The Complete Guide to Understanding CRISPR sgRNA

Before we delve into the depths of our discussion on single guide RNAs (sgRNA), let us first review the CRISPR-Cas9 gene-editing mechanism. The popularity of CRISPR is largely due to its simplicity. As shown in Figure 1, the CRISPR-Cas system relies on two main components: a guide RNA (gRNA) and CRISPR-associated (Cas) nuclease.

• The guide RNA is a specific RNA sequence that recognizes the target DNA region of interest and directs the Cas nuclease there for editing. The gRNA is made up of two parts: crispr RNA (crRNA), a 17-20 nucleotide sequence complementary to the target DNA, and a tracr RNA, which serves as a binding scaffold for the Cas nuclease.
• The CRISPR-associated protein is a non-specific endonuclease. It is directed to the specific DNA locus by a gRNA, where it makes a double-strand break. There are several versions of Cas nucleases isolated from different bacteria. The most commonly used one is the SpCas9 nuclease from Streptococcus pyogenes.

The crRNA part of the gRNA is the customizable component that enables specificity in every CRISPR experiment. But you may have noticed another term, sgRNA, commonly used in CRISPR-related resources. So what exactly is the difference between gRNA and sgRNA?

sgRNA is an abbreviation for “single guide RNA.” As the name implies, sgRNA is a single RNA molecule that contains both the custom-designed short crRNA sequence fused to the scaffold tracrRNA sequence by a linker loop. sgRNA can be synthetically generated or made in vitro or in vivo from a DNA template. In Figure 1, the components of the sgRNA are as follows:

• crRNA - green
• Linker loop - purple
• Scaffold tracrRNA - blue

While crRNAs and tracrRNAs exist as two separate RNA molecules in nature, sgRNAs have become the most popular format for CRISPR guide RNAs with researchers, so the sgRNA and gRNA terms are often used with the same meaning in the CRISPR community these days. However, some researchers are still using guide RNAs with the crRNA and tracrRNA components separate, which are commonly referred to as 2-piece gRNAs or simply as cr:tracrRNAs (pronounced CRISPR tracer RNAs).

The term “sgRNA” has been previously used elsewhere to refer to different types of CRISPR RNAs, including synthetic guide RNA and short guide RNA. In this guide, we have used the conventional definitions to avoid confusion: gRNA is the term that describes all CRISPR guide RNA formats, and sgRNA refers to the simpler alternative that combines both the crRNA and tracrRNA elements into a single guide RNA molecule.

The CRISPR guide RNA sequence directly impacts the on-target DNA cleavage efficiency and unintentional off-target binding and cleavage. Therefore, designing the right guide RNA is a critical step for the success of your CRISPR experiments and there are several important parameters to consider while designing a guide RNA.

What PAM Sequence Does Your Cas Nuclease Use?

Each Cas nuclease binds to its target sequence only in the presence of a specific sequence, called protospacer adjacent motif (PAM), on the non-targeted DNA strand. Therefore, the locations in the genome that can be targeted by different Cas proteins are limited by the locations of these PAM sequences.

Cas nucleases isolated from different bacterial species recognize different PAM sequences. For instance, the SpCas9 nuclease cuts 3-4 nucleotides upstream of the PAM sequence 5′-NGG-3′ (where “N” can be any nucleotide base), while the PAM sequence 5′-NNGRR(N)-3′ is required for SaCas9 (from Staphylococcus aureus) to target a DNA region for editing. For Cas12 variants like hfCas12Max nuclease, these nucleases created staggered cuts of the nucleic acid. Specifically for hfCas12Max whose PAM sequence is 5'-TN-3' and/or 5'-(T)TNN-3', the predicted cut locations are 14-16 nucleotides downstream of the PAM sequence for the non-targeted strand and 24 nucleotides downstream of the targeted strand.

Note that although the PAM sequence itself is essential for cleavage, it should not be included in the single guide RNA sequence.

Using Software to Design CRISPR sgRNAs

Once the target gene and Cas nuclease have been selected, the next essential step is to design the specific guide RNA sequence. Several software tools exist for designing an optimal guide RNA with minimum off-target effects and maximum on-target efficiency. The following are the most popular guide RNA design tools available:

• Synthego Design Tool
• Broad Institute GPP sgRNA Designer
• CRISPOR
• CHOPCHOP
• Off-Spotter
• Cas-OFFinder
• CRISPR-Era
• Benchling CRISPR Guide RNA Design tool

Several of these tools, such as Off-Spotter and Cas-Offinder, are specifically developed for detecting potential off-target editing. Others, like CHOPCHOP, are not only for SpCas9 nuclease but also provide options for alternative Cas nucleases and PAM recognition such as hfCas12Max nuclease. For more details on all the different platforms available, this 2018 paper reviews all the available tools for sgRNA design.

Synthego’s design tool offers fast and easy design of sgRNAs that generate up to 97% editing efficiency and the lowest off-target effects from a library of over 120,000 genomes and over 8,300 species. The tool can also be used to validate guides designed using other methods.

“The Synthego design tool is extremely fast and the user experience is unlike anything I've seen before - very sleek and visually appealing. It allows me to rapidly commence my CRISPR experiments by reducing significant time in the design process.” -Dane Hazelbaker, a researcher at the Broad Institute of MIT and Harvard.

Read more about these design tools on our blog post

Important considerations and limiting factors for sgRNA design

There are several things you should consider when designing sgRNA for CRISPR experiments:

• The GC content of the sgRNA is important, as higher GC content will make it more stable - it should be 40-80%.
• The sgRNA target sequence should be long enough to ensure specificity to the genomic site. While shorter sequences can reduce off-target effects, if too short, they may lose specificity. For example, sgRNA for SpCas9 nuclease and hfCas12Max nucleases typically range from 17-23 nucleotides in length.
• Mismatches between gRNA and target site can lead to off-target effects, depending on the number of mismatches and their positions.
• It may be necessary to design multiple sgRNAs for each gene of interest, due to the fact that activity and specificity can be unpredictable.

Once the guide RNA sequence has been designed, the next step is to actually make it. This can be achieved by synthetically generating the sgRNA or by making the guide in vivo or in vitro, starting from a DNA template. The method used to make the sgRNA influences the experimental editing efficiency.

Plasmid-expressed sgRNA

One of the original methods of making sgRNAs involves expressing the guide RNA sequence in cells from a transfected plasmid. In this method, the sgRNA sequence is cloned into a plasmid vector, which is then introduced into cells. The cells use their normal RNA polymerase enzyme to transcribe the genetic information in the newly introduced DNA to generate the sgRNA.

Cloning the guide RNA plasmid generally requires about 1–2 weeks of lab time prior to the actual CRISPR experiment. The plasmid approach can be prone to off-target effects than other methods because the guide RNA is expressed over longer periods of time. Also, the plasmid DNA can integrate into the cellular genome, which can result in adverse effects and problematic for downstream applications, somtimes resulting in cause cell death.

In Vitro-transcribed sgRNA

Another method for making sgRNA, termed in vitro transcription (IVT), involves transcribing the sgRNA from the corresponding DNA sequence outside the cell. First, a DNA template is designed that contains the guide sequence and an additional RNA polymerase promoter site upstream of the sgRNA sequence. To synthesize the sgRNA, enzymes like T7 RNA polymerase recognize the promoter, initiating transcription and producing the sgRNA complementary to the DNA template. Following transcription, the sgRNA must be purified to remove the unwanted components and byproducts of the synthesis. Lastly, the purified sgRNA should be quantified before use in CRISPR experiments to determine the concentration so appropriate dilutions can be made. During the synthesis process, it is best practice to include an RNase inhibitor to protect the RNA transcripts from being degraded by other enzymes. The whole synthesis process using the IVT approach can take between 1-3 days using T7 RNA polymerase or similar enzymes.

Although synthesizing sgRNA using the IVT method seems straightforward, it can be labor-intensive and prone to error. This results in lower-quality sgRNA that requires additional purification steps to yield sufficient editing efficiencies.

Additional T7 RNA polymerase and RNase inhibitor can be used in the production of mRNA through IVT methods. Similar to sgRNA synthesis, incorporating a T7 promoter sequence and DNA template with T7 RNA polymerase efficiently can transcribe the DNA template into mRNA. Scientists are using this approach to generate mRNA to develop vaccines and therapeutics, and understand gene expression and protein translation.

Synthetic sgRNA

Of the different formats of guide RNA, synthetic sgRNA has been used the most in a wide variety of CRISPR experiment applications. Synthetic sgRNA typically are made using solid-phase chemical synthesis, where individual ribonucleotides are added sequentially to grow the RNA chain. The addition of the ribonucleotides is done through a series of coupling, capping, and oxidation reactions, ensuring that only the desired nucleotides are added to the RNA chain. To prevent unwanted side reactions for each addition of a ribonucleotide, protecting groups are added to the reactions and then removed to enable the next ribonucleotide to be added to the chain. After the full-length sgRNA is assembled, it is cleaved from the solid support and deprotected. The final sgRNA can then undergo purification processes, such as HPLC, to ensure high purity before being used in CRISPR applications.

Synthego’s best-in-class synthetic sgRNA have been cited in +1700 peer-reviewed publications from a variety of research areas including oncology, immunology, genetic disease, and neuroscience. Synthetic sgRNA has several advantages over other sgRNA formats, four of which are listed here:

• Improved Editing Efficiency
• Minimum Risk Delivery System
• Increased Experimental Convenience
• Increased Stability With Chemically Modified Guides

Improved editing efficiency with synthetic sgRNA

The editing efficiency in cells transfected with synthetic sgRNA has been experimentally confirmed to be higher than that of non-synthetically derived sgRNA. Synthego's best-in-class sgRNA achieves up to 99% editing efficiency in immortalized cell lines and greater than 70% in resting human primary cells enabling experimental reproducibility.

Minimum Risk Delivery System

One caveat of DNA-based methods for generating sgRNA, especially the plasmid format of CRISPR components, is the continual expression of guide RNAs inside the cell. This could result in unwanted effects in random or unexpected places in the genome. Introducing the CRISPR machinery in the ribonucleoprotein format into cells alleviates these concerns as the RNP exists transiently inside the cell and shows reduced toxicity and off-target effects (Figure 3).

Increased Experimental Convenience

Even if an experiment is complicated, its preparation does not need to be. Plasmid and IVT-derived RNA, in the best-case scenario, require a few days to a week for preparation time before cell transfection. An important advantage of synthetic sgRNA is that it arrives ready to use, thus saving the valuable time and effort of researchers for the actual experiments - there is no cloning or sequencing required.

Increased stability with chemically modified guides

Stem cells are popular in therapeutics, having been widely applied in studying disease models of different cell types. After the introduction of CRISPR, modifying stem cell genomes was an obvious next step for researchers to test further gene therapy options. However, stem cells, just like primary cells, proved difficult to transfect with regular RNA guides.

“Synthego’s chemically modified sgRNA provides a critical tool for our CRISPR research when it comes to difficult stem cell gene targets. Our research into stem cell-based human therapeutics presents editing challenges that require the highest efficiency guides,” - Andy Scharenberg, MD.

The advantage of synthetic single guide RNA is that it allows chemical modifications that prevent degradation of the CRISPR machinery by the intracellular immune response. Researchers have achieved up to 90% editing efficiency with these challenging cell types using Synthego's chemically modified sgRNAs. Moreover, these modified guides achieve exceptional editing efficiencies even without HPLC treatment, required by other vendors, thus decreasing turnaround time and cost of our product like what is depicted in figure 4. For additional data on how Synthego’s sgRNA are superior to the competition, download our free application note.

Scientists are using synthetic sgRNA in their CRISPR experiments to develop cell and gene therapies due to their higher efficiency and the reproducibility of results. Therefore, there is a great need for high-quality CRISPR solutions to support their work from early discovery to clinical trials.

This means that scientists require CRISPR solutions that enable them to seamlessly transition from using research use only (RUO) to good manufacturing practice (GMP) compliant material to use in their therapeutic development. Synthego’s CRISPR Continuum provides scientists the material they need to scale their therapeutic without sacrificing quality resulting in a seamless transition between development phases.

Research-Grade Synthetic sgRNA

Our research use only (RUO) gRNA and sgRNA supports early discovery work in a variety of applications including cell and gene therapy development. Their ability to achieve editing efficiencies of up to 99% and ease of scaling has played a pivotal role in driving breakthroughs in therapeutics and scientific innovation. If you are using nucleases like SpCas9, hfCas12Max, or other alternatives, our best-in-class RUO gRNA and sgRNA are designed for early-stage discovery and research applications. Get started with your CRISPR experiments today.

IND-enabling sgRNA

Designed for the preclinical phase, IND-enabling (INDe) sgRNA ensure optimal efficiency for safety, toxicity, and efficacy studies. Unlike other GMP-like gRNAs, INDe sgRNAs are produced under GLP guidelines with traceable quality documentation, ensuring comparability with GMP sgRNA reagents. Manufactured in Synthego’s proprietary facilities, these INDe sgRNAs serve as an efficient bridge between RUO and GMP products, facilitating a smooth transition from discovery to clinical development.

GMP-Compliant sgRNA 

Synthesized in a state-of-the-art certified Good Manufacturing Practice (GMP) facility, our GMP sgRNA are synthesized to meet strict regulatory requirements set by regulatory agencies for therapeutic applications in CRISPR-based therapeutic phase 1 clinical trials and beyond. To streamline IND/IMPD submissions, our Drug Master File (DMF) and chemistry, manufacturing, and controls (CMC) documentation for our GMP sgRNA provide the necessary information for the agencies to efficiently evaluate your therapeutic. For more information about our GMP facility and how you can scale your work from RUO to GMP, you can download our RUO to GMP sgRNA flyer for more details.

iPSC gRNA

iPSC gRNA provide a streamlined and efficient pathway for accelerating clinical applications, particularly in the creation of iPSC master cell banks for therapy development. Each iPSC gRNA undergoes rigorous quality control testing, adhering to GMP standards to meet stringent regulatory requirements. Our thorough QC process includes bioburden and sterility testing, mass spectrometry analysis, and HPLC purification, ensuring that every iPSC gRNA meets the highest quality standards necessary for your research and therapeutic development.

CRISPR is not only shaping the future of therapeutics but also enabling innovative research to make groundbreaking discoveries. Through unprecedented precision and efficiency, CRISPR enables faster, more accurate genome editing, opening doors to breakthroughs in drug development and personalized medicine. Advances like best-in-class synthetic sgRNA further simplify experimental workflows, empowering researchers to explore complex genetic questions more easily. As companies like Synthego continue to innovate and expand access to CRISPR solutions, the potential for transformative discoveries grows, driving a new era in medical research and the development of targeted therapies for genetic diseases.