Processing
In a very short period of time, CRISPR (clustered regularly interspaced palindromic repeats) and Cas9 have evolved from an interesting mechanism to the dominant method for simplified genome editing. Unlike methods that require engineering of nucleases for each genomic target sequence, the CRISPR-Cas9 system uses a single nuclease (Cas9) paired with two RNA molecules—a target-specific crRNA and a trans-activating tracrRNA. Variations of these two RNAs can be purchased as chemically synthetized RNA oligonucleotides, and it has been shown that these RNAs can also be fused into a single-guide RNA, expressed by a plasmid or delivered directly into the cells as a double-stranded, linear expression cassette (e.g., as a gBlocks® Gene Fragment) [1–4]. However, not all CRISPR-Cas9 genome editing tools are created equal.
Potent editing performance
The Alt-R CRISPR-Cas9 System is the most potent genome editing solution available that uses experimentally optimized crRNA and tracrRNA as the guide RNA format. Our research team compared several CRISPR technologies and found that our optimized, shorter RNAs consistently improved editing performance when compared to other types of guide RNAs (Figure 1).
Figure 1. Optimized Alt-R CRISPR RNAs improve Cas9 editing efficiency compared to other guide RNA molecules. Alt-R CRISPR RNAs, S. pyogenes native CRISPR RNAs, in vitro transcribed (IVT) single-guide RNAs (sgRNA), and sgRNAs expressed from gBlocks Gene Fragments or a 2.7 kb expression plasmid were designed to recognize the human HPRT 38285-AS site. The RNA duplexes or sgRNAs were reverse transfected using Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher) into a HEK293-Cas9 cell line that stably expresses S. pyogenes Cas9. Optimal doses that give maximal editing were transfected: Alt-R RNAs, S. pyogenes RNAs, and IVT sgRNA (30 nM), gBlocks Gene Fragment sgRNA (3 nM), and sgRNA expression plasmid (100 ng). Genomic DNA was isolated, and editing was measured by PCR amplification of target sites, followed by cleavage with T7EI mismatch endonuclease (New England Biolabs; reagents and protocol now available as the Alt-R Genome Editing Detection Kit) and analysis using the Fragment Analyzer™ (Advanced Analytical). Alt-R CRISPR RNAs outperformed all other gRNA formats at 9 of 12 sites tested. Results from IVT sgRNAs were affected by cellular toxicity.
In fact, we have observed that >80% of Alt-R CRISPR crRNAs have good to excellent performance (measured as >40% editing efficiency by Sanger sequencing, or >20% by the easy-to-use T7EI assay (now available as the Alt-R Genome Editing Detection Kit; Figure 2). With the Alt-R CRISPR-Cas9 System, you can spend less time worrying about whether a particular crRNA site within your gene will work, and more time getting results.
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Figure 2. Potent editing efficiency of Alt-R CRISPR crRNA designs for 92 PAM sites in the STAT3 gene. Alt-R CRISPR crRNAs were generated for 92 distinct PAM sites in the STAT3 locus. Mutation detection using a T7EI assay demonstrated that 93% of the crRNAs provided good to excellent performance in HEK293-Cas9 cells, with editing efficiencies >20% (equivalent to >40% when measured by Sanger sequencing. Note that T7EI does not detect single base deletions or insertions, and therefore underestimates actual editing efficiency). (Reagents and protocol for CRISPR event detection are now available as the Alt-R Genome Editing Detection Kit.)
Reduce cellular toxicity The performance benefits of the Alt-R CRISPR-Cas9 System is not limited to efficiency. Unlike transfections with in vitro transcribed sgRNAs or Cas9 mRNAs, transfections with the Alt-R crRNA and tracrRNA do not trigger the innate cellular immune response, for greater cell viability (Figure 3).
Figure 3. The Alt-R CRISPR-Cas9 system does not trigger a cellular immune response. Alt-R CRISPR-Cas9 RNAs and corresponding in vitro transcribed (IVT) RNAs (triphosphate removed) designed to 12 HPRT1 sites were reverse transfected into HEK293-Cas9 cells that stably express S. pyogenes Cas9. 24 hr after transfection, expression levels of IFIT1 (A) and OAS2 (B), common stress response genes, were assayed. (A) qPCR amplification curves quantifying IFIT1 expression shows strong induction of IFIT1 by IVT RNA, but not Alt-R CRISPR-Cas9 RNA. (B) qPCR amplification data for OAS2 expression shows that IVT RNA cells have measurable induction of OAS2, whereas OAS2 levels in the Alt-R CRISPR-Cas9 RNA cells are at baseline. Similar results were seen for targets in 3 other genes, IFITM1, RIGI, and OAS1.
The Alt-R CRISPR-Cas9 System offers the optimal combination of excellent on-target genome editing performance and low toxicity. If you are looking for the fastest path to publishable results that is also affordable, visit www.idtdna.com/CRISPR for more information or to order the Alt-R CRISPR-Cas9 System.
