Listen to an overview of electrophoresis uses and applications, from gel extraction to analysis.

Webinar content:

• Background on electrophoresis
• Preparative (gel extraction) applications
  • Purification of PCR products
  • Purification of digested DNA
  • Acrylamide gels for single-stranded DNA and RNA
  • Library preparations for next-generation sequencing
• Analytical applications
  • Basic agarose gel electrophoresis
  • Quantitative gel electrophoresis
  • High-throughput DNA electrophoresis
  • Gel shift assays
  • Determining DNA topology
  • Denaturing acrylamide gels
  • Others

With the fast progress and deeper understanding of CAR-T cell therapy, finding a better solution other than lentivirus transduction is more requested, to overcome the functional limitation on random insertion, uncontrolled copy number of CAR insertion and to overcome the capacity limitation of GMP lentivirus manufacture.

In this webinar, we will introduce non-viral solutions for cell therapy development: Starting from how to set up the non-viral working flow, the webinar will present how to use CRISPR-HDR protocol for precise target knock-out and gene knock-in in one step. Additionally, we will explore strategies to further improve the editing efficiency that has been proved on the primary T cell, NK cell, and iPSC. We will also provide solutions for large fragment knock-in via transposon systems. Finally, we will present a few early clinical data about CAR-T therapies which are manufactured by non-viral gene editing methods to illustrate its feasibility for clinical application.

Learning Obejctives: 

1. Discuss the advantages of non-viral cell therapy engineering process.

2. Summarize how to do CRISPR and transposon gene editing on immune primary cells via electroporation.

3. Summarize how to further improve the editing efficiency meanwhile maintain high cell viability.

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.

Coronavirus Disease 2019 (COVID-19) has driven the use of nucleic acid testing technology in clinical testing. There is a growing need for point-of-care testing (POCT) devices that can integrate sample pretreatment and nucleic acid detection in a rapid, cost-effective, and non-labor-intensive manner. The most widely used polymerase chain reaction (PCR)-based nucleic acid assays suffer from complexity, high personnel and laboratory requirements, and contamination. Highly miniaturized microfluidic chips provide the necessary tools to integrate complex nucleic acid detection processes. Various microfluidic chips coupled with clustered regularly interspaced short palindromic repeat (CRISPR) have been developed for the rapid detection of nucleic acids. These tools offer a range of advantages including high sensitivity, high automation, high specificity, and a high level of integration performance, and we have developed a series of nucleic acid detection systems based on CRISPR and microfluidic technologies:

(1) High sensitivity. An integrated assay for nucleic acid enrichment and single-molecule digital detection based on CRISPR/Cas13a and microwell arrays. The design utilizes magnetic beads to capture and enrich targets from a large number of samples, and then target-induced CRISPR/Cas13a cleavage reactions are dispersed and confined to 1 million femtoliter-sized microwells to enhance local signal intensity for single-molecule detection. The limit of this assay for amplification-free detection of SARS-CoV-2 is 2 aM. This "sample-in-answer-out" amplification-free single RNA detection technology improves sensitivity and specificity while reducing detection time.

(2) High automation, rapid detection. The dual CRISPR/Cas12a-assisted RT- recombinase-assisted amplification (RAA) assay and "sample-to-answer" centrifugal microfluidic platform automatically detects 1 copy/μL of SARS-CoV-2 in less than 30 minutes. The chip separates the amplification (RAA) from the detection (CRISPR) to maximize sensitivity and time consumption. The chip separates amplification (RAA) from detection (CRISPR) to maximize sensitivity and reduce time consumption by up to 3-fold. This automated centrifugal microfluidic technology achieved 100% accuracy on 34 clinical SARS-CoV-2 samples compared to the gold standard RT-PCR technique.

(3）High specificity. A universal and high-fidelity genotyping method based on a microfluidic point-of-care device for CRISPR systems. The versatility of the CRISPR / Cas12a-based SNP assay was improved by systematically inserting protospacer adjacent motif (PAM) sequences, and the sensitivity and specificity were improved by removing complementary ssDNA and introducing other nucleotide mismatches.

(4) High integration performance. A centrifugal microfluidic platform that allows for the separation of serum as well as a range of functions such as adsorption, washing, elution, and detection of DNA. The platform combines a variety of signal enhancement systems, including recombinase polymerase amplification (RPA), T7 transcription, and CRISPR, to generate ultra-bright signals, and hepatitis B virus (HBV) has been successfully detected and genotyped from whole blood on an automated centrifugal microfluidic platform. The automated centrifugal microfluidic platform with integrated preprocessing and molecular diagnostics play an important role in clinical testing.

I will also briefly discuss our effort in integrating CRISPR-based therapies with nanomaterials at the end of the talk.

Learning Objectives:

1. Summarize CRISPR/Cas12a-Assisted RT-RAA for Ultrasensitive SARS-CoV-2 Detection.

2. Explain RPA-T7-Cas13a for pathogen diagnosis in 30 min. 

3. Review CRISPR/Cas-assisted assay for single molecule detection of RNA with out amplification on chip.

CRISPR-based diagnostics offer rapid and highly sensitive detection of disease biomarkers. Their ease of use and compatibility with lateral-flow readouts make them suitable for point-of-care applications. In this presentation, we introduce technologies that facilitate the read-out of CRISPR/Cas reactions using nanozymes. Specifically, we present CrisprZyme, which utilizes catalytic metallic nanoparticles to enable Cas13-mediated collateral cleavage of reporter molecules. We also highlight their applications in the detection of disease, including biomarkers relevant to transplantation medicine, heart disease, and cancer.

Learning Objectives: 

1. Discuss the advantages and disadvantages of point-of-care diagnostics that are presently employed in clinical routines.

2. Describe the essential features of CRISPR-based diagnostics.

3. Generate innovative ideas for the application of CRISPR-based diagnostics in disease detection.

In this talk, I will introduce two new tools that we developed based on CRISPR-enabled gene regulation in bacteria, and one method based on P1 phagemid-enabled delivery of Cas9 nuclease. First, I will introduce a eukaryote-like bacterial CRISPRa system based on σ⁵⁴-dependent promoters, which supports long distance, and hence multi-input regulation with high dynamic ranges. Our CRISPRa device can activate σ⁵⁴-dependent promoters with biotechnology relevance in non-model bacteria, and act as a reusable scanning platform for readily optimizing metabolic pathways. Second, I will discuss the programmability of crRNA-tracrRNA hybridization which provides new sources of crRNAs and new biosensing applications for type II CRISPR systems. We show that how these re-engineered gRNA pairings can be implemented as RNA sensors, capable of monitoring the transcriptional activity of various environment-responsive genomic genes, or detecting SARS-CoV-2 RNA in vitro. Lastly, I will describe the use of a broad-host-range P1-derived phagemid to deliver the CRISPR-Cas9 chromosomal-targeting system into Escherichia coli and the dysentery-causing Shigella flexneri to achieve DNA sequence-specific killing of targeted bacterial cells. All these results demonstrate that CRISPR-enabled gene regulation is a powerful and versatile synthetic biology tool for diverse research and biotechnology applications.

Learning Objectives:

1. Design principles and biotech applications of a eukaryote-like CRISPR-enabled gene activation tool in bacteria.

2. Summarize principles and RNA sensing applications of reprogramming tracrRNAs in type II CRISPR system.

3. Design principles of engineering P1 phagemid to deliver CRISPR-Cas9 antimicrobials against Shigella flexneri, a dysentery-causing gut pathogen.

Type V and VI CRISPR/Cas systems have emerged as promising cutting-edge diagnostic tools for nucleic acid detection. Nevertheless, the challenge of low target concentrations necessitates pre-amplification, which constrains their practical use in point-of-care (POC) settings. Our lab has explored multiple strategies to create a platform for detecting nucleic acids that is as quick and simple as rapid antigen tests but has an accuracy of a qPCR test.

Firstly, in pursuit of a unified testing method that combines RT-LAMP and CRISPR at a single operating temperature, we delved into various thermophilic Cas12b orthologs. This exploration led to the discovery of BrCas12b, which possessed exceptional thermostability and enabled the development of a one-pot assay known as SPADE. SPADE successfully identified 5 major SARS-CoV-2 variants at temperatures between 60-62°C. Secondly, employing a combination of structural design and machine learning techniques, we engineered BrCas12b to further enhance its thermophilicity, resulting in the creation of a robust one-pot assay named SPLENDID. The SPLENDID effectively operates within the desirable temperature range of 65-67°C and demonstrates remarkable accuracy in detecting HCV genotype 1 and SARS-CoV-2 RNA clinically.

Thirdly, our research unveiled various CRISPR/Cas12a orthologs capable of partially recognizing native RNAs. This discovery paved the way for the development of SAHARA, a split activator system designed for target RNA detection using CRISPR/Cas12a without the need for reverse transcription. SAHARA represents an amplification-free and reverse-transcription-free one-pot detection technology for the direct analysis of RNAs. Furthermore, our laboratory has been actively investigating methods to develop PAM-less approaches for detecting nucleic acids and single-point mismatches without constraints. These groundbreaking advancements hold the potential to revolutionize the next generation of diagnostic tools, effectively addressing the pressing need for rapid, accurate, and accessible point-of-care tests for infectious diseases.

Learning Objectives: 

1. Recognize the need for a simple and accurate point-of-care test for diagnosing infectious diseases, genetic disorders, and beyond.

2. Identify the limitations of existing platform detection technologies in terms of simplicity, speed, sensitivity, specificity, and stability.

3. Describe how CRISPR/Cas systems can be engineered to develop a simple and robust next-generation point-of-care test for any genetic target.

In the past decade, gene editing has been increasingly emphasized as an essential tool for therapy development. This has led to significant scientific advances in understanding disease mechanisms and developing novel, effective, and safe therapeutic interventions. GenScript offers a wide selection of services and products, enabling CRISPR gene editing for Therapy Development. In this webinar, we will review the options for sgRNA synthesis, HDR knock-in templates, and delivery methodologies and discuss the advantages and disadvantages of each one. And we will also provide an introduction to the GenScript's GMP grade CRISPR reagents capacity. Join us to learn how GenScript’s comprehensive solutions for CRISPR gene editing will enhance your research and therapy development workflows.

Programmable genome integration of large, diverse DNA cargo without DNA repair of exposed DNA double-strand breaks remains an unsolved challenge in genome editing. We present programmable addition via site-specific targeting elements (PASTE), which uses a CRISPR–Cas9 nickase fused to both a reverse transcriptase and serine integrase for targeted genomic recruitment and integration of desired payloads. We discuss how systems such as PASTE can be generated, engineered for higher efficiency, and applied to practical therapeutic applications, especially treatment of monogenic disorders.

Learning Objectives:

1. Understand the current capabilities of DNA-editing modalities, including CRISPR-cas9, Prime editing, and base editors.

2. Discuss the strategies for tool development as it relates to PASTE and similar large genome insertion tools.

3. Develop knowledge on potential applications of large-genomic insertions and the potential drawbacks or advantages when compared to the current state of the art.

Rare diseases pose unique challenges in the medical field, often affecting a small but vulnerable population with limited treatment options. The Rare Disease Challenge (RaDiChal) is a groundbreaking initiative dedicated to conquering these medical mysteries through the power of CRISPR gene editing technology. This presentation abstract delves into the core of RaDiChal's mission, which is to leverage CRISPR's precision and versatility to develop effective therapies for rare diseases. With the ability to target and modify specific genes, CRISPR offers a ray of hope to individuals and families impacted by these often devastating conditions. Our presentation will explore the following key aspects:

1. Understanding Rare Diseases: We will begin by shedding light on the nature of rare diseases, their rarity, and the immense challenges they pose to diagnosis and treatment.

2. The Promise of CRISPR: We will discuss the revolutionary CRISPR technology and how it has transformed genetic research and therapy. Attendees will gain a clear understanding of CRISPR's potential to correct the genetic mutations responsible for rare diseases.

3. RaDiChal's Vision: Participants will learn about RaDiChal's vision to unite researchers, clinicians, and biotech experts in a collaborative effort to tackle rare diseases systematically.

4. Patient-Centered Approach: We will emphasize RaDiChal's commitment to putting patients at the forefront of our efforts, ensuring that therapies developed through CRISPR are safe, effective, and accessible.

5. Success Stories: The presentation will showcase some remarkable success stories where CRISPR-based therapies have provided hope and life-changing results for individuals with rare diseases.

6. Challenges and Ethical Considerations: We will address the challenges and ethical considerations associated with CRISPR-based treatments, highlighting the importance of responsible research and application.

7. Call to Action: In closing, we will invite attendees to join us in our mission to drive innovation, awareness, and funding for rare disease research through RaDiChal.

RaDiChal's pursuit of harnessing CRISPR to cure rare diseases represents a beacon of hope for countless individuals and families affected by these conditions. This presentation aims to inspire, educate, and rally support for this crucial cause, paving the way for a future where rare diseases are no longer insurmountable obstacles but treatable conditions.

Learning Objectives:

1. Demonstrate Proficiency in CRISPR Techniques.

- Develop the ability to perform CRISPR gene editing techniques accurately and efficiently, including designing guide RNAs, transfecting cells, and analyzing results.

- Benefit: Enhanced laboratory skills, making the lab professional proficient in cutting-edge genetic engineering methods.

2. Evaluate Ethical Implications of CRISPR Research. 

- Analyze and critically assess the ethical considerations surrounding CRISPR-based therapies and research.

- Benefit: Increased awareness of ethical issues, enabling the lab professional to make informed decisions and contribute to responsible research practices.

3. Contribute to Rare Disease Research Projects. 

- Actively participate in or initiate research projects focused on rare diseases using CRISPR technology.

- Benefit: Direct involvement in impactful research, potentially leading to novel therapies and advancing the lab professional's career in the field of genetics and rare disease research.

Smartphone-based optical imaging and sensing devices are among the next-generation biosensors that have shown great potential to transform the field of point-of-care (POC) diagnostics. With the rapid improvement of hardware (e.g., lens, image sensor, and CPU), smartphone has become a transformative microscopy and sensing platform that can support various detection or biomedical measurement applications, especially for resource-limited settings. On the other side, smartphone diagnostics can maximize its potential for early disease detection by coupling with specific molecular assays, such as nucleic acid amplification tests and immunoassays. This talk will highlight our recent effort in creating advanced field-portable biosensor platforms based on smartphones and the integration of smartphone device with CRISPR assay to generate a new functional POC diagnostic platform. An unexpected trans-cleavage behavior of Cas12a against double-stranded DNA substrates is discovered, which greatly eases the design of CRISPR-Dx reporter molecules. Promising applications ranging from POC diagnostics of human diseases to rapid assessment of biomanufacturing products will also be illustrated.

Learning Objectives: 

1. Describe what is point-of-care (POC) diagnostics, and how CRISPR can be used for diagnostic applications.

2. Identify the advantages of CRISPR in POC diagnostics.

3. Identify the current challenge and limitations of CRISPR-based diagnostics.

Endonuclease Cas12a, as one key part of the CRISPR/Cas biotechnology, possessed a unique dual enzymatic function which has been widely used for various applications1. However, the limits trans-cleavage efficiency of a single Cas12a ribonucleoprotein (RNP) holds a major barrier for its further growth as a novel biotechnology toolbox2. Unlike any previous studies, we reported the manipulation of Cas12a activity with special circular DNA nanostructure to establish a novel Autocatalytic CRISPR/Cas12a Amplification Reaction (AutoCAR) system, which is able to break the standard reaction pattern of the Cas12a RNP. In our test, >1,000 Cas12a RNPs can be activated with the presence of a single targeted nucleic acid sequence. We carefully investigated the autocatalysis reaction activity of AutoCAR and also its potential mechanism beneath the changed linear to exponential reaction pattern. In addition, to demonstrate the potential value of AutoCAR in bioanalysis, its autocatalysis reaction can be transferred into a groundbreaking approach to existed CRISPR/Cas12a diagnostics, and realized 1 attomole sensitivity to nucleic acid detections at room temperature without the need for any additional amplifications. Furthermore, 1 attomole RNA detection has also been realized by using modified AutoCAR without the need for reverse transcription. These capabilities have been used to detect ~1 copy/μL DNA/RNA targets from H. pylori or SARS-CoV-2 genomes, respectively. The development of AutoCAR served as an exploration to manipulate the Cas enzyme activity patterns, and revealed the under covered potentials of CRISPR/Cas biotechnology for a boarder range of applications, such as biochemistry, bioanalysis and diagnostic applications.

Learning Objectives: 

1. Define the key factors affect the efficiency of Cas12a autocatalysis reaction.

2. Discuss the advantages and disadvantages of the Cas12a autocatalysis reaction developed here.

3. Evaluate the biosensing performance of AutoCAR for nucleic acid detection.

Rather than being famous in gene editing field only, by revealing the collateral cleavage activity of Cas12a, Cas13a, Cas14 effectors, and more, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated (Cas) (i.e., CRISPR/Cas) systems has recently received significant credits in the field of diagnostics with the capability of developing novel CRISPR/Cas medicated biosensors with superior sensitivity and specificity. Exciting development of CRISPR/Cas based biosensors has thrived quickly in recent 2-3 years, and a variety of different biosensing systems have now been developed successfully such as SHERLOCK, SHERLOCKv2, DETECTR, HOLMES, etc. Despite the features of high sensitivity and specificity, all the known CRISPR/Cas involved biosensing systems are specialized to detect nucleic acids. Recent studies have demonstrated that CRISPR/Cas systems are providing excellent biosensing capability for detection of non-nucleic acids, such as small molecules, proteins, exosomes, metal ions, et al. Although the most popular signal read-out in CRISPR/Cas biosensors is fluorescence, recently, various signal read-out modalities such as colorimetric, electrochemiluminescence, electrochemical and electrical have been applied in CRISPR/Cas biosensing systems. Furthermore, they can be multiplexed, enabling a single diagnostic test to identify the presence of multiple targets. Coupling of the CRISPR/Cas detection with lateral flow device-based signal readout, will allow for the highly sensitive and accurate in-field detection of pathogens and viruses.

Preamplification techniques such as rolling circle amplification (RCA), recombinase polymerase amplification (RPA), polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), etc. have significantly boosted the detection sensitivity of CRISPR/Cas. However, they inevitably overshadow Cas effectors and neglect the intrinsic detection capability of Cas effectors due to the exponential format, nonspecific background products that lead to false-positive results. Therefore, research focusing on preamplification based CRISPR/Cas systems is active. Additionally, application of CRISPR/Cas biosensing systems for multiplexed detection and in vivo biosensing is also the spotlight.   The CRISPR/Cas biosensing technology has massive potential in facilitating the development of modern analytical science, and will only be advanced as analytical chemists, engineers and molecular biologists continue to improve collaborative capability.

Learning Objectives:

1. Define the key factors affect the efficiency of Cas12a autocatalysis reaction.

2. Discuss the advantages and disadvantages of the Cas12a autocatalysis reaction developed here.

3. Evaluate the biosensing performance of AutoCAR for nucleic acid detection.

The accurate and timely detection of disease biomarkers at the point-of-care is essential to ensure effective treatment and prevention of diseases. CRISPR-based diagnostics (CRISPR-Dx) is revolutionizing molecular diagnosis by offering exceptional sensitivity, specificity, and simplicity. Research in our laboratory focuses on leveraging CRISPR-Dx and dynamic DNA assembly to create better analytical tools for diagnosing both human and crop diseases. In this talk, I will first introduce a plasmonic CRISPR Cas12a assay that combines the trans-cleavage activity of Cas12a and DNA functionalized gold nanoparticles for the visual, colorimetric detection of grapevine viral infections. I will then describe a molecular translator approach that expands CRISPR-Dx to protein-based biomarkers. Such CRISPR-based ultrasensitive protein assays enabled the detection of attomolar concentrations of SARS-CoV-2 antibodies in serum samples collected from vaccinated kidney transplant recipients who are shown to produce “undetectable” anti-SARS-CoV-2 using standard immunoassay. Finally, I will describe the development of a CRISPR-like assay using RNA-cleaving DNAzyme coupled with isothermal sequence and signal amplification, which demonstrated enhanced accessibility and multiplexity over CRISPR-DX.

Learning Objectives:

1. Discuss possible strategies to enable colorimetric readout for CRISPR diagnostics.

2. Evaluate design approaches that enable CRISPR diagnostics for protein-based biomarkers.

3. Discuss possible roles of CRISPR diagnostics in the face of emerging infectious diseases such as COVID-19.

Molecular diagnostics based on clustered regularly interspaced short palindromic repeats (CRISPR) enzyme systems have been the subject of intense research. CRISPR-associated (Cas) enzyme assays are easily reconfigurable to different nucleic acid target sequences, highly specific, and compatible with simple kits and microfluidic components. We are conducting studies of the basic CRISPR enzyme kinetics. We discovered that the great majority of all CRISPR enzyme kinetics studies show data that are inconsistent and which grossly violate basic rules of mass conservation and rate laws. Further, CRISPR assays which do not use pre-amplification often report limits of detection (LoD) which are difficult to reconcile with CRISPR kinetic rates. This widespread inconsistency makes it difficult to assess the potential of CRISPR as a diagnostics tool. Following up on this, we quantified the kinetics of a range of CRISPR-Cas systems and demonstrated that kinetics fundamentally assay LoD. We also performed a study of CRISPR specificity to small mutations, including single-nucleotide polymorphisms. We are currently developing assays that leverage CRISPR specificity for cancer detection.

Learning Objectives: 

1. Describe the two key reaction steps in CRISPR diagnostic assays.

2. Explain which kinetic rate parameters affect limits of detection?

3. Explain the key advantage of CRISPR diagnostic assays, aside from reconfigurability.