CRISPR Therapeutics: Where the Science Stands and What's Holding It Back

CRISPR is a gene-editing technology used to selectively modify DNA to achieve gene knockout, knock-in, activation, or repression through targeted removal, addition, or alteration of disease-implicated sequences. The technology is based on a naturally-occurring genome-editing defense system found in bacteria, called Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). CRISPR leverages an RNA-guide protein that scans the genome for a precise DNA sequence to target, cuts the target DNA with an enzyme (most famously Cas9), and initiates repair using genetic material altered for therapeutic effect.

Editing the Genome

The CRISPR gene-editing system uses an enzyme called Cas9 and a customized guide RNA to target, cut, alter, and repair particular stretches of the genome. It can disrupt a gene or edit in a new one.

Recent CRISPR Advances

After Dr. Jennifer Doudna and Dr. Emmanuelle Charpentier won the 2020 Nobel Prize in Chemistry for their CRISPR-Cas9 “genetic scissors,” expectations for the technology’s potential grew. According to the Innovative Genomics Institute’s 2023 clinical trial update, CRISPR is currently being studied for a variety of uses, including:

• ​Direct editing of problematic genes: Progress in relatively easy-to-target genetic conditions, such as inherited blood disorders and genetic blindness. Vertex Pharmaceuticals expects a regulatory decision on its CRISPR-based sickle-cell-disease treatment by the end of 2023.
• ​Novel gene editing approaches: Researchers are exploring new approaches, such as reprogramming existing drugs, developing non-viral delivery systems (including lipid nanoparticles), and performing edits without double-strand DNA breaks.
• ​Pathogen-targeted treatments: Treatments are being studied that directly target the genome of hard-to-treat pathogens, such as those implicated in chronic urinary tract infections and HIV.
• ​Cell-therapy modifications: CRISPR is being used to expand cell therapies, such as evolving CAR-T treatments beyond individualized patient-derived treatments by creating and editing cell therapies from healthy donors. It is also being explored to reduce immune response in type-1 diabetes patients treated with stem-cell-derived pancreatic cells.

Beyond therapeutic gene editing, CRISPR is also being used as a diagnostic tool. For example, Sherlock Biosciences is developing CRISPR technology to detect unique genetic fingerprints in virtually any DNA or RNA sequence in any organism or pathogen.

Key Challenges Impeding CRISPR’s Targeted Success

Despite impressive progress, CRISPR’s broader application is impeded by two key issues: off-target effects and delivery challenges.

Off-Target Effects of CRISPR Guide RNA

CRISPR depends on guide RNA (gRNA) to precisely pinpoint locations on a gene sequence for modification. The precision of the guide RNA and its ability to avoid unintended gene locations is critical. Off-target effects are a significant concern, and minimizing these risks requires meticulous design of gRNAs and repair templates, often facilitated by molecular design tools.

For example, the CRISPR-Cas9 system uses gRNA with a 20 base-pair target sequence next to a protospacer-adjacent motif (PAM site) to direct the Cas9 endonuclease. While Cas9 variants have been developed to reduce off-target activity, some risk remains. Researchers must thoroughly scan for gRNA CRISPR sites and potential off-target binding sites, scoring each gRNA for off-target potential. Sequence analysis software can assist in this process. Several techniques are available to scan the genome for mismatches, but none are 100% conclusive; sometimes, whole-genome sequencing is needed for certainty.

CRISPR Delivery Challenges

Another significant limitation is delivering CRISPR components to the intended target, especially in complex eukaryotic systems and therapeutics. Delivery must be optimized to certain cell types while minimizing toxic side effects. Target access is critical; if the CRISPR-Cas9 system cannot bind to the target DNA sequence, it cannot make the needed cleavage. This is particularly challenging in eukaryotic cells, where DNA is tightly packaged with proteins and other molecules.

Factors affecting target access include DNA-binding proteins, sequence-specific effects, and chromatin structure. Chromatin can make it difficult for CRISPR-Cas9 to access the target DNA sequence. Additionally, thousands of DNA-binding proteins compete with CRISPR-Cas9 for binding. Sequence-specific effects, such as GC-rich sequences, can also hinder binding.

Viral delivery systems are commonly used but have issues like carrying-capacity restrictions, potential immune responses, and tissue-specificity limitations. Non-viral alternatives, such as lipid nanoparticle delivery systems or chemical modifications, are being explored to improve delivery without increasing immune response risk.

Overcoming CRISPR Design Hurdles

Researchers are working at higher throughput to study multiple gRNA candidates at once and collaborating across disciplines to explore chemical modification. However, fragmented informatics solutions can make sharing data and optimizing workflows difficult.

Large Batch Guide RNA Design in CRISPR

Large batch gRNA design helps uncover candidates with the lowest potential for off-target effects, especially for complex diseases requiring multiple targets. High-throughput approaches involve designing and assessing hundreds of guides at a time. All potential guides must be interrogated for off-target effects and efficacy. Batch discovery creates abundant data that must be scored for activity and specificity, and researchers may want to build off-target databases, design repair templates, predict editing effects, and analyze sequencing results. This is challenging without integrated R&D platforms.

Chemical Modifications in CRISPR

Chemical modifications to the gRNA or Cas9 system can improve target-binding affinity, nuclease resistance, and reduce off-target effects and immunogenicity. Some researchers are studying whether chemical alterations can help control not only the location but also the timing of CRISPR modifications. Cross-discipline collaboration between biology and chemistry teams is essential but can be difficult due to differing workflows, tools, and data types. Solutions that support both sides can make a significant difference.

Collaborative Cross-Discipline CRISPR R&D with Dotmatics

R&D teams must collaborate to select and analyze gRNA, study off-target effects, explore repair-template editing, design delivery vectors, and test chemical modifications. Dotmatics supports large-scale collaborative CRISPR R&D by uniting various functionalities within a user-friendly, data-centric R&D platform, including:

• ​CRISPR Guide RNA Design Tools: Use specialty tools to design, score, and optimize gRNA, with batch discovery and scoring of CRISPR sites.
• ​Molecular Editing and Sequence Analysis Software: Design repair templates and perform analysis tasks such as PCR, DNA sequence analysis, sequence alignment, and gene expression analysis.
• ​Vector Design and Chemical Modification Tools: Design viral vectors for optimized delivery and explore chemical modifications or non-viral delivery systems.
• ​Inventory and Registration Systems: Manage cells, clones, and samples used in experiments with a registration system accommodating biologic entities with or without chemical modifications.
• ​Flow Cytometry Analysis Software: Measure the impact of gene editing on cell populations.
• ​Electronic Laboratory Notebooks: Track and log all project details with personalizable ELNs supporting cross-discipline CRISPR-design workflows.

Why CRISPR R&D Teams Choose Dotmatics

• ​Supports cross-discipline collaboration: Unites CRISPR design tools on an end-to-end R&D platform, creating a single source of scientific truth.
• ​Optimizes individual productivity: Reduces back-and-forth between software by integrating registration and design tools.
• ​Accelerates innovation: Collates all relevant R&D data and uses advanced analysis tools to analyze, graph, and present data.
• ​Accommodates flexibility: Provides tools optimized for the fluidity and flexibility needed for CRISPR therapeutics.

References

1. 1.What is CRISPR? Innovative Genomics Institute. Accessed September 20, 2023.
2. 2.What are genome editing and CRISPR-Cas9? Medline Plus. Accessed September 20, 2023.
3. 3.The Nobel Prize in Chemistry 2020. The Royal Swedish Academy of Sciences. October 7, 2020.
4. 4.Henderson, H. CRISPR Clinical Trials: A 2023 Update. Innovative Genomics Institute. March 17, 2023.
5. 5.Newman, C. FDA sets decision dates for Vertex, CRISPR gene editing drug. BioPharmaDive. June 9, 2023.
6. 6.What is Sherlock? Sherlock Biosciences. Accessed September 20, 2023.
7. 7.Xu CL, Ruan MZC, Mahajan VB, Tsang SH. Viral Delivery Systems for CRISPR. Viruses. 2019 Jan 4;11(1):28. doi: 10.3390/v11010028.
8. 8.Qiubing Chen, Ying Zhang, Hao Yin. Recent advances in chemical modifications of guide RNA, mRNA and donor template for CRISPR-mediated genome editing. Advanced Drug Delivery Reviews. 2021 Jan; 168;246-258. https://doi.org/10.1016/j.addr.2020.10.014.
9. 9.Rozners, E. Chemical Modifications of CRISPR RNAs to Improve Gene-Editing Activity and Specificity. J. Am. Chem. Soc. 2022, 144, 28, 12584–12594. https://pubs.acs.org/doi/10.1021/jacs.2c02633
10. 10.Zhang H, Kelly K, Lee J, et. al. Self-delivering CRISPR RNAs for AAV Co-delivery and Genome Editing in vivo. bioRxiv [Preprint]. 2023 Mar 23:2023.03.20.533459. doi: 10.1101/2023.03.20.533459.
11. 11.Gene editing improved with chemical process: Case Western Reserve University researchers combine novel chemical method with CRISPR gene-editing tools to target disease-specific versions of genetic code. The Daily. Case Western Reserve University. March 16, 2022.