Genome engineers can leverage an ever-expanding array of biological and computational tools to make targeted genetic changes. Maintaining and optimizing components ranging from reagent design and selection to data analytics has become critical for successful experimental design and intended editing outcomes. In this workshop, we present an end-to-end CRISPR-Cas genome editing paradigm, focusing on applications and improvements at various procedural steps. We will cover key workflows, including target identification through knockout libraries, optimization for targeted genetic changes through homology directed repair (HDR), off-target editing quantification, and more. In addition, we will showcase innovations from IDT’s research and development team.

Learning Objectives:

1. Discover how to optimize oligo and experimental design early in project ideation.

2. Identify ideal methods and reagents to improve the efficiency of generating intended editing outcomes.

3. Discuss the latest IDT R&D developments aimed at improving genome engineering workflows.

Nucleic acid analysis is crucial for the diagnostics of infectious diseases such as COVID-19. CRISPR/Cas technology, beyond its broad application in gene editing, features, in this regard, a simple and powerful tool for the highly sensitive and selective quantification of pathogen-specific oligonucleotides. Besides, single biomarker-based clinical decisions are often inadequate. To improve the accuracy, multiplexing, the measurement of various analytes from a single sample simultaneously, has recently gained great importance in diagnostics.
In this talk, first a short overview of CRISPR-powered biosensing will be given. Then, an electrochemical microfluidic multiplexed biosensor (BiosensorX) platform for on-site COVID-19 management will be introduced: i) CRISPR-powered nucleic-acid amplification-free and multiplexed testing of RdRp and E genes (SARS-CoV-2, Omicron-variant) along with LODs in the lower femtomolar range within a sample-to-result time of around 30 minutes, ii) monitoring ß-lactam antibiotics via an antibody-free bioassay using penicillin-binding proteins, and iii) the combination of both on the same chip to demonstrate the feasibility of the simultaneous measurement of different biomolecule classes. The BiosensorX technology is capable of gauging 4, 6, or 8 (different) analytes or specimen simultaneously via a sequential chip design within a single microfluidic channel.
By eliminating the need for a reverse transcription and target amplification of the pathogen-specific RNAs (necessary for PCR and other detection methods), BiosensorX system features a low-cost, simple-to-use, easily scalable and multiplexed strategy for point-of-care testing of nucleic acids via CRISPR-based bioassays and other biomolecule classes (such as drugs) simultaneously.

Learning Objectives

1. Summarize the fundamentals of CRISPR-powered biosensing.

2. Identify an electrochemical microfluidic multiplexed approach for the simultaneous measurement of nucleic acids and other biomolecules such as drugs or proteins.

3. Apply this knowledge to develop new CRISPR-based biosensors in future.

Clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 13 (Cas13) has been rapidly developed for nucleic-acid-based diagnostics by using its characteristic collateral activity. Despite the recent progress in optimizing the Cas13 system for the detection of nucleic acids, engineering Cas13 protein with enhanced collateral activity has been challenging, mostly because of its complex structural dynamics. Here we successfully employed a novel strategy to engineer the Leptotrichia wadei (Lwa)Cas13a by inserting different RNA-binding domains into a unique active-site-proximal loop within its higher eukaryotes and prokaryotes nucleotide-binding domain. Two LwaCas13a variants showed enhanced collateral activity and improved sensitivity over the wild type in various buffer conditions. By combining with an electrochemical method, our variants detected the SARS-CoV-2 genome at attomolar concentrations from both inactive viral and unextracted clinical samples, without target preamplification. Our engineered LwaCas13a enzymes with enhanced collateral activity are ready to be integrated into other Cas13a-based platforms for ultrasensitive detection of nucleic acids.

Learning Objectives:

1. Discuss the mechanism of CRISPR-Cas13 systems.

2. Explain the principle of using CRISPR-Cas13 systems for nucleic acid detection.

3. Discuss the engineering methods of the  CRISPR-Cas13 systems for nucleic acid detection.

Cancer, as a disease, starts with a change in the genome that results in the downregulation of genes involved in "normal" cell behavior. This genetic change can either be induced by a change in the DNA code (mutation) or a change that does not affect the genetic code but instead influences the way this code can be read. The latter is called an epigenetic alteration. While cytosine methylation is a healthy process, methylation of promoter CpG islands has also been shown to be a critical hallmark in many cancer cells. This is because specific genes on the DNA must always be expressed to prevent a "normal" cell from becoming a cancer cell. With the upcoming field of liquid biopsies and the conformation that these hypermethylated DNA sequences could be found in urine, the development of novel biosensors for fast and reliable detection of these sequences becomes of interest. Integrating CRISPR/Cas technology in miniaturized sensors for on-site analysis is very promising for biomarker detection at the point of care. However, while CRISPR sensing seems a promising alternative to established DNA detection techniques, it is not sensitive to epigenetic changes like hypermethylation. In this presentation, we showcase a method to make Cas12a sensitive to CpG methylation, which is based on the addition of methylation-sensitive restriction enzymes to the CRISPR sensing process. Furthermore, we discuss the effect of DNA fragmentation on the cleavage activity of Cas12a and how this can be used in biosensing.

Learning Objectives:

1. Discuss the mechanism of Cas12a and how this can be used for biosensing of dsDNA sequences.

2. Identifying the difference in Cas12a activity for different target sequence lengths.  

3. Infer that, by selecting the right enzymes and crRNA sequences, Cas12a combined with methylation-sensitive restriction enzymes can be used to determine the methylation state of a single CpG methylation site.

Rapid, accurate and frequent detection of the RNA of SARS-CoV-2 and of serological host antibodies to the virus would facilitate the determination of the immune status of individuals who have COVID-19, were previously infected by the virus, or were vaccinated against the disease. This presentation describes the development and application of a 3D-printed lab-on-a-chip that concurrently detects, via multiplexed electrochemical outputs and within 2 h, SARS-CoV-2 RNA in saliva as well as anti-SARS-CoV-2 immunoglobulins in saliva spiked with blood plasma. The device automatedly extracts, concentrates and amplifies SARS-CoV-2 RNA from unprocessed saliva, and integrates the Cas12a-based enzymatic detection of SARS-CoV-2 RNA via isothermal nucleic acid amplification with a sandwich-based enzyme-linked immunosorbent assay on electrodes functionalized with the Spike S1, nucleocapsid and receptor-binding-domain antigens of SARS-CoV-2. This workflow introduces a new method for CRISPR-based detection of nucleic acids with an electrochemical readout and demonstrates how combining it with antibody detection can allow for a multiplexed electrochemical diagnostic at the point of care that can can facilitate widespread monitoring of infection and immunity in populations.

Learning Objectives:

1. Discuss how multiplexing an assay can improve infectious disease monitoring.

2. Discuss the differing methods used to detect the presence of antibodies and nucleic acids on the electrochemical chip.

3. Discuss how integrating microfluidics improves the CRISPR-based RNA assay workflow.

The CRISPR revolution has begun and shows no signs of slowing down. CRISPR-Cas nuclease-based genome editing strategies hold great promise for the treatment of genetic disorders. However, a major drawback of these approaches is the generation of potentially harmful double-strand breaks (DSBs). Base editing (BE) is a CRISPR-Cas9-based genome editing technology that allows the introduction of point mutations in the DNA without generating DSBs. Two major classes of base editors have been developed: cytidine base editors or CBEs allowing C>T conversions and adenine base editors or ABEs allowing A>G conversions. On the other hand, the prime editing (PE) system contains a prime editing extended guide RNA (pegRNA)-guided reverse transcriptase instead of a deaminase. The development of PE was a breakthrough as it requires no PAM sequence adjacent to the target site and it can accomplish not only all 12 types of point mutations, but also insertions (of up to 44 bp) and deletions (of up to 80 bp), or even combination of substitutions, insertions, and deletions. 

The BEs and PEs are high-precision genome editing tools that can be programmed to alter the desired context of the genome in living cells, without requiring DNA donors. The BE and PE systems have remarkable potential as therapeutic tools to correct disease-causing mutations in the human genome. Over 25% of human pathogenic SNPs can be corrected by targeting the four transition mutations, and in principle, prime-editing could correct up to 89% of known genetic variants associated with human disease. The scope of BE & PE tools has been extensively broadened, allowing higher efficiency, specificity, accessibility to previously inaccessible genetic loci, and multiplexing, while maintaining a low rate of Insertions and Deletions (InDels). The BE and PE toolboxes are promising therapeutic strategies for genetic diseases caused by point mutations and might be more effective than approaches based on the homology-directed repair.

Learning Objectives:

1. Describe CRISPR-Cas Base Editing and Prime Editing systems.

2. Explain the main differences between BE & PE Systems.

3. Discuss applications of BE and PE systems.

Simple and sensitive nucleic acid-based molecular detection plays a crucial role in the early diagnosis of infectious diseases and the rapid screening of cancers.  CRISPR technology has recently emerged as a powerful biosensing tool for point of care molecular diagnostics when coupled with microfluidics technology. In this talk, I will present the recent development of combining CRISPR technology with microfluidic technology for detection of different pathogens. I will also introduce our effort in developing low-cost CRISPR-based microfluidic biosensing systems, enabling simple, rapid, minimally-instrumented, molecular diagnostics at the point of care.

Learning Objectives: 

1. Discuss current challenges in developing simple and rapid nucleic acid-based molecular diagnostics.

2. Introduce recent development of CRISPR-based molecular diagnostic technologies and microfluidic biosensing.

3. Propose CRISPR-based microfluidic biosensing systems for molecular detection at the point of care.

Traditional methods for pathogen detection are often expensive and time-consuming and requires a well-equipped laboratory. Here we present a low-cost, deployable paper-based biosensor device for rapid analysis of pathogens for a wide range of application including rapid diagnosis of infectious disease (such as SARS-CoV-2) and wastewater surveillance for public health. The paper microfluidic device was wax printed with hydrophobic and hydrophilic layer to enable sample processing by folding and unfolding the device. A reverse transcription loop-mediated isothermal amplification (RT-LAMP), together with CRISPR/Cas assay were designed, optimised and integrated into the paper microfluidic device, which shows a comparative sensitivity and specificity. The test results could be read by the naked eye or quantified using a mobile phone camera, as demonstrated for malaria testing in Africa and veterinary diagnosis in an Indian local farm. Furthermore, we show the device for wastewater surveillance, which has emerged as a new monitoring mechanism for early warning of pandemic within population and provides complementary data sets for clinical diagnosis. The device was demonstrated for the field testing in a local quarantine hotel, providing a sample-to-results within less than 1 hours turnround, which is currently developing to trace the source of hotspots for early warning of pandemic within a UK national wastewater surveillance programme.

Learning Objectives:

1. Discuss paper microfluidic device for rapid diagnosis.

2. Discuss molecular diagnosis with isothermal amplification and the assay coupled with CRISPR/Cas.

3. Discuss infectious disease diagnosis and wastewater surveillance for public health.

CRISPR has revolutionized the biosciences over the past decade. The RNA-targeting CRISPR effector protein Cas13, discovered in 2016, opened up a myriad of new applications for CRISPR-based technologies. As a postdoc, I developed Cas13-based technologies for detecting and destroying viral RNA. My talk will discuss both technologies for point-of-care nucleic acid detection (HUDSON, SHINE), and technologies for massively multiplexed nucleic acid detection (CARMEN). During the COVID-19 pandemic, these technologies have rapidly moved from the lab into the clinic. Finally, I will discuss our efforts to use Cas13 as a programmable antiviral therapy for a diverse set of RNA viruses. My lab at Princeton, started in January 2021, is building on this work as we continue to develop new Cas13-based technologies for studying both viral and host RNAs.

The clustered regularly interspaced short palindromic repeat (CRISPR) genome-editing revolution established the beginning of a new era in life sciences. I will report the role of state-of-the-art computations in the CRISPR-Cas9 revolution, from the early refinement of cryo-EM data to enhanced simulations of large-scale conformational transitions. Molecular simulations reported a mechanism for RNA binding and the formation of a catalytically competent Cas9 enzyme, in agreement with subsequent structural studies. Inspired by single-molecule experiments, molecular dynamics offered a rationale for the onset of off-target effects, while graph theory unveiled the allosteric regulation. Finally, the use of a mixed quantum-classical approach established the catalytic mecha- nism of DNA cleavage. Overall, molecular simulations have been instrumental in understanding the dynamics and mech- anism of CRISPR-Cas9, contributing to understanding func- tion, catalysis, allostery, and specificity.

CRISPR/Cas gene editing technology has rapidly evolved over the last decade. Its versatility extends from creating targeted research-use disease models in cells and animals, to high-throughput screening applications, to clinical reagents for cell-based therapies. The choice of CRISPR reagents is integral to the efficiency and outcome of your gene editing experiments. The specificity and precision of CRISPR tools have evolved from generic WT variants to high-fidelity variants, single base editors, and prime editors. The formats for these tools have also evolved from plasmids to ribonucleoprotein complexes and one-part, modified synthetic guides. In this webinar, we’ll discuss how next generation tools, such as PURedit™ CRISPR reagents, can take your research from discovery to the clinic. We’ll provide an overview of the important characteristics of a gene editing experiment to consider when choosing CRISPR reagents for your translational experiments, such as off-target effects and quality grades.
 
Topics include:
- Current and novel approaches to targeted gene editing
- CRISPR tools required for targeted gene editing workflows
- Use of PURedit™ CRISPR reagents in a variety of applications to provide enhanced specificity without sacrificing cutting activity
 
Learning Objectives:

1. Explain the key requirements of CRISPR reagents for preclinical and translational research.

2. Discuss performance and quality characteristics of gene editing reagents that make them ideal for preclinical research.

3. Choosing the right CRISPR reagents for your workflow and application.

My laboratory uses tools from pharmacology, genomics, and cell signaling to study two key aspects of cancer biology. First, we seek to understand how oncogenic signals, altered metabolic states, and drug therapies impact cell death regulation in cancer, with a particular focus on the mitochondrial cell death pathway (for example, Science Translational Medicine 2016, 8, ra175; Nature Communications 2018, 9, 1677; Nature Communications 2018, 9, 3513; Cell Metabolism 2019, 29, 1217; Science Translational Medicine 2022, 14, eabc7480). Second, we seek to understand the mechanisms that shape tumor evolution under the selective pressure of drug treatments, with the long-term goal of creating therapies that select against resistance (for example, Cell Reports 2017, 21, 2796; Cancer Discovery 2020, 10, 1894; Nature Genetics 2020, 52, 408; Nature Cancer 2022, 3, 837). A common theme in nearly all of our studies is the coupling of large-scale, unbiased genomic and pharmacological approaches with mechanistic studies using the classic tools of biochemistry and molecular and cellular biology. Collectively, our studies have led to new, fundamental mechanistic insights into the core survival circuitry operating in defined human tumor subsets as well as novel translational therapeutics. In this talk, I will highlight ways we have used functional genomics, and in particular CRISPR/Cas9-based loss-of-function screens, to define mechanistic determinants of apoptotic commitment and vulnerabilities associated with acquired drug resistance.

Learning objectives:
1. Discuss ways that CRISPR screens can reveal mechanistic determinants of cell fate
2. Relate ways in which drug modifier CRISPR screens can reveal drug mechanisms of action and resistance
3. Highlight translational applications of discoveries made using cancer-focused functional genomics

The COVID-19 pandemic highlights the need for diagnostics that can be rapidly adapted and deployed in a variety of settings. Several SARS-CoV-2 variants have shown worrisome effects on vaccine and treatment efficacy, but no current point-of-care (POC) testing modality allows their specific identification. We have developed miSHERLOCK, a low-cost, CRISPR-based POC diagnostic platform that takes unprocessed patient saliva; extracts, purifies, and concentrates viral RNA; performs amplification and detection reactions; and provides fluorescent visual output with only three user actions and 1 hour from sample input to answer out. miSHERLOCK achieves highly sensitive multiplexed detection of SARS-CoV-2 and mutations associated with variants B.1.1.7, B.1.351, and P.1. Our modular system enables easy exchange of assays to address diverse user needs and can be rapidly reconfigured to detect different viruses and variants of concern. An adjunctive smartphone application enables output quantification, automated interpretation, and the possibility of remote, distributed result reporting.

Learning Objectives:

1. Discuss why it is important to detect nucleic acid from infectious diseases (Basic).

2. Discuss how a CRISPR-based reaction works for diagnostics (Intermediate).

3. Explain the reason why there is an interest in combining nucleic acid amplifiation technologies with CRISPR enzymes (Advanced).

The Cas9 from Francisella novicida (FnCas9) has unique properties of target identification and engagement in terms of specificity, nature of double strand break as well as length of guide RNAs that can be harnessed for precise genome editing and diagnostics. Using a combination of protein and gRNA engineering, we have converted a naturally occuring Cas9 into an engineered version with remarkable prospects of precise therapeutic genome editing. In the past we have harnessed its ability to engage with DNA through FELUDA (FnCas9 Editor Linked Uniform Detection Assay)  based diagnosis of nucleic acids. Currently we are engaged in preclinical studies to proceed towards Phase 1 Clinical trial for hemtological disorders in Indian patients. 

Learning Objectives:

1. Discuss the balance between activity and specificity is inversely related in engineered SpCas9 proteins.

2. Explain how FnCas9 protein engineering was done through modifications in PAM interacting residues- thus the specificity at the level of DNA binding was retained.

3. Explain how FnCas9 and its engineered versions are not tolerant to mismatches in the guide or target. As a result, FnCas9 based diagnostics operate at the level of binding as opposed to diagnostics that work on the basis of transcleavage of reporter molecules.