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Genome-CRISP™ CRISPR Products and Services

Complete Solutions for precision RNA-guided genome editing

Introduction

GeneCopoeia's Genome-CRISP™ CRISPR-Cas9 products and services provide a complete, powerful solution to your genome editing needs. Products and services include:

  • Genome-wide sgRNA clones for human, mouse, and rat. Search our database of more than 45,000 human,  mouse, and rat genes to knock out using CRISPR.
  • HDR donor cloning vectors and custom HDR donor construction. For CRISPR genome editing applications in which homolgous recombination will be used.
  • Cas9 stable cell lines. Premade Cas9-expressing stable cell lines are great for sgRNA validation and library screening.
  • Safe Harbor knockin kits and ORF clones. Easily create knockins of virtually any DNA fragment using our Safe Harbor knockn kits for human AAVS1 and mouse ROSA26. Search our datablase of over 40,000 Safe Harbor knockin ORF clones.
  • IndelCheck™ insertion/deletion detection system. Complete system for prevalidation of sgRNAs and screening for genome modifications. Includes T7 Endonuclease I assay kit and target site PCR kit. Target site-specific PCR primers optional. 
  • sgRNA libraries. Choose from 7 premade, pathway specific, lentiviral-based sgRNA libraries in a variety of formats. Custom libraires also available on request
  • Transgenic mouse services. Let us use CRISPR to generate genome-edited mouse lines for you. Applications include constitutive and conditional knockouts, mutagenesis, gene tagging, and more.
  • VividFISH™ chromosome probes for fluorescence in situ hybridization (FISH). Use FISH to help you with your genome editing workflow in cultured mammalian cell lines.

 

Advantages

  • RNA-guided genomic DNA recognition regardless of the methylation status
  • Similar or greater gene-editing efficiency compared to ZFNs and TALENs
  • Capable of editing multiple genes simultaneously (multiplexing)
  • Simple and fast design process. No need to reengineer the nuclease for each new target

sgRNA and Cas9 clones

sgRNA/Cas9 Expression Clones and Lentiviral Particles

 

sgRNA expression clones and lentiviral particles are available for targeting virtually any gene in any experimental system. sgRNA clones express either sgRNA only, or sgRNA + Cas9 nuclease in an all-in-one format. Lentiviral clones and particles express sgRNA alone. Cas9-expressing lentiviral clones and particles are available separately.

The CRISPR clone options listed below are intended for using 20 nt sgRNAs with the wild-type Cas9 nuclease from Streptococcus pyogenes (SpCas9). GeneCopoeia also provides alternate CRISPR options, such as Cas9 D10A nickase and other high-fidelity versions of Cas9, SaCas9, and shorter sgRNAs. To purchase reagents for these other options, please contact us for a custom quote.

sgRNA Vector Types

Vector Promoter sgRNA and Cas9 Selection Marker/ Reporter Gene Vector Type Vector Map
All–in-one (Cas9 nuclease + sgRNA) clones
pCRISPR-CG01 U6 sgRNA and CMV-driven Cas9 (wild type) in the same vector Neomycin / mCherry Non-viral get_info
pCRISPR-CG02 U6 sgRNA and CBh-driven Cas9 (wild type) in the same vector N/A Non-viral get_info
pCRISPR-CG04 U6 sgRNA and CMV-driven Cas9 (wild type) in the same vector Neomycin / copGFP Non-viral get_info
pCRISPR-CG07 U6 sgRNA and CBh-driven Cas9 (wild type) in the same vector Puromycin / copGFP Non-viral get_info
pCRISPR-CG08 U6 sgRNA and CBh-driven Cas9 (wild type) in the same vector mCherry Non-viral get_info
sgRNA only clones
pCRISPR-SG01 U6 sgRNA only Hygromycin Non-viral get_info
pCRISPR-LvSG03 U6 sgRNA only Puromycin / mCherry Lentiviral get_info

 

Press the "Search CRISPR-Cas9" button below to search our database of sgRNA clones and lentiviral particles for knocking out more than 40,000 human, mouse, and rat genes.

 

Pre-made sgRNA control particles

Buy Catalog# Product Description Vector Price
Pre-made purified sgRNA control lentiviral particles
LPPCCPCTR01L03-025 Purified Lentifect™ Lentiviral Particles of scrambled sgRNA control CCPCTR01-LvSG03 >1×108 TU/ml sgRNA control lentiviral particles ready for transduction, 25 µL (U6/T7/mCherry/Puro) pCRISPR-LvSG03 $295.0
LPPCCPCTR01L03-100 Purified Lentifect™ Lentiviral Particles of scrambled sgRNA control CCPCTR01-LvSG03 >1×108 TU/ml sgRNA control lentiviral particles ready for transduction, 25 µL (U6/T7/mCherry/Puro) pCRISPR-LvSG03 $725.0

 

 

Cas9 Nuclease Expression Clones

Clones expressing human codon-optimized S. pyogenes Cas9 nuclease or D10A nickase are availabe. These clones do not express sgRNAs.

Buy Catalog# Product Promoter Reporter gene / Selection marker Price
Cas9 nuclease and D10A nickase expression clones
CP-C9NU-01 Cas9 nuclease expression clone CMV mCherry / Neomycin $595*
CP-C9NI-01 Cas9 D10A nickase expression clone CBh N/A $595*
CP-C9NI-02 Cas9 D10A nickase expression clone CMV mCherry / Neomycin $595*
Cas9 nuclease lentiviral expression clones
CP-LvC9NU-01 Cas9 nuclease lentiviral expression clone CMV Neomycin $595*
CP-LvC9NU-02 Cas9 nuclease lentiviral expression clone CMV eGFP / Neomycin $595*
CP-LvC9NU-08 Cas9 nuclease lentiviral expression clone EF1a Puromycin $595*
CP-LvC9NU-09 Cas9 nuclease lentiviral expression clone EF1a eGFP/Neomycin $595*
CP-LvC9NU-10 Cas9 nuclease lentiviral expression clone EF1a eGFP/Hygromycin $595*


* Discount applied when purchased together with a sgRNA clone.

 

Pre-made Cas9 nuclease lentiviral particles

Buy Catalog Product Name Description Price
LPP-CP-LvC9NU-01-100-C Cas9 Nuclease Purified Lentifect™ Lentiviral Particles (100 µl x 1 vial) >1x107 TU/ml Cas9 nuclease lentiviral particles ready for transduction,100 µl (CMV/Neomycin) $725
LPP-CP-LvC9NU-02-100-C Cas9 Nuclease Purified Lentifect™ Lentiviral Particles (100 µl x 1 vial) > 1x107 TU/ml Cas9 nuclease lentiviral particles ready for transduction,100 µl (CMV/eGFP/Neomycin) $725
LPP-CP-LvC9NU-08-100-C Cas9 Nuclease Purified Lentifect™ Lentiviral Particles (100 µl x 1 vial) > 1x107 TU/ml Cas9 nuclease lentiviral particles ready for transduction,100 µl (EF1α/Puromycin) $725
LPP-CP-LvC9NU-09-100-C Cas9 Nuclease Purified Lentifect™ Lentiviral Particles (100 µl x 1 vial) > 1x107 TU/ml Cas9 nuclease lentiviral particles ready for transduction,100 µl (EF1α/eGFP/Neomycin) $725
LPP-CP-LvC9NU-10-100-C Cas9 Nuclease Purified Lentifect™ Lentiviral Particles (100 µl x 1 vial) > 1x107 TU/ml Cas9 nuclease lentiviral particles ready for transduction,100 µl (EF1α/eGFP/Hygromycin) $725

 

Pre-made Cas9 stable cell lines

GeneCopoeia also offers stable cell lines constitutively expressing the CRISPR Cas9 nuclease, enabling you to carry out CRISPR genome editing applications with high efficiency.

The Genome-CRISP™ Cas9 stable cell lines are available pre-made in many human cell lines, and in mouse cell line Neuro2a. The CRISPR Cas9 nuclease is stably integrated into either the human AAVS1 or mouse ROSA26 “Safe Harbor” sites (Figure 1), or randomly via lentiviral-mediated transduction.

For more information, visit our Cas9 stable cell line page.

 

Buy Catalog # Cell line Description Selection marker Cas9 integration site Price, Academic Price, Industry
Human Cas9-expressing stable cell lines
SL501 H1299 Human cell line H1299 stably expressing CRISPR Cas9, single clone Puro AAVS1 $1395 $2695
SL502 HEK293T Human cell line HEK293T stably expressing CRISPR Cas9, single clone Puro AAVS1 $945 $1995
SL503 HeLa Human cell line HeLa stably expressing CRISPR Cas9, single clone Hygro AAVS1 $1395 $2695
SL504 A549 Human cell line A549 stably expressing CRISPR Cas9, single clone Hygro AAVS1 $1395 $2695
SL514 MCF-7 Human cell line MCF-7 stably expressing CRISPR Cas9, single clone Hygro AAVS1 $1395 $2695
SL515 MDA-MB-231 Human cell line MDA-MB-231 stably expressing CRISPR Cas9, single clone Hygro Random $1395 $2695
SL518 HepG2 Human cell line HepG2 stably expressing CRISPR Cas9, single clone Puro AAVS1 $1395 $2695
SL520 AGS Human cell line AGS stably expressing CRISPR Cas9, single clone Hygro Random $1395 $2695
SL521 BXPC-3 Human cell line BXPC-3 stably expressing CRISPR Cas9, single clone Hygro Random $1395 $2695
SL522 SNU-475 Human cell line SNU-475 stably expressing CRISPR Cas9, single clone Hygro Random $1395 $2695
SL523 HT-29 Human cell line HT-29 stably expressing CRISPR Cas9, single clone Hygro Random $1395 $2695
SL525 LoVo Human cell line LoVo stably expressing CRISPR Cas9, single clone Hygro Random $1395 $2695
SL527 KATO111 Human cell line KATO111 stably expressing CRISPR Cas9, single clone Hygro Random $1395 $2695
SL535 SK-BR-3 Human cell line SK-BR-3 stably expressing CRISPR Cas9, single clone Hygro Random $1395 $2695
Mouse Cas9-expressing stable cell lines
SL509 Neuro2a Mouse cell line Neuro2a stably expressing CRISPR Cas9, single clone Hygro ROSA26 $1395 $2695
SL510 Neuro2a Mouse cell line Neuro2a stably expressing CRISPR Cas9, single clone Puro ROSA26 $1395 $2695
SL511 Neuro2a Mouse cell line Neuro2a stably expressing CRISPR Cas9, single clone Neo ROSA26 $1395 $2695

 

  MSDS

 

Note: GeneCopoeia also offers custom-built Cas9-expressing stable cell line services. To inquire, please contact inquiry@genecopoeia.com.

How CRISPR works

The clustered, regularly interspaced, short palindromic repeats (CRISPR) system is bacterial immunity mechanism for defense against invading viruses and transposons. This system has been adapted for highly efficient genome editing in many organisms. Compared with earlier genome editing technologies such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR-Cas–mediated gene targeting has similar or greater efficiency. Genome editing has been used for numerous applications, as hown in Table 1.

Application Description
Gene knockout Permanently modify DNA to eliminate gene function.
Gene mutagenesis Introduce point mutations to an endogenous gene
Gene tagging Add a fusion tag (e.g. luciferase, GFP) to track an endogenous promoter activity or an endogenous protein expression and location
Safe harbor knockin Knockin an exogenous ORF or other genetic element to safe harbor sites of human or mouse genome
Gene activation Activate an endogenous gene expression
Gene repression Repress an endogenous gene expression

Table 1. Applications for CRISPR-mediated genome editing.

 

In the type II CRISPR systems, the complex of a CRISPR RNA (crRNA) annealed to a trans-activating crRNA (tracrRNA) guides the Cas9 endonuclease to a specific genomic sequence, thereby generating double-strand breaks (DSBs) in target DNA. This system has been simplified by fusing crRNA and tracrRNA sequences to produce a synthetic, chimeric single-guided RNA (sgRNA). The sgRNA contains within it a 20 nucleotinde DNA recognition sequence (Figure 1).

 

Illustration-of-CRISPR_Cas9-mediated-genome-editing

Figure 1. Mechanism of CRISPR-Cas9:sgRNA target recognition and cleavage.

 

When the Cas9:sgRNA complex encounters this target sequence in the genome followed by a 3 nucleotide NGG PAM (protospacer adjacent sequence) site, the complex binds to the DNA strand complementary to the target site. Next, the Cas9 nuclease creates a site-specific double-strand break (DSB) 3-4 nucleotides 5' to the PAM. DSBs are repaired by either nonhomologous end joining (NHEJ), which is error-prone, and can lead to frameshift mutations, or by homologous recombination (HR) in the presence of a repair template (Figure 2).

 

sgRNA-guided-gene-engineering_A-s
 

Figure 2. CRISPR-Cas9-based gene engineering. Left. DSBs created by sgRNA-guided Cas9-mediated cleavage are repaired by NHEJ. Right. DSBs created by sgRNA-guided Cas9 nuclease are repaired homologous recombination between sequences flanking the DSB site, thereby causing "knock in" of sequences on a donor DNA.

 

While the CRISPR system provides a highly efficient means for carrying out genome editing applications, it is prone to causing off-target indel mutations. Off-targeting is caused by the ability of the Cas9:sgRNA complex to bind to chromosomal DNA targets with one or more mismatches, or non-Watson-Crick complementarity. The propensity of CRISPR for off-target modification is a significant concern for some researchers who want to avoid results that are potentially confounded by off-target mofication, as well as for those who might be interested in developing CRISPR for gene therapy applications.

Several strategies have been employed to mitigate CRISPR's propensity for off-target genome modification. One such strategy is to use double nickases to create DSBs. The Cas9 D10A mutant is able to cleave only one DNA strand, thereby creating a "nick". When two sgRNAs that bind on opposite strands flanking the target are introduced, two Cas9 D10A nickase miolecules together create a staggered-cut DSB, which is then repaired by either NHEJ or HR (Figure 3). The double nickase strategy has been shown to greatly reduce the frequency of off-target modification. However, double nickases are limited in utility by design constraints; the sgRNAs must be on opposite strands, in opposite orientation to one another, and display optimal activity when spaced from 3-20 nucleotides apart. In addition, the cleavage activity of double nickases tends to be lower than that of standard Cas9:sgRNA. Further, nickases can still cause some degree of off-target indel formation.

 

Figure 3. General scheme of Cas9 double-nickase strategy. From Ran, et al. (2013).

 

Two additional strategies, the use of truncated (17-18 nucleotide) sgRNAs, as well as a Cas9-FokI fusion, also dramatically reduce CRISPR-mediated off-target genome modfication. However, these methods suffer from even futher reductions in on-target activity and/or more severe design constraints compared with the double nickase approach.

Recently, two groups demonstrated that engineering Cas9 variants carrying 3-4 amino acid changes virtually eliminates CRISPR off-target genome modification. These variants still retain high on-target activity, without the design constraints of previous approaches, providing a promising alternative for high-fidelity CRISPR-mediated genome editing.

 

References

  • Horvath P, Barrangou R (January 2010). "CRISPR/Cas, the immune system of bacteria and archaea". Science 327 (5962): 167–70.
  • Marraffini LA, Sontheimer EJ (February 2010). "CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea". Nat Rev Genet 11 (3): 181–190.
  • Hale CR, Zhao P, Olson S, et al. (November 2009). "RNA-Guided RNA Cleavage by a CRISPR RNA-Cas Protein Complex". Cell 139 (5): 945–56.
  • van der Oost J, Brouns SJ (November 2009). "RNAi: prokaryotes get in on the act". Cell 139 (5): 863–5. doi:10.1016/j.cell.2009.11.018.
  • Hale CR, Zhao P, Olson S, et al. (November 2009). "RNA-Guided RNA Cleavage by a CRISPR RNA-Cas Protein Complex". Cell 139 (5): 945–56.
  • Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A., and Charpentie E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptiv bacterial immunity. Science 337, 816–821.
  • Jiang, W., Bikard, D., Cox, D., Zhang, F., and Marraffini, L.A. (2013). RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat.Biotechnol. 31, 233–239.
  • Hsu, P.D., Scott, D.A.,Weinstein, J.A., Ran, F.A., Konermann, S., Agarwala, V.,Li, Y., Fine, E.J., Wu, X., Shalem, O., et al. (2013). DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. Published online July 21, 2013.
  • Ran, et al. (2013). Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity. Cell 154, 1380.

Webinars

CRISPR and TALEN

Title: Genome Editing: How Do I Use CRISPR & TALEN?

Presented Wednesday, April 27, 2016

Genome Editing-the ability to make specific changes at targeted genomic sites-is fundamentally important to researchers in biology and medicine. TALEN and CRISPR have become very popular for modifying specific genome sites, and can be used for many applications, including gene knock out, transgene knock in, gene tagging, and correction of genetic defects. However, researchers are often unaware of some of the work required to identify their desired modification in their cell lines. In this webinar, we discuss what you need to do for genome editing in mammalian cell culture after you have obtained your reagents from GeneCopoeia, the so-called “Downstream work”.

Watch recorded webinar / Download slides

Title: GeneCopoeia CRISPR & TALEN Technology For Genome Modification

Presented Wednesday, March 23, 2016

The ability to make specific changes at targeted genomic sites in complex organisms is fundamentally important to researchers in biology and medicine. Over the past decade, researchers have developed chimeric DNA binding proteins complexed with nucleases to stimulate double strand breaks at defined genomic loci, allowing the ability to insert, delete, and replace genetic information at will. These tools can also be used without nucleases to induce or repress gene transcription. While zinc finger nucleases (ZFNs) stimulated tremendous development in the field, they suffer from significant limitations in design capacity and specificity. Recently, bacterial DNA binding proteins, the Transcription Activator Like (TAL) and the Clustered, Regularly Interspersed, Short Palindromic Repeats (CRISPR) systems have gained favor as genome editing tools. In this 45 minute webinar, we discuss the advantages and disadvantages of each system, and provide information on GeneCopoeia's powerful suite of genome editing products and services.

Watch recorded webinar / Download slides

 

Title: GeneCopoeia CRISPR sgRNA Libraries For Functional Genomics

Presented Wednesday, April 29, 2015

Biomedical researchers are enjoying a Renaissance in functional genomics, which aims to use a wealth of DNA sequence information—most notably, the complete sequence of the human genome—to determine the natural roles of the genes encoded by the genome. As a result, biochemical networks and pathways will be better understood, with the hope of leading to improved disease treatments. Researchers are turning increasingly to CRISPR (clustered, regularly interspaced, short palindromic repeats) for functional genomics studies.  Several groups recently adapted CRISPR for high-throughput knockout applications, by developing large-scale CRISPR sgRNA libraries. GeneCopoeia recently launched a number of smaller, pathway- and gene group-focused CRISPR sgRNA libraries, which offer several key advantages over the whole-genome libraries. In this 40 minute webinar, we discuss the merits and applications for CRISPR sgRNA libraries, how to use CRISPR sgRNA libraries, the advantages of using small, pathway- and gene group-focused libraries, and how GeneCopoeia can help you with your high-throughput CRISPR knockout studies.

Watch recorded webinar / Download slides

Publications

2016

 

2015