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gRNA constructs for CRISPR/Cas9-mediated genome editing

gRNA constructs for CRISPR/Cas9 genome editing

CRISPR/Cas9 mediated genome editing is a powerful technique that allows you to create knock-in, knock-out, and mutation of any gene, in any cell, in a highly targeted manner and without introducing foreign DNA. The benefits of CRISPR/Cas9 over previous forms of gene editing, such as TALENs and zinc finger nucleases, are that it is much simpler to implement and has higher efficiency at performing bi-allelic gene modifications. As a leader in gene synthesis technology, GenScript is pleased to offer CRISPR reagents developed in Feng Zhang's laboratory, licensed from the Broad Institute* including:

gRNA construct options

SpCas9 constructs

Cas9 endonucleases derived from the type II CRISPR systems in S. pyogenes (SpCas9) were the first Cas9 enzymes developed for mammalian genome editing. When combined with guide RNA (gRNA) sequences, these enzymes create site-specific double strand breaks (DSBs) in the genome. The CRISPR/Cas9 system accelerated genome editing for its ease of use, specificity, and high efficiency. GenScript is pleased to offer Broad Institute-validated WT SpCas9 constructs for gene editing in mammalian cells. Constructs are available either as all-in-one or dual vector systems, and can be used for non-viral, lenti-viral or adeno-associated virus (AAV) transfection. The lenti-vectors are compatible with 2nd and 3rd generation lentiviral packaging plasmids.

Empty gRNA/SpCas9 vectors

Broad Institute-validated all-in-one gRNA/Cas9 vectors contain a 17bp-1.8kb expressible linker in lieu of gRNA. Guide RNA expression is driven by a U6 promoter.

Vector type Vector name gRNA promoter Selection/Screening marker Price
Non-viral pSpCas9 BB-2A-GFP (PX458) U6 AmpR, GFP $99
pSpCas9 BB-2A-Puro (PX459) U6 AmpR, PuroR $99
Lentiviral plentiCRISPR v2 U6 AmpR, PuroR $99

SpCas9 constructs

Vector type Vector Name gRNA promoter All-in-one vector? Selection/Screening Markers Vector Price*
Lentiviral plentiCRISPR v2 U6 Yes
Driven by EFS promoter, with C-terminal DYK
AmpR, PuroR From $99
plentiGuide-Puro U6 No
pLentiCas9-Blast or pLentiCas9-EGFP are provided free-of-charge for co-delivery
AmpR, PuroR $199
Non-viral pSpCas9 BB-2A-GFP (PX458) U6 Yes
Driven by CBh promoter, with N-terminal DYK & NLS
AmpR, GFP
pSpCas9 BB-2A-Puro (PX459) U6 Yes
Driven by CBh promoter, with N-terminal DYK & NLS
AmpR, PuroR
pGS-gRNA U6 No
pSpCas9 (PX165) is provided free-of-charge for co-delivery
AmpR
pGS-gRNA-Neo U6 No
pSpCas9 (PX165) is provided free-of-charge for co-delivery
AmpR, NeoR
AAV pAAV SpGuide acceptor (PX552) U6 No
pAAV-SpCas9 (PX551) is provided free-of-charge for co-delivery
AmpR, GFP

*Includes gRNA design and synthesis

Delivery Specifications for sgRNA/Cas9 plasmids

  • 4 µg Research Grade* plasmid DNA
  • QC data:
    • Sequence chromatograms covering your gene (electronic)
    • Construct map for the plasmid (electronic)
    • Quality assurance certificate

*Research Grade plasmids are ideal for subcloning and transformation where there is no endotoxin requirement. Transfection-ready plasmids can be requested for an additional fee, starting from $99/prep. For Transfection Grade plasmids, or to receive larger quantities, specify your needs when you request a quote.

How to order:

  • For human and mouse genes, select from pre-validated gRNA sequences that target your gene:
  • For non-human or mouse genes, search for gRNA sequences targeting a 250bp region:
  • For help designing your gRNA sequences:

CRISPR Nickase constructs

While the CRISPR/Cas9 technology is still more specific when compared to other popular gene editing strategies, off-targeting concerns are still a reality. In an effort to improve specificity, the endonuclease activity of Cas9 was modified. WT Cas9 has two catalytic domains, RuvC and HNH, and mutations to catalytic residues within these domains (specifically, D10A in RuvC and H840A in HNH) cause Cas9 to create single strand nicks as opposed to double strand breaks (Ran et al, 2013) (Figure 1). This Cas9 enzyme with nickase activity, or Cas9n, is guided by guide RNAs (gRNA) to opposite sides of the target genomic DNA. Cells will preferentially repair these SSBs by HDR rather than NHEJ. By proceeding through an HDR mechanism, the frequency of unwanted indel mutations from off-target DSBs is minimized. GenScript is pleased to offer Broad-validated nickase vectors for gene editing in mammalian cells types.

Nickase



Figure 1: Increasing on-target specificity with Nickase (Cas9n D10A) pairs


For the nickase system, two gRNAs will need to be designed to target both forward and reverse strands of the target gene.

Empty nickase vectors

The Broad validated all-in-one gRNA/Cas9 vectors contain a 17bp-1.8kb expressible linker in lieu of gRNA. Guide RNA expression is driven by a U6 promoter.

Vector type Vector name gRNA promoter Selection/Screening marker Price
Non-viral pSpCas9n-BB (PX460) U6 AmpR $99
pSpCas9n-BB-2A-GFP (PX461) U6 AmpR, GFP $99
pSpCas9n-BB-2A-Puro (PX462) U6 AmpR, PuroR $99

Nickase constructs

Vector type Vector Name gRNA promoter All-in-one vector? Selection/Screening Markers Vector Price*
Non-viral pSpCas9n-BB (PX460) U6 Yes
Driven by CBh promoter, with N-terminal DYK & NLS
AmpR $199
pSpCas9n-BB-2A-GFP (PX461) U6 Yes
Driven by CBh promoter, with N-terminal DYK & NLS
AmpR, GFP
pSpCas9n-BB-2A-Puro (PX462) U6 Yes
Driven by CBh promoter, with N-terminal DYK & NLS
AmpR, PuroR
Lentiviral pLentiGuide-Puro U6 No
plentiCas9n(D10A)-Blast is provided free-of-charge for co-delivery
AmpR, PuroR

*Includes gRNA design and synthesis

Delivery Specifications for sgRNA/Cas9 plasmids

  • 4 µg Research Grade* plasmid DNA
  • QC data:
    • Sequence chromatograms covering your gene (electronic)
    • Construct map for the plasmid (electronic)
    • Quality assurance certificate

*Research Grade plasmids are ideal for subcloning and transformation where there is no endotoxin requirement. Transfection-ready plasmids can be requested for an additional fee, starting from $99/prep. For Transfection Grade plasmids, or to receive larger quantities, specify your needs when you request a quote.

How to order:

  • From the online quote form:
    • Specify the endogenous target gene(s) for which you wish GenScript to design gRNA, or specify the exact ~ 20bp gRNA sequences you wish GenScript to synthesize.
    • Select cloning into one of our expression-ready vectors, or provide details for a custom vector you will mail us. Cloning into your choice of vector is provided at no additional charge. You may specify cloning sites.
    • Optional: Request plasmid prep services to receive transfection-grade plasmid aliquots ready to use in your experiments.
  • Orders can be placed by phone, email or fax with a formal PO (purchase order) or credit card.

References:

CRISPR SaCas9 constructs

The Cas9 orthologue derived from Staphylococcus aureus, or SaCas9, has similar efficiency to SpCas9; however, SaCas9 is approximately 1kb shorter. The primary advantage of SaCas9 is adeno-associated virus (AAV) packaging: the cargo size of AAV is approximately 4.5kb, and consequently packaging SpCas9 into this vector can be challenging (Ran et al, 2015). The relatively smaller size of SaCas9 makes CRISPR gene editing with AAV vectors possible. Considering the lower immunogenicity of these constructs, SaCas9 is therefore more suited for in vivo editing applications, such as for therapeutics.

Like other Cas9 orthologues, another advantage to SaCas9 vectors are their specificity. The SaCas9 enzyme recognizes a longer PAM sequence, specifically NGRRT or NGRRN. Due to the longer sequence length, this PAM sequence occurs less frequently in the genome – approximately once every 32 bp compared to NGG which appears once in every 8 (Kleinstiver et al, 2015).

SaCas9 all-in-one vectors

GenScript currently offers all-in-one AAV vectors for SaCas9:

Vector type Vector Name gRNA promoter All-in-one vector? Selection/Screening
Markers
Vector Map Vector Price
AAV pX601_AAV U6 Yes
Driven by CMV
promoter with N-terminal NLS
AmpR pX601_AAV vector map $199
10 business day turnaround

Delivery Specifications for SaCas9 vectors:

  • 4 μg of research grade plasmid DNA
  • QC data:
    • Sequence chromatograms covering your gene (electronic)
    • Construct map for the plasmid (electronic)
    • Quality assurance certificate

*Research Grade plasmids are ideal for subcloning and transformation where there is no endotoxin requirement. Transfection-ready plasmids can be requested for an additional fee, starting from $99/prep. For Transfection Grade plasmids, or to receive larger quantities, specify your needs when you request a quote.

How to order:

  • From the online quote form:
    • Specify the endogenous target gene(s) for which you wish GenScript to design gRNA, or specify the exact ~ 20bp gRNA sequences you wish GenScript to synthesize.
    • Select cloning into one of our expression-ready vectors, or provide details for a custom vector you will mail us. Cloning into your choice of vector is provided at no additional charge. You may specify cloning sites.
    • Optional: Request plasmid prep services to receive transfection-grade plasmid aliquots ready to use in your experiments.
  • Orders can be placed by phone, email or fax with a formal PO (purchase order) or credit card.

References:

CRISPR SAM constructs

CRISPR/Cas9 Synergistic Activation Mediator (SAM) is a protein complex engineered to enable robust transcriptional activation of endogenous genes – either a single gene at a time, or up to 10 genes simultaneously in the same cell. SAM takes advantage of the specificity and ease of reprogramming of Cas9 nucleases, which are targeted to a specific locus in the endogenous genome by guide RNA. Through a license with the Broad Institute*, GenScript offers validated SAM gRNA sequences to target any coding region in the human genome, as well as complimentary design of SAM gRNA for any other species. SAM guide RNA sequences are custom-synthesized and cloned into efficient lentiviral vectors, and accompanied by the Cas9-VP64 and MS2-P65-HSF1 components that form the three-part SAM complex.

  • For complete details of SAM design & construction, please see: Konermann et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature, doi:10.1038/nature14136.

SAM Construct Options

GenScript offers SAM sgRNA for any coding gene cloned into the guide RNA expression vector. The sgRNA vectors must be co-delivered with the dCas9-VP64 and MS2-P65-HSF1 lentiviral expression vectors.


sgRNA vectors Selection Marker Price*
pLenti sgRNA(MS2)_zeo Zeo $199
sgRNA(MS2) Amp $199
dCas9-CP64 vectors Selection Marker Price*
pLenti dCas-VP64_Blast Blast $50
pLenti dCas9-VP64_GFP GFP $50
MS2-P65-HSF1 vectors Selection Marker Price*
pLenti MS2-P65-HSF1_Hygro Hygro $50
pLenti MS2-P65-HSF_GFP GFP $50

*Price & Turnaround Time for SAM Constructs

  • $199 for each SAM gRNA sequence synthesized and cloned into the lenti sgRNA(MS2)_zeo vector
  • $50 each for lenti dCas9-VP64_Blast and lenti MS2-P65-HSF1_Hygro, which can be ordered at the same time.
  • Total price for the three-plasmid system: $299; additional gRNA are $199 each 10-day turnaround time

How to order:

  • For human and mouse genes, select from pre-validated gRNA sequences that target your gene:
  • For non-human or mouse genes, search for gRNA sequences targeting a 250bp region:
  • For help designing your gRNA sequences:

gRNA construct FAQs

As CRISPR/Cas9 gene editing becomes increasingly mainstream, the number of vectors available have been increasing as well. The different CRISPR vectors have different advantages, depending on your experimental system and your downstream applications.

How should the gRNA sequences be designed? Read More »

Do I need a single vector (all-in-one) or dual vector system? Read More »

Under what circumstances are lenti- or adeno-associated virus vectors necessary? Read More »

When would I choose nickase vectors? Read More »

Which nickase vector is best for my experiment? Read More »

How are gRNA sequences designed for the transcription activation (SAM) system? Read More »

How does the Synergistic Activation Mediator (SAM) system activate transcription? Read More »

 
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