CRISPR-Cas9 genome editing
IDT가 제공하는 Alt-R® CRISPR-Cas9 System은 2-part RNA CRISPR/Cas9 system으로
기존에 방법에 비해 높은 효율을 제공합니다. 이와 더불어 새로운 S.p.Cas9 Nuclease V3를 이용하여 더 강력한 효과를 볼 수 있습니다.
• RNP 방식의 빠른 transfection efficiency, Lipofection 또는 Electroporation 방식 모두 사용 가능
CRISPR-Cas9 genome editing
Point, click, edit. Guaranteed.*
The Alt-R® CRISPR-Cas9 System includes all of the reagents needed for successful genome editing based on the natural S. pyogenes CRISPR-Cas9 system. Discover what makes the Alt-R CRISPR-Cas9 system best in class.
* See Ordering section for details.
CRISPR-Cas9 crRNA, crRNA XT, and sgRNA
Guide RNAs (gRNAs) contain the target-specific sequence for guiding Cas9 protein to a genomic location. Choose from crRNAs or crRNA XT, which form a gRNA duplex with tracrRNA, or sgRNAs, which are single RNA molecules comprised of both crRNA and tracrRNA sequences. Under challenging experimental conditions (e.g., high nuclease environments or with Cas9 mRNA), use crRNA XT or sgRNA, which have additional chemical modifications for the highest level of stability and performance.
Guaranteed editing with predesigned gRNA designs
We guarantee* our predesigned guide RNAs targeting human, mouse, rat, zebrafish, or nematode genes. For other species, use our proprietary algorithms to design custom guide RNAs. If you have protospacer designs of your own or from publications, use our design checker tool to assess their on- and off-targeting potential before ordering guide RNAs that are synthesized using our Alt-R gRNA modifications.
Use your own designs
If you have protospacer designs of your own or from publications, we recommend using the design checker tool to assess on- and off-targeting potential before ordering guide RNAs that are synthesized using our Alt-R guide RNA modifications. For designs that do not require this analysis, you may directly order your user-defined crRNA or sgRNA with our tube and plate ordering buttons.
Universal 67mer tracrRNA that contains proprietary chemical modifications conferring increased nuclease resistance. Hybridizes to crRNA to activate the Cas9 enzyme. More info »
Custom CRISPR solutions
Don’t see what you’re looking for? We are continually expanding our CRISPR product line, and we may have what you need. If you are interested in custom libraries, other CRISPR enzymes, formulations, or other CRISPR tools, email our CRISPR experts today to discuss customized solutions for your research: CRISPR@idtdna.com.
* We guarantee that predesigned Alt-R CRISPR-Cas9 guide RNAs will provide successful editing at the target site, when delivered as a ribonucleoprotein complex as described in the Alt-R User Guides, using Alt-R CRISPR-Cas9 guide RNAs (crRNA:tracrRNA duplex or sgRNA) and either Alt-R S.p. Cas9 nuclease or Alt-R S.p. HiFi Cas9 nuclease. Analysis of editing must be at the DNA level, such as with the Alt-R Genome Editing Detection Kit or DNA sequencing. If successful editing is not observed for a predesigned guide RNA while an appropriate positive control is successful, a one-time “no-cost” replacement of the predesigned Alt-R CRISPR-Cas9 guide RNA will be approved, upon discussion with our Scientific Applications Support team (email@example.com). This guarantee does not extend to any replacement product, or to any other incurred or incidental costs or expenses.
Note: Performance data for the Alt-R® Genome Editing Detection Kit are available here.
Alt-R® CRISPR-Cas9 RNA triggers are more potent than single guide RNAs
Our optimized Alt-R CRISPR-Cas9 RNAs consistently outperform other CRISPR RNA formats for triggering CRISPR-Cas9 genome modifications, resulting in superior on-target genome editing. Figure 1 shows a comparison of on-target editing efficiency provided by 5 different formats of Cas9 trigger RNAs, as measured by a T7EI assay. Note, that T7EI does not detect single base indels  and underestimates non-homologous end-joining editing events.
Figure 1. Optimized Alt-R CRISPR RNAs improve Cas9 editing efficiency compared to other guide RNA molecules. Alt-R CRISPR RNAs, S. pyogenes native CRISPR RNAs, in vitro transcribed (IVT) single-guide RNAs (sgRNA), and sgRNAs expressed from a 2.7 kb expression plasmid or gBlocks® Gene Fragments were designed to recognize 4 sites within the human HPRT gene (38087 AS, 38509 S, 38285 AS, and 38636 AS). The RNA duplexes or sgRNAs were reverse transfected using Lipofectamine® RNAiMAX™ reagent (Thermo Fisher) into a HEK-293–Cas9 cell line that stably expresses S. pyogenes Cas9. Optimal doses that give maximal editing were transfected: Alt-R RNAs, S. pyogenes RNAs, and IVT sgRNA (30 nM), gBlocks Gene Fragment sgRNA (3 nM), sgRNA expression plasmid (100 ng). Genomic DNA was isolated, and editing was measured by PCR amplification of target sites, followed by cleavage with T7 endonuclease I (Alt-R Genome Editing Detection Kit) and analysis using the Fragment Analyzer™ (Advanced Analytical). Alt-R CRISPR RNAs performed well at all sites tested, while other guide RNA formats performed well at some sites and not others. Results from IVT sgRNAs were affected by cellular toxicity.
Potent editing with the Alt-R S.p. Cas9 V3
The Alt-R CRISPR-Cas9 System includes the potent Alt-R S.p. Cas9 V3. When the Alt-R S.p. Cas9 V3 is combined with the Alt-R CRISPR crRNA and tracrRNA into a ribonucleoprotein (RNP), the system outperforms other editing approaches (Figure 2). RNP transfections also provide optimal control of dose of editing complexes, and the non-renewable Cas9 RNP is cleared after a short duration by endogenous mechanisms, limiting off-target editing.
Figure 2. Lipofection of Alt-R CRISPR-Cas9 System Components as a ribonucleoprotein (RNP) outperforms other transient CRISPR-Cas9 approaches. Alt-R CRISPR HPRT Control crRNA for human, mouse, or rat were complexed with Alt-R CRISPR tracrRNA. Resulting complexes were transfected with Cas9 expression plasmid, Cas9 mRNA, or as part of a Cas9 RNP (containing Alt-R S.p. Cas9 V3, pre-complexed with the crRNA and tracrRNA) into human (HEK-293), mouse (Hepa1-6), or rat (RG2) cell lines. The Cas9 RNP outperformed the other transient Cas9 expression approaches, and performed similar to reference HEK293-Cas9 cells that stably express S. pyogenes Cas9.
View related data shared by Dr Eric Kmiec (Gene Editing Institute, Helen F. Graham Cancer Center and Research Institute, Christiana Care Health System, Wilmington, DE, USA):
Optimizing crRNA:tracrRNA lengths improves gene editing performance
Systematic variation of crRNA and tracrRNA length led to the development of a crRNA:tracrRNA complex that shows improved gene editing in mammalian cell culture with S. pyogenes Cas9 (Figure 3). A 67 nt tracrRNA paired with a 36 nt crRNA (Figure 3, orange arrow) provided the highest editing efficiency. In addition to improved activity, the shorter lengths of the synthetic Alt-R CRISPR RNAs make them more amenable to high throughput manufacturing compared to the longer, native crRNA and tracrRNA. In addition, chemical synthesis offers the opportunity to introduce chemical modifications for additional properties such as increased resistance to nucleases and reduced immunogenicity.
Figure 3. Shorter crRNA:tracrRNA lengths improve on-target genome editing. Varying lengths of crRNAs targeting HPRT 38285-AS were hybridized with tracrRNAs of different lengths. crRNA:tracrRNA complexes were reverse transfected using Lipofectamine® RNAiMAX™ reagent (Thermo Fisher), into a HEK-293–Cas9 cell line that stably expresses S. pyogenes Cas9. Genomic DNA was isolated and editing measured by PCR amplification of target sites, followed by cleavage with T7 endonuclease I (Alt-R Genome Editing Detection Kit), and analysis using the Fragment Analyzer™ (Advanced Analytical).
Robust Alt-R CRISPR-Cas9 System may eliminate the need for a crRNA design tool
While algorithms exist to guide design of the crRNA 19–20 nt protospacer element sequence, few produce designs that correlate consistently with strong genomic editing activity. However, our empirical data demonstrate that the Alt-R CRISPR-Cas9 System is robust, and even without specific design selection, use of these optimized RNAs results in strong on-target editing for the majority of target sites. Figure 4 illustrates the high genome editing function achieved by crRNA:tracrRNA complexes targeting 553 sites across 6 exons.
Figure 4. Alt-R CRISPR-Cas9 System functions well across many sites. Alt-R crRNAs were designed to 553 PAM adjacent sites in 4 distinct genes (HPRT, EMX1, STAT3, and Dicer). Complexes were reverse transfected using Lipofectamine® RNAiMAX™ reagent (Thermo Fisher) into a HEK-293–Cas9 cell line that stably expresses S. pyogenes Cas9. Genomic DNA was isolated and editing measured by PCR amplification of target sites, followed by cleavage with T7 endonuclease I (Alt-R Genome Editing Detection Kit) and analysis using the Fragment Analyzer™ (Advanced Analytical).
Mutation profiles vary by RNA trigger
The type of CRISPR RNA trigger used will affect the proportion of mutation types that result from genome editing. Note that because T7EI often does not detect small indels, the T7EI mismatch endonuclease assays underestimates single-base insertions and deletions. Therefore, we performed Sanger sequencing of PCR amplicons from CRISPR-Cas9 transfections to investigate the editing efficiency and mutation types observed with different Cas9 trigger types (Figure 5). Noteworthy is that single fusion sgRNAs generated more multiple base insertions, including insertions of DNA fragments of several hundred nucleotides, often corresponding to large sections of the sgRNA expression cassette. While large insertions are generally a good method for gene silencing, users may be concerned by the possibility of introducing the U6 promoter usually used in those constructs close to their target gene
Figure 5. Type and proportion of mutations observed differs with CRISPR guide RNA technologies. Four different CRISPR guide RNA formats targeting the HPRT 38285-AS site were transfected into HEK-293–Cas9 cells that stably express S. pyogenes Cas9, or wild-type HEK-293 cells (used only for delivery of the large plasmid). Genomic DNA was isolated and editing measured by PCR amplification of target sites, followed by cleavage with T7 endonuclease I (T7EI; Alt-R Genome Editing Detection Kit), and analysis using the Fragment Analyzer™ (Advanced Analytical). Sanger sequencing was performed for cloned amplicons (“n”) to identify the type of sequence mutation produced by CRISPR editing. Mutation percentages based on T7EI assays are shown for comparison.
Alt-R CRISPR-Cas9 RNAs elicit less toxicity and innate immune response compared to in vitro transcribed guide RNA alternatives
Transfection of long in vitro transcribed (IVT) RNAs has been shown to elicit an innate immune response in our laboratories. This response can result in high cell death due to cytotoxicity. Our use of a particularly robust HEK-293–Cas9 cell line that constitutively expresses Cas9 has allowed us to compare cellular toxicity and immune response activation (Figure 6) by Alt-R RNAs and IVT RNAs. We observed high levels of activation of stress response genes such as IFIT1 (P56) and OAS2 (as well as IFITM1, RIGI, and OAS1; not shown) related to the innate immune response in cells challenged with IVT RNA triggers. These genes were not activated in cells transfected with Alt-R CRISPR-Cas9 RNAs.
Figure 6. The Alt-R CRISPR-Cas9 system does not trigger a cellular immune response. Alt-R CRISPR-Cas9 RNAs and corresponding in vitro transcribed (IVT) RNAs (triphosphate removed) designed to 12 HPRT1 sites were reverse transfected into HEK-293–Cas9 cells that stably express S. pyogenes Cas9. 24 hr after transfection, expression levels of IFIT1 (A) and OAS2 (B), common stress response genes, were assayed. (A) qPCR amplification curves quantifying IFIT1 expression shows strong induction of IFIT1 by IVT RNA, but not Alt-R CRISPR-Cas9 RNA. (B) qPCR amplification data for OAS2 expression shows that IVT RNA cells have measurable induction of OAS2, whereas OAS2 levels in the Alt-R CRISPR-Cas9 RNA cells are at baseline. Similar results were seen for targets in 3 other genes, IFITM1, RIGI, and OAS1.
Protospacer element size is critical for editing efficacy
There are reports in the literature suggesting that CRISPR-Cas9 nuclease specificity can be improved by using truncated guide RNAs . For example, 17-base protospacer elements have been reported to reduce off-target effects. We investigated how shortening protospacer element length would affect CRISPR-Cas9 nuclease specificity (Figure 7). crRNAs with protospacer element lengths of 17–20 bases were designed to 12 distinct HPRT target sites and genome editing efficiency measured using a T7EI cleavage assay (Alt-R Genome Editing Detection Kit). 20-base protospacer elements were optimal, with 19 bases providing similar strong editing efficacy in most cases. Editing efficiency was greatly reduced when 17- and 18-base protospacers were used.
Figure 7. 19–20 nt protospacer element provides optimal genome editing. crRNAs with varying protospacer element lengths (17–20 nt) were designed to 12 distinct HPRT target sites. crRNA:tracrRNA complexes were reverse transfected using Lipofectamine® RNAiMAX™ reagent (Thermo Fisher) into a HEK-293 cell line stably expressing S. pyogenes Cas9. Genomic DNA was isolated and editing measured by PCR amplification of target sites, followed by cleavage with T7 endonuclease I (Alt-R Genome Editing Detection Kit) and analysis using the Fragment Analyzer™ (Advanced Analytical). At all but one of the 12 target sites, crRNAs with 19- and 20-base protospacer elements produced the greatest amount of genomic editing. Each of the 12 data points for each category represent the average of 3 biological replicates, with the exception of one data point in the 19 base category that is composed of 2 biological replicates.
Alt-R CRISPR-Cas9 tracrRNA can be fluorescently labeled without negatively affecting activity of the guide RNA
ATTO™ 550 fluorescent dyes (ATTO-TEC Gmbh) can be added to the tracrRNA without negatively affecting genome editing results.
The labeled tracrRNA is an effective tool for monitoring transfection efficiency and concentrating transfected cells using FACS (fluorescence-activated cell sorting) analysis.
Figure 8. Addition of ATTO 550 fluorescent dye to Alt-R CRISPR-Cas9 tracrRNA does not affect genome editing performance. Guide RNA complexes (30 nM of crRNA complexed to either unlabeled or fluorescently labeled tracrRNA) targeting two sites in the HRPT gene were reverse transfected into Cas9-expressing HEK-293 cells using RNAiMAX™ reagent (Thermo Fisher). 48 hr after transfection, genomic DNA was isolated from cells using QuickExtract™ solution (Epicentre), and total editing efficiency was measured using the Alt-R Genome Editing Detection Kit (T7 endonuclease I assay).
Monitor transfection with a fluorescently labeled tracrRNA
Alt-R CRISPR-Cas9 tracrRNA – ATTO 550 is an effective tool for monitoring transfection efficiency using microscopy. Visual inspection of guide RNA transfection can be helpful when optimizing transfection conditions or should you need to troubleshoot experiments. For tips on using the labeled tracrRNA, see the Application note in the Support section of this web page.
Figure 9. Detection of fluorescently labeled tracrRNA by fluorescence microscopy. HEK-293 cells stably expressing Cas9 nuclease were reverse transfected (RNAiMAX™ reagent, Thermo Fisher Scientific) with Alt-R CRISPR-Cas9 crRNA (unlabeled) complexed with Alt-R CRISPR-Cas9 tracrRNA – ATTO 550 (final concentration of 10 nM). Images were taken 48 hours after transfection. Magnification: 10X.
Increase editing efficiency to simplify downstream analysis of genome editing with a fluorescently labeled tracrRNA
Through use of fluorescence-assisted cell sorting (FACS), Alt-R CRISPR-Cas9 tracrRNA – ATTO 550 allows enrichment of transfected cells, which are more likely to contain CRISPR genome editing. For tips on using the labeled tracrRNA, see the Application note in the Support section of this web page.
Figure 10. Enrichment of sorted cells leads to higher editing efficiencies. HEK-293 and Jurkat cells were transfected (Neon® electroporation system, Thermo Fisher) with 0.5 µM ribonucleoprotein (RNP: Alt-R S.p. Cas9 V3 complexed with Alt-R CRISPR-Cas9 crRNA and Alt-R CRISPR-Cas9 tracrRNA – ATTO 550) and carrier DNA (Alt-R Cas9 Electroporation Enhancer). Cells subjected to RNP, but without electroporation, were used as background controls and were used to set the gates during FACS. Cells were sorted 24 hr post-transfection, and positive cells were re-plated and grown for an additional 48 hr. A population of the cells was not sorted, but simply re-plated, to serve as the unsorted control. Genomic DNA was isolated using QuickExtract™ solution (Epicentre) after cell incubation for 72 hr. Total editing efficiency was measured using the Alt-R Genome Editing Detection Kit (T7 endonuclease I assay).
Optimal fluorescence-activated cell sorting (FACS) occurs 24 hr after transfection
Optimal flow cytometric resolution occurs at 24 hr after transfection with RNP containing fluorescently labeled Alt-R CRISPR-Cas9 tracrRNA – ATTO 550. A slight decrease of positively sorted cells was observed at 48 hr and a significant decrease was observed at 72 hr. Cell can be sorted and re-plated for an additional 24–48 hr to allow time for genome editing to occur. For additional tips on using the labeled tracrRNA, see the Application note in the Support section of this web page.
Figure 11. Flow cytometric resolution decreases over time after transfection. Jurkat and HEK-293 cells were transfected (Neon® electroporation system, Thermo Fisher) with 0.15 or 1.5 µM ribonucleoprotein (RNP: Alt-R S.p. Cas9 V3 complexed with Alt-R CRISPR-Cas9 crRNA and Alt-R CRISPR-Cas9 tracrRNA – ATTO 550) and carrier DNA (Alt-R Cas9 Electroporation Enhancer). Cells were sorted 24, 48, or 72 hr after transfection. Prior to sorting, cells were washed once with PBS containing 1% FBS. Histogram plots show fluorescence intensities and percent positive cells.
Carrier DNA improves CRISPR-Cas9 editing efficiency
The Alt-R Cas9 Electroporation Enhancer is a Cas9-specific, single-stranded, carrier DNA that is optimized to work with the Amaxa® Nucleofector® device (Lonza) and Neon® System (Thermo Fisher) to increase transfection efficiency and thereby increase genome editing efficiency. The enhancer is computationally designed to be non-homologous to human, mouse, or rat genomes.
The amount of improvement in editing efficiency will vary by cell type. In some cases, use of the Alt-R Cas9 Electroporation Enhancer allows you to decrease the amount of Cas9 RNP required for optimal editing efficiency (Figure 12). A reduction in RNP is advantageous because of possible improvements in cell survival and lowered risks of potential off-target editing.
Figure 12. Alt-R™ Cas9 Electroporation Enhancer improves CRISPR editing efficiency in ribonucleoprotein (RNP) electroporation experiments. K562 (A), Jurkat (B), and HEK-293 (C) cells were transfected (Amaxa® System, Lonza) with 0.125–4 µM RNP (Alt-R S.p. Nuclease 3NLS complexed with Alt-R CRISPR-Cas9 crRNA and tracrRNA). Electroporation reactions were performed in the presence (dark blue) or absence (light blue) of 4 µM Alt-R Cas9 Electroporation Enhancer.
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