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CRISPR genome editing

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Genome editing with CRISPR-Cas9

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.

Benefit from the latest improvements in on- and off-target design and chemical modifications, as well as easy ordering of custom or predesigned guide RNAs.
Get optimal editing with high on-target potency and reduced off-target activity with Alt-R HiFi CRISPR-Cas9 nuclease
Precisely control editing with efficient delivery of the RNP by lipofection or electroporation
Protect your cells from toxicity or innate immune response activation

* See Ordering section for details.

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Alt-R HDR Design Tool & Templates

NEW! Streamline your homology-directed repair (HDR) project. Simply provide basic information about your target site, then use the HDR tool to design and visualize your desired edit within the sequence. The HDR Design Tool will provide the recommended gRNA(s) and HDR donor template for your specifications. Note: If you have template designs of your own or from publications, order them as Alt-R HDR Donor Oligos using the ORDER YOUR CUSTOM HDR TEMPLATE button.

Design your HDR template & gRNA

ORDER YOUR CUSTOM HDR TEMPLATE

Ordering

CRISPR-Cas9 gRNA

Guide RNAs (gRNAs) contain the target-specific sequence for guiding Cas9 protein to a genomic location. We offer 3 gRNA formats: crRNA:tracrRNA duplex, crRNA XT:tracrRNA duplex, and single guide RNA (sgRNA).

CRISPR-Cas9 crRNA

Must be used with tracrRNA to form a functional gRNA duplex. Suitable for most applications. Contain chemical modifications to protect from degradation by cellular RNases.

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CRISPR-Cas9 crRNA XT

Must be used with tracrRNA to form a functional gRNA duplex. Suitable for challenging experimental conditions (e.g., high nuclease environments or with Cas9 mRNA). Contain additional chemical modifications compared to crRNA to provide a cost-effective option for increased stability.

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CRISPR-Cas9 sgRNA

Single RNA molecules comprised of both crRNA and tracrRNA sequences. Suitable for challenging experimental conditions (e.g., high nuclease environments or with Cas9 mRNA). Contain chemical modifications for the highest level of stability.

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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.

Select Predesigned

Design Custom

Check your own design

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.

Order in tubes

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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.

Custom CRISPR solutions

For more flexibility in guide RNA ordering, CRISPR Custom Guide RNAs allow a wide range of lengths and modifications to support a wide range of CRISPR systems.

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 (euapplicationsupport@idtdna.com). This guarantee does not extend to any replacement product, or to any other incurred or incidental costs or expenses.

Product details

What makes the Alt-R CRISPR-Cas9 System so great?

Length optimization of the crRNAs for increased performance
Chemical modifications to increase nuclease resistance and reduce innate immune responses
Optimized native Cas9 and HiFi Cas9 variant with increased specificity
Two-part guide RNA (crRNA:tracrRNA) provide high activity at an affordable price
Unique supporting reagents (electroporation enhancer, labeled tracrRNA, controls) ensure successful editing
Complete collection of user-guides and customers-submitted protocols

Alt-R CRISPR-Cas9 System

Simple delivery of ribonucleoprotein complexes (crRNA:tracrRNA:Cas9 or sgRNA:Cas9).

Figure 1. Overview of Alt-R CRISPR-Cas9 System experiments for ribonucleoprotein delivery by lipid-mediated transfection, electroporation, or microinjection using (A) 2-part guide RNAs (crRNA:tracrRNA duplex) or (B) sgRNAs.

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 1A). In an alternative approach, the crRNA and tracrRNA can be delivered as a single RNA oligonucleotide (Figure 1B). 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
Ribonucleoprotein components
Option 1: Alt-R CRISPR-Cas9 crRNA:tracrRNA	Alt-R CRISPR-Cas9 crRNA Must be used with tracrRNA Target-specific RNA oligo, custom synthesized based on your sequence Contains chemical modifications to protect from degradation by cellular RNases crRNA XT contains additional modifications for improved stability and performance when using 2-part gRNAs under challenging conditions (e.g., high nuclease environments or with Cas9 mRNA)
Option 1: Alt-R CRISPR-Cas9 crRNA:tracrRNA	Alt-R CRISPR-Cas9 tracrRNA For use with crRNA or crRNA XT Universal RNA oligo (unlabeled or fluorescently labeled) that forms an RNA duplex with crRNA and subsequently complexes with Cas9 nuclease Contains additional chemical modifications to protect from degradation by cellular RNases
Option 2: Alt-R CRISPR-Cas9 sgRNA	Alternative to using crRNA:tracrRNA A single, long, fusion RNA oligonucleotide containing both crRNA and tracrRNA regions Contains additional chemical modifications for improved stability and performance when using gRNAs under challenging conditions (e.g., high nuclease environments or with Cas9 mRNA)
Alt-R S.p. Cas9 Nuclease/Nickase	Proteins 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) Contain 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 HDR Enhancer	For improved rates of homology-directed repair
Alt-R Genome Editing Detection Kit	For mutation detection and estimating editing efficiency

Ribonucleoprotein components

The Alt-R CRISPR-Cas9 System offers two options for generating synthetic guide RNAs. The two-part system pairs an optimized, shortened universal tracrRNA oligonucleotide (67 nt) with an optimized, shortened, target-specific crRNA oligonucleotide (36 nt) for improved targeting of Cas9 to dsDNA targets (Figure 2). The single guide RNA (sgRNA) option combines the crRNA and tracrRNA segments into one long RNA molecule, reducing the number of components and simplifying the CRISPR workflow.

Figure 2. Components of the Alt-R CRISPR-Cas9 System for directing Cas9 endonuclease to genomic targets. For the 2-part guide RNA format, 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. Alternatively, the sgRNA (not shown), which contains relevant crRNA and tracrRNA sequences, forms a complex with Cas9 endonuclease to guide targeted cleavage of genomic DNA. The cleavage site is specified by the spacer element of the crRNA (light blue bar). The crRNA spacer element recognizes 19 or 20 nt on the strand opposite from the NGG PAM site. 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 of native S. pyogenes crRNA:tracrRNA.

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

Option 1 (2-part guide RNA)

Alt-R CRISPR-Cas9 crRNA

All Alt-R CRISPR-Cas9 crRNAs are 35–36 nt RNA oligos containing the 19 or 20 nt target-specific protospacer region, along with the 16 nt tracrRNA fusion domain. We recommend 20 nt protospacers for most applications. crRNAs must be duplexed with Alt-R CRISPR-Cas9 tracrRNA before RNP complex formation.

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. When using 2-part gRNAs under highly challenging conditions (e.g., high nuclease environments or with Cas9 mRNA), use Alt-R CRISPR-Cas9 crRNA XT, which have additional chemical modifications for the highest level of stability and performance.

We guarantee* our predesigned guide RNAs targeting human, mouse, rat, zebrafish, or nematode genes. For other species, you may 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 guide RNA modifications.

* See Ordering section for details.

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 (Figure 3).

Figure 3. 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 hr after transfection. Magnification: 10X.

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 more information and 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.

Option 2 (single guide RNA)

Alt-R CRISPR-Cas sgRNA

Alt-R CRISPR-Cas9 sgRNAs are long RNA oligonucleotides (99–100 bases) containing the target-specific crRNA region and the Cas9-interacting tracrRNA region within a single molecule (i.e., 19–20 base protospacer region and 80-base universal sgRNA region). Like other Alt-R RNAs, it contains chemical modifications to stabilize the RNA, increasing resistance to nuclease activity. For challenging conditions (e.g., high nuclease environments or with Cas9 mRNA), sgRNAs may provide increased potency.

Cas9 endonucleases

Alt-R S.p Cas9 nuclease

The Alt-R S.p. Cas9 Nuclease V3 enzyme is a high purity, recombinant S. pyogenes Cas9. The enzymes include nuclear localization sequences (NLSs) and C-terminal 6-His tags. The S. pyogenes Cas9 enzyme must be combined with a gRNA to produce a functional, target-specific editing complex. For the best editing, combine the Alt-R S.p. Cas9 Nuclease V3 enzyme with the optimized Alt-R CRISPR gRNA in equimolar amounts.

Alt-R S.p. HiFi Cas9 nuclease

The Alt-R S.p. HiFi Cas9 Nuclease V3 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 V3 with optimized Alt-R CRISPR-Cas9 gRNA (crRNA: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 either Alt-R S.p. Cas9 D10A Nickase V3 or Alt-R S.p. Cas9 H840A Nickase V3, with 2 gRNAs that target two neighboring Cas9 sites, one on either strand of the target region. This functionally increases the length of the recognition sequence from 20 to 40 bases. For more information about using Cas9 nickases, see the application note.

Alt-R S.p. dCas9 protein

Alt-R S.p. dCas9 Protein V3 has mutations that result in the loss of nuclease activity. This protein can form RNP complexes with Alt-R gRNAs and bind to the target region specified by the gRNA without cutting the DNA.

Like the other Alt-R enzymes, Alt-R S.p. dCas9 Protein V3 is provided as 10 mg/mL in 50% glycerol, and it can be diluted in PBS or Opti-MEM^® media (Thermo Fisher) before use.

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.

Cas9 comparison chart

Comparison of Alt-R Cas9 nucleases and nickases. Click here to download PDF version.

Homology-directed repair reagents

Alt-R HDR Donor Oligos

Alt-R HDR Donor Oligos have been developed specifically for insertion into DNA by HDR. These HDR templates have enhanced stability and higher rates of incorporation than standard oligonucleotides.

Exceptional flexibility: multiple species, as well as single or multiple (batch) design modes
Compatible with Cas9 nucleases (double-stranded cleavage) or nickases (single-stranded cleavage)
Accommodates incorporation of desired sequence (such as sequence tags, stop codons, GFP, enhancers, or promoters) into target site

Alt-R HDR Enhancer

Alt-R HDR Enhancer is a small molecule compound that increases homology-directed repair. Alt-R HDR Enhancer exhibits its activity in multiple cell lines, including both adherent and suspension cell lines. Its activity is independent of the enzyme employed; for example, it can be used either with Alt-R S.p. Cas9 nucleases or A.s. Cas12a (Cpf1) nucleases. This versatile reagent is also compatible with electroporation and lipofection methods.

Additional reagents and kits

Alt-R CRISPR-Cas9 Control crRNAs and PCR Assays

Optional controls for human, mouse, and rat are available for the 2-part 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.

For information about sgRNA controls, contact applicationsupport@idtdna.com.

Alt-R Cas9 Electroporation Enhancer

If you are studying primary or 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 (Figure 4).

Figure 4. 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.

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 >>

Performance

Homology-directed repair (HDR)

Alt-R HDR Enhancer improves HDR efficiency

The simple addition of the HDR enhancer to culture media in the CRISPR-Cas9 editing protocol increases HDR efficiency in multiple cell types (Figure 1) and with multiple targets (Figure 2).

Figure 1. Alt-R HDR enhancer improves HDR efficiency across multiple cell types. 4 µM of RNP complex (Alt-R S.p. Cas9 Nuclease V3 complexed with Alt-R CRISPR-Cas9 crRNA and tracrRNA) targeting human HPRT site was delivered into multiple human cell lines via electroporation using the Amaxa Nucleofector System (Lonza), along with 4 µM of Alt-R Cas9 Electroporation Enhancer and 3 µM of IDT Ultramer oligonucleotides as HDR template. After electroporation, cells were plated in media that contained either 30 µM of Alt-R HDR enhancer (gray bar), or the same volume of DMSO solution as the negative control (light blue bar). The HDR template contains a 6-base Eco RI recognition site that enables assessment of HDR efficiency by restriction fragment length polymorphism (RFLP). Genomic DNA was isolated 48 hr (HEK-293 and HeLa) or 72 hr (Jurkat and K562) after electroporation, and the target locus was amplified by PCR. Amplicons were digested with EcoRI enzyme to determine the rate of HDR insertion. HDR = homology-directed repair; DMSO = dimethyl sulfoxide.

Figure 2. Alt-R HDR Enhancer improves HDR mediated by S.p. Cas9. 4 µM of Alt-R Cas9 RNP targeting multiple sites in the human genome were delivered into human Jurkat cells via electroporation using the Neon Transfection System (Thermo Fisher), along with 4 µM of Alt-R Cas9 Electroporation Enhancer and 3 µM of single-stranded DNA template (Ultramer oligo, IDT). After electroporation cells were plated in media that contained either 30 µM of Alt-R HDR Enhancer (gray bar), or equal volume of DMSO solution as the negative control (light blue bar). A “no treatment” control group which received media alone was also included in the experiment (dark blue bar). HDR efficiency was assessed 48–72 hr after electroporation by EcoRI cleavage of the PCR products amplified from the target locus.

Alt-R HDR Enhancer also improves HDR efficiency in cells transfected via lipofection (please see supplemental data).

Guide RNAs

Alt-R guide RNA variants generate similar editing rates when used as part of ribonucleoprotein (RNP) complexes

Guide RNAs (gRNAs) targeting 12 sites in the HPRT gene were delivered as RNP. Genome editing using 3 gRNA formats (crRNA:tracrRNA duplex, crRNA XT:tracrRNA duplex, and sgRNA) at two doses were examined. The comparable results are shown in a box-and-whiskers plot (Figure 3).

Figure 3. Similar genome editing by gRNA variants when delivered as RNP complexes. gRNA variants targeting 12 HPRT sites were precomplexed to Alt-R Cas9 nuclease. The resulting RNP [4 or 0.25 µM of RNP (1:1.2 molar ratio of protein: gRNA)] and 2 µM of Alt-R Cas9 Electroporation Enhancer were electroporated into HEK-293 cells. Genomic DNA (gDNA) was isolated after 48 hr, and total editing was assessed using next-generation sequencing.

When co-delivering Cas9 mRNA with gRNA, the stability of the Alt-R guide RNAs becomes important for targeted genome editing

Genome editing activity of 3 gRNA formats (crRNA:tracrRNA duplex, crRNA XT:tracrRNA duplex, and sgRNA) was compared. The gRNA was co-delivered with Cas9 mRNA into cultured cells. The results show that at two HPRT sites, gRNA format can affect the rate of genome editing (Figure 4).

Figure 4. At some genome editing sites, increased chemical modification was needed in the gRNA when co-delivered with Cas9 mRNA. gRNA variants (4 µM) targeting 2 HPRT sites were electroporated into HEK-293 cells with 1 µg of Cas9 mRNA. Genomic DNA (gDNA) was isolated after 48 hr, and total editing was assessed using next-generation sequencing.

Alt-R guide RNA variants generate identical on-target repair profiles when used as part of RNP complexes

Guide RNAs (gRNAs) targeting the EMX1 gene were delivered as RNP that included either wild type or HiFi Cas9 nuclease. Indel size after genome editing using 3 gRNA formats (crRNA:tracrRNA duplex, crRNA XT:tracrRNA duplex, and sgRNA) were analyzed by next generation sequencing. The comparable results are shown in Figure 5.

Figure 5. gRNA variants produce identical on-target repair profiles. gRNA complexes targeting EMX1 were precomplexed with either wild type Alt-R Cas9 nuclease (blue, green, and orange bars) or Alt-R HiFi Cas9 nuclease (light blue, green, and orange bars). The resulting RNP [4 µM of RNP (1:1.2 molar ratio of protein:gRNA)] and 2 µM of Alt-R Cas9 Electroporation Enhancer were electroporated into HEK-293 cells. Genomic DNA (gDNA) was isolated after 48 hr, and total editing was assessed using next-generation sequencing.

Alt-R CRISPR-Cas9 RNAs elicit less toxicity and lower 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 [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 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.

Cas9 nuclease

For additional data focused on Cas9 nucleases, visit the Alt-R CRISPR enzymes page.

References

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