Read time: 7 minutes

Prime editing (PE) is a next-generation gene editing technology that utilizes a Cas9 protein fused to a prime editing guide ribonucleic acid (pegRNA) to achieve high CRISPR/Cas9 editing efficiency and precision. However, the length requirement of pegRNAs at 120–250 nucleotides (nt) and their high level of secondary structure formation present analytical challenges for the purity analysis of chemically synthesized pegRNAs during development and quality control (QC).

In a recent webinar, Dr. Tingting Li, Manager, Cell and Gene Therapy Application at SCIEX and Ashley Jacobi, Director, Applications and Market Development at Integrated DNA Technologies (IDT) gave insights into state-of-the-art pegRNAs, their advantages and future outlook. In addition, they discussed how to break through analytical boundaries for purity assessment using capillary electrophoresis to enable next-generation CRISPR/Cas9 gene editing approaches.

In this blog, I summarize their answers to pressing questions from the webinar.

pegRNA purity assessment with CGE

Can you summarize the details of your CGE method?

Tingting Li: Yes, you can find materials and methods summarized in the technical note here. Please do not hesitate to contact us if you have further questions.

Can you comment on the typical lifespan of the capillaries used for pegRNA analysis?

Tingting Li: The capillary used for this assay is the coated DNA capillary for the PA 800 Plus system available in the ssDNA 100-R kit (PN 477480). It is validated for 100 runs. However, my personal experience is that more runs can be performed. For the pegRNA analyses, I conducted >150 runs and the capillary can still be used for further analyses. I used the same capillary to develop the method presented in the webinar and to analyze the pegRNAs of different lengths, from different lots on different instruments and on different days. The key to achieving a long capillary lifetime is to avoid exposing the capillary ends to air for more than 3 minutes. Additionally, it is important to store the capillary appropriately after usage.  When not in use, always rinse and store the capillary in unused gel buffer at 2°C to 8°C with both capillary ends submerged in Tris-Borate-Urea buffer.

What resolution did you achieve for the CGE workflow?

Tingting Li: For the optimized CGE workflow we tested pegRNAs ranging from 120 to 229 nucleotides. For these samples, we achieved a resolution of three to seven bases. This level of resolution allowed us to accurately separate and analyze the pegRNA samples, providing valuable information about their purity and integrity. If higher resolution is required for specific applications or if further fine-tuning is desired, there are several potential optimization strategies that can be explored. These may include, but are not limited to, using a longer separation capillary, employing a lower separation voltage or using a less diluted gel matrix. Recently, our team worked on an application achieving single-base resolution to enable the separation of different poly-A tail lengths of messenger RNA (mRNA). The technical note describing the method can be found here.

Have you tried adding a denaturation agent to the separation buffer to maintain the denatured state of the pegRNA during the CGE analysis?

Tingting Li: Yes, we have explored the use of the denaturant in the separation buffer for pegRNA analysis. The gel buffer of the kit we used—ssDNA 100-R kit (PN 477480)—contains 7M urea. However, due to the highly complementary structure of the pegRNA molecules, we found that the denaturant urea alone was not sufficient to maintain the denatured status of the molecules during the CGE separation process. As a result, we needed to further optimize the method to achieve a suitable peak shape for the pegRNAs. One of the key parameters we implemented was elevating the capillary temperature. The higher capillary temperature kept the hydrogen bound disrupted as the pegRNA molecules migrated through the separation capillary and prevented the formation of the secondary structures, ensuring that the pegRNA molecules remained in their denatured state during the analysis. Hence, achieving great peak shapes and accurate purity assessments.

Can you provide further insights into your strategy for addressing the challenge of pegRNA peak shape?

Tingting Li: Optimizing the peak shape of pegRNA was indeed a challenging task. Our hypothesis is that highly complementary primer binding sites (PBS) and protospacer sequences in pegRNA molecules caused the formation of hairpin and secondary structures, resulting in a very broad peak when analyzed with CE under standard conditions. To confirm this, we synthesized 4 pegRNAs with varying lengths and their corresponding non-complementary RNAs (NC RNAs) with the same PBS sequence but with a non-targeting protospacer. We analyzed all 8 samples and found that the NC RNAs exhibited a singular sharp peak while we observed broad, unresolved peaks for all pegRNAs. This further indicated that the broad, undesired peak shape observed for pegRNAs was likely due to the formation of high-order structures caused by their highly complementary sequence design. For the next step of method development, we focused on a robust denaturing technique for CGE purity analysis of pegRNAs with high levels of secondary structure. We explored various denaturation methods during the sample preparation. However, we did not achieve significant improvements of the peak shape using these different sample preparation procedures. We then focused on optimizing the conditions during the separation process. The breakthrough came with heat-based denaturation during separation in the CE capillary. We maintained a capillary temperature of 50⁰C throughout the separation process. Based on the peak profiles achieved, we concluded that this step prevented the formation of inter- and/or intramolecular hydrogen bonds of pegRNA molecules. With the temperature-controlled capillary at 50⁰C, we can achieve a single sharp peak for each of the pegRNA samples.

Could you please elaborate on the temperature control feature of your CE system and its role in improving the accuracy of pegRNA purity analysis?

Tingting Li: Certainly. The unique capillary temperature control feature of the PA 800 Plus system is a critical aspect that ensures precise and stable temperature control during the separation process. This is achieved by an inert liquid circulating through the cartridge around the separation capillary. Compared to air used in other systems, the inert liquid can maintain temperature more precisely and consistently throughout the analysis. For the analysis of oligonucleotides like pegRNA, the stable temperature control is vital as it minimizes the variations in migration time and peak shape, resulting in highly reproducible results for purity analyses.

How can the denaturing technique and gel matrix be adapted for other analyses with high levels of secondary structures? Can you provide any advice?

Tingting Li: The denaturing technique presented in the webinar and the gel matrix can indeed be adapted for the analysis of other oligonucleotides with high levels of secondary structures. I recommend testing different denaturation agents and temperatures for sample preparation and during the CE separation process while considering the characteristics of your oligonucleotides, such as heat and pH stability for example. In addition, the gel matrix, separation voltage and separation time can be optimized to achieve the needed resolution and peak shape. I can see the flexibility of this method and system opening up possibilities for investigating various oligonucleotides and their functions in different research areas.

Molecular weight determination with LC-MS

Which MS instrumentation did you use for the molecular weight determination?

Tingting Li: We used a time-of-flight instrument. For this type of analysis, the X500B QTOF system and the ZenoTOF 7600 system are both great options for achieving high-quality MS data used for deconvolution and MW determination. For the data presented in the webinar, we used the ZenoTOF 7600 system.

Did you use LC-MS for molecular weight confirmation of RNAs larger than 122 nt?

Tingting Li: Yes, we have conducted molecular weight (MW) confirmation experiments for pegRNAs larger than 122 nucleotides. We tested samples up to 167 nucleotides in length. While we noticed an increase in impurities, as expected for longer synthetic oligonucleotides, we were able to achieve high accuracies for MW determination. The difference between experimental and theoretical MW was <5 ppm. You can find example data for pegRNA of up to 167 nt in the SCIEX CRISPR solution guide here.

Future outlook for synthetic guide RNAs

How do you foresee the CGE purity workflow impacting future research and advances in CRISPR technologies?

Ashley Jacobi: High quality synthetic guide RNAs with high purity is very important for the efficiency of your gene editing experiments. This holds true not only for prime editing but for any CRISPR technology. As these technologies move from research towards therapeutic use, the purity of the starting material is of the utmost importance. Having analytical technology that provides accurate purity determination is a necessity to achieve desired outcomes and bring safe gene editing technologies to market.

Can you comment on modalities beyond pegRNAs in your pipeline?

Ashley Jacobi: We are always looking for improvement and innovation within IDT. The data we showed in the webinar was up to about 200 nucleotides. However, we are investigating even longer guide RNAs. Another avenue we are exploring is different chemical modifications. How do those modifications affect editing efficiency and/or the stability of these compounds? It is something we are actively looking into.