CRISPR-Cas9 Genome Editing

Increase efficiency of genome editing using the
Alt-R™ CRISPR-Cas9 System. Now powered by the new S.p. 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

New! Alt-R CRISPR crRNAs with increased nuclease resistance

IDT has updated the Alt-R CRISPR-Cas9 System with the addition of proprietary chemical modifications to the Alt-R CRISPR crRNA. These modifications protect the crRNA from degradation by cellular RNases, and further improve on-target editing performance. The modifications are included automatically to the final Alt-R CRISPR crRNA oligonucleotide sequence and do not require any changes to the ordering process.

IDT recommends using the Alt-R S.p. Cas9 Nuclease 3NLS combined with the Alt-R CRISPR crRNA and tracrRNA to generate a ribonucleoprotein editing complex for very high editing efficiency across most target sites. View the Cas9 Ribonucleoprotein User Guide for guidance on transfection reagents and materials for delivery of the RNP into your cell lines. We also highly recommend use of the controls listed here.

CRISPR 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 crRNA, 2 nmol$95.00 USD19 - 20 BasesOrder
Alt-R™ CRISPR crRNA, 10 nmol$125.00 USD19 - 20 BasesOrder
ProductPricingLength96 Well384 Well
Alt-R™ CRISPR crRNA, 2 nmol, Plate$80.00 USD / well19 - 20 BasesOrderOrder
Alt-R™ CRISPR crRNA, 10 nmol, Plate$105.00 USD / well19 - 20 BasesOrderOrder

Alt-R™ CRISPR-Cas9 System

CRISPR-Cas9 genome editing uses a Cas9 endonuclease to generate double-stranded breaks in DNA. The cleaved DNA is then repaired by non-homologous end-joining or homology-directed recombination, resulting in a modified sequence. The native Streptococcus pyogenes system uses a 42 nt CRISPR RNA (crRNA) and an 89 nt transactivating CRISPR RNA (tracrRNA) to guide and activate the Cas9 nuclease for target specific cleavage of double-stranded DNA (dsDNA). 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 1).


Figure 1. 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 crRNA (thick green bar). The crRNA protospacer element recognizes 19 or 20 nt on the opposite strand of the NGG PAM site (see Figure 2 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).

The shortened crRNA and tracrRNA oligos in the Alt-R CRISPR-Cas9 System show improved on-target Cas9 editing activity compared to the native, longer RNAs from S. pyogenes. The Alt-R CRISPR-Cas9 System also shows less activation of cellular immune response, resulting in reduced toxicity, when compared to in vitro transcribed RNAs.

IDT recommends using the Alt-R S.p. Cas9 Nuclease 3NLS combined with the Alt-R CRISPR crRNA and tracrRNA into a ribonucleoprotein (RNP) complex for very high editing efficiency across most target sites. Using the RNP complex, instead of Cas9 mRNA or DNA expression constructs, has been shown to solve some of the challenges associated with these other methods [1, 2]. For example, using the Alt-R S.p. Cas9 Nuclease allows researchers to precisely control how much Cas9 is introduced, and the non-renewable Cas9 enzyme limits the duration of Cas9 activity. Both of these factors help to reduce off-target editing. In addition, using the RNP eliminates issues of genomic incorporation from DNA constructs, and the toxicity issues associated with transfecting long mRNA.

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™ 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 molecular weight of the nuclease is 163,700 g/mol. 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.

Product specifications:

Alt-R™ S.p Cas9 Nuclease 3NLS 

  • Amount provided: 100 µg or 500 µg
  • Molecular weight: 163,700 g/mol
  • Concentration: 10 µg/µL in 50% glycerol, [61 µM]
  • Endotoxin tested: <2 EU/mg
  • Shipping conditions: dry ice Store at –20°C

Dilute S.p Cas9 Nuclease to working concentration in 20 mM HEPES, 150 mM KCI, pH 7.5, or Opti-MEM® (Thermo Fisher) before use.

Alt-R™ CRISPR crRNA and Design

The Alt-R CRISPR crRNA is a chemically modified 35–36 nt RNA oligo containing the 19 or 20 nt target-specific protospacer region, along with the 16 nt tracrRNA fusion domain. Cas9 endonuclease requires a crRNA to specify the DNA target sequence, and the Alt-R CRISPR crRNA must be combined with the transactivating Alt-R CRISPR tracrRNA in order to activate the endonuclease and create a functional editing ribonucleoprotein complex. New! Alt-R CRISPR crRNAs with increased nuclease resistance

Through extensive research, IDT has updated the Alt-R CRISPR-Cas9 System with the addition of proprietary chemical modifications to the Alt-R CRISPR crRNA. These modifications protect the crRNA from degradation by cellular RNases, and further improve on-target editing performance. The modifications are included automatically to the final Alt-R CRISPR crRNA oligonucleotide sequence and do not require any changes to the ordering process. In combination with the Alt-R CRISPR tracrRNA and the Alt-R Cas9 protein, this modified crRNA provides the highest on-target editing performance available.

For use 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 crRNA will bind to 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: How to design gene disruption experiments using the Alt-R™ CRISPR-Cas9 System.

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 2). These additional bases and modifications are necessary to create a complete Alt-R CRISPR crRNA. The crRNA must also be combined with an Alt-R CRISPR tracrRNA, and S. pyogenes Cas9 in order to form an active editing complex, as described below.

crRNA Do's and Dont's ORDERING

Figure 2. How to enter your 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 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. The Alt-R CRISPR tracrRNA also contains proprietary chemical modifications that confer increased nuclease resistance. Cas9 endonuclease requires a crRNA and tracrRNA to form an active editing complex. The Alt-R CRISPR crRNA described above must be combined with the transactivating Alt-R CRISPR tracrRNA in order to activate the endonuclease.

The Alt-R CRISPR tracrRNA ships with Nuclease-Free Duplex Buffer for forming the complex between crRNA and tracrRNA oligos. The 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™ CRISPR Controls 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 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 Negative Control. The kit also includes the Alt-R CRISPR 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 T7 Endonuclease I assays (Figure 3).

CRISPR Phase 2 controls

Figure 3. T7EI sample data for Alt-R CRISPR HPRT Positive Controls. Alt-R CRISPR HPRT Positive Controls for human, mouse, and rat were used to edit HEK293 (human), Hepa1-6 (mouse), and RG2 (rat) cell lines. Genomic DNA from the CRISPR-Cas9 edited cells was PCR amplified, digested using T7 Endonuclease I, and run on the Fragment Analyzer™. Reference standard bands at 5000 and 35 bp are used to align the gel and analyze the results. Estimated band sizes for the cut and uncut positive control amplicons are listed in the table. Cell lines used were HEK293 (human), Hepa1-6 (mouse), and RG2 (rat).

Alt-R™ S.p. Cas9 Expression Plasmid

In some cases, transfections of an RNP or the creation of stably transfected cells is not possible. In those applications, the 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.

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.

Alt-R™ CRISPR-Cas9 RNA triggers are more potent than single guide RNAs

Our optimized Alt-R CRISPR 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.

CRISPR RNA trigger comparison (by type) GEN V7

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 Transfection Reagent (Thermo Fisher) into a HEK293-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 T7EI mismatch endonuclease (New England Biolabs) 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 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 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.

RNP vs other transient methods (expanded)

Figure 2. Lipofection of Alt-R™ CRISPR-Cas9 System Components as a ribonucleoprotein outperforms other transient CRISPR-Cas9 approaches. Alt-R CRISPR HPRT Control crRNA complexes 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 (HEK293), 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.

Case study

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.

Length optimization data

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 Transfection Reagent (Thermo Fisher), into a HEK293-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 T7EI mismatch endonuclease (New England Biolabs), 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.

6 ski slopes

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 Transfection Reagent (Thermo Fisher) into a HEK293-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 T7EI mismatch endonuclease (New England Biolabs) 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
Sanger vs T7EI

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 HEK293-Cas9 cells that stably express S. pyogenes Cas9, or wild-type HEK293 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 T7EI mismatch endonuclease (New England Biolabs), 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 HEK293-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.

IFIT1 and OAS2 immune response data

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 HEK293-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. 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 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 Transfection Reagent (Thermo Fisher) into a HEK293 cell line stably expressing S. pyogenes Cas9. Genomic DNA was isolated and editing measured by PCR amplification of target sites, followed by cleavage with T7EI mismatch endonuclease (New England Biolabs) 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.

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.