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:
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.
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.
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.