March 1, 2016 (Vol. 36, No. 5)
Mark Behlke, M.D., Ph.D.
Ashley Jacobi Staff Scientist Integrated DNA Technologies
Accurate and Efficient CRISPR Technology Is Paving the Way for Therapeutic Genome Editing
Advances in gene-editing technologies have enabled scientists to push forward their understanding of human genetics, especially in the context of genetic disorders. A number of different tools exist for making targeted genome modifications. The CRISPR/Cas9 system is a naturally occurring bacterial antiviral defense mechanism that has become one of the most prominent and versatile gene-editing tools available.
As our understanding of genetic diseases continues to grow, CRISPR/Cas9 has the potential to become key for therapeutic purposes. It could enable the direct correction of genetic disease-causing mutations. Although it has evolved at a dramatic pace, even moving beyond genome-editing applications, therapeutic use requires more progress. Here we describe a method by which CRISPR/Cas9 editing can attain greater levels of performance, reliability, and ease of use, creating a cleaner gene-editing system.
The Development of CRISPR Single Guide RNA
In general, gene-editing tools nick or break DNA at specific locations, activating a DNA repair pathway that enables modification of the sequence, such as insertion of new sequences or deletion of existing sequences. Useful for creating cell lines and animal models of disease, many CRISPR/Cas9 reagent systems rely on a version of the S. pyogenes Cas9 endonuclease for cleavage and a single guide RNA (sgRNA) for targeting.
Since its discovery, scientists have refined the natural system, developing sgRNA in the process. It is a fusion of the native crRNA (CRISPR RNA) and tracrRNA (trans-activating CRISPR RNA), providing both target site recognition and recruitment of the Cas9 enzyme in one. Using sgRNAs has simplified gene-editing protocols, primarily by reducing the expression cassette requirements to a single sgRNA-encoding unit rather than separate cassettes for crRNA and tracrRNA that must later associate and anneal to be functional (Figure 1). However, this simplicity comes at a cost, particularly when there is a view toward future therapeutic use.
The Challenges of sgRNA
Studying multiple genes requires at least one sgRNA to be synthesized for testing at each editing site. Several options for this exist, each with its own limitations:
- In vivo transcription: Synthesizing sgRNAs in vivo using a promoter-driven expression vector is common practice. However, Cas9-sgRNA expression plasmids are large—upwards of 9 kb—which decreases transfection efficiency and increases the probability of undesired DNA insertions into the genome.
- In vitro transcription (IVT): Often laborious, this method requires synthesis, quantification, and purification. Products often have unwanted base additions, and most labs do not have the ability to perform proper QC.
- Chemical synthesis: Although only the 20 base targeting sequence varies between sgRNAs, the full ~100 base length must be synthesized for each variation, which is not cost-efficient.
The size and complexity of sgRNAs also has intracellular effects: The multidomain secondary structure can activate the innate immune system, potentially leading to interferon pathway activation and cell death. Any future therapeutic use would require combating these issues.
Taking a Cue from Nature
Revisiting the natural bacterial CRISPR/Cas9 system, which has evolved over millions of years, has provided alternative methods to achieving gene-editing goals.
Recent research from Harvard’s Church Lab identified the ability to modulate CRISPR/Cas9 by truncating the guide RNA target-specific region. Scientists at Integrated DNA Technologies (IDT) have further worked on optimizing the lengths of universal regions of the crRNA and tracrRNA.
The natural CRISPR/Cas9 system consists of a 42 nt crRNA and an 89 nt tracRNA. Through empirical length studies (Figure 2), IDT has shortened the crRNA and tracrRNA components of the guide complex to 36 and 67 nt respectively. These lengths are more amenable to high-quality chemical synthesis and unexpectedly result in more efficient genome editing. Furthermore, this two-part RNA system allows use of a single large-scale lot of tracrRNA to pair with unique crRNAs, making it cost-effective for studying multiple sites.
Chemical synthesis of RNA oligos also enables chemical modifications to be made to RNA bases. IDT has empirically determined regions of the crRNA and tracrRNA where modifications improve stability of RNA in the cellular environment while maintaining a high level of gene editing.
The editing efficiency of IDT’s optimized crRNA:tracrRNA complex—components of its Alt-R™ CRISPR/Cas9 System—was compared with native crRNA:tracrRNA; IVT sgRNA; and plasmid- and gBlocks® Gene Fragments-expressed sgRNA. The optimized complex performed equal to or better than native crRNA:tracrRNA and sgRNA at multiple sites within the HPRT gene (Figure 3).
The experiments demonstrated that:
- The shorter target-specific crRNA, which pairs with the shorter universal tracrRNA in equimolar amounts, makes chemical synthesis easier and more cost-effective
- The shortened crRNA would be amenable to forming screening libraries for high-throughput arrayed use.
- A standardized tracrRNA can be bulk manufactured, providing consistent quality and reduced costs.
Design and Use
The two-part Alt-R CRISPR-Cas9 System only requires design of the target-specific region of the crRNA. As with sgRNA, general design rules, such as targeting coding regions or exon/intron junctions and avoiding 5’ and 3’ UTRs, apply to ensure optimal on-target effects. Tools such as MIT’s CRISPR Design make this straightforward, enabling the selection of targets with minimal predicted off-target effects, saving cost and effort. Designing crRNAs to target two or three sites following these general guidelines will produce effective results for most applications.
A particularly useful benefit of the optimized crRNAs and tracrRNAs is compatibility with existing protocols and numerous variations of the CRISPR/Cas9 system. Their versatility allows use with Cas9 delivered as a plasmid, mRNA, or protein across various cell types. Delivery of the Alt-R crRNA:tracrRNA and Cas9 as a ribonucleoprotein complex further improves efficiency and eliminates unwanted integrations.
Many tools are available for gene editing, with novel developments emerging all the time. For example, the recently characterized CRISPR/Cpf1 system provides a new tool to target additional sites for editing. An ideal tool should be easy to use and provide accurate, efficient, and clean gene editing. While the Cpf1-based system is in its infancy, the Cas9-based system continues to mature, taking steps toward therapeutic use.
In re-examining the native crRNA and tracrRNA, IDT created cleaner, more effective, and cost-efficient CRISPR/Cas9 genome editing in the form of the Alt-R system. Bringing CRISPR into the therapeutic frame, this work highlights the importance of considering alternative routes to advance gene-editing technology from which scientists can benefit immediately.