CRISPR sgRNA for Successful Gene Editing: Discovery to Clinic

The famous CRISPR-Cas9 gene editing system has two key parts: the CRISPR-associated (Cas) nuclease, and the single guide RNA (sgRNA). While the Cas9 nuclease creates double-stranded breaks in DNA, the sgRNA directs Cas9 where to create these cuts. This makes the single guide RNA sequence super important for successful CRISPR experiments.

At Synthego, we are an industry leader in the synthesis of sgRNAs for CRISPR editing, supplying researchers with high-quality synthetic guides for the clinical development of CRISPR cell and gene therapies. In this blog, we will elaborate on the definition of sgRNA, why the sgRNA sequence is so important, and the sgRNA grades that Synthego offers, from discovery to delivery. Read on to learn how we can partner with you to accelerate the development of your CRISPR cell and gene therapy.

What is sgRNA?

The single guide RNA (sgRNA) is one of the key components of successful CRISPR gene editing, as it identifies the correct place for the Cas nuclease to perform editing. This can easily be confused with guide RNA (gRNA), so let’s briefly explore the difference between these two guide formats.

In bacterial CRISPR immune systems, the guide RNA is comprised of two RNA molecules, the CRISPR RNA (crRNA) and the trans-activating crRNA (tracrRNA). The crRNA is a ~20 nucleotide sequence complementary to the target DNA region, while the tracrRNA acts as a scaffold that binds the crRNA to the Cas nuclease.

sgRNA is a guide in which the crRNA and tracrRNA are fused into one chimeric RNA molecule. This format is now the most popular for CRISPR-based researchers because it is simpler and more easily customizable compared to the cr:tracrRNA format. While sgRNAs can be in vitro transcribed or encoded in plasmids or lentiviral vectors, synthetic sgRNA is the best performer.

Why is the sgRNA Sequence so Important?

If we think about the Cas nuclease being like a pair of scissors, the single guide RNA is the hand holding those scissors and directing them where to cut the DNA. In order to have the highest chance of successful CRISPR editing, the sgRNA design and how the molecule is produced are absolutely crucial. 

The sgRNA design should be complementary to a region of the genome where editing is desired, with the correct protospacer adjacent motif (PAM) upstream of the target site. Since even slight mismatches between the target and the guide sequence can lead to off-target editing, it’s important to sequence the target region of the cell line or organism to be edited rather than using a reference genome to design the guide molecule. 

Synthetic CRISPR sgRNA sequences can be generated at high purity and with additional chemical modifications that make the molecule more stable and prevent activation of cellular innate immune responses and subsequent degradation of the molecule. Synthetic sgRNAs with chemical modifications achieve consistently high editing efficiencies with a lower risk of off-target editing in the majority of cell types compared to other available guide formats.

Synthego's Offerings of CRISPR sgRNA Grades

At Synthego, we supply a full spectrum of high-quality single guide RNA products to support you at every step of the clinical development process for CRISPR cell and gene therapies. Let’s explore the full range of Synthego’s sgRNA offerings, and what each is best suited for.

Research-use only sgRNA for discovery studies

Research-use only (RUO) sgRNA is the best option to support your discovery and proof-of-concept studies. In these early stages of clinical development, it’s not necessary to use GLP or GMP-grade guides, but you will need a molecule that can produce superior editing results in any cell type, at scale. For functional genomics, drug discovery, or target validation studies, Synthego’s high quality synthetic RUO guides can be designed to target any gene, and produced at large scales. Site-specific chemical modifications can be added to make the molecule more stable for superior editing efficiencies.

INDe sgRNA for IND-enabling research

For IND-enabling research, such as toxicity and safety studies, you’ll need an sgRNA product that comes with QA oversight and documentation but may not yet require a full GMP-grade reagent. At this stage of clinical development, INDe sgRNA is the ideal type of guide. As an intermediate product between RUO and GMP-grade, INDe guides are produced in a controlled manufacturing environment, are compliant with key regulatory requirements such as 21 CFR part 58, and comes with QA oversight and documentation.

 INDe sgRNAs are manufactured in accordance with GLP guidelines for nonclinical studies with traceable quality documentation and oversight, enabling comparability between INDe and GMP sgRNA reagents. Built on Synthego’s proprietary Halo™ platform and our innovative gRNA design capabilities, Synthego’s INDe sgRNAs integrate seamlessly as an intermediary between RUO and GMP products, providing an efficient and convenient solution for researchers transitioning between discovery, preclinical, and clinical development stages.

GMP sgRNA for clinical studies

For clinical use, CRISPR guides must be produced according to good manufacturing practice (GMP) requirements. GMP sgRNA molecules are of extremely high purity, have quality assurance oversight throughout the entire manufacturing process, are produced with detailed product specifications and quality agreements, and are compliant for CMC module 3 submission. 

For GMP sgRNA, Synthego also provides full support from our regulatory team for FDA interactions and documentation. Importantly, our increased manufacturing capacity means that we can produce GMP-grade molecules at scale.

The Synthego Difference: Supporting Your Journey from Discovery to Delivery

Changing vendors throughout the clinical development process can cause significant setbacks. As the leadingCRIS vendor that provides a full continuum of sgRNA products, choosing Synthego as your vendor from day one means that you can ensure reliable results and cost-effectiveness while minimizing regulatory delays. With our new state-of-the-art GMP production facility, you can rely on us for a secure supply chain of guide molecules to suit demanding clinical timeframes. 

As a trusted industry leader, Synthego does more than simply provide high-quality single guide RNAs. We partner with you to accelerate the clinical development process for CRISPR cell and gene therapy products and maximize your chances of success. This includes expert regulatory support, such as assistance with CMC module 3 submission and related documentation, and support during FDA meetings and interactions. Our teams of CRISPR chemistry and biology experts are also available around the clock to answer your questions and provide support. 

We hope this blog helped you come to grips with the importance of single guide RNAs in CRISPR cell and gene therapy development and how Synthego’s RUO, INDe, and GMP sgRNA offerings can support your clinical journey. For more information on CRISPR guides, or to request a consultation, you can contact one of our clinical experts.