Scott Pritchett

CRISPR gene editing has proven to be an efficient therapeutic approach, as evidenced by the recent approval of CASGEVY™, a cell therapy utilizing CRISPR/Cas9 for the treatment of sickle cell disease. However, generating double strand breaks (DSBs) using the CRISPR/Cas9 system introduces the risk off-target effects. ¹ Developing novel editing systems that do not require DSBs is an ever-expanding area of genetic research.

Introducing prime editing

Prime editing was developed as a “search-and-replace” genome editing technology.² It comprises three key components (Figure 1):

• 1. Prime Editing Guide RNA (pegRNA): Guides the target sequence to be edited and includes the desired sequence for replacement.
• 2. Cas9 H840A Nickase: Introduces a single-strand break (or ‘nick’) without creating a double-strand break.
• 3. M-MLV Reverse Transcriptase: Generates the desired DNA sequence from the RNA template in pegRNA.

PegRNAs are generally composed of four parts: a spacer, scaffold crRNA, reverse transcription (RT) template, and a primer binding site (PBS) (Figure 2). Due to this intricate design, pegRNAs usually exceed 150 nucleotides (nt), making manufacturing and quality control challenging.

Manufacturing strategies for ultra-long RNAs

There are three main methods for preparing ultra-long RNA (≥150 nt).

• (1) In vitro transcription (IVT)
  IVT is conventionally used for synthesizing ultra-long RNA at both research and commercialization stages. However, it cannot introduce chemical modifications such as 2-OMe and phosphorothioate, which significantly improve editing efficiency.³ Additionally, IVT can result in extra Gs at the 5’ end, which can adversely affect the guide region in CRISPR/Cas9 systems.⁴
• (2) Enzymatic ligation
  Previously, the enzymatic ligation of oligonucleotides was a useful approach for synthesizing long RNA when the achievable synthetic length via intact chemical synthesis was limited. Assembling and ligating two 80 nt fragments to get a 160 nucleotide (nt) full length product was, in theory, easier than intact synthesis of a single 160 nt product. In addition, without the use of a high amount of organic solvent to wash the solid support, enzymatic ligation was more environmentally- friendly when scaling up to hundred kgs to tons scale, especially for producing small nucleic acid drugs.⁵ However, the relatively high cost of enzymes and long turnaround time for enzymatic reactions make this approach less effective for high-throughput screening.
• (3) Solid phase synthesis
  Traditionally, solid phase synthesis of oligonucleotides is limited to about 150 nt due to reaction efficiency constraints. However, it is very efficient during screening stages, allowing hundreds of sequences to be synthesized simultaneously. The manufacturing process is heavily machine-oriented, making it controllable during scale-up. Also, without introducing any recombinant proteins, chemically-synthesized oligonucleotides can meet very low endotoxin levels and maintain a very low level of residual proteins. Thus, regulatory requirements can be met more easily without the need to introduce additional controlling strategies.

We believe that developing a solid phase synthetic approach for ultra-long RNAs is crucial for providing the GCT community with high-quality, cost-effective materials for early-phase screening and future regulatory purposes.

Key aspects for the success of ultra-long RNAs

Maintaining a high conversion ratio (efficiency, or eff) for each synthesis cycle is crucial for producing ultra-long RNAs. Using a 150 nt sequence as an example, if the efficiency for producing a 150 nt sequence drops from 99.5% to 98.5%, the full-length product (FLP) percentage drops from 47.1% to 10.4%, increasing purification difficulty and decreasing yield (Figure 3). To address this, we improved crude purity and optimized a cost-effective approach specific to ultra-long RNAs.

(1) Improving crude purity through optimized solid supports

All oligonucleotides are synthesized on the solid support. Features such as bed-height, swell-ability, loading amount, and pore size are crucial to the synthesis outcome.⁶ In order to get the best synthetic performance, we made solid supports that are extremely suitable for synthesizing ultra-long RNAs (Figure 4). When a commercial support (from Vendor C) was applied as comparison, significantly higher amounts of early elute (EE) impurities were observed. We believed the relatively low coupling efficiency on commercial support resulted in more N-x impurities during the synthesis. These impurities usually eluted earlier than the FLP on the chromatography. However, our optimized solid support significantly lowered the presence for early elute (EE) impurities without increasing the late elute (LE) impurities during the synthesis of a 173 _nt guide RNA.

(2) Improving cost effectiveness through post-synthetic processes

HPLC purification, while ensuring the highest purity of ultra-long RNAs, is less effective at early screening stages due to low recovery, higher cost and longer turnaround time. With good crude purity, the desalt process can provide a satisfactory option for early screening. We optimized the production process by screening various solid supports and eluting conditions (Figure 5). Based on our model study, the early elute (EE) impurities and late elute (LE) impurities are further controlled with our process.

Case Study-Reliably delivering ultra-long RNAs of 218 nt

Prime editing, while promising for precise editing and low off-target risk, initially showed relatively low editing efficiency across different targets, even in immortalized cell lines.⁷ Installing a secondary structure (tevopreQ1) onto the 3’ end of the pegRNA significantly improves editing efficiency.⁸ However, introducing an additional 37 nucleotides during the synthesis of ultra-long RNA was very challenging, as the crude purity and recovery decreased drastically when the synthetic length increased.

With our newly optimized production procedure, we are now capable of producing ultra-long RNA up to 231 nt in both desalt and HPLC-purified grades. The high quality of our pegRNA has been verified by HPLC and MS spectrometry (Figure 6a). These ultra-long RNA also showed good editing efficiency in cellular platforms (Figure 6b). An increase in editing efficiency was observed when 3’ tevopreQ1 was introduced during chemical synthesis.

GenScript’s ultra-long oligonucleotide synthesis service

GenScript has over 21 years of experience in producing long oligonucleotides. Our team of experts has developed optimized production processes for both desalt and HPLC-purified grade ultra-long oligonucleotides, incorporated within a ISO9001 quality system.

• Delivery scale: µg~g
• Purity: ≥85% by internal UPLC method
• Modification: Default modification on the first and last three nucleotides with PS and 2’-methoxyl

Learn more about our pegRNA synthesis service here.

Reference

• (1) Zhang, X.-H.; Tee, L. Y.; Wang, X.-G.; Huang, Q.-S.; Yang, S.-H. Off-Target Effects in CRISPR/Cas9-Mediated Genome Engineering. Molecular Therapy - Nucleic Acids 2015, 4, e264. https://doi.org/10.1038/mtna.2015.37.
• (2) Anzalone, A. V.; Randolph, P. B.; Davis, J. R.; Sousa, A. A.; Koblan, L. W.; Levy, J. M.; Chen, P. J.; Wilson, C.; Newby, G. A.; Raguram, A.; Liu, D. R. Search-and-Replace Genome Editing without Double-Strand Breaks or Donor DNA. Nature 2019, 576 (7785), 149–157. https://doi.org/10.1038/s41586-019-1711-4.
• (3) Hendel, A.; Bak, R. O.; Clark, J. T.; Kennedy, A. B.; Ryan, D. E.; Roy, S.; Steinfeld, I.; Lunstad, B. D.; Kaiser, R. J.; Wilkens, A. B.; Bacchetta, R.; Tsalenko, A.; Dellinger, D.; Bruhn, L.; Porteus, M. H. Chemically Modified Guide RNAs Enhance CRISPR-Cas Genome Editing in Human Primary Cells. Nat Biotechnol 2015, 33 (9), 985–989. https://doi.org/10.1038/nbt.3290.
• (4) Gallo, S.; Furler, M.; Sigel, R. K. O. In Vitro Transcription and Purification of RNAs of Different Size. Chimia 2005, 59 (11), 812. https://doi.org/10.2533/000942905777675589.
• (5) Andrews, B. I.; Antia, F. D.; Brueggemeier, S. B.; Diorazio, L. J.; Koenig, S. G.; Kopach, M. E.; Lee, H.; Olbrich, M.; Watson, A. L. Sustainability Challenges and Opportunities in Oligonucleotide Manufacturing. J. Org. Chem. 2021, 86 (1), 49–61. https://doi.org/10.1021/acs.joc.0c02291.
• (6) Guzaev, A. P. Solid-Phase Supports for Oligonucleotide Synthesis. Curr Protoc Nucleic Acid Chem 2013, Chapter 3, 3.1.1-3.1.60. https://doi.org/10.1002/0471142700.nc0301s53.
• (7) Zhao, Z.; Shang, P.; Mohanraju, P.; Geijsen, N. Prime Editing: Advances and Therapeutic Applications. Trends in Biotechnology 2023, 41 (8), 1000–1012. https://doi.org/10.1016/j.tibtech.2023.03.004.
• (8) Nelson, J. W.; Randolph, P. B.; Shen, S. P.; Everette, K. A.; Chen, P. J.; Anzalone, A. V.; An, M.; Newby, G. A.; Chen, J. C.; Hsu, A.; Liu, D. R. Engineered PegRNAs Improve Prime Editing Efficiency. Nature Biotechnology 2022, 40 (3), 402–410. https://doi.org/10.1038/s41587-021-01039-7.