To modify genes, scientists need a way to direct mechanisms to the right spot. That’s the job of guide RNA (gRNA). Getting the job done efficiently, though, depends on engineering these molecules for a specific task and putting them in the proper place. A variety of tools can be used to simplify these processes.
“The guide RNA sequence selection can influence both activity and specificity,” says Laurakay Bruhn, section manager of biological chemistry at Agilent Research Laboratories . “Not all guide RNA sequences prove to have equivalent activity when tested empirically, so the choice of guide RNA sequence can influence the fraction of cells that are edited in a population of cells.” Bruhn adds, “Long chemically synthesized guides, such as those in Agilent’s SureGuide sgRNA portfolio, enable researchers to customize sequence selection up to 120mer and longer, and incorporate chemical modifications to ensure optimal performance.”
Much of the engineering of gRNA depends on how it will be used. In an article for Addgene about designing gRNAs for CRISPR genome editing, John Doench, associate director of the genetic perturbation platform at the Broad Institute, noted that “the ‘best’ gRNA depends an awful lot on what you are trying to do: gene knockout, a specific base edit, or modulation of gene expression.”
So, scientists need a variety of ways to design and make, or obtain, the needed gRNA.
Design tips
To edit genes, says Shawn Zhou, senior scientist at GenScript , the “two most important factors that need to be taken into consideration are gRNA cleavage efficiency and gRNA targeting sites.”
The targeting needs to be as specific as possible. “Several design algorithms exist to help predict the target-site specificity and on-target editing efficiency for a particular gRNA sequence,” says Ashley Jacobi, staff scientist at Integrated DNA Technologies. “IDT has a web-based design tool that automatically selects gRNAs with high on-target activity or can alternatively provide an on-target quality score for an existing gRNA.” Jacobi notes that off-target prediction algorithms exist, but they are not very reliable. So Jacobi recommends empirical determination of that feature.
Zhou offers other tips for a functional knockout, including using validated gRNA or designing gRNA near a published ZFN/TALEN targeting site. He adds, “To make gRNA design easier, GenScript licensed its CRISPR gRNA design tool and gRNA/Cas9 plasmids from the Feng Zhang’s lab at the Broad Institute of MIT and Harvard.
Design for delivery
Like designing the gRNA for targeting and cleavage, the best design for delivery also depends on the application. The best method of delivery “really depends on what you’re trying to do, what your cells will ‘allow’ and how concerned you are or are not with off-target effects,” Doench explains. “There’s really no generic answer, but rather a menu of choices, and the ‘right’ answer will be very context-specific.”
For example, adding unmodified RNA to mammalian cells can trigger an innate immune response. In addition, the RNA can be degraded by nucleases. Some chemically synthesized gRNAs can avoid being degraded and do not set off an immune response.
“The gRNA can effectively be delivered either in its native 2-part form—containing two RNA molecules, the crRNA and tracrRNA, annealed together to form the active gRNA complex—or as a single-guide RNA molecule that has the crRNA and tracrRNA fused together by a linker loop, and IDT offers chemically modified forms of both,” Jacobi explains. “The chemical modification pattern used in these RNAs has been empirically determined by IDT scientists to maintain full on-target potency.”
The nuclease that the gRNA works with in CRISPR also impacts the results. “Introducing a high-fidelity nuclease provides even lower risk for non-specific editing,” Jacobi explains. “IDT has developed a high-fidelity S.p. Cas9 nuclease that greatly reduces the off-target editing while maintaining on-target activity.”
Other experts agree that delivery is application-dependent. As Zhou says, the “gRNA/Cas9 delivery method is highly dependent on cell types.” For mammalian cell lines, for example, gRNA/Cas9 is usually delivered by electroporation as a ribonucleoprotein (RNP). “RNP edits genes with improved efficiency and lowered off-target effect when compared to other methods,” Zhou says. “In addition, RNP presents low toxicity to cells, especially for suspension cell lines, such as THP-1, U937, NK92, etc.” For more information, see GenScript’s CRISPR Ribonucleoprotein (RNP) User Manual.
The RNP method is not easy in all respects. According to Zhou, “The challenges of this method include: stability of gRNA and Cas9 protein during transporting and storing; higher equipment requirements for transfection methods; and higher cost compared to using CRISPR plasmids.” He adds, “To meet various research application needs, GenScript provides synthetic gRNAs, Cas9 proteins, as well as CRISPR plasmids.”
For cells in culture, plasmid or viral vectors can be used to deliver gRNA. “These methods can provide advantages for some situations—for example when sustained delivery of the guide RNA is advantageous and/or when libraries of guide RNAs, such as the SureGuide libraries provided by Agilent Technologies, are employed in pooled functional genomics screens,” says Bruhn. “Viral vectors are also commonly used for guide RNA and Cas protein delivery in vivo—in whole organisms—including viral strains that target specific tissue types.”
The range of delivery methods is already increasing. As an example, Bruhn notes, “Researchers are also developing other in vivo delivery methods—for example, lipid nanoparticles, which have the potential for delivery of RNP or RNA forms of the guide RNA and Cas9 and can also be targeted to specific tissue types.”
The use of gRNAs is really just getting started, especially in terms of the products that make this technology easier to use and more effective. More techniques will be developed to design better gRNAs for specific jobs and to deliver the molecules. Furthermore, scientists will be able to grow increasingly confident that a process makes the desired modifications in a genome. As a result, even more scientists will use gRNAs to accomplish more tasks.
Hero image: Various methods, such as CRISPR-Cas9, use guide RNA as part of a process to modify an organism’s genome. Image courtesy of Ernesto del Aguila III, National Human Genome Research Institute, NIH.