To address concerns of off-target editing commonly observed in CRISPR-Cas9 applications, IDT has developed a high-fidelity, S. pyogenes Cas9 enzyme variant. The recombinant Alt-R® S.p. HiFi Cas9 Nuclease 3NLS offers improved specificity over wild-type Cas9, greatly reducing the risk of off-target cutting events during CRISPR genome editing. This Cas9 variant also preserves the high level of editing efficiency at intended on-target sites expected from a Cas9 nuclease, maintaining 90–100% on-target editing activity at most sites.
For best results, we recommend combining the Alt-R S.p. HiFi Cas9 Nuclease with optimized Alt-R CRISPR-Cas9 crRNA and Alt-R CRISPR-Cas9 tracrRNA, and delivering them in a ribonucleoprotein (RNP) format. Figure 1 demonstrates on-target vs off-target genome editing at 3 loci for wild-type Cas9 (dark blue) compared to 3 Cas9 variants, all delivered in an RNP format, in this case, by lipofection. (Note that as for Alt-R S.p. Cas9 Nuclease 3NLS, RNPs containing Alt-R S.p. HiFi Cas9 Nuclease are also effective when delivered by electroporation or microinjection.) All of the Cas9 variants showed strong discrimination between on-target and off-target sites, however the Alt-R S.p. HiFi Cas9 Nuclease 3NLS enzyme (orange) also consistently provided the highest on-target editing, comparable to that of wild-type Cas9.
Figure 1. Alt-R S.p. HiFi Cas9 Nuclease 3NLS provides consistently high on-target performance, while reducing off-target editing, when delivered as a ribonucleoprotein (RNP). RNP complexes (1 µM) were formed with wild-type Cas9 protein (Alt-R S.p. Cas9 Nuclease 3NLS; dark blue) and 3 high-fidelity Cas9 variants, Alt-R HiFi Cas9 (orange), SpCas9-HF1 (light blue; Kleinstiver et al., Nature 529:490–495), or eSpCas9 (gray; Slaymaker et al., Science 351:84–88), combined with an Alt-R crRNA:tracrRNA gRNA complex targeting the EMX1, HEKSite4, or VEGFA3 loci. Complexes (10 nM) were delivered into HEK-293 cells by reverse transfection using Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher), and DNA was extracted after 48 hr. Editing was measured by PCR amplification of the indicated on- and off-target sites, followed by cleavage with T7EI and analysis using the Fragment Analyzer™ system (Advanced Analytical). Error bars represent the standard errors of the means. The sequence of the on- and off-target sites for each crRNA are indicated at the bottom (red = mismatch in protospacer; blue = mismatch in PAM site).
The Alt-R S.p. HiFi Cas9 Nuclease 3NLS is purified from an E. coli strain expressing codon optimized Cas9. It contains 1 N-terminal nuclear localization sequence (NLS), 2 C-terminal NLSs, and a C-terminal 6-His tag, and is available in 100 and 500 µg aliquots (100 µg Cas9 nuclease = 610 pmol).
Learn more about Alt-R S.p. HiFi Cas9 Nuclease 3NLS.