Benefits of Using Synthetic sgRNA for CRISPR-based Editing

Since its initial discovery as an innate bacterial immune system, the CRISPR-Cas9 system from S. pyogenes has been developed to be the most popular and efficient technology for engineering genomes. Compared to previous protein-based technologies, such as ZFNs and TALENs, the RNA-based CRISPR technology is simple to use—researchers can make targeted changes to genetic sequences both in vivo and in vitro at relatively low cost. The simplicity and robustness of gene editing made possible with CRISPR has resulted in its use in a myriad of applications, from basic research and drug discovery to gene therapy.

But there are still some limitations of the technology to be addressed, including variations in delivery efficiency, off-target activity, and potential immunogenic effects, which mean editing can be unreliable in some primary cells and tissues—particularly important when considering the use of CRISPR in a clinical setting. Advances in nucleic acid chemical synthesis mean that high-quality CRISPR guides can now be chemically synthesized with high-fidelity, and crucially with the addition of modifications that enhance the efficiency of CRISPR editing, reduce immunogenicity, and improve specificity. This article discusses the application of such chemically synthesized synthetic guide RNAs, the modifications that can improve guide efficiency, and the benefits they bring to a CRISPR gene-editing workflow.

RNA-guided nuclease for robust gene editing

The CRISPR-Cas9 nuclease system is inherently simple, requiring only two components—a RNA portion that ‘guides’ a Cas9 endonuclease to the loci of interest where it triggers endogenous DNA repair pathways to facilitate site-specific genome modifications. The guide RNA molecule itself consists of two domains—a tracrRNA that binds and activates the Cas9 protein, and a customizable crRNA that directs the RNA-Cas9 complex to the matching DNA sequence. The tracrRNA and crRNA can be delivered separately as a dual guide (gRNA), also known as a two-piece system, or as a single guide (sgRNA) where the crRNA and tracrRNA are combined into a continuous sequence by a short loop. The majority of publications use sgRNA as it facilitates easier expression and transcription, with comparable efficiency of editing.¹

The limitations of in vivo and in vitro methods to create sgRNA

In vivo expression of the sgRNA from a transfected plasmid was the original method of generating sgRNA for gene editing—the sgRNA is cloned into a plasmid vector and then transfected into cells where expression of the sgRNA inside the cell is driven by cellular machinery, such as RNA polymerase III promotors U6 or H1. While effective for editing due to the high levels of sgRNA in the cell, this approach is associated with a higher risk of off-target activity, as the longer an sgRNA is expressed in a cell line or model, the more likely it is that an edit will be introduced to a location other than the intended target.

For this reason, scientists moved toward transfecting cells with sgRNA directly, generated by in vitro transcription (IVT). This cost-effective method can generate relatively large amounts of sgRNA using a DNA template containing the sgRNA sequence, which is transcribed using recombinant RNA polymerases. Directly transfected sgRNA will have a shorter half-life, reducing the likelihood of off-target edits—they can be used in combination with a stable Cas9-expressing cell line, plasmids expressing Cas9 or, again to further reduce half-life and therefore off-target risk, precomplexed with purified Cas9 to produce a ribonucleoprotein (RNP).

But despite their widespread use, both plasmid-expression and IVT methods come with several disadvantages:

• Significant technical burden: Plasmid-expression of sgRNA takes around 1–2 weeks before CRISPR gene editing can even begin and comes with multiple preparation steps. IVT sgRNA is a quicker process, taking around 1–3 days, but it is labor-intensive and requires sequencing of the DNA template for accuracy
• Generation of sgRNA is error-prone: IVT products can be of variable length and structure, plus after transcription the sgRNA requires additional purification steps to remove unincorporated triphosphates, as well as any protein and DNA
• Deleterious cellular effects: Integration of plasmid DNA into the cellular genome can cause unintended changes to gene expression, or even genomic instability and cell death. IVT sgRNA has also been shown to be immunogenic, with the 5’triphosphate group of IVT sgRNA activating an innate immune response in some cell types leading to cell death.²

The limitations of plasmid-expression and IVT to generate sgRNA can lead to variable editing efficiency, off-target activity, as well as potential cellular effects such as immune response or cell death.

Why you should consider synthetic sgRNA

Alternatively, sgRNA can be generated through direct chemical synthesis, which uses nucleoside phosphoramidite building blocks to create sgRNA with defined sequences and structures. The quality of the guide in this instance is only limited by the fidelity of the synthesis process and can also be enhanced with secondary chemical modifications to improve guide efficiency.³

Previously, chemical synthesis of sgRNA came with high cost and nucleotide length was limited—but recent developments in nucleic acid chemistry plus technological advancements now mean that sgRNAs can now be chemically synthesized with high-fidelity, at high consistency, and at an affordable cost. Synthetic sgRNA come with significantly less hands-on time for the CRISPR researcher—oligonucleotides simply arrive ready to go for an experiment with no additional cloning and sequencing steps required.

A big advantage of chemically synthesized sgRNA is it allows for chemical modification of the sgRNA to improve in vivo editing capability. For example, modifying the RNA backbone, such as 3’-phosphorothioate internucleotide linkages, and the addition of 2’-O-methyl modifications at the 5’ and 3’ terminal ends of the sgRNA improves resistance to nucleases and editing efficiency, decreases off-target activity, as well as reduces the innate immune response to the sgRNA.⁴

Enhanced CRISPR editing with synthetic sgRNA

CRISPR-Cas9 genome editing is highly efficient in human cell lines but can still be a challenge in some primary cells due to inefficient delivery of the CRISPR components, off-target activity, as well as the activation of innate immune responses in some cells. Synthetic sgRNA with chemical modifications have increased stability with resistance to nucleases, enhanced targeting efficiency, reduced off-target effects, and minimized immunogenicity—all with no preparation time required. They also provide an option for completely DNA-free gene editing—combining synthetic sgRNA with Cas9 mRNA or Cas9 protein to create a RNP complex, allows for transient expression of the CRISPR system to improve specificity and reduce off-target activity.⁵ And with many tool companies providing GMP-grade synthetic sgRNA, their use could take your CRISPR experiments from early-phase research to process development right through to the clinic.

The benefits of synthetic sgRNA

Why you should consider them for your CRISPR experiments

• Enhanced sgRNA activity and editing efficiency over transcribed sgRNA
• Addition of chemical modifications can improve stability and reduce immunogenicity
• Quick and easy to use—synthetic sgRNA come ready to use, with no cloning, sequencing, or in vitro transcription steps required
• More consistency and higher quality, meaning less validation and more reproducible results
• DNA-free editing with RNPs for reduced off-target effects and workflow, crucial for therapeutic applications

References

1. Jinek, Martin et al. “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Science (New York, N.Y.) vol. 337,6096 (2012): 816-21. doi:10.1126/science.1225829

2. Kim, Sojung et al. “CRISPR RNAs trigger innate immune responses in human cells.” Genome research, vol. 28,3 367–373. 22 Feb. 2018, doi:10.1101/gr.231936.117

3. Chen, Qiubing et al. “Recent advances in chemical modifications of guide RNA, mRNA and donor template for CRISPR-mediated genome editing.” Advanced drug delivery reviews vol. 168 (2021): 246-258. doi: 10.1016/j.addr.2020.10.014

4. Hendel, Ayal et al. “Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells.” Nature biotechnologyvol. 33,9 (2015): 985-989. doi:10.1038/nbt.3290

5. Kim, Sojung et al. “Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins.” Genome research vol. 24,6 (2014): 1012-9. doi:10.1101/gr.171322.113