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.
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
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 Alt-R S.p. Cas9 Nuclease 3NLS enzyme with 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
- Storage conditions: –20°C
Dilute S.p. Cas9 Nuclease to working concentration in 20 mM HEPES, 150 mM KCI, pH 7.5, or in Opti-MEM® medium (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.