The widespread application of CRISPR-Cas technology in the gene editing field is driving rapid growth in the demand for sgRNA synthesis. However, the traditional SPOS (Solid-Phase Oligonucleotide Synthesis) method struggles to keep pace with this increasing demand. The reason is that when SPOS is used for sgRNAs approximately 100 nt in length, it is difficult to ensure the fidelity of the sgRNA guide region. Furthermore, due to the high density of chemical modifications and complex secondary structures of sgRNAs, the SPOS method also finds it challenging to achieve high purity and yield.
Through careful strategic planning, Hongene has deeply developed enzymatic ligation synthesis technology, which has already been applied to the high-purity synthesis of bivalent siRNA (see Part 1 for details). This article, as the second part of the technology application case series, will continue to introduce Hongene's sgRNA synthesis case study, aiming to demonstrate how the two-step enzymatic ligation method ensures the high fidelity of the sgRNA guide region and highlight the potential of enzymatic ligation technology in gene editing drug development!
The sgRNA developed by Intellia Therapeutics can guide CRISPR-Cas editing of the TTR (transthyretin) gene for the treatment of hATTR (hereditary transthyretin amyloidosis). This case study focuses on the Intellia G211 sgRNA. Its structure comprises a guide region complementary to the target gene, a repeat/anti-repeat region, and a structural stability region containing multiple stem-loops (Figure 1). A key feature is the introduction of 2'-OMe modifications at multiple positions and PS (phosphorothioate) modifications at both ends. These modifications can significantly enhance its in vivo stability and activity.
However, the complexity of G211 sgRNA presents the following challenges for the SPOS method: (i) Because SPOS synthesis proceeds from the 3' end to the 5' end, the synthesis of the 20 nt guide region occurs at the final stage of the SPOS process. The accumulation of reaction steps makes it difficult to control the fidelity of this crucial sequence, implying potential off-target risks; (ii) Synthesizing the 100 nt G211 sgRNA via SPOS requires approximately 400 reaction steps. This leads to an extremely complex impurity profile for the produced sgRNA and reduces the sgRNA yield.
Unlike SPOS which synthesizes the 100 nt strand in one go, Hongene's two-step enzymatic ligation method divides the sgRNA into 3 fragments (Figure 1), each requiring SPOS to synthesize only about 40 nt at once. Therefore, the impurity profile for each fragment is simpler, and its purity and yield are higher. Moreover, splitting the sgRNA into 3 fragments for separate synthesis also reduces interference from secondary structure during SPOS. Most importantly, the two-step enzymatic ligation method includes an independent enzymatic ligation step for the guide region to ensure its high fidelity.
The enzymatic ligation synthesis process for the guide region includes the following steps:
- First, synthesize two guide region fragments (Fragment 1A and 1B) and one 20 nt DNA template (Template 1*) via SPOS (Figure 2, A).
- Second, Fragments 1A and 1B hybridize complementarily with Template 1* to form the ligation site (Figure 2, B).
- Third, use a ligase to connect 1a and 1b, forming the complete Fragment 1 (Figure 2, C).
- use DNase to degrade the DNA Template 1*, leaving the RNA Fragment 1 (Figure 2, D).
As shown in Figure 3, A, after completing the synthesis of Fragment 1, synthesize the other two fragments (Fragments 2 and 3) via SPOS, then synthesize 2 DNA templates (Templates 1*2* and 2*3*) to ligate Fragments 1, 2, and 3. With the assistance of the templates, the enzymatic ligation of the three fragments is completed in a one-pot reaction. Finally, degrade the two templates with DNase to obtain the complete sgRNA.
Fragments 1 and 2 are fully complementary to their templates, while Fragment 3 is only partially complementary to its template. Reasons include: (i) Full complementarity would require Template 2*3* to be 60 nt long, which would reduce its synthesis purity and yield; (ii) The current length of Template 2*3* is sufficient to ensure complementary specificity with Fragment 3 and exclude interference from impurities.
Hongene selected different purification strategies for the templates and fragments. As shown in Figure 3, B, all RNA fragments were purified using HPLC, while the DNA templates were purified only by UF/DF (ultrafiltration/diafiltration). The rationale is that the final purity of the sgRNA product is primarily determined by Fragments 1-3, and the template impurities will ultimately be degraded by DNase. It can be seen that even though the purity of Template 1*2* is only 70.4%, the purity of the sgRNA product still reaches as high as 96.8% (after DNase degradation and HPLC purification). Omitting column purification for the templates helps reduce synthesis costs.
In this case, the enzymatic ligation synthesis of bivalent exNA siRNA demonstrated the following characteristics and advantages:
- High fidelity of the guide region. The two-step enzymatic ligation method uses the independent ligation of the guide region as the first step and the assembly ligation of the complete sgRNA as the second step. This strategy shortens the SPOS synthesis length for the guide region to about 10 nt, thereby achieving very high purity (>98%, Figure 3, B) and, consequently, high fidelity for the guide region.
- Higher sgRNA purity. The two-step method achieved a final product purity of 96.8%, significantly higher than the purity typically achievable with SPOS (60-80%). Furthermore, by drastically reducing the single-run SPOS synthesis length (100 nt → 40 nt), the enzymatic ligation method also reduces the complexity of the sgRNA impurity profile.
- Higher yield and lower cost. Similar to the case with bivalent siRNA (see Part 1 for details), using enzymatic ligation for sgRNA synthesis results in a higher final product yield. Additionally, the enzymatic ligation method can significantly reduce the consumption of SPOS reagents, thereby lowering production costs.
- Flexible modular synthesis characteristics. The two-step enzymatic ligation method used in this case validates the feasibility of modular synthesis. If necessary, independent enzymatic ligation steps can be applied to other fragments to ensure high purity and fidelity of specific segments. This strategy has good scalability and holds promise for application in the synthesis of more complex RNA structures, such as pegRNA (prime editing guide RNA).
Today, as gene editing technology rapidly evolves from the laboratory to clinical applications, enzymatic ligation technology is becoming a key pathway to break through the synthesis bottleneck of long-chain sgRNAs. Through its stepwise synthesis strategy, Hongene's enzymatic ligation technology not only significantly improves the purity and yield of sgRNA but also ensures high fidelity of the guide region.
Hongene is further expanding the application of enzymatic ligation technology to longer sgRNA molecules, such as pegRNAs which can reach up to 250 nt in length. These RNAs incorporate a reverse transcription template fused to the guide region, demanding even higher synthesis precision and structural integrity. We believe the high flexibility of the two-step enzymatic ligation method will make it one of the most promising solutions for the industrial-scale synthesis of pegRNA. As CRISPR-based therapeutic approaches diversify in the future, the two-step enzymatic ligation method will unlock broader application spaces in the field of gene editing!
In September 2024, Hongene entered into a strategic partnership with the US-based CDMO service provider ReciBioPharm. Hongene's enzymatic ligation technology for synthesizing high-purity sgRNA will empower ReciBioPharm to bring higher quality products and more efficient solutions to gene editing drug development!