CRISPR-based genome editing has accelerated biological research and holds great potential for studying and treating human diseases. The CRISPR-Cas9 system requires a Cas9 nuclease and a guide RNA, which may consist of either a CRISPR RNA (crRNA) coupled with a trans-activating crRNA (tracrRNA), or a single guide RNA (sgRNA) that combines the crRNA and tracrRNA in a single molecule. Both guide RNA formats can be chemically synthesized, offering advantages over expression systems. Synthetic guide RNAs are amenable to chemical modifications for increased stability, eliminate time-consuming steps of cloning and sequencing, and do not have the inherent immune response and cytotoxicity of in vitro transcribed guide RNAs. They can be readily delivered into cells for high-throughput arrayed screening applications and expand the types of phenotypic readouts that can be leveraged. In this webinar we will present data around the development and application of Horizon Edit-R synthetic sgRNA reagents for gene specific CRISPR knockout. We will discuss the importance of rational design, chemical modifications, delivery methods, and comparison of functionality with existing reagents. The functionality of individual guide RNA’s as well as a pool of all three guide RNAs, was tested in U2OS and primary human T cells. We performed viability assays for the knockout of essential genes and immuno-fluorescence analysis to demonstrate protein knockout. We also measured the phenotypic effects of gene knockout by flow cytometry showing depletion of cell surface proteins. Both sgRNA and crRNA:tracrRNA synthetic guide RNAs efficiently knockout the targeted proteins as measured by functional assays and sequence analysis. Pooling of the synthetic guide RNAs provides robust functional gene knockout and further simplifies high-throughput loss-of-function screening in arrayed format.

Learning Objectives:

1. Understand the usefulness of CRISPR Cas9 gene editing 

2. Understand the different tools available for gene silencing, knockout and editing

3. Understand the potential research and therapeutic applications for gene edited cell lines

While transformative, first-generation CRISPR technologies remain limited across multiple important dimensions including scalability, editing efficiency, types of modifications available, and ease of use. Inscripta’s Onyx™ platform addresses these limitations by offering push-button, high efficiency, massively parallel, and precise delivery of diverse edit types across the genomes of S. cerevisiae and E. coli. The platform simplifies previously complex CRISPR strain engineering workflows by offering an end-to-end solution from design through engineered strain library and analytics, including software, reagents and a benchtop instrument. Join us to learn more about this platform and see examples of engineering genomes at unprecedented scale end efficiency.

Learning Objectives:

1. Introduction to Inscripta’s Onyx™ Digital Genome Engineering Platform

2. Examples of  genome-wide strain engineering application in E. coli and S. cerevisiae

Genome Engineering allows the easy manipulation of genomes down to the nucleotide level.  Targeted deep sequencing enables the detection and quantification of low-frequency editing events.  However, the large amounts of data generated by targeted deep sequencing can be difficult to interpret and quickly analyze.  We have developed a Python-based computer program called CRIS.py that allows the easy analysis of multiple types of editing events.  We will show examples of how rapid deep sequence analysis has guided experimental design leading to high-efficiency genome editing in a broad range of applications.

Learning Objectives:

1. Discuss assays for evaluating on-target genome editing outcomes

2. Explain CRIS.py NGS pipeline

3. Discuss best practices for creating custom genome edited cell lines and animals

Genome editing with an expanded CRISPR-Cas tool set increases the opportunity to make intentional, targeted changes in the genome. Furthermore, improved specificity of genome editing and ability to fully characterize and quantify these events is a centerpiece of preclinical studies. The CRISPR Research and Development team at IDT remains focused on delivering reagents, tools, and methods to allow scientists to optimize the performance of genome editing workflows. In this webinar, I will focus on optimized parameters to obtain high rates of homology-directed repair (HDR), which allows the generation of specific mutations or insertions in the genome. This includes editing methods with both Cas9 and Cas12a proteins and strategies to introduce short insertions or SNPs as well as large insertions or knock-ins. There will be an interactive demonstration introducing our new NGS data analysis tool for CRISPR applications: CRISPAltRations. The presentation of our cloud-hosted data analysis tool will lead into a discussion of a workflow for complete characterization of on- and off-target editing efficiency and specificity. In addition, I will discuss the expanded genome editing space afforded by the dramatically improved activity of A.s. Cas12a Ultra and L.b Cas12a Ultra. These engineered Cas12a mutant proteins present advantages for genome editing in mammalian cells and plants. We will also introduce fluorescently tagged Cas proteins that allow for visualization in uptake experiments and maintain potent editing activity. Finally, I will introduce expanded gRNA manufacturing capabilities that maintain quality and rapid turnaround times and allow for genome engineering work in high throughput experiments or in animal models that demand more material made possible with large-scale synthesis capabilities. Additionally, our new custom gRNA ordering interface will allow for easy design and ordering of a gRNA to suit any targeted Cas nuclease of interest from a diversity of species.

Learning Objectives:

1. Our latest developments and customizations for guide RNAs, including pegRNAs and CRISPR libraries, and our newest CRISPR nucleases with fluorescently labeled Cas9 variants

2. Improved design tools, enhancing reagents, and analysis tools used to efficiently generate SNPs and large knock-ins

3. Simplified methods for high-throughput detection and analysis of CRISPR-induced off-target effects, including a demonstration of our new, biologically informed CRISPR NGS analysis pipeline, CRISPAltRations

The discovery of CRISPR technology has revolutionized the fields of transcriptional activation and repression, genome editing, gene-based therapeutics and diagnostics. The applications of this technology have been rapidly expanding as researchers continue to discover new Cas enzymes, engineer high fidelity Cas orthologs and modify and synthesize guide RNAs to efficiently direct these Cas enzymes to their targets. Despite these new discoveries and advancements in the CRISPR space, these developments can impact the Cas complex stability and efficiency and limit the efficient use of CRISPR technology. Therefore, quality control tools are needed to evaluate and understand the stability and functionality of these newly discovered or engineered Cas enzymes and synthetic gRNAs to better inform their design and applications in various environmental conditions. For example, the stability of various CRISPR-Cas complexes may vary at altered pH and temperatures making them less or more suitable for their intended applications. We have recently developed a CRISPR-based transistor technology, termed CRISPR-Chip™, that can monitor CRISPR’s efficiency in terms of Cas-gRNA binding, the stability of the Cas-gRNA complex, and the efficiency of DNA/RNA binding and cleavage under different environmental conditions. This talk will focus on the expansion of CRISPR-Chip technology being integrated into an automated platform that allows the user to perform multiple CRISPR quality control assays simultaneously. This technology is also paired with sophisticated software analysis and machine learning capabilities to further identify the relevant factors affecting CRISPR performance.

Learning Objectives:

1. Using an optics- and label-free approach to CRISPR research

2. Explain current approaches to selecting the most robust gRNA for CRISPR assays

3. The opportunities that come with automated CRISPR quality control straight at your lab bench

A repeat expansion in the C9orf72-SMCR8 complex subunit (C9orf72) represents the most common genetic cause of two fatal neurodegenerative diseases: frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). To determine the length of this repeat expansion, we used No-Amp sequencing (Pacific Biosciences [PacBio]), a targeted amplification-free long-read sequencing technology. In total, we examined 28 fully characterized C9orf72 expansion carriers for whom cerebellar tissue was available. We obtained more than 3500 circular consensus sequencing (CCS) reads, including 814 reads that spanned the C9orf72 repeat expansion. Repeat lengths obtained using No-Amp sequencing significantly correlated with those obtained using Southern blotting. Importantly, we demonstrated that smaller expansions are associated with prolonged survival after onset. Moreover, we revealed that smaller expansions associate with higher levels of transcripts containing the expansion and of dipeptide repeat (DPR) proteins, a hallmark of C9orf72-linked diseases. These findings suggest that, during transcription, it might be easier to cover small expansions than long expansions, which could explain why we observed elevated levels of expansion-containing transcripts in patients with smaller expansions. Since these expansion-containing transcripts can serve as templates for repeat-associated non-ATG (RAN) translation, this could account for the increased levels of DPR proteins we detected. Investigation of the expansion itself uncovered that its GC-content is high (100%). The expansion appears to be relatively pure, while interruptions are only occasionally encountered (<5%). In conclusion, we have shown that No-Amp sequencing is a powerful tool to examine the length and purity of repeat expansions, which enabled us to identify relevant clinico-pathological associations for C9orf72-related diseases.

Learning Objectives:

1. Learn about methods that can be employed to detect and size C9orf72 repeat expansions

2. Understand that targeted long-read sequencing accurately measures the C9orf72 repeat length and presence of interruptions

Combinatorial inhibition of effector and feedback pathways is a promising treatment strategy for KRAS mutant cancers. However, the particular pathways that should be targeted to optimize therapeutic responses are unclear. Using CRISPR/Cas9, we systematically mapped the pathways whose inhibition cooperates with drugs targeting the KRAS effectors MEK, ERK, and PI3K. By performing 70 screens in models of KRAS mutant colorectal, lung, ovarian, and pancreas cancers, we uncovered universal and tissue-specific sensitizing combinations involving inhibitors of cell cycle, metabolism, growth signaling, chromatin regulation, and transcription. Furthermore, these screens revealed secondary genetic modifiers of sensitivity, yielding a SRC inhibitor based combination therapy for KRAS/PIK3CA double- mutant colorectal cancers (CRCs) with clinical potential. Surprisingly, acquired resistance to combinations of growth signaling pathway inhibitors develops rapidly following treatment, but by targeting signaling feedback or apoptotic priming, it is possible to construct three-drug combinations that greatly delay its emergence.

Learning Objectives:

1. Learn a scalable method to systematically define combination therapies in tissue culture models

2. Identify challenges with two-body combination therapies in cancer

3. Explain rational ways to design higher order combination therapies that forestall the emergence of resistance

The adenosine analogue remdesivir has emerged as a front-line antiviral treatment for SARS-CoV-2, with preliminary evidence that it reduces the duration and severity of illness. Prior clinical studies have identified adverse events, and remdesivir has been shown to inhibit mitochondrial RNA polymerase in biochemical experiments, yet little is known about the specific genetic pathways involved in cellular remdesivir metabolism and cytotoxicity. Through genome-wide CRISPR-Cas9 screening and RNA sequencing, we show that remdesivir treatment leads to a repression of mitochondrial respiratory activity, and we identify five genes whose loss significantly reduces remdesivir cytotoxicity. In particular, we show that loss of the mitochondrial nucleoside transporter SLC29A3 mitigates remdesivir toxicity without a commensurate decrease in SARS-CoV-2 antiviral potency and that the mitochondrial adenylate kinase AK2 is a remdesivir kinase required for remdesivir efficacy and toxicity. This work elucidates the cellular mechanisms of remdesivir metabolism and provides a candidate gene target to reduce remdesivir cytotoxicity.

Learning Objectives:

1. Learn about the use of CRISPR-Cas9 system for high-throughput analysis

2. Explain mechanism of action of remdesivir and what is important for it's metabolism

3. Discover how to use CRISPR genome wide screens and RNA sequencing to interpret cellular mechanisms of drug metabolism

Cas9 based therapeutics have the potential to revolutionize the treatment of genetic diseases.  However, safe and effective methods for delivering Cas9 protein, gRNA and donor DNA need to be developed before the therapeutic potential of CRISPR based therapeutics can be realized.  In this presentation, I will describe a non-viral Cas9 delivery vehicle, termed CRISPR-Gold, which can induce homology directed DNA repair (HDR) in vivo by directly delivering Cas9 protein, gRNA, and donor DNA.  CRISPR-Gold is composed of gold nanoparticles assembled with the Cas9/gRNA ribonucleoprotein (RNP) complex, donor DNA, and an endosomal disruptive polymer.  We have been able to demonstrate that CRISPR-Gold can correct the DNA mutation that causes Duchenne muscular dystrophy (DMD) in mdx mice via HDR, with an efficiency of 5.4% after an intramuscular injection and with minimal levels off-target DNA damage.  In addition, CRISPR-Gold was able to deliver Cas9 RNP into the brain, after an intracranial injection, and rescue mice from FMR1 related autism, via deletion of the mGluR5 gene via non-homologous end joining (NHEJ).  CRISPR-Gold is a non-viral delivery vehicle that can generate HDR and NHEJ in vivo and has potential for treating muscle and neurological diseases.

Learning Objectives:

1. Strategies for delivering the Cas9 protein in vivo

2. New ways to conjugate polymers to proteins

3. Therapeutic applications of gene editing

An extensively debated concern about CRISPR-Cas9 genome editing is that unspecific guide RNA (gRNA) binding may induce off-target mutations. However, accurate prediction of CRISPR-Cas9 off-target sites and activity is challenging. We have developed a new protocol for detection of gRNA binding and Cas9 cleavage, based on amplification-free long-read sequencing. This method, which is named “off-target sequencing” (OTS), was assessed using a human cell line that was first re-sequenced using long and highly accurate reads to get a detailed view of all on- and off-target binding regions. The OTS method detected many Cas9 cleavage sites that were not reported by off-target prediction software, including in “dark” regions of the human genome inaccessible by standard short-read sequencing. We are currently investigating whether in vivo off-target editing can occur at these sites.

Relevant publications:

• Amplification-free long read sequencing reveals unforeseen CRISPR-Cas9 off-target activity. Höijer I, Johansson J, Gudmundsson S, Chin CS, Bunikis I, Häggqvist S, Emmanouilidou A, Wilbe M, den Hoed M, Bondeson ML, Feuk L, Gyllensten U, Ameur A. bioRxiv 2020.02.09.940486; doi: https://doi.org/10.1101/2020.02.09.940486

• Single-Molecule Sequencing: Towards Clinical Applications. Ameur A, Kloosterman WP, Hestand MS. Trends Biotechnol. 2019 Jan;37(1):72-85. https://www.ncbi.nlm.nih.gov/pubmed/30115375

Learning Objectives:

1. Introduction to long-read sequencing technologies

2. New methods study CRISPR-Cas9 off-target activity

CRISPR experiments are a powerful tool which are easy to carry out, however it is more difficult to determine the outcome of these experiments, and to ensure that only the desired targets were addressed and were so correctly. I will describe new methods to evaluate the outcomes of CRISPR experiments and highlight their importance for characterizing the results and lay a foundation for future optimizations. In addition, I will describe new methods to leverage the specificity of Cas9 to target genomic regions without amplification for sequencing with high accuracy and multiplex capabilities.

Learning Objectives:

1. Understand the importance of measuring outcomes of CRISPR experiments, limitations of previous approaches, and the benefits of new methods

2. Learn about new methods to leverage Cas9 to target genomic regions for sequencing without amplification with high specificity, accuracy and multiplex