CRISPR 2018

The CRISPR-Cas9 system has proven to be a powerful tool for genome editing allowing for the precise modification of specific DNA sequences within a cell. Many efforts are currently underway to use the CRISPR-Cas9 system for the therapeutic correction of human genetic diseases. The most widely used homologs of the Cas9 protein are derived from the bacteria Staphylococcus aureus (S. aureus) and Streptococcus pyogenes (S. pyogenes). Based on the fact that these two bacterial species cause infections in the human population at high frequencies, we looked for the presence of pre-existing adaptive immune responses to their respective Cas9 homologs, SaCas9 (S. aureus homolog of Cas9) and SpCas9 (S. pyogenes homolog of Cas9). We have identified the presence of pre-existing humoral immune responses to SaCas9 and SpCas9 by both Western Blot and ELISA. In addition we have also identified pre-existing cellular responses to Cas9 by intracellular cytokine staining and ELISPOT. 

Learning Objectives: 

1. Pre-existing adaptive immunity to the most commonly used homologous of Cas9 is prevalent
2. Potential immune responses to any new therapeutic should be considered when considering any kind of in-vivo therapy

The National Institute of Standards and Technology (NIST), is the measurement institute of the U.S., supplying unbiased expertise in measurements and standards to support growth in all sectors of economy and commerce including healthcare and the bio-economy.  Confidence and comparability of measurements are critical to underpinning any scientific effort including the genome editing field.  Through a newly launched NIST-led Genome Editing Consortium, NIST is working with experts in the genome editing field to identify and develop solutions for pre-competitive needs for establishing confidence in assessing the results of genome editing process.  Leading pre-competitive needs the consortium will address are: shared process control samples, reference data and standard data formats, minimum information norms, and standard terminology. This talk will give an introduction measurement assurance and overview of the newly launched NIST-led Genome Editing Consortium.

Learning Objectives: 

1. Gain a better understanding of how community norms and standards can help give confidence in data and accelerate the genome editing field 
2. Gain knowledge of the NIST Genome Editing Consortium and the ongoing efforts to develop genome editing community standards and norms

The most recently developed genome editing system, CRISPR-Cas9 has greater inherent flexibility than prior programmable nuclease platforms. Because of its simplicity and efficacy, this technology is revolutionizing biological studies and holds tremendous promise for therapeutic applications. However, imperfect cleavage specificity of CRISPR-Cas9 nuclease within the genome is a cause for concern for its therapeutic application. To facilitate the adoption and improvement of this technology, we have developed CRISPRseek for designing target specific gRNAs, and GUIDEseq for identifying genome-wide offtarget sites from GUIDE-seq and CIRCLE-seq experiments to assess the precision of engineered CIRSPR-Cas9 nucleases. In this talk, I will give an introduction to the CRISPR genome editing, GUIDE-seq and CIRCLE-seq technologies, followed by an overview of the functionalities of CRISPRseek and GUIDEseq. By the end of talk, the participants should be able to design target-specific gRNAs for various cas9 nucleases and genomes using CRISPRseek, and analyze GUIDE-seq and CIRCLE-seq data using GUIDEseq.

Learning Objectives: 

1. Understand how the CRISPR-Cas9 Genome-Editing system is recolutionizing biological studies
2. Understand how to design target-specific gRNAs 

Learn how to optimize CRISPR-Cas9 editing efficiencies in cell lines and primary cell types using modified synthetic sgRNA. Includes tips for achieving maximum knockout and knock-in efficiencies, experimental examples of how to optimize iPSC editing and a detailed explaination of CRISPR design and ICE analysis tools. 

Learning Objectives: 

1. Learn how to utilize free software tools to design CRISPR guide RNAs and analyze CRISPR editing.  
2. Learn some key experimental parameters for optimizing CRISPR in cell lines and primary cells. 

There have been an increasing number of successful human gene therapy clinical trials, and in particular gene delivery vehicles or vectors based on the adeno-associated virus (AAV) have enabled success in trials for hemophilia B, spinal muscular atrophy, X-linked myotubular myopathy, and Leber’s congenital amaurosis type 2 (LCA2). The LCA2 trials led to the first FDA approval of a gene therapy for rare disease in the US in December, 2017.  That said, vectors based on natural versions of AAV face a number of delivery challenges that limit their efficacy and will thus preclude the extension of these successes to the majority of human diseases.  These delivery limitations arise since the parent viruses upon which these vectors are based were not evolved by nature for our convenience to use as human therapeutics.  We have been developing and implementing a high throughput approach termed directed evolution – involving the iterative genetic diversification of a viral genome and functional selection for desired properties – to engineer highly optimized variants of AAV for a broad range of cell and tissue targets.

In parallel, the advent of genome editing technologies such as the CRISPR/Cas9 system raise the possibility of using gene delivery not only for gene replacement but for repair or knockout of endogenous genes.  We have thus been combining engineered AAVs with CRISPR/Cas9 for a range of applications.  For example, delivery of a Cas9 targeting mutant superoxide dismutase (SOD1) in a murine model of amyotrophic lateral sclerosis delayed the onset of disease symptoms and significantly extended animal lifespan.  In addition, we engineered an AAV for enhanced transport along neuronal axons, and delivery of Cas9 enabled the knockout of specific genes in vivo within projection neurons from a distance, work with both basic and translational applications.  Finally, we have combined AAV delivery of donor constructs with Cas9 ribonucleoproteins to effect efficient homologous recombination within in genomic target loci.  The integration of these new technologies – Cas9 cargo with AAV delivery – can thus enable a broad range of basic and therapeutic applications.

Learning Objectives: 

1. Adeno-associated viral vectors have the capacity to mediate efficient delivery of CRISPR/Cas9, leading to effective genome editing in vivo.
2. As a result, CRISPR/Cas9 is an effective tool to knock out autosomal dominant alleles that underlie human disease.

All forms of life require immune systems to stave off infection from viruses and other pathogens. In bacteria and archaea, clustered regularly interspaced short palindromic repeats (CRISPR) arrays and CRISPR-associated (Cas) proteins provide adaptive immunity against invading DNA from bacteriophages and plasmids. During infection, host cells adapt by storing short segments of foreign DNA in CRISPR arrays, providing heritable molecular memories of the infection. CRISPR-derived RNAs guide Cas complexes to bind and cleave the specific matching region of the foreign nucleic acids, thus neutralizing the infection. The unique, programmable DNA cleavage activity of CRISPR-Cas systems has been widely adapted for genome editing. In addition to providing revolutionary biotechnological tools, fundamental research into CRISPR/Cas systems has deepened our understanding of the complex molecular warfare that occurs between bacteria and their invaders. I will present recent mechanistic insights into CRISPR-Cas systems that reveal how bacteria adapt to rapidly evolving viruses.

Learning Objectives: 

1. An overview of the basic biology of CRISPR-Cas adaptive immune systems
2. The importance of specificity during CRISPR-Cas immunity
3. How guide RNA sequences can affect immunity and how these sequences are acquired in bacteria

Decoding human genetic disease allows us to develop models of the pathology that can be directly tested with gene correction or targeted drug therapy.  Dominant negative mutations are particularly promising therapeutic targets since they are resistant to traditional therapies, yet precise excision of disease-causing allele could provide a cure. We are using patient-derived induced pluripotent stem cells (iPSCs) to model diseases in tissues that are particularly susceptible to dominant negative mutations: cardiomyocytes, motor neurons and retinal pigment epithelial (RPE) cells. By developing CRISPR genome surgery in human cells, we hope to devise improved cellular models as well as human therapies.  By focusing on allele-specific gene excision we can select gene mutations that are highly penetrant, with clear phenotypes in cell types that can be readily derived from iPSCs.  We use whole genome sequencing to identify common genetic polymorphisms that can be used to selectively inactivate the disease allele with CRISPR nucleases.  The diseased cell types allow us to decode the cellular signatures of disease and determine if the excision of the disease allele restores cellular functioning.  Genome surgery is a rapidly advancing field that uses state-of-the-art techniques that pushes the boundaries of cell and molecular biology. We use advanced microscopy, tissue engineering and single cell genomics to optimize precise editing. We are developing computational methods to select optimal CRISPR/Cas9 combinations in diverse populations. We aim to produce therapies that are safe and cost effective so that they can benefit the maximal number of people. In collaboration with clinical scientists and the Innovative Genomics Institute (https://innovativegenomics.org/) we are preparing large animal models and clinical grade reagents to prepare for human clinical trials.  

Learning Objectives: 

1. Specific examples of where genome surgery could be used
2. Future directions in genome surgery

Gene editing using CRISPR is a very promising technology, and it has already had a significant impact on a number of research fields. However, while CRISPR makes targeted modifications easier to achieve, designing suitable guides is not a trivial task. It is crucial to ensure both the efficiency and the safety of the selected target sites. In this presentation, we discuss how to implement relevant selection criteria into an algorithm for whole-genome detection and evaluation of candidate sites. We report on the impact of specific design rules and on results to date. We also outline outstanding computational challenges.

Learning Objectives:

1. Learn how to improve the efficiency of CRISPR knock-outs
2. Understand how computational tools can accelerate gene editing projects
3. Learn about outstanding challenges in making these tools more practical and more widely applicable