CRISPR-Cas9 genome editing is changing the entire landscape of genomic research. The CRISPR-Cas9 system uses the bacterial-derived Cas9 endonuclease to generate double-stranded breaks in DNA. Cas9 is guided to specific sites by a short CRISPR RNA (crRNA) and a slightly longer transactivating CRISPR RNA (tracrRNA). The cleaved DNA is then repaired by non-homologous end joining (NHEJ) or homologous recombination, resulting in a modified sequence. Through a series of rational experiments, IDT scientists have improved the efficiency of this popular tool and developed a set of potent CRISPR tools that are now offered as the Alt-R® CRISPR-Cas9 System.
The Alt-R CRISPR-Cas9 System includes optimized, 36 base crRNA and 67 base tracrRNA, and a potent S.p. Cas9 Nuclease 3NLS. The following experiments address factors that influenced the development of the Alt-R CRISPR-Cas9 System components and their delivery. These results also inform recommendations for designing more effective genome editing experiments of your own:
- Does delivery format of Cas9 nuclease affect editing efficiency?
- What are optimal crRNA and tracrRNA lengths that yield the best gene editing performance, while providing cost-effective manufacturer?
- Which types of CRISPR guide RNA formats result in the most efficient on-target genome editing?
- Is protospacer site selection critical for editing efficacy?
- Is protospacer size critical for editing efficacy?
- Are there CRISPR RNA designs that elicit less toxicity and innate immune responses?
1. Potent editing with Cas9 Nuclease delivered as a ribonucleoprotein
IDT scientists conducted experiments to determine whether delivery of Cas9 protein complexed with CRISPR RNAs or as an expression plasmid or mRNA affected the efficiency of genome editing. When the Alt-R® S.p. Cas9 Nuclease 3NLS is combined with the Alt-R CRISPR crRNA and tracrRNA into a ribonucleoprotein (RNP), the system outperforms those using the other Cas9 formats (Figure 1). RNP transfections also provide optimal control of dose of editing complexes, and the non-renewable Cas9 RNP is cleared after a short duration by endogenous mechanisms, limiting off-target editing.
Figure 1. Lipofection of Alt-R CRISPR-Cas9 System Components as a ribonucleoprotein outperforms other transient CRISPR-Cas9 approaches. 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 part of 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 approaches, and performed similar to reference HEK293-Cas9 cells that stably express S. pyogenes Cas9.
2. Optimizing crRNA and tracrRNA lengths improves gene editing performance
Manufacturing long RNA oligos is costly and limited by the sequential coupling efficiencies of each RNA base. In developing the Alt-R CRISPR-Cas9 System, our research team conducted empirical length studies to develop the shortened crRNA and tracrRNA components (36 nt and 67 nt, respectively) that would be more amenable to affordable, high quality chemical synthesis, while not affecting gene editing performance. In addition, chemical synthesis offers the opportunity to introduce chemical modifications that confer benefits, such as resistance to nucleases and reduced immunogenicity. Furthermore, the resulting shortened RNAs significantly improve gene editing efficiency with S. pyogenes Cas9 in mammalian cell culture (Figure 2).
A 67 nt tracrRNA paired with a 36 nt crRNA (Figure 2, orange arrow) provided the highest editing efficiency. (Editing efficiency was analyzed using a convenient T7EI mismatch cleavage assay. T7EI assessment underestimates editing events by approximately 50% compared to Sanger sequencing, as it does not detect single-base indels . However, it is a quick and cost-effective assay that provides a consistent readout relative to Sanger sequencing data, available as the Alt-R Genome Editing Detection Kit.)
These results were the rationale for the optimized CRISPR RNA oligonucleotides, now available as components of the Alt-R CRISPR-Cas9 System, that produce superior on-target genome editing, consistently outperforming other CRISPR RNA formats.
Figure 2. Shorter crRNA and tracrRNA lengths improve on-target genome editing. crRNAs of different lengths targeting HPRT 38285-AS were hybridized with tracrRNAs of different lengths. crRNA:tracrRNA complexes were reverse transfected using Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher), into a HEK293-Cas9 cell line that stably expresses S. pyogenes Cas9. Genomic DNA was isolated, and editing was measured by PCR amplification of target sites, cleavage with T7EI mismatch endonuclease (New England Biolabs), and analysis using the Fragment Analyzer™ (Advanced Analytical).
3. Alt-R CRISPR-Cas9 RNA triggers are more potent than single guide RNAs (sgRNAs)
Numerous methods have been described for expressing Cas9 and the required crRNA and tracrRNA in cells. In developing our Alt-R CRISPR-Cas9 System we looked at several popular mechanisms for generating the crRNA and tracrRNA to determine the most effective approach. Figure 3 shows a comparison of on-target editing efficiency resulting from several different formats of Cas9 trigger RNAs:
- Alt-R CRISPR crRNA (36 nt) and tracrRNA (67 nt)
- S. pyogenes native CRISPR crRNA (42 nt) and tracrRNA (89 nt)
- in vitro transcribed (IVT) sgRNA
plasmid sgRNA (2.75 kb)
- gBlocks® Gene Fragments sgRNA
The data demonstrate the high cleavage efficiency of Alt-R CRISPR RNAs (green; editing efficiency was analyzed using a T7EI mismatch cleavage assay). In fact, the Alt-R CRISPR RNAs outperformed all other guide RNA formats tested.
Figure 3. 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 a 2.7 kb expression plasmid or gBlocks® Gene Fragments were designed to recognize 4 sites within the human HPRT gene (38087 AS, 38509 S, 38285 AS, and 38636 AS). 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), 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) and analysis using the Fragment Analyzer™ (Advanced Analytical). Alt-R CRISPR RNAs had the highest editing efficiency when compared to the other gRNA formats.
4. The robust Alt-R CRISPR-Cas9 System may eliminate the need for a crRNA design tool
While several free software programs exist to design the 19–20 nt crRNA protospacer sequence, few produce designs that correlate consistently with strong genomic editing activity. However, our empirical data demonstrate that the Alt-R CRISPR-Cas9 System is robust. Figure 4 illustrates the high genome editing function achieved by crRNA:tracrRNA complexes targeting all 553 PAM sites across 6 exons, where the majority of crRNAs produced good to excellent results. This means that even without specific design selection, use of these optimized RNAs will show strong on-target editing for the majority of target sites.
Figure 4. Alt-R CRISPR-Cas9 System functions well across many sites. Alt-R crRNAs were designed to target all 553 PAM adjacent sites in 6 exons selected from 4 distinct genes (HPRT, EMX1, STAT3, and Dicer). Complexes were reverse transfected using Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher) into a HEK293-Cas9 cell line that stably expresses S. pyogenes Cas9. Genomic DNA was isolated, and editing was measured by PCR amplification of target sites, cleavage with T7EI mismatch endonuclease (New England Biolabs), and analysis using the Fragment Analyzer™ (Advanced Analytical).
5. Protospacer element size is critical for editing efficacy
There are also reports in the literature suggesting that CRISPR-Cas9 off-target activity can be reduced by using truncated guide RNAs . For example, 17 base protospacer elements have been reported to reduce off-target effects. While the shortened protospacer may reduce off-target effects, we investigated how shortening protospacer element length would affect CRISPR-Cas9 nuclease on-target performance (Figure 4). crRNAs with protospacer element lengths of 17–20 bases were designed to 12 distinct HPRT target sites, and genome editing efficiency was measured using a T7EI cleavage assay. 20 base protospacer elements were optimal, with 19 bases providing similar strong editing efficacy in most cases. Editing efficiency was greatly reduced when 17 and 18 base protospacer lengths were used.
Figure 5. 19–20 nt protospacer element provides optimal genome editing. crRNAs with varying protospacer element lengths (17–20 nt) were designed to 12 distinct HPRT target sites. crRNA:tracrRNA complexes were reverse transfected using Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher) into a HEK293 cell line stably expressing S. pyogenes Cas9. Genomic DNA was isolated, and editing was measured by PCR amplification of target sites, cleavage with T7EI mismatch endonuclease (New England Biolabs), and analysis using the Fragment Analyzer™ (Advanced Analytical). At all but 1 of the 12 target sites, crRNAs with 19 and 20 base protospacer elements produced the greatest amount of genomic editing. Each of the 12 data points for each category represent the average of 3 biological replicates, with the exception of one data point in the 19 base nt category that is composed of 2 biological replicates.
6. Alt-R CRISPR-Cas9 RNAs elicit less toxicity and innate immune response compared to in vitro transcribed guide RNA alternatives
Transfection of long IVT RNAs has been shown to elicit an innate immune response in our laboratories. This response can result in high cell death due to cytotoxicity. We compared cellular toxicity and immune response activation by Alt-R RNAs and IVT RNAs using our HEK293-Cas9 cell line that constitutively expresses Cas9 (Figure 5). We observed high levels of activation of stress response genes such as IFIT1 (P56) and OAS2 (as well as IFITM1, RIGI, and OAS1; not shown) related to the innate immune response in cells challenged with IVT RNA triggers. These genes were not activated in cells transfected with Alt-R CRISPR-Cas9 RNAs.
Figure 6. 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-treated cells have measurable induction of OAS2, whereas OAS2 levels in the Alt-R CRISPR-Cas9 RNA-treated cells are at baseline. Similar results were seen for targets in 3 other genes: IFITM1, RIGI, and OAS1.
Potent editing performance
Through the use of experimentally optimized crRNA and tracrRNA as the guide RNA format, and delivery of the CRISPR RNAs and Cas9 nuclease as an RNP, the Alt-R® CRISPR-Cas9 System provides the most potent genome editing solution available. The IDT research team compared several CRISPR technologies and found that our optimized, shortened RNAs consistently improved editing performance when compared to other types of guide RNAs. Genome editing efficiency was further improved by complexing these optimized RNAs with the Cas9 Nuclease and delivering the resulting RNP to the cells being edited. The Alt-R CRISPR-Cas9 System allowed the scientists to spend less time worrying about whether a particular crRNA site would work and more time getting results.