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GEN Protocols

Genome Editing

Genome Editing

Protocol

CRISPRmod dCas9-VPR or dCas9- SALL1-SDS3 mRNA and synthetic guide RNA transfection protocol

The following is a protocol for transfecting CRISPRmod dCas9-VPR or dCas9-SALL1-SDS3 mRNA with synthetic guide RNA into cultured mammalian cells using DharmaFECT™ Duo transfection reagent (Cat #T-2010-xx). The protocol is written for transfection into 96-well tissue culture plates.... More


Protocol

Dharmacon Edit-R Lentiviral sgRNA glycerol stocks

The Dharmacon™ Edit-R™ Lentiviral sgRNA vector is part of the Edit-R CRISPR-Cas9 system for genome engineering. The purpose is to provide the researcher with the most effective tools to deliver a gene-specific sgRNA and, together with Cas9 expression, allow gene editing in cells. In addition to the ... More


Protocol

Enrichment of transfected cells with Dharmacon Edit-R Fluorescent dCas9-VPR mRNA

Edit-R Fluorescent dCas9-VPR mRNA enables both transfection optimization and enrichment for gene modulation experiments. For optimal enrichment of edited cells, sorting the cells for high EGFP expression levels (selecting the top 10% fluorescence) in addition to negative and dim fluorescence for com... More


Protocol

Dharmacon Edit-R crRNA Libraries

Libraries of predesigned synthetic crRNAs provide an opportunity to apply the CRISPR-Cas9 system for functional gene knockout analysis, or dCas9-VPR system for transcriptional gene activation, in an arrayed format. Dharmacon crRNA libraries for functional gene knockout consist of Edit-R synthetic cr... More


Protocol

Massively parallel genome engineering followed by pooled growth selections for rapid target discovery in microbes

The ability to engineer the entire genome of microorganisms at a massively parallel scale using the Onyx platform will reshape the manner in which researchers perform pooled cultivation experiments (such as ALE) and generate data sets. Here, Onyx technology was used to rapidly generate genomewide k... More


Protocol

Multiplexed, trackable CRISPR-based genome engineering for optimization of heterologous protein expression and activity in E. coli

This work demonstrates the use of the Onyx platform for interrogation and optimization of heterologous proteins in E. coli. Compared to traditional methods for protein engineering, such as random mutagenesis or targeted site-directed mutagenesis approaches, the Onyx technology dramatically increases... More


Protocol

Lentiviral titering by crystal violet staining

This protocol provides an example for titering lentiviral particles using crystal violet staining in a 6-well format to obtain transducing units per mL (TU/mL). Once lentiviral particles are packaged and collected, small aliquots should be frozen at –80°C. One aliquot should be thawed on ice and use... More


Protocol

Detergent-Free Nuclei Isolation

A novel spin column-based approach to isolate intact single nuclei from tissues (fresh or frozen) and cells without using a detergent. The protocol is fast, easy, and reliable, producing high-quality single nuclei with minimized clumping and possible leakage of the nuclear envelope. The protocol is ... More


Protocol

Gene Therapy for Hemoglobinopathies

Gene therapy for β-thalassemia and sickle-cell disease is based on transplantation of genetically corrected, autologous hematopoietic stem cells. Preclinical and clinical studies have shown the safety and efficacy of this therapeutic approach, currently based on lentiviral vectors to transfer a β-gl... More


Protocol

Advances in Gene Therapy for Diseases of the Eye

Over the last few years, huge progress has been made with regard to the understanding of molecular mechanisms underlying the pathogenesis of neurodegenerative diseases of the eye. Such knowledge has led to the development of gene therapy approaches to treat these devastating disorders. Challenges re... More


Protocol

Transfecting Edit-R™ Cas9 mRNA with Synthetic Guide RNA in Cultured Mammalian Cells

This is an abbreviated protocol for transfecting Edit-R™ Cas9 mRNA (Cat #CAS11195, #CAS11859, or #CAS11860) with synthetic guide RNA into cultured mammalian cells using DharmaFECT™ Duo transfection reagent (Cat #T-2010-xx). Synthetic guide RNA can be either synthetic single guide RNA, or synthetic c... More


Protocol

AAV Aggregate Quantitation and Identification with the Aura System

Adeno-associated viruses (AAVs) have made the promise of gene therapy a reality by enabling safe, targeted delivery of curative genetic payloads. The formation of aggregated viral vector is an important critical quality attribute to measure because they lead to adverse immunogenic events and are an ... More


Protocol

IsoCode Single-Cell Adaptive Immune: TCR-T Protocol

A. Overview of ProtocolDay 1: Cryopreserved TCR-T cells are thawed and cultured overnight in the presence of IL-2. Cryopreserved target cell lines are thawed, cultured and passaged several days prior to beginning the experiment.Day 2: Enrichment, Staining, and Antigen Stimulation of CD4+ and/or CD8+... More


Protocol

CRISPRmod dCas9-VPR or dCas9- SALL1-SDS3 mRNA and synthetic guide RNA transfection protocol

The following is a protocol for transfecting CRISPRmod dCas9-VPR or dCas9-SALL1-SDS3 mRNA with synthetic guide RNA into cultured mammalian cells using DharmaFECT™ Duo transfection reagent (Cat #T-2010-xx). The protocol is written for transfection into 96-well tissue culture plates.... More


Protocol

Dharmacon Edit-R Lentiviral sgRNA glycerol stocks

The Dharmacon™ Edit-R™ Lentiviral sgRNA vector is part of the Edit-R CRISPR-Cas9 system for genome engineering. The purpose is to provide the researcher with the most effective tools to deliver a gene-specific sgRNA and, together with Cas9 expression, allow gene editing in cells. In addition to the ... More


Protocol

Enrichment of transfected cells with Dharmacon Edit-R Fluorescent dCas9-VPR mRNA

Edit-R Fluorescent dCas9-VPR mRNA enables both transfection optimization and enrichment for gene modulation experiments. For optimal enrichment of edited cells, sorting the cells for high EGFP expression levels (selecting the top 10% fluorescence) in addition to negative and dim fluorescence for com... More


Protocol

Dharmacon Edit-R crRNA Libraries

Libraries of predesigned synthetic crRNAs provide an opportunity to apply the CRISPR-Cas9 system for functional gene knockout analysis, or dCas9-VPR system for transcriptional gene activation, in an arrayed format. Dharmacon crRNA libraries for functional gene knockout consist of Edit-R synthetic cr... More


Protocol

Massively parallel genome engineering followed by pooled growth selections for rapid target discovery in microbes

The ability to engineer the entire genome of microorganisms at a massively parallel scale using the Onyx platform will reshape the manner in which researchers perform pooled cultivation experiments (such as ALE) and generate data sets. Here, Onyx technology was used to rapidly generate genomewide k... More


Protocol

Multiplexed, trackable CRISPR-based genome engineering for optimization of heterologous protein expression and activity in E. coli

This work demonstrates the use of the Onyx platform for interrogation and optimization of heterologous proteins in E. coli. Compared to traditional methods for protein engineering, such as random mutagenesis or targeted site-directed mutagenesis approaches, the Onyx technology dramatically increases... More


Protocol

Lentiviral titering by crystal violet staining

This protocol provides an example for titering lentiviral particles using crystal violet staining in a 6-well format to obtain transducing units per mL (TU/mL). Once lentiviral particles are packaged and collected, small aliquots should be frozen at –80°C. One aliquot should be thawed on ice and use... More


Application

Knock-out cell lines and their use in signaling pathways

Knock-out (KO) cell lines are powerful tools to study disease mechanisms, such as the effect of gene disruption on downstream pathways. In this application note, we highlight the effectiveness of Abcam KO cell lines that can be used to investigate signaling pathways, in this case, the TRAIL pathway.... More


Application

Rebuilding evolutionary success: Using Inscripta’s Onyx Digital Genome Engineering Platform to ease and enhance Adaptive Laboratory Evolution experiments

Adaptive Laboratory Evolution (ALE) is a powerful approach to leverage biology’s evolutionary capacity to develop strains that thrive in challenging growth environments, with applications to biotechnology and bioproduction and for basic biochemical discovery. ALE experiments have been enabled by nex... More


Application

Benchling for Gene Therapy & Gene Editing R&D

Modern gene therapy requires investigation of a wide variety of genes of interest, engineering of delivery vectors, and optimization of vector production processes. This necessitates a strong digital foundation to comprehensively characterize gene therapy candidates to bring the best products into t... More


Application

Benchling & uniQure: Bringing a gene therapy pioneer from paper to the cloud

uniQure is rapidly advancing a pipeline of AAV-based gene therapies to treat patients with hemophilia, Huntington’s disease, and other severe genetic diseases, but they needed a digital solution that could track their gene therapy workflows end-to-end and enable international collaboration. Learn ho... More


Application

Benchling & Beam Therapeutics: A Two-Pronged Approach to Automation for Gene Therapy Research

Beam Therapeutics is developing a new class of genetic medicines using novel base editing technology that enables them to edit precise sets of base pairs without introducing a double-stranded break in the DNA. Beam needs to evaluate thousands of biological samples each week, but their digital infras... More


Application

Benchling for Industrial Biotech R&D

Industrial biotechnology is transforming traditional manufacturing technologies by providing more eco-friendly approaches like bioprocessing and biomanufacturing. However, there are several hurdles for industrial biotech companies seeking to develop next-gen strains, novel processes, and new applica... More


Application

Benchling for CRISPR Screening

CRISPR screening is a revolutionary methodology to identify genes of interest underlying a specific phenotype. CRISPR can be scaled up for screening applications because of the versatility of CRISPR/Cas9 in disrupting target DNA and the ease of gRNA/plasmid generation. However, large-scale use of th... More


Primer

Selecting a Functional Oligonucleotide from a Random Mix

SELEX (Systematic Evolution of Ligands by EXponential enrichment) was first described and used in 1990 to isolate a functional oligonucleotide from a random pool of differently-shaped DNA or RNA molecules.1-2SELEX-derived oligonucleotides (RNA or single-stranded DNA) known as aptamers, bind with hig... More


Primer

Solvent Precipitation Method Increases Protein Recovery for Proteomics

Study designs in proteomics aim for inclusivity—to characterize every protein in a sample. However, extracting proteins from complex biological samples involves the use of harsh buffers containing detergents and salts that are incompatible with the proteomics workflow. More proteins are lost in atte... More


Primer

Orthogonal Validation in Genome Editing Discovery Work

Simply put, orthogonal validation uses additional methods that provide very different selectivity to the primary method to confirm or refute a finding. All methods are independent approaches that can answer the same question and are used to evaluate and verify the findings. According toClarence Mill... More


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Audio

GEN Protocols Expert Exchanges: Next Generation Techniques to Develop Genetic Medicines

The past decade has seen a shift in drug discovery to include targeting the root cause of genetic diseases: loss-of-function, gain-of-function, and change-of-function mutations in the genome. Although first generation genetic medicines like gene therapies heralded a new approach to treating previous... More


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