NEW YORK (GenomeWeb) – Duke University researchers reported today in a new study that they were able to increase the specificity of CRISPR-based gene editing by several orders of magnitude by engineering a hairpin secondary structure onto the spacer region of single guide RNAs (hp-sgRNAs) and combining them with various CRISPR effectors.
In its paper published in Nature Biotechnology, the team demonstrated that designed hp-sgRNAs can tune the activity of a transactivator based on Cas9 from Streptococcus pyogenes (SpCas9). The researchers also showed that hp-sgRNAs increased the specificity of gene editing using five different Cas9 or Cas12a variants, demonstrating that RNA secondary structures can tune the activity of diverse CRISPR systems.
In order to increase the specificity of class 2 CRISPR systems and reduce off-target effects, the researchers began with the general strategy of reducing the energetics of DNA interrogation by the Cas9-sgRNA complex. They hypothesized that engineering the sgRNA might serve to regulate diverse CRISPR systems, and they specifically engineered an RNA secondary structure onto the spacer by extending a designed hairpin on the 5' end of the sgRNA.
"The resulting hairpin structure could then serve as a steric and energetic barrier to R-loop formation," senior author Charles Gersbach and his colleagues wrote. "We hypothesized that by adjusting the strength of the secondary structure, R-loop formation could proceed to completion at the on-target site but could be impeded at off-target sites, which have reduced energetics due to RNA-DNA mispairing."
The RNA hairpin is a fundamental structural unit in many RNA molecules and is composed of stems and loops. In order to engineer hp-sgRNAs, the researchers began by extending the protospacer-adjacent motif (PAM)-distal end of the spacer. They took into account all the variables they could use to create different structures with similar stability profiles, including placement of the stem along any area of the 20-nucleotide spacer, stem lengths, and tetraloop utilization. They also designed non-structured sgRNAs (ns-sgRNAs), which have extensions to the spacer but whose extensions are not predicted to form any secondary structures, to control for any effects of sgRNA length.
The team then tested the effect of predicted hp-sgRNAs structures on Cas9 binding to DNA. Specifically, as they wanted to analyze this interaction in human cells, they decided to utilize nuclease-inactive dCas9-based transcriptional activators where endogenous gene activation can serve as a sensitive measure of dCas9 binding to target DNA.
"For our initial hp-sgRNA designs, we used a tetraloop that is external to the 20-nucleotide spacer and placed the hairpin stems on the PAM-distal end of the spacer using canonical Watson-Crick base pairing. We used a spacer that targets the endogenous promoter of IL1RN, a gene we have previously activated with high efficiency," the authors wrote. "Transfecting sgRNA variants and a dCas9-P300 transactivator into human cells, we observed that hp-sgRNAs can tune gene activation at the target locus, suggesting modulation of dCas9 binding. We observed a generally regular relationship between length of the hp-sgRNA spacer extension and impact on dCas9 binding."
This underscores the need to control for guide length when measuring the effects of sgRNA secondary structure, they added.
The team then went on to assess the effect of spacer secondary structure on SpCas9 nuclease activity. The researchers generated a variety of hp-sgRNAs, and measured indel frequency at on-target and off-target sites for each spacer. They observed a number of hp-sgRNA designs with on-target activities comparable to wild-type sgRNAs and reduced off-target activity, comparable to truncated sgRNAs (tru-sgRNAs). They concluded that hp-sgRNAs can increase the specificity of SpCas9 nuclease by multiple orders of magnitude.
"Comparing with wild-type sgRNA, the tru-sgRNA eliminated 77 off-target sites but also had 25 unique off-target sites that were reproducibly detected using CIRCLE-seq," the authors wrote. "In contrast, the hp-sgRNA eliminated 124 off-target sites found with the WT-sgRNA and generated no unique off-target sites."
Importantly, further experiments found that similar to high-fidelity Cas9 variants, hp-sgRNAs do not mediate specificity increases through a decrease in binding. The researchers also found that hp-sgRNAs increased the specificity of gene editing with SaCas9 and AsCas12a proteins as well.
"At the off-target sites, hp-sgRNAs showed decreases of 91 percent, 79 percent, and 67 percent [for SpCas9, SaCas9 and AsCas12a, respectively] relative to WT-sgRNAs, compared with decreases of 88 percent, 38 percent, and 0 percent for the ns-sgRNAs," the researchers wrote. "These analyses showed that only hp-sgRNAs, and not ns-sgRNAs, robustly and reproducibly decreased occupancy at off-target sites relative to the on-target site."
The team concluded that R-loop formation is likely the central process governing CRISPR nuclease activity, and that its modulation allows for more specific genome editing. Improvements to the modeling of this process would be significantly useful for researchers as they attempt to create better predictions of off-target effects and design functional hp-sgRNAs.
"In this study, we demonstrate a method to increase specificity across diverse CRISPR systems. Future studies will be useful to determine whether hp-sgRNAs can similarly regulate new Cas12, Cas13 or Cas14 effectors," the authors added. "The hp-sgRNA secondary structures that regulate specificity may be combined with other methods of sgRNA engineering to modulate activity, specificity and orthogonality."