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Genome Editing – CRISPR/Cas9 – Now available with Nuclear Localization Signal

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. This follows several attempts over the years to manipulate gene function, including homologous recombination and RNA interference. RNAi, in particular, became a laboratory staple enabling inexpensive and high-throughput interrogation of gene function, but is hampered by providing only temporary inhibition of gene function and unpredictable off-target effects. Other recent approaches to targeted genome modification — zinc-finger nucleases [ZFNs] and transcription-activator like effector nucleases [TALENs]– enable researchers to generate mutations by introducing double-stranded breaks to activate repair pathways. These approaches are costly and time consuming to engineer, limiting their widespread use, particularly for large scale, high-throughput studies.

CRISPR (Clustered Regulary Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) genes are essential in adaptive immunity in select bacteria and archaea, enabling the organisms to respond to and eliminate invading genetic material.

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.

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For in vitro applications NEB offers recombinant Streptococcus pyogenes Cas9 Nuclease.

in vivo Gene Editing in Zebrafish

– NEBs Cas9 in microinjection in customer experiment

„„The experiments with NEB Cas9 are going well, seems very effective with a variety of sgRNAs. The high conc. Cas9 protein (Anm.: #M0386 M) is extremely efficient in vivo!““

James A. Gagnon, Department of Molecular and Cellular Biology, Harvard University, USA

cas9_zebrafish

Mutations in the tyrosinase gene block melanocyte pigmentation in zebrafish: 1-cell embryos were injected with 10 sgRNAs targeting tyrosinase and NEB 18 μM Cas9 protein. Categories of pigmentation at 3 days post fertilization. At 2nl injection volume, >90% of embryos completely lack pigmentation.

Images and data by courtesy of James A. Gagnon, Department of Molecular and Cellular Biology, Harvard University, USA

Further online ressources:

D O W N L O A D S

Article
“CRISPR/Cas9 and Targeted Genome Editing:
A New Era in
Molecular Biology”

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Download PDF (1.1 MB)

Application Note
“Protocol for using recombinant Cas9 Nuclease to assess locus modification in genome editing experiments”

Icon_AppNote_LocusModification
Download PDF (0.5 MB)

Application Note
“Construction of an sgRNA-Cas9 expression vector via single-stranded DNA oligo bridging of double-stranded DNA fragments”

Icon_AppNote_ConstructionsgRNA
Download PDF (0.4 MB)

P R O D U C T T A B L E

Products for the CRISPR/Cas9 Workflow

PRODUCT Prod.Nr.: SIZE PRICE
Cas9 Nuclease, Streptococcus pyogenes M0386 S 50 pmol (50 rxns) 155 € shop_icon
M0386 L 250 pmol (250 rxns) 675 €
M0386 M 500 pmol (500 rxns) 877 €
Cas9 Nuclease NLS, Streptococcus pyogenes M0641 S 50 pmol (50 rxns) 187 € shop_icon
M0641 L 250 pmol (250 rxns) 815 €
M0641 M 500 pmol (500 rxns) 1.058 €
Selected NEB Products for use in CRISPR Workflows:
HiScribe T7 High Yield RNA Synthesis Kit E2040 S 50 rxns 210 € shop_icon
HiScribe T7 Quick High Yield RNA Synthesis Kit E2050 S 50 rxns 259 € shop_icon
HiScribe T7 ARCA mRNA Kit (with Tailing) E2060 S 20 rxns 368 € shop_icon
HiScribe T7 ARCA mRNA Kit E2065 S 20 rxns 315 € shop_icon
Q5 Site-directed Mutagenesis Kit (with or without comp. E. coli) E0554 S 10 rxns
(+ comp. E.coli)
180 € shop_icon
E0552 S 10 rxns 125 €
Q5 High-Fidelity DNA Polymerase M0491 S 100 u 92 € shop_icon
M0491 L 500 u 410 €
Q5 Hot Start High-Fidelity DNA Polymerase M0493 S 100 u 108 € shop_icon
M0493 L 500 u 490 €
NEBuilder HiFi DNA Assembly Master Mix E2621 S 10 rxns 158 € shop_icon
E2621 L 50 rxns 632 €
E2621 X 250 rxns 2525 €
NEBuilder HiFi DNA Assembly Cloning Kit
(incl. comp. E. coli)
E5520 S 10 rxns 185 € shop_icon
T7 Endonuclease I M0302 S 250 u 66 € shop_icon
M0302 L 1,250 u 264 €