Obtain higher on-target CRISPR genome editing with optimized Cas9 nucleases

Broadened Cas9 cleavage

As a leading developer of CRISPR genome editing solutions, IDT continues to expand its family of Alt-R CRISPR-Cas9 nucleases with Alt-R S.p. Cas9 Nuclease V3. This CRISPR-Cas9 protein is designed to maximize the efficiency of genome editing across a broad number of sites. Alt-R S.p. Cas 9 Nuclease V3 is a recombinant variant, derived from native Streptococcus pyogenes Cas9 protein, that has been purified from an E. coli strain expressing codon optimized Cas9. Modification of the expression construct facilitates nucleus-targeted delivery, resulting in enhanced cleavage, particularly at difficult targets (Figure 1).

Figure 1. Alt-R S.p. Cas9 Nuclease V3 maximizes genome editing efficiency even at challenging target sites. Ribonucleoprotein (RNP) complexes were formed with 1 of the 2 wild-type Cas9 proteins—Alt-R S.p. Cas9 Nuclease 3NLS (light blue) or Alt-R S.p. Cas9 Nuclease V3 (dark blue), combined with an Alt-R crRNA:tracrRNA complex targeting one of 11 loci on the human HPRT gene. RNP complexes (4 µM) were delivered into HEK-293 cells by nucleofection. Total editing at the on-target loci was calculated by NGS.

HiFi variant for experiments that require potent, specific editing

The IDT market-leading Alt-R HiFi Cas9 Nuclease V3 also benefits from these improvements. This high-fidelity Cas9 variant provides consistent, high editing efficiency with very low off-target activity. When delivered as part of a ribonucleoprotein (RNP) complex, other high-fidelity Cas9 enzymes have shown reduced on-target activity compared to wild-type enzymes. However, Alt-R HiFi Cas9 Nuclease V3 not only provides superior cutting specificity, but also achieves similar potency to the wide-type enzyme on the intended sites (Figure 2).

Figure 2. Alt-R S.p. HiFi Cas9 Nuclease V3 facilitates near-WT on-target editing potency and significantly reduces off-target site editing. RNP complexes were formed with either Alt-R S.p. Cas9 Nuclease V3 or Alt-R S.p. HiFi Cas9 Nuclease V3, combined with an Alt-R crRNA:tracrRNA complex targeting the EMX1 gene. RNP complexes (4 µM) were delivered into HEK293 cells via nucleofection. INDEL formation at the on-target locus as well as 9 known off-target sites were measured by NGS (indicated on the y axis in log scale).

Advantages of a ribonucleoprotein delivery format

For best results, we recommend assembling Cas9 V3 nucleases with optimized Alt-R CRISPR-Cas9 crRNA and Alt-R CRISPR-Cas9 tracrRNA, and delivering them in an RNP format. Unlike conventional vector-based methods that require transcription and translation inside cells, the RNP complex can function immediately upon delivery, and is compatible with lipofection or electroporation using commercially available reagents. Moreover, the use of RNP complexes mitigates the risk of off-target cleavage by shortening the duration of Cas9 presence in cells, a major determinant of precision genome editing. Both Alt-R S.p. Cas9 Nuclease V3 and HiFi Cas9 Nuclease V3 are available in 100 and 500 µg aliquots (100 µg Cas9 nuclease = 610 pmol). Both enzymes are shipped in ready-to-use buffer at a high concentration (10 µg/µL), allowing use in a wide range of applications, including electroporation and microinjection.

Learn more about Alt-R S.p. Cas9 Nuclease V3 and Alt-R S.p. HiFi Cas9 Nuclease V3.