A recombinant Cas9 enzyme that drastically reduces CRISPR off-target effects

Learn about the Cas9 variant, Alt-R® S.p. HiFi Cas9 Nuclease 3NLS, that greatly reduces off-target cutting events during CRISPR genome editing. At the same time, it maintains the high level of on-target editing efficiency seen with wild-type Cas9 nuclease.

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.

Stanford University researcher compares high fidelity Cas9 enzymes

“We performed an unbiased evaluation of several versions of high fidelity Cas9 enzymes in primary human stem cells. We have been very impressed with the characteristics of this new IDT enzyme. Unlike other versions, this version consistently gives us high on-target editing activity while having low off-target activity. Because of the retained excellent on-target activity and improved specificity profile, we are excited to use this version in our future experiments focused on developing novel genome editing based therapies for severe diseases with unmet medical needs.”
– Matthew Porteus, MD, PhD
Division of Stem Cell Transplantation and Regenerative Medicine
Stanford University
Stanford, CA USA

Published Aug 17, 2017