CRISPR-Cas9 genome editing is changing the landscape of genomic research. The CRISPR-Cas9 mechanism relies on the Cas9 endonuclease to generate double-stranded breaks at specified sites in double-stranded DNA (dsDNA). The native Streptococcus pyogenes Cas9 protein that is most commonly used requires a 42 nt CRISPR RNA (crRNA) and an 89 nt transactivating CRISPR RNA (tracrRNA) to guide and activate the nuclease for target specific cleavage of dsDNA. The cleaved DNA is then repaired by one of two available cellular mechanisms: the error-prone non-homologous end-joining that is used to introduce frameshift mutations into protein coding sequences, leading to gene disruptions; and homology-directed recombination that is use to introduce foreign DNA into the genome.
Through extensive testing, scientists at IDT have developed the Alt-R® CRISPR-Cas9 System. The system combines a high quality S. pyogenes Cas9 protein with experimentally optimized, 36 nt crRNA and 67 nt tracrRNA oligonucleotides that outperform traditional CRISPR genome editing methods.
The Alt-R S.p. Cas9 Nuclease 3NLS further improves genome editing
For optimal on-target editing efficiency, IDT offers the Alt-R S.p. Cas9 Nuclease 3NLS. The addition of this Cas9 protein gives researchers a powerful new tool that can be combined with Alt-R CRISPR crRNA and tracrRNA into a ribonucleoprotein (RNP). The RNP is active upon delivery and is compatible with lipofection with commercially available reagents (see User Guide) or electroporation methods, and outperforms methods that rely on transfection of DNA expression constructs or Cas9 mRNA (Figure 1).
Figure 1. Alt-R CRISPR-Cas9 System ribonucleoprotein outperforms other transient CRISPR-Cas9 editing methods. Alt-R CRISPR HPRT Control crRNAs for human, mouse, or rat were complexed with Alt-R CRISPR tracrRNA. Resulting complexes were transfected with Cas9 expression plasmid, Cas9 mRNA, or as a Cas9 RNP (containing Alt-R S.p. Cas9 Nuclease 3NLS pre-complexed with the crRNA and tracrRNA) into human (HEK293), mouse (Hepa1-6), or rat (RG2) cell lines. The Cas9 RNP outperformed the other transient Cas9 expression methods, and performed comparably to reference HEK293-Cas9 cells that stably express S. pyogenes Cas9.
In addition to improved editing performance, using RNP complexes, instead of DNA expression constructs or Cas9 mRNA, mitigates some of the problems associated with other methods [1,2]. For example, RNPs eliminate genomic incorporation issues associated with introducing double-stranded DNA expression constructs, and greatly reduces or eliminates toxicity observed when delivering long, in vitro transcribed RNAs. Also, because RNPs are non-renewable, this method allows for precise control over the dose of editing complexes, for more consistent editing results.
Updated Alt-R CRISPR crRNAs with increased nuclease resistance
Alt-R CRISPR crRNAs have also been updated for improved performance through the addition of proprietary chemical modifications. These modifications provide further protection from degradation by cellular RNases, which improves on‑target editing performance in many applications. The modifications are included automatically in the final Alt-R CRISPR crRNA oligonucleotide sequence and do not require any changes to the ordering process.
The addition of the Alt-R S.p. Cas9 Nuclease 3NLS and the modified Alt-R CRISPR crRNA build on the already market leading Alt-R CRISPR-Cas9 system. The changes further improve performance in many applications and give researchers more options for implementing high performance genome editing without affecting current protocols. For more information, visit www.idtdna.com/CRISPR.