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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“Genome Editing”!

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

Further online ressources:

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

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

Products for the CRISPR/Cas9 Workflow

PRODUCT	Prod.Nr.:	SIZE	PRICE
Cas9 Nuclease, S. pyogenes	M0386S	70 pmol
	M0386M	400 pmol
	M0386T	2000 pmol
EnGen™ Spy Cas9 Nuclease NLS	M0646T	400 pmol
	M0646M	2000 pmol
EnGen Spy Cas9 Nickase	M0650S	70 pmol
	M0650T	400 pmol
EnGen Spy dCas9 (SNAP-Tag)	M0652S	70 pmol
	M0652T	400 pmol
EnGen Sau Cas9	M0654S	70 pmol
	M0654T	400 pmol
EnGen Lba Cas12a (Cpf1)	M0653S	70 pmol
	M0653T	2000 pmol
EnGen sgRNA Synthesis Kit, S. pyogenes	E3322V	10 rxns
	E3322S	20 rxns
EnGen Mutation Detection Kit	E3321S	25 rxns
Selected NEB Products for use in CRISPR Workflows:
HiScribe T7 High Yield RNA Synthesis Kit	E2040 S	50 rxns
HiScribe T7 Quick High Yield RNA Synthesis Kit	E2050 S	50 rxns
HiScribe T7 ARCA mRNA Kit (with Tailing)	E2060 S	20 rxns
HiScribe T7 ARCA mRNA Kit	E2065 S	20 rxns
Q5 Site-directed Mutagenesis Kit (with or without comp. E. coli)	E0554 S	10 rxns (+ comp. E.coli)
	E0552 S	10 rxns
Q5 High-Fidelity DNA Polymerase	M0491 S	100 u
	M0491 L	500 u
Q5 Hot Start High-Fidelity DNA Polymerase	M0493 S	100 u
	M0493 L	500 u
NEBuilder HiFi DNA Assembly Master Mix	E2621 S	10 rxns
	E2621 L	50 rxns
	E2621 X	250 rxns
NEBuilder HiFi DNA Assembly Cloning Kit (incl. comp. E. coli)	E5520 S	10 rxns
T7 Endonuclease I	M0302 S	250 u
	M0302 L	1,250 u