crispr cas9 presentation

CRISPR-Cas9 Mechanism & Applications

• DNA & RNA
• Biotechnology
• Gene Expression & Regulation
• Genetic Disease

Resource Type

• Click & Learn

Description

This interactive module explores how CRISPR-Cas9 technology works and the many ways in which scientists are using it in their research.

Since it was first described in 2012, CRISPR-Cas9 (often shortened to “CRISPR”) has generated much interest and excitement. This Click & Learn allows students to explore and learn about a biotechnology tool that is at the forefront of scientific research and hear directly from leading scientists about how they use CRISPR. The Click & Learn comprises a self-paced interactive animation and a series of short videos of various scientists who are using CRISPR-Cas9 technologies for basic research, medical, and agricultural applications.

The “Web Assets” ZIP file contains the full animation and a set of related images and GIFs for use on social media, in the classroom, and for presentations to the public. Please credit HHMI BioInteractive if you use them.

Estimated Time

base pairing, cleavage, complementary sequence, DNA repair, enzyme, guide RNA, homology-directed repair (HDR), laboratory technique, nonhomologous end joining (NHEJ), nuclease

Terms of Use

Please see the Terms of Use for information on how this resource can be used.

Accessibility Level (WCAG compliance)

Version history, curriculum connections, ngss (2013).

HS-LS1.A, HS-LS3.A

AP Biology (2019)

IST-1.K, IST-1.L, IST-1.P, IST-2.A, IST-2.E, IST-4.B

IB Biology (2016)

2.6, 2.7, 3.5, 7.2, B.4, B.5

VIsion and Change (2009)

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Gene editing

• Gene editing product finder
• sgRNA design tools
• Tools for successful CRISPR/Cas9 genome editing
• Gene editing posters
• Customer data for Guide-it products
• How to design sgRNA sequences

Introduction to the CRISPR/Cas9 system

• Gene editing of CD3+ T cells and CD34+ HSCs
• Mutation detection kit comparison
• Screening for effective guide RNAs
• Monoallelic versus biallelic mutants
• Indel identification kit for mutation characterization

Choosing an HDR template format

• Homology-directed repair FAQs
• Mouse CRISPR knockin protocol
• Site-specific gene knockins using long ssDNA
• Efficient CRISPR/Cas9-mediated knockins in iPS cells
• Oligo design tool user guide (insertions)
• CRISPR library screening

CRISPR library screening webinar

• Phenotypic screen using sgRNA library system
• SNP detection with knockin screening kit
• Oligo design tool user guide (SNPs)
• Sign up: SNP engineering webinar

Guide-it SNP Screening Kit FAQs

• Electroporation-grade Cas9 for editing in diverse cell types
• CRISPR/Cas9 gene editing with AAV

CRISPR/Cas9 gesicles overview

• Cas9 Gesicles—reduced off-target effects
• sgRNA-Cas9 delivery to many cell types
• Tet-inducible Cas9 for gene editing
• Control your Cre recombinase experiments
• Fast Cre delivery with gesicle technology

A powerful method for engineering your gene of interest

Although recently developed programmable editing tools, such as zinc finger nucleases and transcription activator-like effector nucleases, have significantly improved the capacity for precise genome modification, these techniques have limitations. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 technology represents a significant improvement over these other next-generation genome editing tools, reaching a new level of targeting, efficiency, and ease of use. The CRISPR/Cas9 system allows for site-specific genomic targeting in virtually any organism.

The type II CRISPR/Cas system is a prokaryotic adaptive immune response system that uses noncoding RNAs to guide the Cas9 nuclease to induce site-specific DNA cleavage. This DNA damage is repaired by cellular DNA repair mechanisms, either via the non-homologous end joining DNA repair pathway (NHEJ) or the homology-directed repair (HDR) pathway.

The CRISPR/Cas9 system has been harnessed to create a simple, RNA-programmable method to mediate genome editing in mammalian cells, and can be used to generate gene knockouts (via insertion/deletion) or knockins (via HDR). To create gene disruptions (Figure 1), a single guide RNA (sgRNA) is generated to direct the Cas9 nuclease to a specific genomic location. Cas9-induced double strand breaks are repaired via the NHEJ DNA repair pathway. The repair is error-prone, and thus insertions and deletions (INDELs) may be introduced that can disrupt gene function.

The principle of CRISPR/Cas9-mediated gene disruption.  A single guide RNA (sgRNA), consisting of a crRNA sequence that is specific to the DNA target, and a tracrRNA sequence that interacts with the Cas9 protein (1), binds to a recombinant form of Cas9 protein that has DNA endonuclease activity (2). The resulting complex will cause target-specific double-stranded DNA cleavage (3). The cleavage site will be repaired by the nonhomologous end joining (NHEJ) DNA repair pathway, an error-prone process that may result in insertions/deletions (INDELs) that may disrupt gene function (4).

CRISPR/Cas9 technology has revolutionized genome editing, allowing a previously unattainable level of genomic targeting, efficiency, and simplicity. Guide-it products further improve the usability of the CRISPR/Cas9 system by providing a streamlined method for:

• Producing sgRNAs  in vitro
• Testing the cleavage efficiency of sgRNAs
• Monitoring the efficiency of genome editing in cultured cells

CRISPR/Cas9 information

Choosing sgRNA design tools

Browse a collection of sgRNA design tools for Cas9-based genome editing experiments.

Choosing a target sequence for CRISPR/Cas9 gene editing

Learn how to design sgRNA sequences for successful gene editing.

The CRISPR/Cas9 system for targeted genome editing

Overview of CRISPR/Cas9 system for genome editing.

CRISPR/Cas9 genome editing tools

An overview of tools available for each step in a successful genome editing workflow.

Gene editing technical notes

Delivery of Cas9 and sgRNA to mammalian cells using a variety of innovative tools.

SNP engineering application note

Learn about a simple assay for sensitive detection of single-nucleotide substitutions in bulk-edited or clonal cell populations.

Learn about Guide-it CRISPR/Cas9 Gesicle Production System components and workflow.

Watch this webinar to learn how you can perform genome-wide lentiviral sgRNA screens easily.

Watch a webinar on how to choose the right HDR template for knockin experiments.

Get answers to frequently asked questions and view a video explaining the enzymatic assay.

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CRISPR-Cas Gene Editing Teaching Resources

Using CRISPR in your research?

See Bio-Rad’s Solutions for Gene Editing Workflows

One of the most exciting recent developments in genetic engineering is CRISPR-Cas9 (CRISPR) gene editing. CRISPR technology allows scientists to edit genes and manipulate gene expression within living organisms. This allows the use of CRISPR gene editing in a far-reaching range of applications from basic research to the development of novel therapies and other biotechnology products.

CRISPR-Cas9 gene editing is now accessible to students from high school through college in the form of a hands-on CRISPR gene editing lab . This page provides links to background information explaining how CRISPR works as both a microbial immune system and a gene editing system. It also features a selection of free resources to support you in bringing this exciting topic to your classroom.

What Is CRISPR?

A Microbial Immune System and a Gene Editing Technology

CRISPR gene editing technology is the result of decades of research into genome sequences and the adaptive immune system of bacteria immune system of bacteria and archaea. Read below to learn what CRISPR sequences are and how they work with Cas enzymes to incorporate snippets of viral DNA into the bacterial genome. Then extend your understanding to how these elements work with host cell DNA repair mechanisms in the CRISPR-Cas9 gene editing workflow.

CRISPR-Cas — a Microbial Immune System​

CRISPR gene editing technology is the result of decades of research into genome sequences and the adaptive immune system of bacteria and archaea. The following are some highlights:

A CRISPR region within a microbial genome

This video from The Franklin Institute summarizes how research in multiple areas came together to give the world CRISPR-Cas9 technology.

• In the late 1980s and early 1990s, researchers including Francisco Mojica noticed unusual repetitive DNA sequences in prokaryotic genomes. These sequences were short, palindromic (read the same forward and backward), and repeated many times with "spacer" sequences between the repeats. These unique sequences were called "clustered regularly interspaced short palindromic repeats" (CRISPR) sequences
• The function of CRISPR sequences remained a mystery until the early 2000s, when Alexander Bolotin and colleagues identified the CRISPR-associated (Cas) protein called Cas9 and proposed its role as a DNA-cutting enzyme in the bacterial immune system
• In 2007, Philippe Horvath and his team demonstrated that CRISPR sequences work with Cas proteins to provide adaptive immunity against viruses

CRISPR sequences and Cas enzymes work together to incorporate snippets of viral DNA into the bacterial genome. This allows bacteria to recognize and defend against future infections in three phases:

• Cutting and capture — When bacteria are infected by a virus, they use their CRISPR system to cut up the invading viral DNA and insert pieces of it (spacers) into their own genome as a "memory" of the infection
• Monitoring — Bacteria transcribe the spacer DNA into CRISPR RNA (crRNA); Cas9 and the crRNA form a complex and monitor the cell for any DNA sequence complementary to the RNA
• Defense — If matching (viral) DNA is encountered, the crRNA and RNA-Cas9 complex binds to and cuts the viral DNA to prevent it from replicating. This halts the viral infection

Using this system, bacteria can collect sequences from many different infecting viruses to create a "library." Since the CRISPR sequence is contained in genomic DNA, it is passed on to each generation, and the library continues to change and adapt to more common threats over time.

The CRISPR-Cas9 microbial defense system. 1.    The Cas1-Cas2 enzymes of the microbe recognize and cut out a segment of foreign DNA. 2.   The Cas1-Cas2 enzymes insert the DNA segment into the CRISPR region of the bacterial genome as a spacer. 3. A spacer sequence is transcribed and then linked to a Cas9 protein.  4. Upon reinfection by the same invader, the CRISPR-Cas9 complex can recognize the foreign DNA sequence and cut it to prevent complete reinfection .

CRISPR-Cas9 — A Gene Editing System

In 2012, Jennifer Doudna and Emmanuelle Charpentier demonstrated that the CRISPR-Cas9 system could be repurposed to edit DNA in living organisms. They earned the 2020 Nobel Prize for Chemistry for their work. In 2017, Feng Zhang and colleagues demonstrated the use of CRISPR-Cas9 to edit mammalian genomes.

The CRISPR-Cas9 system uses modified components of the bacterial CRISPR system to direct target-specific cutting of double-stranded DNA. DNA repair mechanisms then take over to fix the break in a manner that modifies the genetic sequence that has been cut.

Step 1: Cutting the DNA

Visit our YouTube CRISPR Gene Editing playlist for videos that depict the CRISPR-Cas9 method for genome editing.

• Cas9 enzyme (Cas9) — an endonuclease that cuts both strands of DNA at a specific site. Multiple types of Cas enzymes are found in nature, but Cas9 is commonly used in the laboratory
• Guiding region — part of the CRISPR RNA or crRNA in nature, a 20-nucleotide region that is complementary to the target region and defines the target DNA sequence that Cas9 cuts. Scientists customize this sequence for their own targets
• Scaffold region — the trans-activating CRISPR RNA or tracrRNA in nature, this region forms a multi-hairpin loop structure (scaffold) that binds in a crevice of the Cas9 protein
• Protospacer adjacent motif (PAM) — required for Cas9 function, this sequence motif is immediately downstream of the target sequence. Cas9 recognizes the PAM sequence 5’-NGG, where N can be any nucleotide (A, T, C, or G). When Cas9 binds the PAM, it separates the DNA strands of the adjacent sequence to allow binding of the sgRNA. If the sgRNA is complementary to that sequence, Cas9 cuts the DNA

5 Steps of Cas9 DNA Cleavage

5. the complex releases from the dna.

The Cas9-sgRNA complex releases the cut DNA and is ready to repeat the process.

1. Cas9 Binds an sgRNA

Cas9 recognizes and binds the scaffold (tracrRNA) region of a sgRNA. The nucleotide sequence of the scaffold region determines its structure, which is tailored to fit within the Cas9 protein as a key fits into a lock.

2. The Cas9-sgRNA complex binds to a PAM site on the target DNA

Cas9 requires a particular PAM sequence (5’-NGG) to be present directly adjacent to the protospacer sequence. When the Cas9-sgRNA complex recognizes and binds a PAM, it separates the DNA strands of the adjacent sequence to allow binding of the sgRNA.

3. The guiding region of the sgRNA binds to the target DNA sequence

The guiding region of the sgRNA attempts to base-pair with the DNA. If a match is found, the process continues. Otherwise, the complex releases and attempts to bind another PAM and target DNA sequence.

4. Cas9 makes a double-stranded break in the DNA three base pairs upstream of the PAM

Step 2: repairing the break to engineer the change.

Researchers can use the cell's own DNA repair machinery to modify, insert, or delete a nucleotide sequence. The repair can happen in two ways:

This video from Science Communication Lab explains homology directed repair (HDR).

• Non-homologous end joining (NHEJ) — enzymes reconnect the ends of the double-stranded break back together. This process may randomly insert or delete one or more bases and can cause mutations that can disrupt gene function or expression
• Homology directed repair (HDR) — proteins patch the break using donor template DNA. Researchers design the donor template DNA that may include a desired sequence flanked on both sides by "homology arms" that match the sequence upstream and downstream of the cut. A complementary DNA strand is created during the repair

DNA repair via homology directed repair and non-homologous end joining (Scroll right to view image)

Hands-On CRISPR Gene Editing Lab for the Classroom

Out of the Blue CRISPR and Genotyping Extension Kits

Use CRISPR-Cas9 gene editing to edit a bacterial chromosome! These lab kits use familiar — and safe — reagents, techniques, and organisms to bring students to the cutting edge of molecular biology. Using carefully designed bacterial strains and plasmids, students see and learn how the CRISPR-Cas9 system works with homology-directed repair (HDR) to introduce a stop codon into the chromosomal lacZ gene of E. coli . Order Kits

CRISPR Paper Model & Bioinformatics Activity

CRISPR Paper Model

Use this free CRISPR paper model to enhance understanding of how the CRISPR-Cas9 system directs precise cuts of DNA.

CRISPR Bioinformatics Activity

Use this activity to explore the design of Cas9 target sites in the human genome and determine risk for off-target effects.

CRISPR Videos & Other Resources

YouTube CRISPR Gene Editing Playlist

Access technique videos, recorded webinars, and perspectives about CRISPR from leading experts.

PowerPoint Presentation for Classroom Use

Use this student-facing slide deck to help guide your students through the CRISPR-Cas9 gene editing lab activity.​

Out of the Blue CRISPR Kit Student Activity Video Quick Guide

Use this overview video to prepare for the CRISPR gene editing lab for the Out of the Blue CRISPR Kit.

CRISPR in the Classroom

Looking to add CRISPR into your curriculum? We've got you covered with the essentials you'll need to introduce this cutting-edge topic to your students. ​

New to CRISPR? Join us at a webinar or view on-demand CRISPR-related webinars.

Join us at a hands-on workshop or attend an upcoming webinar.​ ​Gain insights on how to teach about CRISPR and its many applications.

CRISPR Poster (Free)

The poster features medical breakthroughs enabled by CRISPR gene editing technology, as well as a timeline of this and other Nobel prizes earned by women in science.

CRISPR Infographic (Free)

This infographic provides an overview of the CRISPR timeline and the regulatory, legal and ethical debates it has led to – great topics of discussion in your classroom!

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