CRISPR-Cas Gene Editing Teaching Resources

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?

  • 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:

crispr bacterial chromosome

A CRISPR region within a microbial genome

franklin institute youtube

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.

  • Illustration of how CRISPR-Cas9 acquires foreign DNA sequences
  • Illustration of how CRISPR-Cas9 defends against future infection

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

hq crispr youtube

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
  • Single guide RNA (sgRNA) — an engineered RNA that forms a complex with Cas9. The sgRNA is a fusion of two regions that occur as separate RNAs in nature:
    • 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

  • cas9 dna cleavage wf step5

    5. The complex releases from the DNA

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

  • cas9 dna cleavage wf step1

    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.

  • cas9 dna cleavage wf step2

    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.

  • cas9 dna cleavage wf step3

    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.

  • cas9 dna cleavage wf step4

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

  • cas9 dna cleavage wf step5

    5. The complex releases from the DNA

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

  • cas9 dna cleavage wf step1

    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.

 

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:

homology directed repair

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
 
Illustration showing DNA repair via homology directed repair and non-homologous end joining

DNA repair via homology directed repair and non-homologous end joining

 

Hands-On CRISPR Gene Editing Lab for the Classroom

  • out of the blue crispr and genotyping extension kits pdp

    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 Videos & Other Resources

  • CRISPR gene editing

    YouTube CRISPR Gene Editing Playlist

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

  • out of the blue CRISPR Kit

    PowerPoint Presentation for Classroom Use

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

  • CRISPR kit student activity video guide

    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

    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. ​

  • webinars

    Webinars

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

  • feature image bioed workshops

    Workshops

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

  • pdf crispr poster

    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.


    Add to Cart
  • pdf bulletin 7289

    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!


    Add to Cart