M0493S | 100 u | |
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| M0493L | 500 u | ||||
| Einfache, isothermale Generierung Ihres Cas9-sgRNA Konstrukts und HDR Templates | NEBuilder HiFi DNA Assembly Master Mix | E2621S | 10 rxns | |
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| E2621L | 50 rxns | ||||
| E2621X | 250 rxns | ||||
| NEBuilder HiFi DNA Assembly Cloning Kit (inkl. comp. E. coli) |
E5520S | 10 rxns | |
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As of: 01.01.2024
Further information can be found in our Technical Resources section or at neb.com. Information on trademarks can be found here.
