Identifying and verifying low frequencies of genomic alterations resulting from off-target editing, gRNA synthesis errors, cross-contamination, or other unintended gRNA activity is critical to preventing unexpected genotoxic effects for the gene editing therapeutic community. However, assembling the necessary components and expertise for genotoxicity characterization studies is expensive and labor intensive. To better enable the gene editing community, we demonstrate a series of tools, workflows, and services that can be leveraged to perform genotoxicity characterization of CRISPR reagents. First, we demonstrate an empirical in vivo tag-based off-target nomination service rooted in our RNaseH2-dependent amplification technology as a method to generate an off-target list for a target gRNA. Then, we demonstrate how outputs of this service can be fed into rhAmpSeq to identify and classify indels and chromosomal re-arrangements at on/off-target loci with increased effectivity compared to existing methods. Finally, we demonstrate a method to sensitively identify gRNA cross-contamination down to sub-0.1% gRNA contamination levels by leveraging unique NHEJ DNA repair fingerprint information of select gRNAs. Using this method, we demonstrate that editing attributable to a characterized gRNA activity can be identified at frequencies far below detection limits of canonically utilized tools, and we further use this method to characterize the ability of IDT’s synthesis platform to prevent cross-contamination reproducibly. From this work, we demonstrate the importance of manufacturing processes in mitigating unintended genomic alterations and present new tooling/services to improve genotoxicity characterization in gene therapy workflows.

Learning Objectives:

• Discuss key challenges in CRISPR genotoxicity characterization and their impact on CRISPR-based gene editing therapies

• Explore advanced tools and workflows for detecting off-target effects and genomic alterations.

• Demonstrate methods to identify gRNA cross-contamination in CRISPR workflows.

CRISPR has emerged as a promising alternative to viral-based gene editing by allowing scientists to modify genes with unprecedented ease and flexibility. 
The discovery and development of a diverse range of CRISPR enzymes, each with its own specificities and modalities (such as CAS-9, CAS-12, CAS-13, and others), is a testament to the robustness of this technology. The new developments in Base and Prime editing further enhance the performance of CRISPR-based technologies, increasing their specificity and efficiency.
GenScript has an extensive portfolio of high-quality services and products for CRISPR gene editing. 
In this webinar, we will discuss GenScript's CRISPR solutions for the different stages of the therapeutics development process, from discovery to lead generation and optimization, as well as pre-clinical and clinical development.

Learning Objectives:

• Identify the different modalities available for CRISPR gene editing 

• Compare the advantages and disadvantages of each modality

• Discover the complete toolbox of technologies available from GenScript

Join this year's poster presenters in the Poster Hall during the Poster Discussion, Wednesday, October 23rd, from 1:00pm – 2:00pm PDT, to chat live about their posters and latest research and findings.

Directing nucleobase deamination or removal can generate precise genomic edits without double-stranded DNA breaks. However, a much broader spectrum of DNA chemical modifications is available that could further expand gene-editing capabilities. In this talk, I will describe my group's effort to evaluate the impact of appending rather than removing a chemical moiety using the bacterial DNA ADP-ribosyl transferase DarT2. DarT2 naturally exists within an anti-phage toxin-antitoxin system that appends a bulky ADP-ribosyl moiety to thymine in single-stranded DNA, interfering with phage replication. Fusing an attenuated DarT2 to a Cas9 nickase, we program site-specific ADP-ribosylation of thymine. Remarkably, this combination drives editing outcomes that depend on the domain of life and are distinct from base deamination and removal. In tested bacteria, site-specific ADP-ribosylation drives efficient homologous recombination with a provided repair template, offering flexible and scar-free genome editing without base replacement or counterselection. In tested eukaryotes, including yeast, plants and human cells, site-specific ADP-ribosylation preferentially drives replacement of the modified thymine with a bias towards adenine or cytosine, with limited insertions or deletions. Altogether, our approach expands current modalities for precision gene editing by appending chemical moieties to DNA, creating distinct editing opportunities in bacteria and eukaryotes.

Learning Objectives:

• Specify why DarT2 was chosen to explore append editing

• Describe the outcome of targeted DNA ADP-ribosylation in bacteria and eukaryotes

• Compare and contrast append editing to base editing

This talk will provide an overview of our work in the design of self-assembled, functionalised peptide and protein nanoparticles, hydrogels and bio-interfaces for applications in healthcare. These hybrid materials are of growing importance with potential applications including drug delivery and disease detection. We are developing high-throughput synthesis techniques to diversify peptide libraries and quickly survey properties of interest such as fluorescence and supramolecular behaviour [1]. We design nanomimicking approaches for virus and parasite host cell entry inhibition recently demonstrated for malaria, HSV-2 and SARS-CoV-2 [2], high molecular weight polymer carriers for enhanced delivery of saRNA therapeutics [3], and photo-responsive nanoreactors inspired in the circadian rhythms [4]. We are exploiting the sensing capabilities of functionalised nanoparticles to engineer nanoprobes for in vivo disease diagnostics that produce a colorimetric response ideal for naked eye read-out [5] and for CRISPR-based preamplification free detection of ncRNAs (CrisprZyme) [6]. I will present advances in Raman spectroscopy for high-throughput label-free characterization of single nanoparticles (SPARTA™) that allow us to integrally analyse a broad range bio-nanomaterials without any modification [7]. Finally, I will explore how these versatile technologies can be applied to transformative biomedical innovations and will discuss our efforts in establishing effective translational pipelines to drive our innovations to clinical application while actively engaging in efforts towards the democratisation of healthcare [8].

[1] Y. Lin, … M. M. Stevens. ACS Nano. 2021. 15(3); 4034-4044.

[2] A. Najer, … M.M. Stevens. ACS Central Science. 2022. 8(9): 1238–1257

[3] A. Blakney, … M. M. Stevens. ACS Nano. 2020, 14(5): 5711-5727.

[4] O. Rifaie-Graham, … M.M. Stevens. Nature Chemistry. 2023. 15: 110–118.

[5] C. N. Loynachan, … M. M. Stevens. Nature Nanotechnology. 2019. 14: 883-890.

[6] M. Broto, … M. M. Stevens. Nature Nanotechnology. 2022. 10: 1038.

[7] J. Penders, … M. M. Stevens. Nature Communications. 2018, 9: 4256.; J. Penders, … M. M. Stevens. ACS Nano. 2021, 15, 11, 18192–18205; H. Barriga, … M. M. Stevens. Advanced Materials. 2021, 34(26):2200839.

[8] A. T. Speidel, … M. M. Stevens. Nature Materials. 2022. DOI: 10.1038/s41563-022-01348-5.

Learning Objectives:

1. Define the steps involved in the CrisprZyme assay.

2. Describe the range of nanomaterials that can be studied with SPARTA technology.

3. Describe the advantages of SPARTA technology.

The development of acid degradable linkers that rapidly hydrolyze at endosomal pHs is a central problem in the field of drug delivery. Acid degradable linkages are challenging to develop because of their instability. In this report we present a new acid degradable linker based on an azide-acetal, which rapidly hydrolyzes at endosomal pHs but has exceptional stability at pH 7.4. The azide-acetal linkage hydrolyzes via a two-step mechanism that requires reduction and acid hydrolysis and has a unique combination of stability and rapid triggerable hydrolysis. The azide-acetal has a hydrolysis half-life of days at pH 7.4 and can be conveniently synthesized and incorporated into delivery vectors, however after in situ reduction with DTT it hydrolyzes with a t1/2<15 minutes at pH 6.0. We used the azide-acetal linker to synthesize acid degradable analogs of the lipid components of lipid nanoparticles and demonstrate that these new lipids are significantly better at delivering mRNA to mice than traditional lipids. Lipid nanoparticles containing acid degradable lipids transfected multiple non-liver organs after an intravenous injection and efficiently transfected brain tissue after an intracranial injection. The azide-acetal linkage has the potential to solve the instability problems associated with acid degradable linkers and has numerous applications in drug delivery.

Learninf Objectives:

• Identify the advantages of using azide-acetal linkers for acid degradable lipid nanoparticles in drug delivery
• Compare the stability and hydrolysis rates of azide-acetal linkers at different pH levels
• Analyze the efficacy of acid degradable lipid nanoparticles in mRNA delivery across various tissues in animal models.

The Myhrvold lab develops CRISPR-based technologies for studying RNA. Our primary interest is RNAs involved in host-pathogen interactions. We use a variety of RNA viruses and other pathogens as model systems. In collaboration with others, we also apply our technologies in a diverse array of biological contexts. Our goal is to quickly, cheaply, and easily quantify thousands of nucleic acid sequences in hundreds of samples simultaneously. To achieve this goal, we are building on our massively multiplexed nucleic acid detection technology CARMEN (e.g., mCARMEN, qCARMEN, bbCARMEN).We develop CRISPR-based diagnostic assays that are easier to deploy widely, allowing people to perform testing in a distributed fashion. We are inspired by the utility of rapid antigen tests during the SARS-CoV-2 pandemic.

Learning Objectives:

• Describe the CRISPR-based technologies developed for detecting RNA involved in host-pathogen interactions.
• Differentiate between CARMEN, mCARMEN, qCARMEN, and bbCARMEN in terms of their applications for multiplexed nucleic acid detection.
• Discuss the potential of CRISPR-based diagnostics for widespread, distributed pathogen testing.

Gene therapy has experienced an increasing number of successful human clinical trials, leading to 6 FDA approved products using delivery vectors based on adeno-associated viruses (AAV). These successes were possible due to the identification of specific disease targets for which natural variants of AAV were sufficient. However, vectors face a number of barriers and shortcomings that preclude their extension to most human diseases, including limited delivery efficiency to target cells, pre-existing antibodies against AAVs, suboptimal biodistribution, limited spread within tissues, and/or an inability to target delivery to specific cells. These barriers are not surprising, since the parent viruses upon which vectors are based were not evolved by nature for our convenience to use as human therapeutics. Unfortunately, for most applications, there is insufficient mechanistic knowledge of underlying virus structure-function relationships to empower rational design improvements.

As an alternative, for over two decades we have been implementing directed evolution – the iterative genetic diversification of the viral genome and functional selection for desired properties – to engineer highly optimized, next generation AAV variants for efficient and targeted delivery to any cell or tissue target. We have genetically diversified AAV using a broad range of approaches from fully random (e.g. error prone PCR) to computationally guided (e.g. by machine learning). The resulting large (~109) libraries are then functionally selected for substantially enhanced delivery, yielding AAVs capable of highly efficient therapeutic gene delivery. Our variants have been effective in both animal models and in 6 human clinical trials to date, and results from both will be discussed.

Learning Objectives:

• Describe the process of directed evolution for optimizing AAV vectors used in gene therapy.
• Differentiate between random and computationally guided approaches for AAV genetic diversification.
• Discuss the clinical implications of using evolved AAV variants in gene therapy based on results from animal models and human trials.

Originally developed for gene editing, CRISPR-Cas systems have now become powerful molecular diagnostic tools. However, their widespread adoption in diagnostics has relied heavily on traditional biochemical methods, such as real-time PCR and plate readers, which, while effective, face challenges in sensitivity, speed, and ease of use. To push the boundaries of molecular diagnostics, particularly in cancer detection, more advanced tools are necessary.

This lecture will explore the synergy between CRISPR-Cas diagnostics and Lab-on-a-chip (LOC) technology, showcasing how their combination can significantly improve detection limits, usability, and accessibility for clinical settings. LOC technology, with its potential for miniaturization, automation, and integration, allows CRISPR-based diagnostics to be conducted more efficiently and with greater sensitivity, using minimal sample volumes. These enhancements pave the way for point-of-care applications and more accessible diagnostics, particularly in cancer care.

Highlighting the work done in our lab, we will demonstrate how we’ve utilized microfluidic platforms to develop CRISPR diagnostics specifically for early cancer detection. By leveraging the strengths of both CRISPR and LOC technologies, we aim to create highly sensitive, rapid, and portable diagnostic tools that can revolutionize cancer biomarker detection in liquid biopsies, offering better patient outcomes.

Learning Objectives:

• Describe the limitations of traditional diagnostic platforms (e.g., cuvettes, 96-well, and 384-well plates) under the condition of increasing demands for multiplex detection and lower detection limits, and determine why transitioning to Lab-on-a-Chip technology is necessary for modern diagnostics.
• Explain how Lab-on-a-Chip technology enhances CRISPR-Cas-based diagnostics when addressing the need for lower sample volumes, automation, and integration in point-of-care applications, and perform a comparative analysis between traditional and LOC-based diagnostics.
• Evaluate a real-world case of Lab-on-a-Chip used in cancer diagnostics, under the condition of balancing detection sensitivity with quantification, and assess the impact of droplet microfluidics on detection limits without losing quantification accuracy.