CRISPR-Cas9 genome editing

Increase efficiency of genome editing using the
Alt-R® CRISPR-Cas9 System. Now available with Alt-R S.p. HiFi Cas9 Nuclease 3NLS!

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, the Alt-R CRISPR-Cas9 System offers numerous advantages over alternative methods:

  • Higher on-target potency than other CRISPR systems
  • Precise control of editing complex delivery with Cas9 ribonucleoprotein
  • Efficient delivery of the RNP with lipofection or electroporation
  • No toxicity or innate immune response activation as observed with in vitro transcribed Cas9 mRNA and sgRNAs

Alt-R CRISPR-Cas9 RNAs with increased nuclease resistance

Alt-R CRISPR-Cas9 crRNA and tracrRNA include proprietary chemical modifications that protect the RNA from degradation by cellular RNases and further improve on-target editing performance. The modifications are included automatically and do not require changes to the ordering process.

New! Alt-R HiFi Cas9 Nuclease with improved specificity

The new Alt-R HiFi Cas9 Nuclease has similar on-target potency to wild-type S.p. Cas9, but with significantly reduced off-target effects, allowing for incredibly precise genome editing, even under challenging conditions. More info »

We highly recommend using the following:

  • One of the Alt-R S.p. Cas9 nucleases combined with the Alt-R CRISPR-Cas9 crRNA and tracrRNA to generate a ribonucleoprotein (RNP) editing complex for very high editing efficiency across most target sites. View the Cas9 Ribonucleoprotein User Guides for guidance about delivery of the RNP complex into your cell lines by transfection or electroporation.
  • The positive and negative controls.
  • The electroporation enhancer, if your cells require electroporation.

CRISPR-Cas9 crRNA

Customer-defined crRNA that will bind to 20 bases on the DNA strand that is opposite to the NGG, PAM sequence. Prices shown are for each crRNA and plates require a minimum order of 24. More info »
ProductPricingLength
Alt-R® CRISPR-Cas9 crRNA, 2 nmol$95.00 USD19 - 20 BasesOrder
Alt-R® CRISPR-Cas9 crRNA, 10 nmol$125.00 USD19 - 20 BasesOrder
ProductPricingLength96 Well384 Well
Alt-R® CRISPR-Cas9 crRNA, 2 nmol, Plate$80.00 USD / well19 - 20 BasesOrderOrder
Alt-R® CRISPR-Cas9 crRNA, 10 nmol, Plate$105.00 USD / well19 - 20 BasesOrderOrder

Heading

Note: Ordering information for the Alt-R® Genome Editing Detection Kit is available here.

Additional reagents and controls

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 contact our CRISPR experts today to discuss customized solutions for your research: CRISPR@idtdna.com.

Alt-R® CRISPR-Cas9 System

Simple, 3-step delivery of ribonucleoprotein complexes (crRNA:tracrRNA:Cas9)

Alt-R CRISPR-Cas9 System using ribonucleoprotein transfection

Figure 1. Overview of Alt-R CRISPR-Cas9 System experiments for ribonucleoprotein delivery by lipid-mediated transfection, electroporation, or microinjection.



CRISPR-Cas9 genome editing methods use a Cas9 endonuclease to generate double-stranded breaks in DNA. Cas9 endonuclease requires a CRISPR RNA (crRNA) to specify the DNA target sequence, and the crRNA must be combined with the transactivating crRNA (tracrRNA) to activate the endonuclease and create a functional editing ribonucleoprotein complex (Figure 1). After cleavage, DNA is then repaired by non-homologous end-joining (NHEJ) or homology-directed recombination (HDR), resulting in a modified sequence. Alt-R CRISPR-Cas9 reagents and kits provide essential, optimized tools needed to use this pathway for genome editing research.

Alt-R® CRISPR-Cas9 System
Required ribonucleoprotein components
Alt-R CRISPR-Cas9 crRNA • Target-specific RNA oligo, custom synthesized based on your sequence
• Contains proprietary chemical modifications to protect from degradation by cellular RNases
Alt-R CRISPR-Cas9 tracrRNA • Universal RNA oligo (unlabeled or fluorescently labeled) that forms an RNA duplex with the crRNA and subsequently complexes with Cas9 nuclease
• Contains proprietary chemical modifications to protect from degradation by cellular RNases
Alt-R S.p. Cas9 Nuclease/Nickase • Protein that binds the crRNA:tracrRNA duplex, creating an experiment-ready, active ribonucleoprotein (RNP) complex
• Available as Cas9 wild-type, HiFi, or nickase variants (D10A and H840A)
• Contains 3 nuclear localization signals (NLSs) for optimal performance

Additional reagents and kits
Alt-R CRISPR-Cas9 Control Kits • Human, rat, or mouse
• Contains positive control crRNA, negative control crRNA, tracrRNA, PCR primers for analysis of samples transfected with the positive control crRNA
Alt-R CRISPR-Cas9 Electroporation Enhancer For primary and difficult-to-transfect cells
Alt-R Genome Editing Detection KitFor mutation detection and estimating editing efficiency


Click on More Info for additional descriptions and design tips for various Alt-R CRISPR-Cas9 products.


Heading

The Alt-R CRISPR-Cas9 System pairs optimized, shortened 67 nt universal tracrRNA oligonucleotide with an optimized, shortened, target-specific 36 nt crRNA oligonucleotide for improved targeting of Cas9 to dsDNA targets (Figure 2). Use of the Alt-R CRISPR-Cas9 crRNA and tracrRNA also shows less activation of cellular immune responses, resulting in reduced toxicity when compared to in vitro transcribed RNAs.

Figure 2. Components of the Alt-R CRISPR-Cas9 System for directing Cas9 endonuclease to genomic targets. The crRNA:tracrRNA complex uses optimized Alt-R crRNA and tracrRNA sequences that hybridize and then form a complex with Cas9 endonuclease to guide targeted cleavage of genomic DNA. The cleavage site is specified by the protospacer element of the crRNA (thick green bar). The crRNA protospacer element recognizes 19 or 20 nt on the opposite strand of the NGG PAM site (see Figure 3 for design guidance). The PAM site must be present immediately downstream of the protospacer element for cleavage to occur. Research by IDT scientists has shown that the Alt-R CRISPR-Cas9 System provides the highest percentage of on-target genome editing when compared to competing designs, including both native S. pyogenes crRNA:tracrRNA and single fusion sgRNA triggers (see the Performance tab for data).

While delivering Cas9 nuclease as part of an RNP is the preferred method, the Alt-R CRISPR-Cas9 System is also compatible with S.p. Cas9 from any source, including cells that stably express S. pyogenes Cas9 endonuclease, or when Cas9 is introduced as a DNA or mRNA construct.

Alt-R CRISPR-Cas9 crRNA and design tips

Alt-R CRISPR-Cas9 crRNA is a 35–36 nt RNA oligo containing the 19 or 20 nt target-specific protospacer region, along with the 16 nt tracrRNA fusion domain. All Alt-R CRISPR-Cas9 crRNAs are synthesized with proprietary chemical modifications, which protect the crRNA from degradation by cellular RNases and further improve on-target editing performance.

For crRNAs used with S. pyogenes Cas9, identify locations in your target region with the PAM sequence NGG, where N is any DNA base. Your Alt-R CRISPR-Cas9 crRNA will bind to 19–20 bases on the DNA strand opposite to the NGG, PAM sequence (Figure 1). Do not include the PAM sequence in your crRNA design. An example of a correct crRNA sequence is shown in Figure 2. For more information on how to design your crRNA, see the Application Note.

Once you enter your 19 or 20 base target sequence, 16 additional bases and the necessary modifications will automatically be added by the order entry system for a total of 35–36 RNA bases. The system will also convert the final sequence to RNA—enter DNA bases only into the ordering tool (Figure 3). These additional bases and modifications are necessary to create a complete Alt-R CRISPR crRNA.

crRNA Do's and Dont's ORDERING

Figure 3. How to enter your Cas9 crRNA target sequence. Because the crRNA recognizes and binds 20 bases on the DNA strand opposite from the NGG sequence of the PAM site, order your crRNA by entering the 20 bases upstream of the PAM site, in the forward orientation as shown. Enter only DNA bases into the order entry tool. If you are pasting your CRISPR target site from an online design tool, make sure you verify the correct strand orientation. Do not include the PAM site in your design. Common incorrect design examples are shown in red.

Alt-R CRISPR-Cas9 tracrRNA

The 67 nt Alt-R tracrRNA is much shorter than the classical 89 bases of the natural S. pyogenes tracrRNA. We find that shortening the tracrRNA increases on-target performance. Alt-R CRISPR tracrRNA also contains proprietary chemical modifications that confer increased nuclease resistance.

Alt-R CRISPR-Cas9 tracrRNA labeled with ATTO™ 550 (ATTO-TEC) provide the same function as their unlabeled counterparts. However, the fluorescent dye allows you to monitor transfection or electroporation efficiency during preliminary experiments to optimize transfection conditions in your cell types. Labeled tracrRNAs can also help concentrate transfected cells via FACS (fluorescence-activated cell sorting) analysis, which can simplify your screening process for cells with CRISPR events. (For tips on using Alt-R CRISPR-Cas9 tracrRNA – ATTO 550, see the Application Note.)

Alt-R CRISPR tracrRNA orders include Nuclease-Free Duplex Buffer for forming the complex between crRNA and tracrRNA oligos. Alt-R tracrRNA can be ordered in larger scale and paired with all of your target specific crRNAs, allowing for an easy and a cost-effective means of studying many CRISPR sites.

Alt-R S.p Cas9 Nuclease 3NLS

The Alt-R S.p. Cas9 Nuclease 3NLS enzyme is a high purity, recombinant S. pyogenes Cas9. The enzyme includes 1 N-terminal nuclear localization sequence (NLS) and 2 C-terminal NLSs, as well as a C-terminal 6-His tag. The S. pyogenes Cas9 enzyme must be combined with a crRNA and tracrRNA in order to produce a functional, target-specific editing complex. For the best editing, combine the Alt-R S.p. Cas9 Nuclease 3NLS enzyme with the optimized Alt-R CRISPR crRNA and tracrRNA in equimolar amounts.

Alt-R S.p. HiFi Cas9 Nuclease 3NLS

The Alt-R S.p. HiFi Cas9 Nuclease offers improved specificity over wild-type Cas9, greatly reducing the risk of off-target cutting events. This Cas9 variant also preserves the high level of editing efficiency expected from a Cas9 nuclease, maintaining 90–100% on-target editing activity at most sites. For applications that are sensitive to off-target events, combining the Alt-R S.p. HiFi Cas9 Nuclease with the optimized Alt-R CRISPR-Cas9 crRNA and Alt-R CRISPR-Cas9 tracrRNA is highly recommended.

Alt-R S.p. Cas9 Nickases

Cas9 nickases allow specific cutting of only one strand at the DNA target site. Cuts to both strands of DNA are accomplished by using both the Alt-R S.p. Cas9 D10A and the Alt-R S.p. Cas9 H840A Nickase 3NLS, and targeting two neighboring Cas9 sites, functionally increasing the length of the recognition sequence from 20 to 40 bases.

Alt-R S.p. Cas9 Expression Plasmid

In some cases, transfection of RNP or the creation of stably transfected cells is not possible. In those applications, Alt‑R S.p. Cas9 Expression Plasmid is designed to provide expression of Cas9 endonuclease under CMV promoter control. Note that the plasmid contains no eukaryotic selectable marker, making expression of S.p. Cas9 transient. The Alt-R CRISPR-Cas9 System Plasmid User Guide provides instructions for using this plasmid.

Ribonucleoprotein components

Heading

Cas9 nuclease comparison table

Comparison of Alt-R Cas9 Nucleases and Nickases. Click here to download PDF version.

Cas9 comparison table

Heading

  • Alt-R CRISPR-Cas9 Control crRNAs and PCR Assays

Optional controls for human, mouse, and rat are available for the Alt-R CRISPR-Cas9 System.

We recommend using the appropriate Alt-R CRISPR-Cas9 Control Kit for studies in human, mouse, or rat cells. The control kits include an Alt-R CRISPR HPRT Positive Control crRNA targeting the HPRT (hypoxanthine phosphoribosyltransferase) gene and a computationally validated Alt-R CRISPR-Cas9 Negative Control crRNA. The kit also includes the Alt-R CRISPR-Cas9 tracrRNA for complexing with the crRNA controls, Nuclease-Free Duplex Buffer, and validated PCR primers for amplifying the targeted HPRT region in the selected organism. The inclusion of the PCR assay makes the kits ideal for verification of HPRT modification using the Alt-R Genome Editing Detection Kit.

Alt-R control kit components can also be ordered individually.

  • Alt-R Cas9 Electroporation Enhancer

If you are studying hard-to-transfect cells, electroporation is often a viable alternative to lipid-based transfection in CRISPR experiments. The Alt-R Cas9 Electroporation Enhancer is a Cas9-specific 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.

  • Alt-R Genome Editing Detection Kit

Use this kit to detect on-target genome editing and estimate genome editing efficiency in CRISPR experiments. Learn more >>

Additional reagents and kits



References

  1. Zuris JA, Thompson DB, et al. (2015) Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotech, 33(1):73–80.
  2. Ramakrishna S, Kwaku Dad AB, et al. (2014) Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res, 24(6):1020–1027.

User guide


Related protocols


Selected webinars and videos


Posters

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 [1] 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.

Increased specificity using Alt-R S.p. HiFi Cas9 Nuclease 3NLS

To address concerns of off-target editing commonly observed in CRISPR-Cas9 applications, IDT has developed a high-fidelity, S. pyogenes Cas9 nuclease. This novel Cas9 variant drastically reduces activity at off-target sites, while maintaining the high potency of wild-type Cas9 at the intended on-target site (Figure 2 and 3).

Cas9 wt vs HiFi

Figure 2. Alt-R S.p. HiFi Cas9 Nuclease 3NLS provides consistently high on-target performance, while reducing off-target editing, when delivered as a ribonucleoprotein (RNP). RNP complexes (1 mM) were formed with wild-type Cas9 protein (Alt-R S.p. Cas9 Nuclease 3NLS; dark blue) and 3 high-fidelity Cas9 variants, Alt-R HiFi Cas9 (orange), SpCas9-HF1 (light blue; Kleinstiver et al., Nature 529:490–495), or eSpCas9 (gray; Slaymaker et al., Science 351:84–88), combined with an Alt-R crRNA:tracrRNA gRNA complex targeting the EMX1, HEKSite4, or VEGFA3 loci. Complexes (10 nM) were delivered into HEK-293 cells by reverse transfection using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher), and DNA was extracted after 48 hr. Editing was measured by PCR amplification of the indicated on- and off-target sites, followed by cleavage with T7EI and analysis using the Fragment Analyzer system (Advanced Analytical). Error bars represent the standard errors of the means. The sequence of the on- and off-target sites for each crRNA are indicated at the bottom (red = mismatch in protospacer; blue = mismatch in PAM site).

Alt-R® HiFi Cas9 similar editing efficiency to wild-type Cas9 v2

Figure 3. Alt-R S.p. HiFi Cas9 Nuclease 3NLS maintains similar on-target editing efficiency to wild-type Cas9. Alt-R crRNAs targeting 12 sites within the human HPRT gene were annealed with Alt-R tracrRNA, then complexed to form ribonucleoprotein (RNP) editing complexes with wild-type Cas9 (Alt-R S.p. Cas9 Nuclease 3NLS; dark blue) or one of 3 high-fidelity Cas9 variants, Alt-R HiFi Cas9 (orange), SpCas9-HF1 (light blue; Kleinstiver et al., Nature 529:490–495), or eSpCas9 (gray; Slaymaker et al., Science 351:84–88). The RNP complexes were reverse transfected using Lipofectamine RNAiMAX Reagent (Thermo Fisher) into HEK-293 cells at a concentration of 10 nM. After 48 hr, genomic DNA was isolated, and editing at the intended on-target site was measured by a T7 endonuclease I assay (Alt-R Genome Editing Detection Kit). The Alt-R S.p. HiFi Cas9 maintains higher levels of editing across different target sites when compared to previously published, enhanced specificity Cas9 mutants.

Potent editing with the Alt-R S.p. Cas9 Nuclease 3NLS

The Alt-R CRISPR-Cas9 System includes the potent Alt-R S.p. Cas9 Nuclease 3NLS. When the Alt-R S.p. Cas9 Nuclease 3NLS is combined with the Alt-R CRISPR crRNA and tracrRNA into a ribonucleoprotein (RNP), the system outperforms other editing approaches (Figure 4). 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 4. 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 Nuclease 3NLS, 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.

Improved homology-directed gene repair using the Alt-R CRISPR-Cas9 System RNP

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 5, 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 5. 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 6 illustrates the high genome editing function achieved by crRNA:tracrRNA complexes targeting 553 sites across 6 exons.

Figure 6. 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 7). 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 7. 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 8) 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 8. 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 [2]. 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 9). 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.

CRISPR protospacer boxplot

Figure 9. 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-RGenome 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.

Dye-labeled Alt-R CRISPR-Cas9 tracrRNA has same performance as unlabeled tracrRNA

Figure 10. 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.

Dye-labeled Alt-R CRISPR-Cas9 tracrRNA: monitor transfection and cellular localization

Figure 11. 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.

Use FACS to enrich for CRISPR edited cells that have dye-labeled Alt-R CRISPR-Cas9 tracrRNA

Figure 12. 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. Nuclease 3NLS 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.

Optimal FACS analysis after 24 hr transfection using dye-labeled Alt-R Cas9 tracrRNA

Figure 13. 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. Nuclease 3NLS 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.

Alt-R Cas9 Electroporation Enhancer improves CRISPR editing efficiency

Figure 14. 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. 

References

  1. Vouillot L, Thélie A, et al. (2015) Comparison of T7E1 and Surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases. G3: Genes|Genomes|Genetics, 5(3):407–415.

  2. Fu Y, Sander JD, et al. (2014) Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotech, 32(3):279–284.