- CRISPR Plasmids
- CRISPR Lentivirus
- CRISPR AAV
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
Product Information
CRISPR Plasmids
sgRNA expression plasmids are available for targeting virtually any gene in any experimental system. sgRNA plasmids express either sgRNA only, or sgRNA + Cas9 nuclease in an all-in-one format. Lentiviral clones express sgRNA alone. Cas9-expressing lentiviral clones are available separately.
The CRISPR plasmid options listed below are intended for using 20 ntsgRNAs 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.
Custom sgRNA Expression Plasmids
| Vector | Promoter | sgRNA and Cas9 | Selection Marker/ Reporter Gene | Vector Type | Vector Map |
|---|---|---|---|---|---|
| All-in-one (Cas9 nuclease + sgRNA) clones | |||||
| pCRISPR-CG02 | U6 | sgRNA and CBh-driven Cas9 (wild type) in the same vector | N/A | Non-viral |  |
| pCRISPR-CG04 | U6 | sgRNA and CMV-driven Cas9 (wild type) in the same vector | Neomycin / copGFP | Non-viral |  |
| pCRISPR-CG07 | U6 | sgRNA and CBh-driven Cas9 (wild type) in the same vector | copGFP | Non-viral |  |
| pCRISPR-CG08 | U6 | sgRNA and CBh-driven Cas9 (wild type) in the same vector | mCherry | Non-viral |  |
| pCRISPR-CG12 | U6 | sgRNA and CMV-driven Cas9 (wild type) in the same vector | Neomycin / mCherry | Non-viral |  |
| sgRNA only clones | |||||
| pCRISPR-SG01 | U6 | sgRNA only | Hygromycin | Non-viral |  |
| pCRISPR-LvSG03 | U6 | sgRNA only | Puromycin / mCherry | Lentiviral |  |
Cas9 Nuclease Expression Plasmids
Clones expressing human codon-optimized S. pyogenes Cas9 nuclease or D10A nickase are available. These clones do not express sgRNAs. Click here to search our database of sgRNA plasmids for knocking out more than 45,000 human, mouse, and rat genes.
CRISPR Lentivirus
Custom sgRNA Lentiviral Particles
sgRNA for human, mouse, and rat packaged in high-titer lentivirus.| Vector | Promoter | sgRNA and Cas9 | Selection Marker/ Reporter Gene | Vector Type | Vector Map |
|---|---|---|---|---|---|
| pCRISPR-LvSG03 | U6 | sgRNA only | Puromycin / mCherry | Lentiviral |  |
Pre-made sgRNA Control Particles
Pre-made Cas9 Nuclease Lentiviral Particles
CRISPR AAV
sgRNA and Cas9 AAV Particles
| Vector name | CRISPR | Description | Reporter gene |
|---|---|---|---|
| pCRISPR-AD01 | SaCas9 & sgRNA | All-in-one AAV vector with SaCas9 and sgRNA | N/A |
| pCRISPR-AV20 | sgRNA only | AAV vector expressing sgRNA driven by U6 promoter | N/A |
| pCRISPR-AV22 | sgRNA only | AAV vector expressing sgRNA driven by U6 promoter | eGFP |
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 shown in Table 1.
Table 1. Applications for CRISPR-mediated genome editing.
| 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 knock-in | Knock-in 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 |
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 nucleotide DNA recognition sequence (Figure 1).
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 non-homologous 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).
 |
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 is 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 complementary. 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 modification, 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 molecules 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 modification. However, these methods suffer from even further 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”. Cell139 (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 Marrafï¬ni, 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 speciï¬city 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.
Resources
Technical Notes
- Knockout By TALEN Or CRISPR vs. Knockdown By shRNA or siRNA
- CRISPR-Cas9 Specificity: Taming Off-target Mutagenesis
- Genome Editing: Which Should I Choose, TALEN Or CRISPR?
- Genome Editing In Mammalian Cells: What Do I Do Next?
- Genome Editing: HDR Donors For Gene Knockout, Mutagenesis, Tagging, and Safe Harbor Knock-in
- Genome Editing: Applications For GeneCopoeia CRISPR sgRNA Libraries
- IndelCheck™: A Powerful CRISPR/TALEN Validation & SCreening Tool
- Genome Editing: Cas9 Stable Cell Lines for CRISPR sgRNA Validation, Library Screening, and More
- CRISPR-Cas9 Protein for Highly Efficient Genome Editing With Minimal Off-Target Activity
- Why You Should Know Your Gene’s Accession Number
Webinars
Watch recorded webinar / Download slides Title: Strategies For Effective CRISPR-Mediated Gene Modification Presented Monday, December 3, 2018
CRISPR genome editing-the ability to make specific changes at targeted genomic sites-has transformed research in biology and medicine, due to its precision, ease of use, and relatively low cost. CRISPR’s many applications include gene knock out, gene tagging, correction of genetic defects, and gene activation. In this webinar, we discuss how to use GeneCopoeia’s CRISPR technology for efficient genome editing in mammalian cell lines.
Watch recorded webinar Download slides Title: Harnessing CRISPR for Activation of Gene Expression Presented Wednesday, November 14, 2018
CRISPR-Cas systems have been successfully re-purposed from bacterial adaptive immune mechanisms for the modification of genes. Most CRISPR-mediated gene modifications include permanently changing the genetic code for such applications as gene knockout, mutagenesis, and fusion tagging. However, CRISPR-Cas has also been adapted for stimulating gene transcription, without modifying coding sequences. In this webinar, we discuss how GeneCopoeia CRISPR products and services can help you get more from your transcriptional activation applications.
Watch recorded webinar / Download slides Title: Applications For CRISPR-Cas9 Stable Cell Lines Presented Wednesday, March 22, 2017
The CRISPR-Cas9 system has become greatly popular for genome editing in recent years, due to its ease-of-design, efficiency, specificity, and relatively low cost. In mammalian cell culture systems, most genome editing is achieved using transient transfection or lentiviral transduction, which works well for routine, low-throughput applications. However, for other applications, it would be beneficial to have a system in which one component, namely the CRISPR-Cas9 nuclease, was stably integrated into the genome. In this webinar, we introduce GeneCopoeia’s suite of Cas9 stable cell lines, and discuss the great utility that these cell lines provide for genome editing applications.
Watch recorded webinar / Download slides Title: Safe Harbor Transgenesis in Human & Mouse Genome Editing Presented Wednesday, April 19, 2017
Insertion of transgenes in mammalian chromosomes is an important approach for biomedical research and targeted gene therapy. Traditional lentiviral-mediated transgenesis is effective and straightforward, but its random integration can often be unstable and harm cells. “Safe Harbor” sites in human and mouse chromosomes have been employed recently as an alternative to random, viral-mediated integration because they support consistent, stable expression, and are not known to hamper cell fitness or growth. In this webinar, we will discuss the merits of Safe harbor transgenesis approaches, and how GeneCopoeia’s CRISPR tools for Safe Harbor knock-in can greatly benefit your research.
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.
FAQ
Frequently Asked Questions about CRISPR
Answer: If you are doing simple gene knockouts in humans or mice, you can order CRISPR sgRNAs on our website. All you need to do is go to the TALEN/CRISPR search page, search for your gene, and then choose the appropriate clones that will work for your system. You can also order donor clones for these knockouts from the search results page. Please note that these CRISPR sgRNAs are designed by default to target the protein coding-region as closely as possible to the initiator ATG of the splice variant (accession number) you select. This design strategy does not consider other possible variants of the gene. If the gene has multiple variants and you would like to target one particular exon, one unique variant, multiple variants, or all variants, or if you are doing a different application, such as introducing a point mutation, then you will need to contact us and, after determining what you need, we will send you a custom quote.
Answer: For sgRNA clones (including both all-in-one Cas9/sgRNA clones and sgRNA-only clones) as well as TALE clones, the default delivery format is bacterial stock. You have the option of ordering purified DNA for these clones for an additional charge. For HDR donor clones, the default delivery format is purified DNA. In addition, we offer ready-to-use lentiviral particles for Cas9 and sgRNA delivery.
Answer: The turnaround time for sgRNA clones (including both all-in-one Cas9/sgRNA clones and sgRNA-only clones) is 2-3 weeks. The turnaround time for HDR donor clones depends greatly on the nature of the modification that the clone is being used for. For HDR donor clones used for simple knockout, the turnaround time is 2-4 weeks. Other HDR donor clones, such as those used for fusion tagging or mutagenesis, can take 6-8 weeks, but can also take longer.
Answer: Yes. We sequence the inserts of each CRISPR sgRNA, and provide you with datasheets that show the full sequence of each clone (including HDR donor clones), a map, restriction enzyme digestions sites, and suggested sequencing primers. To obtain these datasheets, you just need to visit our datasheet download page on our website. You will need an account on our website, your catalog number(s), and your sales order number.
Answer: In the presence of drug, the only way for cells to survive is to integrate the plasmid into the chromosome, so it is possible to get drug-resistant clones that were transfected only with the donor plasmid. However, such integration is random. CRISPR increases donor targeting frequency by several orders of magnitude, and CRISPR-mediated donor integration is usually targeted.
Answer: Yes. For this you will need a donor that is homologous to your locus on either side of the base you want to mutate. The donor can be either a plasmid or a single-strand oligonucleotide. The donor is co-transfected with Cas9 and sgRNA. Generation of a double strand break (DSB) leads to repair of the break by homology-directed repair (HDR) using the donor as a template.
GeneCopoeia recommends our donor plasmid design and construction service. We will construct a donor plasmid that contains a defined modification, flanked by a selectable marker such as puromycin resistance, and homologous arms from your target region. The donor may or may not also include a fluorescent reporter such as GFP. The markers can be flanked by loxP sites, to permit Cre-mediated removal, if desired. Use of a GeneCopoeia-designed donor plasmid allows you to select for edited clones and reduces the number of clones required for screening. You can also purchase our donor cloning vectors for do-it-yourself donor clone construction.
Answer: There are two strong reasons to use HDR for genome editing: 1) It is precise and controllable. Any desired change can be implemented; 2) You will be able to use a knocked-in marker (such as drug resistance or fluorescence), which will greatly facilitate your ability to identify candidate clones that have the modification you want. In addition, some applications, such as fusion tagging, require using HDR.
GeneCopoeia recommends our donor plasmid design and construction service. We will construct a donor plasmid that contains a defined modification, flanked by a selectable marker such as puromycin resistance, and homologous arms from your target region. The donor may or may not also include a fluorescent reporter such as GFP. The markers can be flanked by loxP sites, to permit Cre-mediated removal, if desired. Use of a GeneCopoeia-designed donor plasmid allows you to select for edited clones and reduces the number of clones required for screening. You can also purchase our donor cloning vectors for do-it-yourself donor clone construction.
Answer: Our genome editing products can be used for virtually all species. Our standard plasmids for CRISPR are designed for work in mammalian cells. In addition, these plasmids can be used as templates for T7 promoter-driven in vitro transcription, for introduction into mice, zebrafish, Drosophila, and many other model organisms. Further, we can generate custom constructs that can be used in a wide variety of organisms.
Answer: Yes. The donor must be present when the double strand break (DSB) is generated in order to be used as a repair template. Otherwise, the cell must use non-homologous end joining (NHEJ) to repair the DSB, because unrepaired DSBs are lethal.
Answer: Our CRISPR plasmids typically only integrate into the host genome at very low frequency in transfection experiments. However, after clonal selection for edited cells, we recommend screening clones for those which have lost the plasmids. This can be done by testing clones to see if they have become sensitive to the antibiotic of the resistance gene on the plasmid, or if they no longer express the plasmid’s fluorescent marker (where applicable). Our lentiviral clones, when packaged into lentiviral particles and used to infect cells, are expected to integrate randomly into chromosomes.
Answer: The presence of the CMV or other promoters driving expression of the Cas9 or TALE coding sequences permits expression from the plasmid DNA. sgRNA transcription is driven by the U6 promoter. We recommend that you use the most efficient method for your cell type of interest. The following are all acceptable approaches for delivering our genome editing tools into cells:
1. Standard transfection. GeneCopoeia recommends our EndoFectin™ transfection reagents. Transfection can be used for either DNA, RNA, or Cas9 nuclease protein.
2. Electroporation, which can be used for either DNA, RNA, or Cas9 nuclease protein.
3. Lentiviral transduction (cannot be used with HDR donors).
4. Micro-injection of either DNA, mRNA, .or Cas9 nuclease protein. The plasmids can be used as templates for in vitro transcription of RNA, or synthetic RNA can be used.
Answer: If you are using CRISPR to create mutations in your gene of interest without the use of a homologous donor, then you will need to undertake much time-consuming, labor-intensive downstream work in order to identify edited clones. After transfection, you will need to isolate many colonies grown up from single cells. Next, the screening procedure depends on the nature of the modification, as described below:
1. If you are making an insertion or deletion, the easiest way to screen your cells is by PCR using primers flanking the modified site, provided that the insertion or deletion is large enough to detect by standard agarose gel electrophoresis.
2. For very small insertions or deletions, you can screen your clones using GeneCopoeia’s IndelCheck™ T7 endonuclease I assay, which is a method that detects mutations by cleaving double stranded DNA containing a mismatch. You can also screen using Sanger sequencing of PCR products. Ultimately, Sanger sequencing needs to be done to verify the presence of the modification.
3. If you are introducing a point mutation, then you can use either real-time PCR or Sanger sequencing to detect the modification.
4. If the modification you are introducing creates or destroys a restriction enzyme site, then enzyme cleavage of PCR products can be used to distinguish between modified and unmodified alleles.
5. Finally, either Sanger sequencing of PCR products or Next Generation sequencing of whole genomes can be used to screen for modifications. Regardless of which screening method you choose, it is also important that you are able to determine whether only a portion or all of the alleles have been modified.
In order to reduce the amount of time and effort required to identify edited clones, GeneCopoeia recommends our donor plasmid design and construction service. We will construct a donor plasmid that contains a defined modification, flanked by a selectable marker such as puromycin resistance, and homologous arms from your target region. The donor may or may not also include a fluorescent reporter such as GFP. The markers can be flanked by loxP sites, to permit Cre-mediated removal, if desired. Use of a GeneCopoeia-designed donor plasmid allows you to select for edited clones and reduces the number of clones required for screening. You can also purchase our donor cloning vectors for do-it-yourself donor clone construction.
Answer: For safe harbor integration, we recommend PCR using primers designed to amplify the recombination loci, or Southern blotting, which can rule out random integration. All GeneCopoeia Safe Harbor Gene knock-in Kits contain PCR primer pairs intended for this exact purpose. For other editing experiments using HDR, junction PCR can be be used to identify edited sites. The junction PCR method will will only yield product if donor integration occurred at the correct genomic site. Southern blotting or PCR using primers recognizing the plasmid backbone can also be used to rule out random integration.
Answer: Yes. Even though frameshifts are not possible with miRNAs and other noncoding RNAs, an indel occurring in a critical region, such as the mature sequence of a miRNA, should be enough to abolish its function.
Answer: The vector backbones of our CRISPR sgRNAs are designed to not replicate in the host. These plasmids, which are transiently transfected, will typically be lost after several rounds of cell division and will not further affect the host cell. After transfection, cells are plated at low density to promote the formation of single colonies. These colonies should be screened to ensure that they have lost the plasmid(s). This can be done by testing clones to see if they have become sensitive to the antibiotic of the resistance gene on the plasmid, or if they no longer express the plasmid’s fluorescent marker (where applicable). However, even if the CRISPR plasmid integrates, it can no longer cut the site after it is edited, because NHEJ destroys the sgRNA recognition site. To be completely assured that the transfection is transient, we recommend delivering RNA or Cas9 nuclease protein instead of plasmid DNA. If you are using HDR, we recommend engineering synonymous mutations into the donor to destroy the sgRNA recognition site without changing the amino acid sequence.
Answer: Yes. CRISPR has been shown to be able to disrupt multiple copies at once. The efficiency varies depending on different factors, such as cell type, transfection efficiency and CRISPR activity.
Answer: Yes. We have the reagents for the Cas9 D10A nickase, and have successfully tested our double nickase designs. However, in order to create mutagenic DSBs, the nickase requires the correct targeting of two appropriately-spaced sgRNAs on opposite strands, flanking the break site. Because proper sgRNA targeting requires the presence of the N-G-G “PAM” site immediately following the recognition site, it might not always be possible to use the nickase for DSB formation. There are also “high-fidelity” variants of Cas9 nuclease that edit genes with greater specificity than wild type Cas9, but sometimes with reduced efficacy and with increased design constraints. However, since these high fidelity variants use only one sgRNA, they are easier to work with than Cas9 nickases. In addition, using Cas9 nuclease protein instead of a DNA plasmid could help reduce off-target activity.
Answer: Yes. To create a DSB, the nickase requires the correct targeting of two appropriately-spaced sgRNAs on opposite strands, flanking the break site. This is sufficient to stimulate HDR between the target site and the donor. While this method has the advantage of potentially fewer off-target NHEJ-mediated mutations, since single strand nicks are repaired with higher fidelity than DSBs, it is not without limitations. Proper sgRNA targeting requires the presence of the N-G-G “PAM” site immediately following the recognition site. Therefore, it might not always be possible to use the nickase for HDR. There are also “high-fidelity” variants of Cas9 nuclease that edit genes with greater specificity than wild type Cas9, but sometimes with reduced efficacy and with increased design constraints. However, since these high fidelity variants use only one sgRNA, they are easier to work with than Cas9 nickases. In addition, using Cas9 nuclease protein instead of a DNA plasmid could help reduce off-target activity.
Answer: Yes. We offer Cas9 nuclease protein that can be used to form a ribonucleoprotein (RNP) complex with either synthetic or in vitro transcribed sgRNA. The RNP can then be used to transfect cells with our CRISPR-Fectin™ transfection reagent.
Answer: We only sell plasmids containing our custom-designed CRISPR sgRNAs. If you need a negative control, we also sell a CRISPR plasmid containing a scrambled sgRNA.
Answer: Yes.
Answer: Yes. There is a double mutant of the Cas9 nuclease that completely abolishes nuclease activity. This mutant can be fused to a transcriptional modulator such as VP64 and targeted to specific genes. You can also use the catalytically dead Cas9 with properly-designed sgRNAs to repress, or interfere with, gene expression. For more information, please visit our pages on CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi).
Answer: Yes. We have both non-viral and lentiviral formats. We also have custom lentiviral particle production services, in which we can provide you with lentiviral particles expressing both Cas9 and sgRNAs.
Answer: Unfortunately, no. Lentiviruses enter cells as RNA, but HDR donors must enter the cells as DNA at the same time as Cas9 and the sgRNAs.
Answer: Cas9 stable cell lines are primarily useful for lentiviral CRISPR delivery and CRISPR sgRNA library screening. sgRNA library screening is typically done with lentivral delivery. In principle, it is possible to either co-infect your cell line with one Cas9-expressing lentivirus and with an sgRNA-expressing lentivirus, or with a single “all-in-one” lentivirus (Cas9 and sgRNA expressed in the same virus). However, in practice, due to the large size of the Cas9 gene (4.4 kb), the titers of Cas9-expressing lentiviruses tend to be much lower than sgRNA-expressing lentiviruses. Therefore, we recommend using a cell line that has previously had Cas9 stably integrated in the genome for infection with lentiviral-based sgRNA libraries. GeneCopoeia carries a large collection of pre-made Cas9-expressing stable cell lines, as well as stable cell lines expressing the machinery for CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi). You can also purchase reagents that you can use to build a Cas9 stable cell line yourself, or you can have GeneCopoeia create a custom Cas9-expressing stable cell line as a custom cell service project.
sgRNA Libraries-related Questions
Answer: Lentiviral particles, transfection-ready DNA, and bacterial stock.
Answer: Yes. Currently, our sgRNA libraries are available standard as pools. However, if you wish to receive your libraries as individually arrayed clones, simply for a custom quote.
Answer: Yes. However, we strongly recommend that you use a cell line that is stably expressing Cas9 to transduce sgRNA-expressing lentiviral particles with, in order to best maintain consistent representation of each sgRNA in the library. To save you time and effort, GeneCopoeia provides a large collection of Cas9-expressing stable cell lines that are ideally suited for sgRNA library screening. If you would like to create a Cas9-expressing stable cell line yourself, you should first transduce your cells with Cas9-expressing lentiviral particles. Once you have established the stable Cas9-expressing cell line, use the sgRNA libraries for transduction.
Answer: Yes. The lentiviral plasmids are “dual-use”, so that they can either be packaged into lentiviral particles or transfected into cells by standard transfection methods.
Answer: Our sgRNA representation does not need to be validated by Next Generation Sequencing. Each library is small compared with the genome-wide libraries, and each sgRNA clone is constructed individually, cultured in E. coli individually, then pooled as E. coli in approximately equal amounts. From those pools we prepare DNA and then, if necessary, lentiviral particles.