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.
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.
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).
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.