CRISPR Systems

Santa Cruz Biotechnology now offers target-specific CRISPR/Cas9 Knockout (KO) Plasmids, CRISPR Double Nickase Plasmids, CRISPR/ dCas9 Activation Plasmids and CRISPR Lenti Activation Systems for over 18,910 human and 18,340 mouse protein encoding genes.

History of CRISPR/Cas9

The CRISPR/Cas system is an adaptive immune defense mechanism used by Archea and bacteria for the degradation of foreign genetic material. In these organisms, the foreign genetic material from a bacteriophage is acquired and integrated into the CRISPR loci (1,2). This new material, also known as a spacer, creates a sequence-specific fragment used for future resistance against a bacteriophage infection. These sequence-specific fragments are translated into short CRISPR RNAs (crRNAs) and function as a guide to direct cleavage of complementary invading DNA via the nuclease activity of CRISPR-associated (Cas) protein also encoded by the CRISPR loci (1,2). Cas9 nuclease of the type II CRISPR system has an RNA binding domain, an alpha helix recognition lobe (REC), a nuclease lobe that include the RuvC and HNH for DNA cleavage, and a protospacer adjacent motif (PAM) interacting site (1,2). crRNA forms a complex with the Cas9 nuclease by binding to the bridge helix within the REC lobe, and forms multiple salt bridges with the backbone of the crRNA (1,2,3).

Once the crRNA binds to the Cas9 the conformation of the Cas9 nuclease changes and creates a channel that allows for DNA binding (1,2,3). The Cas9/crRNA complex scans the DNA for a PAM (5'-NGG) site (4,5,6). Recognition of a PAM site leads to unwinding of the DNA, and allows the crRNA to check for complementary DNA adjacent to the PAM site. When Cas9 binds to a PAM site adjacent to a DNA sequence that is complementary to the crRNA, the bridge helix within the REC lobe creates an RNA-DNA heteroduplex with the target DNA (3,4,7). The PAM site recognition is involved in activating the nucleolytic HNH and RuvC domains which create a double-stranded break (DSB) in the target DNA, leading to DNA degradation (1,2,5,8). If the crRNA is not complementary, then Cas9 releases and searches for another PAM site (7). Targeted genome strand breaks in the DNA can be repaired via the nonhomologous end-joining (NHEJ) repair pathway, which introduces insertion or deletions creating errors, or through the homologous directed repair (HDR) pathway, that can be used to recombine selected markers at specific sites in the genome (2,9,10). This CRISPR/Cas9 mechanism can be repurposed for genomic engineering of various systems, including mammalian cells.

Genome editing via introduction of DSBs can be performed with meganucleases, zinc finger nucleases (ZF), or transactivator-like effectors (TALEs), which recognize DNA sequences, however, each has their limitations. When using meganuclesases it is difficult to clearly show site-specific recognition between nuclease and DNA (2).The other options, ZFs and TALEs, have proven difficult to design and recognize up to 3 nt of DNA (2). Single guide RNAs (sgRNAs) that act like crRNAs are easily designed and can be expressed along with Cas9 nuclease in the same vector to target specific DNA sites for genome editing. The CRISPR/Cas 9 system also has higher sensitivity and is more efficient when used for screening than small hairpin RNAs.

A significant advantage of using the CRISPR/Cas9 system to induce DSB in genomic DNA is its high level of efficiency. However, this efficiency can be clouded by a number of off-target effects, thereby reducing the specificity of CRISPR/Cas9 editing. Specificity can be improved by using a CRISPR double nickase system, whereby a pair of plasmids, each encoding a Cas9 (D10A) nickase mutant (Cas9n) are directed to a distinct, site specific region in the genomic DNA by a target-specific guide RNA (12). Each Cas9n/sgRNA complex creates only one nick in the DNA strand that is complementary to the guide RNA (12). Each pair of guide RNAs are offset by approximately 20 bp and recognize target sequences located on opposite strands of the target DNA. The double nick created by the pair of Cas9n/sgRNA complexes mimics a DSB (12). Thus, the use of paired-guide RNAs allows for increased specificity of Cas9-mediated gene editing, while maintaining a high level of efficiency (12).

In addition to genome editing, the CRISPR system has been engineered to allow for robust activation of endogenous gene expression (13). Several components of the CRISPR system have been modified to generate the synergistic activation mediator (SAM) complex that results in a highly efficient and specific transcription activation system (13). One component of the SAM complex that is modified is the Cas9 nuclease. In the SAM system, the catalytic domains of Cas9 have been deactivated and the resulting dCas9 is fused to a transcription activation domain (VP64). Directed by a target specific guide RNA (sgRNA), the dCas9-VP64-sgRNA complex targets the -200 bp region from the Transcriptional Start Site (TSS) of endogenous genes to upregulate gene expression (13). To further enhance transcription, the sgRNA has been modified by appending a minimal hairpin aptamer to the tetraloop and stem loop 2 (13). This aptamer on the sgRNA selectively binds dimerized MS2 bacteriophage coat proteins (13). Fusing the MS2 proteins to p65 and HSF1 transactivation domains allows the resulting MS2-P65-HSF1 fusion protein to enhance the recruitment of transcription factors, thereby improving the potency of dCas9-mediated gene activation (13). Activation products are supplied as both standard plasmids for transfection and lentiviral plasmids for lentiviral packaging and transduction, for efficient delivery of the SAM Transcription Activation System into all cell types (13).

CRISPR/Cas9 Products offered by Santa Cruz Biotechnology, Inc.

CRISPR/Cas9 Plasmids

CRISPR/Cas9 Plasmid product details:

  • 20 µg, up to 20 transfections
  • CRISPR/Cas9 Knockout (KO) Plasmids consist of a pool of three plasmids each encoding the Cas9 nuclease and a target-specific 20 nt guide RNA (gRNA) designed for maximum knockout efficiency
  • gRNA sequences are derived from the GeCKO (v2) library and direct the Cas9 protein to induce a site-specific double strand break (DSB) in the genomic DNA ()
  • CRISPR/Cas9 KO Plasmids available for human and mouse genes are indicated by the (h) or (m) designation in the product name. Example: p53 CRISPR/Cas9 KO Plasmids (h) for human or p53 CRISPR/Cas9 KO Plasmids (m) for mouse
  • Provided as transfection-ready, purified plasmid DNA

Support Products for CRISPR/Cas9 Plasmids:

  • Suitable control antibodies are available
  • Ultracruz Transfection Reagent, sc-395739
  • Plasmid Transfection Medium, sc-108062
  • Control CRISPR/Cas9 Plasmid, sc-418922
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How do CRISPR/Cas9 Plasmids work?

Download CRISPR / Cas9 Protocols

HDR Plasmid

HDR Plasmid description:

  • HDR plasmids provide a specific DNA repair template for a DSB, and are only used when co-transfected with CRISPR/Cas9 KO Plasmids.
  • When co-transfected with the CRISPR/ Cas9 KO Plasmid, the HDR Plasmid incorporates a Puromycin resistance gene for selection of cells where Cas9-induced DNA cleavage has occurred.

HDR Plasmid product details:

  • 20 µg, up to 20 transfections
  • Target specific HDR Plasmids are recommended for co-transfection with the CRISPR/Cas9 KO Plasmid for the same gene target and species
  • HDR Plasmid consists of a pool of 2-3 plasmids, each containing a homology-directed DNA repair (HDR) template corresponding to the cut sites generated by the corresponding CRISPR/Cas9 KO Plasmids
  • Each HDR template contains two 800 bp homology arms designed to specifically bind to the genomic DNA surrounding the corresponding Cas9-induced double strand DNA break site
  • Each HDR Plasmid inserts a puromycin resistance gene to enable selection of stable knockout (KO) cells
  • Each puromycin resistance gene is flanked by two LoxP sites to allow for further processing by the Cre vector
  • Each HDR Plasmid also contains RFP to visually confirm transfection
  • Provided as transfection-ready, purified plasmid DNA

Support Products for HDR Plasmids:

How do HDR Plasmids work?

Allelic Expression

Diploid cells have two homologous copies of each chromosome, carrying two copies of each gene. These alternate forms of the same gene, which don’t need to be identical, are referred to as alleles and at each specific chromosomal locus, an individual possesses two alleles for the same gene. Within a population, one allele occurs most frequently and is therefore designated the dominant wild-type allele and is usually responsible for the wild-type phenotype. Mutations can create new alleles, and each new allele can lead to changes in phenotype.

Since diploid cells contain two alleles for most genes, additional rounds of transfection and selection may be needed in order to knock out every targeted gene, to produce a homozygous knockout cell population. Using only the CRISPR/Cas9 Knockout Plasmid will lead to NHEJ repair, introducing indels into the gene of interest. After a CRISPR/Cas9 Plasmid transfection, knockout of the gene of interest will occur in both alleles (bi-allelic knockout) in some cells, while in other cells, knockout will occur in only one allele (mono-allelic knockout). The CRISPR/Cas9 transfected population of cells will therefore include both bi-allelic and mono-allelic knockout cells.

Performing a Western Blot experiment after transfection with a CRISPR/Cas9 Knockout Plasmid is one way to determine if you have bi-allelic or mono-allelic knockout in your cell population. By isolating single cell colonies of cells, it is possible to grow a population of cells that are either mono-allelic or bi-allelic. Once these populations are grown to confluency, a Western Blot experiment can show which colonies have bi-allelic or mono-allelic knockdown of the gene of interest. A population of cells with bi-allelic knockout would show 100% knockdown of the protein of interest on a Western Blot while a population of cells with a mono-allelic knockdown would show a 50% knockdown of protein expression.

Successful co-transfection of a specific CRISPR/Cas9 Plasmid with a Homology Directed Repair (HDR) Plasmid will lead to gene disruption and homologous directed repair (HDR) of the gene of interest. After co-transfection, selection for cells that have successfully incorporated the puromycin resistance gene can be performed at this point. This selection will also show a mixed cell population of mono-allelic and bi-allelic cells. A Western Blot will again be useful in showing which colonies show a bi-allelic knockout of the gene of interest.


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Cre Vector

Cre Vector description:

  • The Cre Vector expresses Cre recombinase, a bacteriophage p1 enzyme that catalyzes site-specific DNA recombination between two LoxP sites
  • When the CRISPR/Cas9 Knockout Plasmid is co-transfected with the HDR Plasmid, cells containing the edited DNA can be isolated using the selection marker inserted during homology-directed repair
  • Following selection, cells can be transfected with the Cre Vector to excise the genetic material inserted during homology-directed repair, such as the puromycin resistance gene

Cre Vector product details:

  • 20 µg, up to 20 transfections
  • Recommended for DNA repair of selected cells successfully edited by CRISPR/Cas9 KO Plasmid and HDR Plasmid
  • Cre Vector contains a CMV promoter to drive expression of Cre recombinase
  • Provided as a transfection-ready, purified plasmid DNA
  • Cre Vector, sc-418923

Support Products for Cre Vector:

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How does the Cre Vector work?

Download Cre Vector Protocols

Double Nickase Plasmids

Double Nickase Plasmid product details:

  • 20 µg, up to 20 transfections
  • Double Nickase Plasmids consist of a pair of plasmids each encoding a D10A mutated Cas9 nuclease and a target-specific 20 nt guide RNA (gRNA) designed to knockout gene expression with greater specificity than its CRISPR/Cas9 KO counterpart
  • Paired gRNA sequences are offset by approximately 20 bp to allow for specific Cas9-mediated double nicking of the genomic DNA, which mimics a DSB
  • One plasmid in the pair contains a puromycin-resistance gene for selection; the other plasmid in the pair contains a GFP marker to visually confirm transfection
  • Double Nickase Plasmids available for human and mouse genes are indicated by the (h) or (m) designation in the product name. Example: p53 Double Nickase Plasmid (h) for human or p53 Double Nickase Plasmid (m) for mouse
  • Provided as transfection-ready, purified plasmid DNA

Support Products for Double Nickase Plasmids:

  • Suitable control antibodies are available
  • Ultracruz Transfection Reagent, sc-395739
  • Plasmid Transfection Medium, sc-108062
  • Control Double Nickase Plasmid, sc-437281
  • Puromycin dihydrochloride, sc-108071
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How do Double Nickase Plasmids work?

Download Double Nickase Protocols

Activation Plasmids

CRISPR/dCas9 Activation Plasmid product details:

  • 20 µg, up to 20 transfections
  • CRISPR/dCas9 Activation Plasmids are a synergistic activation mediator (SAM) transcription activation system designed to specifically upregulate gene expression
  • CRISPR/dCas9 Activation Plasmids consist of the following three plasmids at a 1:1:1 mass ratio: a plasmid encoding the deactivated Cas9 (dCas9) nuclease (D10A and N863A) fused to the transactivation domain VP64; a plasmid encoding the MS2-p65-HSF1 fusion protein; and a plasmid encoding a target-specific 20 nt guide RNA
  • gRNA sequences are derived from the CRISPR/Cas9 Synergistic Activation Mediator (SAM) pooled human library and direct the SAM complex to bind to a site-specific region approximately 200-250 nt upstream of the transcriptional start site of the target gene
  • The SAM system provides robust recruitment of transcription factors for highly efficient activation of the target gene
  • CRISPR/dCas9 Activation Plasmids available for human and mouse genes are indicated by the (h) or (m) designation in the product name. Example: p53 CRISPR/dCas9 Activation Plasmid (h) for human or p53 CRISPR/dCas9 Activation Plasmid (m) for mouse
  • Provided as transfection-ready, purified plasmid DNA

Support Products for CRISPR Activation Plasmids:

  • Suitable control antibodies are available
  • Ultracruz Transfection Reagent, sc-395739
  • Plasmid Transfection Medium, sc-108062
  • Control CRISPR/dCas9 Activation Plasmid, sc-437275
  • Blasticidin S HCl solution, sc-495389
  • Hygromycin B, sc-29067
  • Puromycin dihydrochloride, sc-108071
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How do Activation Plasmids work?

Download Activation Protocols

Lentiviral Activation Particles

CRISPR/dCas9 Lentiviral Activation Particles product details:

  • Lentiviral Activation Particles encode a synergistic activation mediator (SAM) transcription activation system designed to specifically and efficiently upregulate gene expression via lentiviral transduction of cells (13)
  • The SAM complex binds to a site-specific region approximately 200-250 nt upstream of the transcriptional start site and provides robust recruitment of transcription factors for highly efficient gene activation
  • Lentiviral Activation Particles are supplied frozen in 200 µl of Dulbecco's Modified Eagle Medium with 25 mM HEPES pH 7.3, containing the critical elements in the three CRISPR/dCas9 Activation plasmids, that are required for targeted gene upregulation:
    1. Deactivated Cas9 (dCas9) nuclease (D10A and N863A) fused to the transactivation domain VP64, and blasticidin resistance genes
    2. MS2-p65-HSF1 fusion protein, and hygromycin resistance genes
    3. Target-specific 20 nt. guide RNA, and a Puromycin resistance gene
  • Recommended for use with hard-to-transfect cells
  • CRISPR/dCas9 Lentiviral Activation Particles are available for human and mouse genes, indicated by the (h) or (m) designation in the product name. Example: p53 Lentiviral Activation Particles (h) for human or p53 Lentiviral Activation Particles (m) for mouse

Support Products for CRISPR Lentiviral Activation Particles:

Shop for Lentiviral Activation products now

How do Lentiviral Activation Particles work?

Download Lentiviral Protocols

Frequently Asked Questions

  • What are the advantages of using the CRISPR/Cas9 system versus RNAi?

    The CRISPR/Cas9 system knocks out a protein of interest by targeting and editing genomic DNA rather than using RNA interference, which targets and degrades mRNA. Once the genomic DNA is edited, the change is permanent.

  • Why do researchers prefer using the CRISPR/Cas9 genome-editing tool over zinc-finger nucleases (ZFNs) and TALENs?

    The CRISPR/Cas9 system is less expensive, more efficient and has higher sensitivity.

  • How did you design the gRNA sequence?

    SCBT uses a pool of 3 gRNA target sequences published from the GeCKO v2 library. The published sequences have been shown to have high modification efficiency and low off target effects.

  • Do you provide the gRNA sequence?

    Yes, these sequences are available to customers. Please contact Technical Service.

  • Why does the CRISPR/Cas9 KO Plasmid come as a pool of three target RNA sequences?

    We designed our CRISPR/Cas9 KO Plasmid as a pool of three gRNA sequences to ensure sufficient gene disruption.

  • Does the HDR Plasmid come as a pool of three?

    Yes, each HDR Plasmid is designed to work with its corresponding gRNA.

  • Can the CRISPR/Cas9 KO Plasmid alone be used to knockout a gene function?

    Yes, but this will result in non-homologous end-joining (NHEJ), which may lead to InDels (insertions/deletions). InDels alter the Open Reading Frame (ORF) and can significantly change the amino acid sequence downstream of the double strand break (DSB) or introduce a premature stop codon. InDels created by NHEJ may lead to random mutations, so the type and extent of gene disruption will need to be determined experimentally.

  • How do I know if my cells were successfully transfected with the CRISPR/Cas9 KO Plasmid?

    Cells may be assayed for GFP expression to determine transfection efficiency. Further assays are necessary to determine the degree of gene knockdown.

  • How do I know if my cells were successfully transfected with the HDR Plasmid?

    Cells may be assayed for RFP expression to determine transfection efficiency. Further assays are necessary to determine the degree of gene knockdown.

  • How do I know successful gene editing occurred using the CRISPR/Cas9 KO Plasmid and HDR Plasmid?

    Successfully edited cells will contain a puromycin resistance gene in their genomic DNA and puromycin selection may be used.

  • How long are the left homology arms (LHA) and right homology arms (RHA) of the HDR Plasmid?

    They are 800 base pairs in length to ensure proper alignment with genomic DNA and homology directed repair.

  • Do you offer a CRISPR control?

    We offer a negative control, which is a CRISPR/Cas9 KO Plasmid with a single scrambled gRNA sequence, Control CRISPR/Cas9 Plasmid sc-418922. The scrambled gRNA sequence will not bind to genomic target DNA and the Cas9/gRNA complex will not bind to or create a DSB.

  • What is a PAM sequence?

    The protospacer adjacent motif (PAM) sequence must be present at the 3’ end of the DNA target sequence, but is not present in the gRNA sequence. For Cas9 to successfully bind to DNA, the target sequence in the genomic DNA must be complimentary to the guide RNA sequence and it must be immediately followed by the PAM sequence at the 3’ end.

  • Where does the Cas9/gRNA complex cut the genomic DNA?

    Cas9 will cut the genomic DNA approximately 3-5 base pairs downstream of the 3’ end of the target sequence.

  • Will the CRIPSPR/Cas9 system work on cells that are dividing and non-dividing?

    Yes. Cells that are dividing may use either the NHEJ or HDR pathways where as cells that are non-dividing may only use the NHEJ pathway.

  • Is there any variability in efficiency between different cell lines?

    Yes, results will vary between cell lines and target sequences.

  • How do you determine if the gene of interest was successfully knocked-down by the CRISPR/Cas9 system?

    There are a few different ways to verify gene knockdown. Puromycin will allow for the selection of cells that have been successfully edited by the HDR Plasmid. WB analysis will show protein knockdown. Genomic PCR will detect genomic integration. Mutations may be detected by amplifying the genomic sequence, cloning the PCR fragment, and then sequencing it.

  • What do you recommend for Western Blot and/or Immunofluorescence verification of transfection?

    Western Blot and Immunofluorescence analysis will determine that the Cas9 protein is expressed. You may use the RFP marker to visualize fluorescence under the microscope or use a Cas9 antibody, which is currently in production.

  • How many genes can I knockout at once?

    The maximum amount of genes that can be knocked-out at once has not been determined. Research suggests you can knockout several genes at once, depending on how well the cells do with the co-transfection. We recommend and warranty the use of our CRISPR/Cas9 KO Plasmids one at a time.

  • Which support products and transfection reagents should I purchase from SCBT in order to use your CRISPR/Cas9 KO plasmid?

    Control CRISPR/Cas9 Plasmid sc-418922, Ultracruz Transfection Reagent sc-395739, Plasmid Transfection Medium sc-108062, Puromycin dihydrocholoride sc-108071. We also offer control antibodies for each target-specific CRISPR/Cas9 KO Plasmid.

  • Can we provide custom CRISPR products?

    Please contact Technical Service for further details.

  • What is the ETA for CRISPR products that are not yet in stock?

    10-14 days

References

  1. Van der Oost J., et al. 2014. Unraveling the Structural and Mechanistic Basis of CRISPR-Cas Systems. Nat. Rev. Microbiol. (7):479-92. PMID 24909109.

  2. Hsu, P., et al. 2014. Development and Applications of CRISPR-Cas9 for Genome Editing. Cell. 157(6):1262-78. PMID 24906146.

  3. Nishimasu, H., et al. 2014. Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA. Cell. 156(5):935-49. PMID 24529477.

  4. Deltcheva, E., et al. 2011. CRISPR RNA Maturation by Trans-Encoded Small RNA and Host Factor RNASE III. Nature. 471: 602-607. PMID 21455174.

  5. Jinek, M., et al. 2012. A Programmable Dual-RNA Guided DNA Endonuclease in Adaptive Immunity. Science. 337(6096):816-21. PMID 22745249.

  6. Deveau, H., et al. 2010. CRISPR/Cas System and its Role in Phage-Bacteria Interaction. Annu. Rev. Microbiol. 64: 475-493. PMID 20528693.

  7. Sternberger, SH., et al. 2014. DNA Interrogation by the CRISPR RNA-guided Endonuclease Cas9. Nature. 507(7490):62-7. PMID 24476820.

  8. Gasuinas, G., et al. 2012. Cas9-crRNA Ribonucleoprotein Complex Mediates Specific DNA Cleavage for Adaptive Immunity in Bacteria. Proc. Natl. Acad. Sci. 109(39):E2579-86. PMID 22949671.

  9. Mali, P., et al. 2013. RNA-Guided Human Genome Engineering via Cas9. Science. 339 (6121): 823-6. PMID 23287722.

  10. Ran, FA., et al. 2013. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. (11): 2281-308. doi: 10.1038/nprot.2013.143. PMID 24157548.

  11. Shalem O., et al. 2014. Genome-scale CRISPR-Cas9 Knockout Screening in Human Cells. 343(6166):84-7. PMID 24336571.

  12. Ran, F.A., et al. 2013. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154: 1380-1389.

  13. Konermann, S., et al. 2015. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature. 517: 583-588. PMID 25494202

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