The advent of CRISPR-Cas (Clustered regularly interspaced short palindromic repeats-CRISPR associated) makes it feasible to target/edit the genome with precision. Most of the CRSIPR-Cas systems show a trans nuclease activity upon binding their specific target, which is absent in FnCas9 (Cas9 derived from Francisella novicida). This high specificity along with an extremely high mismatch sensitivity (binding is abolished if there is more than a single base pair mismatch between guide RNA and the substrate DNA) of FnCas9 makes it possible to target and edit SNPs with precision. The proof-of-concept for which has been shown by the correction of the Sickle Cell Disease (SCD) point mutation (in patient derived iPSCs, induced pluripotent stem cells) and the detection of SCD/COVID-19 by FELUDA (FnCas9 linked uniform detection assay is a robust, rapid, and inexpensive point-of-care test).

Learning objectives:

1. Discuss orthogonal Cas systems for precise genome manipulation.

2. Break down affinity mediated CRISPR diagnostics, their adaptation and translation to on-field assays.

Rapid, accurate, and wide-spread COVID-19 testing is essential for contact tracing, timely triaging of patients, and viral monitoring. The current gold standard for COVID-19 testing is RT-PCR. However, such nucleic acid amplification tests (NAATs) require a laboratory, severely limiting large-scale implementation. Our lab thus focused our efforts on this pandemic challenge, with the goal of engineering a decentralized CRISPR-based NAAT solution that can be used by anyone without the need for complex laboratory instruments. We have designed, developed, and validated a point-of-care lyophilized COVID-19 NAAT sensor that can be integrated into any face mask for detecting the virus from a user’s breath. The sensor employs isothermal amplification and Cas12a-based SHERLOCK reactions to achieve a detection sensitivity on par with that of current CDC-approved RT-PCR COVID-19 tests. Breath aerosols collected on the interior surface of the face mask are used for analysis and all reactions operate at ambient temperatures. The freeze-dried synthetic biology reactions are shelf-stable, eliminating the need for cold-chain distribution or storage. The system executes autonomously without any power through passive capillary forces, eliminating operator steps such as liquid handling. All the user needs to do is press a button to start the sensor analysis and the output is a simple visual result. Importantly for practical implementation, the sensor is compact (smaller than a business card), lightweight (equal in weight to a penny), and inexpensive (currently at $5 per sensor). We envision our face mask sensors can be used as a widespread disposable COVID-19 diagnostic, combining the protective functions of a mask with the capabilities of NAATs for sensitive, specific and convenient high-frequency testing. 

Learning Objectives:

1. Discuss the key functional steps for the COVID-19 face mask sensor.

2. Discuss some of the advantages of integrating COVID-19 diagnostics into a face mask.

The recent outbreak of the Coronavirus Disease 2019 (COVID-19) has spread all over the world and raised global concerns. Rapid and early detection of the novel coronavirus SARS-CoV-2, the causative agent of the COVID-19, plays a crucial role in reducing transmission of the virus and facilitating early intervention and treatment. In this talk, I will present our CRISPR-based nucleic acid detection technology for SARS-CoV-2 testing. I will also introduce our effort on developing low-cost CRISPR-based diagnostic devices and instrument-free detection platforms, enabling simple, rapid, affordable, mobile, point of care molecular diagnostics.

Learning Objectives:

1. Discuss current challenges in developing simple and rapid molecular diagnostic assay to fight COVID-19.

2. Introduce new CRISPR-based molecular diagnostic technologies.

3. Present CRISPR-based microfluidic diagnostic system for SARS-CoV-2 detection at the point of care.

The COVID-19 pandemic demonstrated the need for rapid, accurate diagnostic tests that could be developed and deployed quickly. Since the company’s founding in 2018, Mammoth has been advancing our CRISPR-based DETECTRTM technology to enable diagnostic solutions across the continuum of molecular testing. By programming a Cas protein with a sequence specific gRNA, DETECTRTM couples a highly specific target recognition event with cleavage of a reporter molecule, generating a measurable signal. In response to the pandemic, Mammoth rapidly reconfigured our DETECTRTM platform to detect SARS-CoV-2, demonstrating the speed and accuracy of CRISPR-based diagnostics for COVID-19. Mammoth has further implemented its CRISPR technology into device formats that enable high-throughput and decentralized testing solutions. The platform nature of the DETECTRTM platform will continue to democratize diagnostics for SARS-CoV-2 and beyond. 

Learning Objectives:

1. Define how CRISPR proteins enable disease diagnostics.

2. Explain the application of CRISPR diagnostics to the COVID-19 pandemic.

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. Discuss in vivo gene editing via the delivery of the Cas9 ribonucleoprotein.
2. Discuss new strategies for conjugating polymers to proteins.

Over the past ten years, CRISPR technology has revolutionized genome engineering, as reflected by the recently awarded Nobel Prize. Nevertheless, the process of performing and characterizing editing events can be challenging for both novices and experts. Suboptimal gRNA design and delivery of the CRISPR components into the target cell can severely affect editing efficiency. This, in turn, can make the already laborious process of characterizing the editing event even more time-consuming and inefficient.

To help you detect and validate your gene edits more effectively, we’ve developed an optimized workflow. The new workflow offers everything you need to characterize your editing event faster and easier, from sample preparation to target amplification and analysis.

Learning Objectives:

1. Verify your editing efficiency with confidence using the new sequencing analysis tool.

2. Design PCR and Sanger sequencing primers with ease.

3. Break down technical failures and input quality.

Multi-target screening can offer great insight into the genetic pathways in drug resistance and disease. RNAi and the fast pace of CRISPR technology development has brought sophisticated options for library screens including the possibility of genome-wide, epigenetic target gene modulation. CRISPRi allows for targeted suppression of gene function while CRISPRa offers a gain-of-function approach to screening. Both complement existing loss-of-function and gain-of-function technologies such as RNAi and ORFs; and have several distinct advantages, including efficient targeting of non-coding genes. You can also take your screens to the single-cell level by using our 10X Genomics compatible CRISPR guide vectors containing capture sequences that allow efficient feature barcoding of individual cells for transcriptomics across pools of guides, allowing functional genomic analysis of top screen hits for single-cell genomic exploration. 

In this webinar, we will explore screening technologies for gene knockout, activation, and repression to help you choose the right format and approach for your screening applications.

Learning Objectives:

1. Review gene knockout, activation, and repression to help you choose the right format and approach for your screening applications.

2. Indentify how orthogonal validation via multiple technologies lends to additional gene pathway insights.

3. Differentiate between delivery options and formats for screening tools for different applications.

Performance. Automation. Convenience. Versatility. Those three elements exemplify the Miltenyi Biotec family of flow cytometers. Designed to support the field of flow cytometry, the MACSQuant® Flow Cytometer offer exclusive features that help untangle the complexities of cell analysis for both beginners and experts. 

By incorporating a yellow 561 nm laser and ten optical detection channels, the MACSQuant VYB Flow Cytometer offers researchers a versatile option of fluorescent proteins and conjugates to perform sophisticated multicolor experiments.  The combination of violet, yellow, and blue lasers is ideal for CRISPR applications utilizing fluorescent protein reporters and for detecting a wide range of fluorescent proteins. 

Laboratories across the globe are using the MACSQuant VYB Flow Cytometer for numerous and diverse applications including cell characterization, bacteria evaluation for bioproduction, nucleosome research, and investigations of new tools for synthetic biology.
 

Learning Objectives:

1. Break down how automated features that contribute to ease of use for all levels of flow cytometry.

2. Explain how simultaneous detection of multiple fluorescent proteins can support complex cell analysis.

3. Discuss laboratories that are performing flow cytometric assays with the MACSQuant VYB Flow Cytometer today.

Nucleic acid testing is crucial for the prevention, diagnosis and treatment monitoring of many diseases in medicine. Over the last years, microRNAs (miRNAs), short non-coding RNAs, as biomarkers have become increasingly important since a dysregulation of specific miRNA levels in human body fluids are linked to many diseases, such as Alzheimer of different types of cancer. Beyond its broad use in gene editing, CRISPR/Cas technology features a powerful tool for the highly sensitive and selective quantification of nucleic acids. In this talk, first a brief summary of CRISPR powered biosensing will be given. Then, a CRISPR/Cas13a powered electrochemical microfluidic multiplexed biosensor (CRISPR-Biosensors X) for on-site miRNA detection will be introduced. Without any target amplification, CRISPR-Biosensor X offers a low-cost, easily scalable, and multiplexed approach for nucleic acid diagnostics.

Learning Objectives:

1. Review the fundamentals and different modes of CRISPR-enabled testing of nucleic acids.

2. Identify an electrochemical microfluidic approach for the simultaneous quantification of multiple miRNAs.

3. Differentiate future aspects of CRISPR/Cas technology with respect to biosensors.

4. Apply this knowledge to create new CRISPR-powered biosensing devices in future.

To find out more visit here CRISPR genome editing with CRISPR-Cas9 technology | IDT (idtdna.com)