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A Definitive Guide to CRISPR for Research Scientists

The CRISPR Research Hub brought to you by Sigma-Aldrich® Advanced Genomics

Whether you need help designing your first CRISPR experiment, you're looking for troubleshooting tips, or you want to learn about how to apply CRISPR in your research, the CRISPR Research Hub has something for you.

CRISPR is best known as the gene-editing tool that allows you to easily and precisely edit DNA both in vitro and in vivo.

However, CRISPR technology has evolved beyond basic gene editing, with the development of tools and techniques that allow you to edit the epigenome or even activate or inhibit gene expression without altering the underlying DNA sequence of your target gene.

There's so much information available on CRISPR that it can feel overwhelming. No matter what you want to do with CRISPR, our carefully crafted resources allow you to find and digest the information you need.

Your Complete Guide to CRISPR
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    CRISPR Explained

    How Does CRISPR Gene-Editing Work?

    If you are new to performing CRISPR or if you want to refresh your background knowledge on how CRISPR gene editing works, this is the section for you. You can discover the history and learn the components of this gene-editing system, from protospacer-adjacent motifs (PAMs) to guide RNA (gRNA). If you are unsure if CRISPR is the right gene-editing system for your experiments, you can compare it with other gene-editing systems, including zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs).

    Image of two hands altering DNA to depict CRISPR genome editing

    A Brief History of CRISPR-Cas9 Genome-Editing Tools

    Learn how the CRISPR prokaryote immune response systems were first discovered and the development of the CRISPR-Cas9 gene-editing tool.

    Image of scientists editing DNA as in CRISPR technology

    CRISPR Technology Explained: Towards a CRISPR Genome!

    Grab an overview of CRISPR technology from its roots as a bacterial defense system to how it can be utilized in health and research.

    Weight scales represent weighing up the pros and cons of crispr cas9

    CRISPR-Cas9 Genome Editing: Weighing the Pros and Cons

    Discover the pros and cons of CRISPR as a genome-editing tool in your research.

    Advanced CRISPR

    Take CRISPR to the Next Level with Advanced Applications

    If you're looking to do something more than just basic gene editing, come and discover the amazing adaptations that have been made to CRISPR technology. With modified versions of Cas9, you can perform epigenome editing and activate or inhibit gene expression (without changing the DNA) with CRISPRa and CRISPRi.

    DNA being modified using a tool to represent modified CRISPR nuclease formats

    How to Understand CRISPR Formats and Their Applications

    Find out how modified variants of CRISPR nucleases provide gene editing with reduced off-target effects and can even control gene expression without altering the DNA sequence.

    Image of DNA transcriptio to show how CRISPR can modify gene expression using CRISPRa and CRISPRi

    Why You Should Consider Adding CRISPRa and CRISPRi to Your Toolbox

    Find out how CRISPR-mediated gene activation (CRISPRa) and repression (CRISPRi) works and why you should consider using them in addition to your CRISPR knockouts.

    A light switch depiciting how CRISPRa allows you to swtich gene expression on

    CRISPR-Based Activation (CRISPRa): A How-To Guide

    CRISPRa allows you to activate or overexpress genes in a more endogenous manner. Find out the steps to getting started.

    A zombie holding a spanner to depict the usefulness of dead Cas9 for epigenome editing

    Dead Useful: CRISPR-Cas9 Epigenome Editing

    Want to do some epigenome editing? Discover the usefulness of catalytically inactive (dead) Cas9.

    CRISPR Applications

    Discover Various Applications of CRISPR 

    Do you have a specific application of CRISPR in mind, and are looking for help and guidance? Discover the various ways this technology is used in research and get inside tips on how to use it in specific applications, such as performing CRISPR experiments in hard-to-transfect cells like T cells We also uncover what you need to know to scale up to CRISPR screens.

    Image of amultiplex theatre to represent multiplex CRISPR experiments

    Multiplex CRISPR Gene Editing: What You Need to Know

    If you need a multi-gene knockout or large-scale genomic modification, or want reduced off-target effects, then multiplex CRISPR is for you!

    Image of a microplate use to show how CRISPR can be scaled to undertaking CRISPR screening.

    Level Up Your Drug Screening With CRISPR

    Discover how CRISPR can be scaled up for drug screening applications.

    Image of T cells and cancer cells highlighting how CRISPR can be used to enhance T cells anti cancer abilities.

    A CRISPR Approach to Immuno-Oncology

    T cells can be tricky to engineer with CRISPR. Find out the key considerations when editing these cells and how you can overcome any associated challenges.

    Image of a fruit fly to represent CRISPR-Cas9 editing in Drosophila

    A Crash Course in CRISPR-Cas9 Editing in Drosophila

    Get tips and tricks for performing CRISPR-Cas9 editing in Drosophila.

    Experimental Design

    Tips and Tricks for Setting up Your CRISPR Experiments

    Are you ready to get started with CRISPR in your lab? This section takes you through all aspects of CRISPR experimental design and setup, from designing gRNAs to choosing the right method for delivering CRISPR reagents. We also cover the various delivery formats, including DNA, plasmids, and ribonucleoprotein (RNP) complexes, and advise you on how to choose the right delivery format for your experiment.

    Image of CRISPR-Cas complex targeting DNA with gRNA

    CRISPR Gene Editing: Considerations and Getting Started

    Discover how to get started with CRISPR gene editing in your experiments with our key considerations.

    CRISPR Research Hub

    CRISPR Nucleases: The Ultimate Guide To Selection

    Confused about CRISPR nucleases? Read this guide to discover the various CRISPR nucleases available and what they are best suited for.

    Image of interior designing to represent how to design a CRISPR experiment

    How to Design a CRISPR Cas9 Experiment and Start Genome Editing

    Designing a CRISPR experiment can be daunting. We’ve got tips and pointers to help you get off on the right foot.

    image of microinjection as one way to deliver CRISPR

    Four Ways to Get CRISPR Reagents Into Your Cells

    Read up on the various methods for delivering CRISPR reagents and how to choose between them.

    A road showing the path to get started with CRISPR

    CRISPR Genome Editing: What You Need to Know to Get Started

    Get some ideas on what CRISPR can do for you and what using it involves.

    Troubleshooting

    Troubleshooting Help for When CRISPR Goes Wrong

    If things keep going wrong in your experiments, or you've got results you can't explain don't panic. We've got guides to walk you through how to troubleshoot your CRISPR experiment, including what controls you need to use to ensure you can effectively troubleshoot, and how to interpret confusing results.

    Image of sequencing data to depict validating CRISPR

    How to Validate a CRISPR Experiment  

    Discover how to validate your CRISPR gene editing, from the successful delivery of CRISPR reagents to the confirmation of desired genetic and phenotype changes.

    Image of the word Valid to highlight importance of determining CRISPR success

    How to Confirm Your CRISPR-cas9 Genome Editing Was Successful

    Level-up your troubleshooting ability by determining the success of failure of each stage of your CRISPR experiment.

    Latest CRISPR Articles

    Keep Up-to-date with the Latest CRISPR Articles

    Image of amultiplex theatre to represent multiplex CRISPR experiments

    Multiplex CRISPR Gene Editing: What You Need to Know

    If you need a multi-gene knockout or large-scale genomic modification, or want reduced off-target effects, then multiplex CRISPR is for you!

    Weight scales represent weighing up the pros and cons of crispr cas9

    CRISPR-Cas9 Genome Editing: Weighing the Pros and Cons

    Discover the pros and cons of CRISPR as a genome-editing tool in your research.

    Image of two hands altering DNA to depict CRISPR genome editing

    A Brief History of CRISPR-Cas9 Genome-Editing Tools

    Learn how the CRISPR prokaryote immune response systems were first discovered and the development of the CRISPR-Cas9 gene-editing tool.

    A detective dressed like a detective to symbolize the DETECTR and SHERLOCK CRISPR methods for Viral diagnosis.

    How CRISPR Can Be Used to Detect Emerging Viral Pathogens

    Discover two CRISPR-based viral diagnostic strategies, DETECTR and SHERLOCK.

    Image of T cells and cancer cells highlighting how CRISPR can be used to enhance T cells anti cancer abilities.

    A CRISPR Approach to Immuno-Oncology

    T cells can be tricky to engineer with CRISPR. Find out the key considerations when editing these cells and how you can overcome any associated challenges.

    A zombie holding a spanner to depict the usefulness of dead Cas9 for epigenome editing

    Dead Useful: CRISPR-Cas9 Epigenome Editing

    Want to do some epigenome editing? Discover the usefulness of catalytically inactive (dead) Cas9.

    A light switch depiciting how CRISPRa allows you to swtich gene expression on

    CRISPR-Based Activation (CRISPRa): A How-To Guide

    CRISPRa allows you to activate or overexpress genes in a more endogenous manner. Find out the steps to getting started.

    Image of the word Valid to highlight importance of determining CRISPR success

    How to Confirm Your CRISPR-cas9 Genome Editing Was Successful

    Level-up your troubleshooting ability by determining the success of failure of each stage of your CRISPR experiment.

    CRISPR Terminology

    Glossary of CRISPR Terminology

    Is there a CRISPR term you're not quite sure about? We've compiled a glossary of CRISPR terminology to help you out. Just click on the arrow next to the term to see the definition.

    CRISPR-associated protein. This is the nuclease component of the CRISPR complex, the most common of these proteins is Cas9.

    CRISPR associated protein 9. One of several identified CRISPR nucleases. The Cas9 nuclease is the most widely used CRISPR nuclease. Different variants of Cas9 have been identified from different species. The PAM sequences that are recognized differ between the Cas9 variants.

    CRISPR from Prevotella and Francisella 1. A CRISPR endonuclease that is now more commonly called Cas12a. Cas12a differs from Cas9 in several ways: it is smaller and simpler, it creates staggered rather than blunt cuts, it uses a different PAM, and cleaves distal to the recognition site. These differences make it a useful alternative to Cas9.

    Clustered, Regularly Interspaced, Short Palindromic Repeats. The name given to the sequences that CRISPR technology was first identified from. Is now used to refer to the gene-editing system that uses CRISPR nucleases.

    CRISPR activation. A technology using catalytically inactive Cas9 (dCas9) to target transcriptional activators to specific DNA/genes to activate target gene expression.

    CRISPR inhibition. A technology using catalytically inactive Cas9 (dCas9) to target transcriptional repressors to specific DNA/genes to inhibit target gene expression.

    CRISPR RNA. A component of the gRNA containing the variable targeting sequence responsible for the CRISPR complex's specificity for the target DNA.

    Catalytically dead Cas9. This is a catalytically inactive form of Cas9, created by point mutations in the two endonuclease domains (RuvC and HNH). These point mutations are D10A and H840A and render the nuclease unable to cleave DNA. With the help of gRNA, the nuclease can still be targeted to specific DNA and is often coupled with transcriptional or epigenetic regulators to modify gene expression.

    Donor DNA is required when using homology-directed repair (HDR) in CRISPR. HDR allows precise gene editing, such as specific insertions and deletions or base substitutions. 

    Double-strand break. This is where both strands of a DNA molecule are cut, leaving a complete break. 

    Gain of function. This refers to an additional function being conferred or a current function being enhanced by mutation/gene editing. This can refer to activating or increasing the expression of a gene/non-coding RNA (ncRNA).

    Guide RNA. This is the RNA that targets the nuclease to a specific DNA sequence. gRNA is composed of a scaffold RNA (tracrRNA) and the variable targeting RNA (crRNA), supplied as either as two individual RNAs or as a single guide RNA in which the two components are fused.

    Homology-directed repair is one of the two cellular DNA repair pathways. HDR can be used in CRISPR gene editing where very precise gene editing is required, including for specific insertions and deletions, or base substitutions. Donor DNA is required to act as a template in the repair.

    Loss of function. This describes a mutation or gene edit that results in the loss of the native function of a protein or ncRNA. In CRISPR gene-editing this can refer to a loss/reduction of expression of proteins/ncRNA or the expression of an inactive form (e.g. truncated proteins missing an active domain).

    CRISPR nickases. These are modified forms of Cas9 with a point mutation in one of the two endonuclease domains, which results in them 'nicking' one strand of the DNA rather than making a complete double-strand break. Two nickases are paired together to create the desired double-strand break. Paired nickases effectively eliminate off-target effects, as different gRNAs are used with each nickase.

    Non-homologous end-joining is one of the two cellular DNA repair pathways. NHEJ is the more commonly used pathway in CRISPR gene editing because it is more efficient than HDR. However, NHEJ is also more prone to error than HDR.

    Protospacer-adjacent motif. A sequence of ~3–8 nucleotides downstream of the target site that is required for the nuclease to successful cleave target DNA. Different nucleases recognize different PAM sequences.

    Ribonucleoprotein complex. A complex consisting of a CRISPR nuclease protein and gRNA that can be delivered into cells.

    Single-guide RNA. A common format of gRNA in which the tracrRNA and the crRNA are fused into a single RNA molecule.

    Trans-activating CRISPR RNA. A component of the gRNA that acts as a scaffold between the crRNA and the nuclease.

    Got a Question or a Suggestion?

    Didn't find what you were looking for? Or perhaps you have some CRISPR tips and tricks that we haven't covered here? Get in touch and let us know so we can continue to improve the information we share!

    Contact us here.

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