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Applications include guide RNAs (for directing CRISPR-like programmable nucleases) and donor templates for gene editing. Oligos used for this purpose in CRISPR must be chemically synthesized and modified appropriately for use as therapeutics.
Fig. 1 CRISPR mechanism.1,5
Targeting oligonucleotides and donor template oligonucleotides are types of oligonucleotides used for CRISPR genome editing. Some modifications that improve CRISPR targeting oligonucleotides and donor template oligonucleotides properties are described below. Guide RNAs in CRISPR must be structurally sound enough for the ribonucleoprotein to form, yet also resist cellular breakdown. Donor template oligonucleotides used as repair templates for HDR require careful sequence engineering.
| Oligonucleotide Type | Primary Function | Key Structural Features |
| Single guide RNA (sgRNA) | Directs Cas nuclease to target DNA | 20 nt target sequence + scaffold |
| CRISPR RNA (crRNA) | Target recognition (dual-RNA systems) | 20 nt variable region |
| trans-activating crRNA (tracrRNA) | Scaffold for Cas9 recruitment | Constant hairpin structure |
| Donor DNA oligonucleotides (ssODN) | Template for homology-directed repair | Homology arms flanking modification |
Table 1 Types of Oligonucleotides in CRISPR Applications
Guide RNAs are the targeting moiety of CRISPR tools that recruit Cas nucleases to locations in the genome complementary to their twenty nucleotide spacer sequence adjacent to a protospacer adjacent motif (PAM), via Watson-Crick interactions with target DNA. To bind target DNA and recruit Cas scaffolds, guide RNAs must be chemically intact to form a complex that cleaves target DNA. Single-stranded oligonucleotides called donors may be used as external repair templates during homology directed repair. Donor oligonucleotides are external nucleotide sequences used by cellular DNA repair enzymes to repair double-strand breaks instead of using the natural sequence adjacent to the cut site. If donor templates are supplied externally, they typically consist of homology arms up to 60 nt in length on either side of the edit. These edits can consist of single nucleotide substitutions, epitope tag insertion, or mutation correction. Homology directed repair is more efficient the closer the distance between the cut site and the edit on the donor template. Ideally, the edit should be no more than 10 nt away from the cut site. Therefore, it is important to strategically choose where your guide cuts as well as your donor template layout.
Guide RNA is an important factor contributing to the effectiveness of CRISPR techniques. Important considerations in designing guide RNA sequences include the rate and strength of binding to the target sequence, discrimination against similar sequences, guide RNA scaffold folding and its effects on complex formation with Cas proteins, DNA target accessibility, and possible improvements by chemical modifications. Modifications should ideally increase resistance to nucleases and improve binding affinity, though must be placed such that protein interactions are not disturbed. Guide RNAs should allow for target cleavage with minimal off-target effects.
Guide RNA sequences can be designed to have specific lengths, preferred structural elements, and target specific genomic locations. The twenty-nucleotide target sequence typically is enough to identify a unique genomic site. Shorter guide RNAs of 17–19 nucleotides can be synthesized to weaken the interaction with the target DNA. Alterations can also be made to the constant region but should avoid altering parts of the RNA that interact with Cas to form the ribonucleoprotein complex and activate cleavage. Furthermore, only regions of DNA that are accessible will be able to bind the guide RNA. Therefore sequence targets should be computationally screened to identify sequences likely open enough for hybridization. The guide sequence can also be screened for secondary structure that would hide the target sequence or scaffold sequence from interacting with their targets.
Avoidance of off-target effects is often cited as one of the first goals when designing guide RNAs for therapeutic use. Any off-target changes could potentially knock-out genes with unwanted consequences or trigger tumor suppressor genes. By matching the sequence of the guide RNA to other regions in the genome, off-target activity can be determined. Guide RNAs will tolerate mismatches depending on their location and identity. Several algorithms exist that utilize sequence homology to predict potential off-target sites which can be used to design guide RNAs with non-overlapping specificity. Additionally, increasing the activation energy required for mismatched binding events can improve fidelity. Modifications such as bridged nucleic acids or other chemical backbones can improve fidelity. By using high-fidelity Cas nucleases with engineered recognition domains, specificity can be further increased by limiting activity to sequences perfectly matching the protospacer adjacent motif (PAM) proximal to the guide RNA.
Synthesizing CRISPR oligos chemically is nontrivial. Guide RNAs and donor DNA templates require exact chemical specification to fold correctly, bind to proteins, and function in the cell. RNA itself is less stable than DNA, which can require special handling and may involve chemical modification post-synthesis. Secondary structure formation can also impact solid phase synthesis of the DNA leading to decreased yields and more difficulty with purification. These factors can become larger problems with increased sequence length and needed modifications. Synthesis of these molecules can take advanced chemistry and analytical knowledge to confirm correct product.
Chemically, RNA oligonucleotides are much less stable than DNA oligonucleotides. This is because RNA contains a 2'-hydroxyl group making the phosphodiester backbone more prone to hydrolysis under basic conditions. Exposure to even mildly basic conditions during solid phase synthesis and purification/handling will increase strand cleavage by nucleophilic attack of the hydroxyl ions onto the phosphorus atom. Another common cause of instability is contamination by ribonucleases (RNases), which are very common in most lab environments and notoriously difficult to inactivate. When applied to guide RNAs, these factors must be considered carefully. Degradation will often result in products that still have partial activity but with different specificity or lower activity. Production methods should ensure that buffers and storage conditions are RNase free, as well as maintaining a mild pH and making any needed chemical modifications to preserve activity.
Guide RNAs must fold into a particular secondary structure in order to function, especially in the region used to bind Cas proteins (sometimes referred to as the scaffold). These stable secondary structures hinder synthesis by temporarily sequestering the 3' end of the growing oligonucleotide from reactive phosphoramidites, resulting in deletions. This becomes a serious issue for the scaffold portion of sgRNAs since this region must be a stable stem-loop to bind proteins but this structure prevents successful phosphoramidite reactions from elongating the guide sequence. Sometimes very high temperatures, alternative coupling reagents, or chemical modifications that disrupt the secondary structure during synthesis are necessary. Synthesis might need to be split into fragments such that the structured portion of the RNA is chemically synthesized separately and then ligated to the targeting sequence.
Chemical modifications of CRISPR guide RNAs and/or donor DNA molecules address limitations of naturally occurring nucleic acids such as nuclease sensitivity, immunogenicity, binding affinity and specificity. Modifications can include altered backbones, sugars, or conjugation to molecules at the ends of guide RNAs and donor DNAs. Modified CRISPR nucleic acids can exhibit increased resistance to nucleases and serum, altered binding kinetics, reduced off-target binding, and improved cellular uptake. Modifications to guide RNAs can be utilized to increase complex formation with the guide and binding to the target DNA sequence while maintaining structural dynamics required for Cas recruitment. Modifiers to donor DNA can help increase stability of the molecule while it is within the cell undergoing HDR. Chemical modification sites and the type of modification need to be optimized as too many modifications can hinder protein binding or hybridization to the target DNA.
Terminal protection is one approach to shielding CRISPR ONs from exonucleases, the main mode of nucleic acid clearance in serum and within cells. Linkages between phosphorothioates at the 3' and 5' ends yield sulfur-substituted backbone that is nuclease resistant but still capable of binding to Cas proteins to form active complexes. Terminal modifications are especially important for gRNAs and donor templates that need to remain stable in vivo for successful editing. 2' methoxy or fluoro modifications of terminal nucleotides can also protect against degradation and increase DNA-binding affinity. The level of modification required must be weighed against potential toxicity/altered pharmacokinetics; typically only a few terminal linkages are required for stability.
Chemical modifications to the guide sequence itself can also improve editing efficiency. This can occur either by increasing binding affinity to the genomic target or through increased discrimination to improve specificity. Two-prime-O-methyl (2'-OMe) modifications to the guide sequence increase duplex stability and nuclease protection along the entire targeting region. Locked nucleic acid (LNA) monomers within a guide RNA sequence also confer conformational rigidity, which increases melting temperature with improved mismatch discrimination. Both of these modifications can be placed within the targeting sequence of a guide avoiding modifications to the constant scaffold sequence necessary for Cas binding. Modifications that affect hybridization kinetics, such as bridged nucleic acids (BNAs) or constrained ethyl (cEt) residues, can also be used to tune activity for desired applications.
| Modification Type | Position in Oligonucleotide | Primary Functional Benefit |
| Phosphorothioate linkages | Terminal positions | Exonuclease resistance and extended half-life |
| Two-prime-O-methyl | Internal targeting sequence | Enhanced binding affinity and specificity |
| Locked nucleic acids | Selective internal positions | Increased duplex stability and mismatch discrimination |
| Bridged nucleic acids | Targeting region | Optimized hybridization kinetics |
| Phosphorothioate backbone | Throughout donor template | Protection during cellular delivery and nuclear persistence |
Table 2 Chemical Modifications in CRISPR Oligonucleotides
Fig. 2 Genome editing efficiencies of gRNAs with an engineered tracrRNA backbone.2,5
Guide RNAs, like any other oligonucleotide intended for use as a delivery method for CRISPR-based drugs, need to be purified and fully characterized. You need to know what oligonucleotides you are putting into humans to understand the potential downstream effects editing-wise and toxicity-wise. If the guide is chemically synthesized the traditional phosphoramidite solid phase route, it will be present in a crude reaction mixture that contains the full length product as well as shorter oligonucleotide species, truncated deprotection byproducts, and other analogs made during synthesis. These criteria include set amounts of full length oligonucleotide as well as identification of any impurities. These purity guidelines can be satisfied with efficient chromatographic purification techniques that can resolve the full-length guide RNA from other structurally similar species followed by extensive analysis to confirm identity and purity.
Guide RNAs should be free of contaminants that reduce the specificity and efficiency of the edit. For clinical applications, a highly pure guide RNA should be used to ensure high levels of activity at the intended target (on-target activity) and a lack of activity at unintended targets (off-target activity) as off-target activity can cause deleterious effects and reduce the safety of the therapy. Some contaminants that could be introduced during synthesis include guide RNAs that do not contain the full targeting sequence or contain chemically modified bases. Truncated guide RNAs could compete with the guide RNA for Cas protein binding or cause DNA cuts at off-target sites. Impurities can decrease on-target editing activity by decreasing the concentration of the guide RNA. Mixtures of guide RNAs can also lead to the formation of RNP complexes with different target specificities. Truncated guide RNAs that are capable of targeting their sequence but lack catalytically important domains could lead to indels at unintended sites. The FDA's Center for Biologics Evaluation and Research guidances state that the purity of the full guide RNA should be at least 80% and that any impurities greater than 1% should be well characterized.
Guide RNA impurities can be difficult to fully characterize. Synthetic impurities can be complex and the separation of guide RNA from guide RNAs that differ by only 1 or 2 nucleotides can be difficult depending on the length of the guide sequence and analysis technique used. Analytical techniques with sufficient resolution may be necessary to separate full-length guide RNA from truncated forms. For longer guide sequences (>100 nt) strong secondary structure can impact chromatographic properties. Ion-pairing reversed-phase chromatography and strong anion exchange chromatography are orthogonal and should be used in combination to get a better understanding of impurities present. Mass spectrometry should be used to confirm molecular weight and possible identities of impurities. Guide RNA isomers/stereomers may not be fully resolved by any analytical technique. Standard methods with set resolution parameters would allow comparison of purity between sources.
The delivery of chemically synthesized guide RNA versus in vitro transcribed gRNA is another consideration that can greatly affect efficiency, reproducibility, and safety of CRISPR therapies. Synthetic gRNAs are generated using solid phase phosphoramidite chemical synthesis, allowing for precise customization of sequence and site-specific modifications that may increase stability. IVT gRNAs on the other hand are synthesized via enzymatic transcription using bacteriophage RNA polymerases (like T7 RNA polymerase). These polymerases lack the proofreading ability of cellular polymerases resulting in variation from transcription errors. In addition, polymerases such as T7 can cause heterogeneity with premature termination and addition of non-templated nucleotides. Transcripts synthesized via IVT are less expensive than synthetic transcripts and can be used for early stage discovery. However, since IVT methods utilize RNA polymerases, the resulting RNAs include a 5'-triphosphate that can be recognized by the immune system. In addition, synthesis via IVT generates double-stranded RNA byproducts that can activate the innate immune response. Chemically synthesized RNAs lack these immunostimulatory characteristics and have more batch-to-batch consistency with defined chemical structure.
In general, chemically synthesized gRNAs have been shown to work better than in vitro transcribed (IVT) gRNAs with higher editing levels across many cell lines and targets. Chemically synthesized gRNAs consistently yield editing efficiencies of over 75%, whereas IVT gRNAs are often heterogeneous and vary greatly in quality batch to batch. Chemically synthesized gRNAs are synthesized on a solid support which allows absolute control over stoichiometry and modifications ensuring that each molecule has the exact same sequence targeting your gene of interest as well as chemical structure necessary for efficient incorporation into ribonucleoprotein complexes. These attributes make synthetic gRNAs ideal for high throughput and therapeutic applications where consistency is key. On the other hand, IVT gRNAs can be produced in milligram to gram quantities from a single transcript easily scaling up experiments that require a lot of material at a low cost. Synthesized gRNAs can also be produced at clinical scale with appropriate quality control and documentation under GMP conditions for therapeutic use where batch consistency and characterizations of impurities are tracked.
Scale-up considerations also become important as gene editing advances from being a tool for basic research to one that can be used in clinical and commercial applications. There is often considerable variation in editing efficiency and outcomes depending on experimental variables. Maintaining consistent editing results can be a challenge when scaling experiments to include more cell types or increasing sample sizes. Differences in reagent quality, variations in delivery methods and efficiencies, as well as inherent differences between cell types can all contribute to variations in genome editing performance. Being able to reproduce editing results is extremely important when transitioning from proof-of-concept experiments in one cell type to screening applications involving dozens of conditions or to manufacturing clinical products. Ensuring consistent practices and rigorous quality control of key materials like guide RNAs and donor DNA templates will be important for proving reproducibility of results. Additionally, keeping detailed records of all variables in your experiments will be helpful for your own validation studies as well as for sharing your results with the broader scientific community.
Batch-to-batch consistency is critical for therapeutic-grade gene editing components produced for clinical applications and essential for preclinical experiments to ensure reproducibility of results. Consistency of chemical synthesis: chemically synthesized guide RNAs, Cas proteins or donor templates may differ from batch to batch, leading to varying on-target activity, off-target activity, or toxicity of the editing components. For example, varying coupling efficiencies during solid phase synthesis of synthetic guide RNAs can lead to variations in the percentage of intact molecules and thus the true concentration. Enzymatically generated components such as RNA transcripts can also vary in length and modification state or contain varying levels of immunostimulatory impurities. Rigorous characterization by mass spectrometry, purity analysis by chromatography, and functional assays ensure batch-to-batch consistency. Facility certifications and quality management protocols offer documentation of manufacturing practices for therapeutic use cases.
When catalog oligos do not meet your technical, purity, or regulatory needs, custom oligo synthesis services are available for CRISPR applications. Catalog guide RNAs (desalted) are suitable for standard screening applications and proof-of-concept work. For more advanced work, such as gene editing programs that require higher purity materials, chemically modified guide RNAs, or in vivo experiments, primary cell editing, or translation into therapeutics, custom oligo synthesis will be required. Custom oligos are also critical for materials intended for therapeutic development in the clinic. These custom molecules must be made available with current good manufacturing practice (cGMP) quality and have extensive documentation, are lot-to-lot consistent, and have validated quality control assays which are not provided with research-grade oligos. Custom oligos should be considered when the complexity of the application requires it or the target is sensitive. Custom oligos allow you to have tighter control over the sequence and chemical structure of your product.
| Application Requirement | Standard Catalog Product | Custom Synthesis Recommendation |
| Research screening | Generally sufficient | Required for specialized modifications |
| In vivo or primary cell editing | Often insufficient | High purity and specific modifications essential |
| Therapeutic development | Insufficient | GMP-grade manufacturing and documentation required |
| Complex modifications | Limited availability | Custom chemistry for stability and specificity |
Table 3 Decision Criteria for Custom CRISPR Oligonucleotide Synthesis
Synthesis of guide RNAs for CRISPR applications is essential for preclinical studies and therapeutic development programs. Validating your targets often begins with immortalized cell lines, but once you are moving towards a therapeutic development program, custom oligonucleotide synthesis becomes necessary to produce guide RNAs with the quality and consistency necessary for therapeutic use. Preclinical validation will move you towards using primary cells and in vivo models. Guide RNA reagents must be of the highest purity for efficient and specific editing with minimal cytotoxicity and immunogenicity. Therapeutic development programs demand reproducible guide RNAs produced under a quality management system with well-defined and documented synthetic methods, analytics and batch release criteria. Additionally, guide RNA sequences are program-specific and thus will often be designed de novo, rather than using predesigned guide RNAs. For therapeutic applications you will need custom synthesis aligned with your therapeutic development stage. When moving towards IND enabling studies and clinical trials you will need GMP quality reagents that are appropriate for the phase of your program. Custom synthesis services can provide you with the exact quantity, purity and modification needs for your therapeutic development program and have the flexibility to scale up or modify as your program advances.
There are other CRISPR modifications that are not covered by existing products that can be made through custom synthesis. This includes prime editing guides (pegRNAs) which include a longer reverse transcriptase template, CRISPR-Cas13 guide RNAs which target RNA instead of DNA, and other CRISPR systems that recognize different protospacer adjacent motifs (PAMs). Custom chemical modification positions can also be specified for phosphorothioate bridges, 3' or 5' capping groups, and ribose modifications. These chemistries can make your sgRNA more nuclease resistant, increase binding affinity, and decrease immunogenicity. Customization allows for these chemistries to be placed at user defined locations which is useful if you are targeting a certain delivery system or cell type/tissue that you know unmodified sgRNAs will not work in. This is especially useful for in vivo or therapeutic applications where you want your guide RNA to have a longer half life.
CRISPR and gene editing workflows demand oligonucleotides with exceptional sequence accuracy, structural integrity, and reproducibility. Guide RNAs (gRNAs), crRNAs, tracrRNAs, and donor repair templates must be synthesized with strict control over length, purity, and chemical modification to ensure reliable editing efficiency. Our capabilities in CRISPR oligonucleotide synthesis include:
By integrating precise synthesis chemistry with tailored purification and analytical control, CRISPR-related oligonucleotides can be produced to meet the performance demands of research and translational programs.
Low editing efficiency, inconsistent results, or unexpected off-target activity can sometimes be traced back to guide RNA design, structural instability, or oligonucleotide quality limitations. If your CRISPR program involves:
a technical review of your oligonucleotide design and synthesis approach may help identify optimization opportunities. Contact our scientific team to discuss your CRISPR and gene editing oligonucleotide requirements and determine the most appropriate synthesis and purification strategy for your application.
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