CUT! How Does CRISPR Work?

Background Information for Teachers

Reliable and inexpensive gene editing, used either to "fix" genetic defects in patients or to introduce genomic changes for further study in a research lab, is a much-desired technology. Since its development around 2012, CRISPR (short for clustered regularly interspaced short palindromic repeats) systems have gained recognition as powerful gene editing techniques. The resources and videos provided in the Additional Background section cover much of the information on how CRISPR works. CRISPR harnesses the natural immune system bacteria use to fight off viruses. The antiviral defense mechanism is based on the incorporation of DNA fragments from viruses into the bacterial DNA. This part of the DNA is called the CRISPR array. Within the CRISPR array, the acquired virus DNA fragments are separated by conserved short nucleotide sequence repeats (Figure 1). The bacteria can then use this acquired virus DNA to identify and defend themselves against new viral threats in the future.

Image Credit: Svenja Lohner, Science Buddies / Science Buddies
Figure 1. Schematic view of a CRISPR repeat-spacer array.

Several different types of CRISPR systems have been identified, but the one most studied is the CRISPR-Cas9 system. In this system, the genes next to the CRISPR repeat-spacer array encode a unique defense mechanism consisting of a single-guide RNA and an endonuclease (Cas9), a protein that is able to cut double-stranded DNA. The single-guide RNA (sgRNA or gRNA) helps target the dangerous virus DNA, and the Cas9 endonuclease degrades foreign nucleic acids by inducing a double-strand break. The sgRNA contains one or more acquired CRISPR sequences from the CRISPR repeat-spacer array. In the event of a virus infection, the sgRNA combines with the Cas9 nuclease to build a sgRNA-Cas9 complex and guides it to the appropriate target in the virus DNA (Figure 2).

Because the CRISPR mechanism can cut anywhere in the DNA, the bacteria must protect its own DNA from being damaged. The PAM (protospacer adjacent motif) region is a short DNA sequence that is an essential targeting component that allows the bacteria to distinguish its own DNA from foreign DNA. The sgRNA will only adhere to and disable a DNA sequence that contains a PAM sequence nearby. The sgRNA locks onto the PAM sequence and starts unzipping the DNA double-strand to test if the sgRNA matches the target DNA. Once a matching sequence is found, the sgRNA binds to the target genomic site through complementary base pairing, and the Cas9 nuclease cuts the double-stranded virus DNA, inactivating the virus.

Image Credit: Wikimedia commons user marius walter / Creative Commons Attribution-Share Alike 4.0 International
Figure 2. The single-guide RNA combines with Cas9 and guides the nuclease to the target within a double-stranded genomic DNA. The sgRNA locks onto the DNA target directly next to the required PAM sequence. If the sgRNA matches with the target DNA, the Cas9 nuclease cuts the DNA double-strand 3 base pairs upstream of the PAM motif. (Image credit: Marius Walter [CC BY-SA 4.0], Wikimedia Commons, 2017)

Researchers have now found a way to manipulate the nucleotide sequence of the sgRNA so the Cas9 system can target any DNA sequence for cleavage. Once the Cas9 nuclease cuts the target DNA, the cell's natural DNA repair mechanisms kick in. There are two main pathways within a cell that result in the repair of DNA double-strand breaks. The first pathway is the non-homologous end joining (NHEJ) pathway, which is error-prone and can lead to insertion or deletion mutations within the DNA. The second pathway is the homologous direct repair (HDR) mechanism, which allows for insertion of a specific DNA template (single or double-stranded) at the target site. Currently, the two most common applications of the CRISPR technology are the targeted mutation of specific genes resulting in functional gene knockouts, and the replacement of a gene variant with another. Both strategies harness the natural CRISPR mechanism. Knockouts and gene replacement have made it much easier to probe gene functions and establish causal linkages between genetic variations and biological phenotypes.

The CRISPR-Cas9 technology has been successfully used in bacterial and mammalian cells, allowing for the creation of transgenic animals with targeted mutations. Most CRISPR research has focused on treating diseases by introducing genetic changes into blood, lung, or brain cells. Researchers have high hopes that this technology will one day enable scientists to repair disease-causing gene variants in patients with certain genetic diseases. This approach is especially promising in diseases such as sickle cell anemia, cystic fibrosis, or Huntington's disease, each of which is linked to a single gene variant. In 2019, the first gene-editing clinical trials began, targeting patients with sickle cell anemia. In the future, CRISPR might also significantly impact agriculture and the environment, including their impact on human health. For instance, we may one day be able to modify crops to make them more drought- or pest-resistant, or eradicate disease-spreading insects such as mosquitoes.

Although genome editing brings significant potential benefits, it also raises profound ethical questions. What CRISPR safety standards are appropriate? Where are the limits of this technology? Does gene editing bring us closer to "designer babies"? What about potential genetic discrimination? How can we grant equitable access to gene editing technologies? In 2018, Chinese researcher He Jiankui claimed to have edited the genomes of two human embryos, which then developed into two human babies, highlighting the need for ongoing public discourse to address these fundamental ethical issues related to CRISPR.

Additional Background Links

• Genome editing using the CRISPR-Cas9 system, National Institutes of Health National Library of Medicine
• Development and Application of CRISPR-Cas9 for Genome Editing, National Institutes of Health National Library of Medicine
• CRISPR-Cas guides the future of genetic engineering, National Institutes of Health National Library of Medicine
• Sickle Cell Gene Therapy with CRISPR, Synthego
• CRISPR enters its first human trials, Science News
• Experiments that led to the first gene-edited babies: the ethical failings and the urgent need for better governance, National Center for Biotechnology Information
• Why gene editing is so controversial, ABC News (Australia)

Prep Work (15 minutes)

1. Print out one Brainstorming Worksheet, Storyboarding Template, and one Peer Work Group Evaluation Form for each student.
2. Bookmark these videos and the interactive simulation, so you can access them quickly in class.
   • Video: NIH Common fund Somatic Cell Genome Editing Program
   • Video: Genome Editing with CRISPR-Cas9
   • Simulation: How CRISPR Works
   • CRISPR-Cas9 in Action: A Real-Time Video
   • Video: How to Storyboard Your Animation
   • Video: How to Make Stop Motion Videos
   • Video: About Lulu and Nana: Twin Girls Born Healthy After Gene Surgery As Single-Cell Embryos
3. Do a brief internet search and scan the most recent news articles and debates about CRISPR. This will ensure you are better-informed if any students ask related questions.

Engage (15 minutes)

Remote learning adaptation: This lesson plan can be conducted remotely. Students can work independently (individually or in virtual groups) to brainstorm, storyboard, and film using the Brainstorming Worksheet, the Storyboard Template, and the Grading Rubric for guidance. The videos can then be shared online on a class drive or classroom sharing apps like Flipgrid. The Engage and Reflect sections can either be dropped entirely, done individually remotely, or be conducted over a video chat.

1. Tell students that today they will explore the most recent developments in gene editing.
2. Start by introducing genes and genetic diseases as the driver for the creation of gene editing tools.

   Before we talk about gene editing, let's briefly talk about genes. What do you know about genes and why are they important?

   Listen to what students know about genes and why they are important. Remind them that genes are a distinct sequence of nucleotides that encode instructions for the synthesis of a protein that performs a function. Our human DNA encodes for more than 22,000 different genes. Many of these genes control our physical traits. Not every person has exactly the same genes, a gene can come in different variations. These genetic variations make each person unique and are responsible for genetic diversity.

   In some occasions, a specific gene variation can cause a disease. Do you know any examples of genetic diseases?

   Listen to students' replies. Examples they might mention are breast cancer caused by BRCA1 and BRCA2 gene variants, cystic fibrosis caused by a CFTR variant, sickle cell anemia caused by a hemoglobin (HbS) variant, or Huntington's disease caused by an HTT gene variant. Other diseases such as Alzheimer's, Parkinson's, or diabetes are linked to not just one but several genes. As these mutated genes are passed on from generation to generation, some diseases tend to run in families.

3. Tell students that a lot of research in biotechnology is focusing on finding ways to cure such genetic diseases. Show the video "NIH Common Fund Somatic Cell Genome Editing Program" to your students in order to introduce gene editing as a potential therapy to cure genetic diseases.
   

   https://www.youtube.com/watch?v=27o3s3TktHI

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4. Point out that one of the most recent developments in gene editing is the CRISPR technology. Gauge students' knowledge on CRISPR.

   Has anybody heard of or read about the CRISPR technology before? What do you know about CRISPR?

   Have students share their knowledge on gene editing and the emerging CRISPR technology. Together with students, define CRISPR as a gene editing technology that allows changes to DNA bases within a gene at precisely predetermined locations in a living cell. Thus, CRISPR holds great potential for curing genetic diseases and engineering desirable genetic traits.

Explore (165 minutes)

1. Explain to students that today they will explore the molecular mechanisms of the CRISPR technology in more detail to find out how it can be used for gene editing.
2. Show students the video "Genome editing with CRISPR-Cas9" that demonstrates how gene editing with the CRISPR-Cas9 technology works on a molecular level.
   

   https://www.youtube.com/watch?v=2pp17E4E-O8

   Alternatively, you can have them explore these interactive simulations about how CRISPR-Cas9 works.

   • This simulation shows how CRISPR-Cas9 initiates the DNA double-strand break in the target region.
   • This simulation shows the two alternative pathways for how the created double-strand break in the DNA is repaired.
3. Point out that to visualize molecular processes, we have to use simulations or animations like the ones they have just watched, as it is impossible to see what happens on a molecular level with your naked eye. Even if we use equipment that is able to look at DNA with high magnification, we wouldn't be able to make sense of what is happening in detail. To demonstrate this, show the "CRISPR-Cas9 in Action: A Real-Time Video", which shows how the Cas9-sgRNA complex cleaves DNA in real-time using atomic force microscopy.
   

   https://www.youtube.com/watch?v=aSbVC_3NQuk
4. Briefly ask students about their thoughts on the real-time CRISPR-Cas9 video.

   What could you see in the video?

   Were you able to infer the molecular mechanism of CRISPR-Cas9 from this real-time video?

   Collect students' replies. Most likely, they were able to see how the DNA double-strand breaks due to the CRISPR-Cas9 action. However, from the visuals in the video it would not be possible to infer the actual CRISPR-Cas9 mechanism.

5. Tell students that their task will be to create their own model of the molecular mechanism of CRISPR-Cas9. They will produce and shoot a stop-motion animation/video that demonstrates how the CRISPR-Cas9 technology works on a molecular level. Their end product should be 2-4 minutes long.
6. If you do this project in class, divide students into groups of 3. If you decide that students will do this project remotely, students can either work in groups of 3 online or work individually.
7. Briefly walk students through the individual steps of their stop-motion animation-making process. First, they will have a brainstorming session that includes a deep dive into the molecular mechanism of CRISPR-Cas9. The second step is for them to storyboard their animation before they continue with the actual video shoot as their last step. Provide each student with the Grading Rubric, so they know what the grading criteria for the video are. At the same time, distribute a Peer Work Group Evaluation Form and tell students they will need to fill this form out at the end of their project.

Part 1: Brainstorming

1. Distribute the Brainstorming Worksheet and have students discuss the posed questions in their groups. Circulate between groups and check in with students to ensure they are making progress. Make sure students have a good understanding of each component involved in the CRISPR-Cas9 system, as well as each component's function in the gene editing process. Table 1 and Table 2 provide an overview for you to assist students with answering the questions in the Brainstorming Worksheet if they are struggling. A Brainstorming Teacher Answer Key is also available. Ask additional questions that might prompt more discussion among the group, such as:
   • What other ways can you show that...?
   • Have you thought about using...?
   • How are you going to...?
   • Who is going to be in charge of...?

2. Give students time until the end of the class period to fill out their Brainstorming Worksheet. If they haven't completed the document at the end of the class period, assign the rest of the worksheet as homework.

Part 2: Storyboarding

1. After completing their Brainstorming Worksheet, students should have a good working knowledge of the CRISPR-Cas9 mechanism and should have initial ideas about how to create their stop-motion animation.
2. Tell students that in the next step, each group will need to storyboard their animation. Briefly introduce them to the concept of storyboarding by showing students the "How to Storyboard Your Animation" video.
   

   https://www.youtube.com/watch?v=ji2nwkH2JRo
3. Provide each group with several copies of the Storyboarding Template and give students the rest of the class period to work on their storyboards.
4. Again, circulate between groups and check in with students regularly. Remind them to indicate motion in their storyboard and to create a video description, as well as an audio description, for each of their frames. Also reiterate that the video should be 2-4 minutes long.
5. Have students finalize their storyboard at home if they haven't completed it during class.
6. Remind students that the final day of their project will be dedicated to their video shoot. This means they have to gather and bring all the materials they need to create their stop-motion animation.
7. Suggest students use the stop-motion application Stop Motion Studio (iOS Stop Motion app, Android Stop Motion app) to make their video. They should have a stop-motion application and an electronic device ready to go for their next class.

Part 3: Animating

1. The last day of the project is dedicated to video work. Before students start, show them the "How to Make Stop Motion Videos" video, which provides useful tips and tricks for their video shoot.
   

   https://www.youtube.com/watch?v=_ppedXZHhE0
   Remind students that they don't have to create a professional video. They should do their best, but they should not get slowed down by perfection or small details.
2. Next, have students familiarize themselves with the stop-motion app that they have installed on their device.
3. Once they are ready, let students start creating their stop-motion animations. Remind them to use their storyboard as a basis for their animation. Circulate amongst groups and help students troubleshoot if they encounter any problems.
4. If students need more time, have them finish their video as homework. Once the video is done, have students export it from their device and send it to you. All videos should be uploaded to a joint class drive so students can share their work with you and each other.
5. Once students have successfully submitted their videos, remind them to fill out the Peer Work Group Evaluation Form. This will allow you to assess the engagement of each student within their groups.

Reflect (30 minutes)

1. If you have time, watch the videos of each student group together in class. Otherwise, ask students to watch the videos at home.
2. Gather all students for a discussion on the potential applications of CRISPR. Ask students what they think the major applications of this technology are.

   What applications for CRISPR do you think are most promising?

   Students might mention that CRISPR or gene editing can be used to cure genetic diseases that are caused by DNA mutations. Point out that the first clinical trial to treat human patients with CRISPR gene editing is currently underway. In summer 2019, a patient with sickle cell anemia started her gene therapy treatment based on CRISPR. Sickle cell anemia is a disease that is caused by the mutation of one single gene. Other potential CRISPR applications are basic research, drug development or agriculture (engineering crops). Environmental and human health applications are also possible (for example, eradicating pests or insects that transmit diseases, such as mosquitos).

3. Engage students in a discussion about the ethical challenges of the CRISPR technology. Tell them that in 2018, Chinese researcher He Jiankui claimed that he used the CRISPR technology to edit the genome of two human embryos, which went on to develop into twin girls. He modified their genome in a way that made them naturally immune to HIV. To support your explanations, you can show the video "About Lulu and Nana: Twin Girls Born Healthy After Gene Surgery As Single-Cell Embryos."
   

   https://www.youtube.com/watch?v=th0vnOmFltc
4. Point out that when he announced his experiment to the public, he faced massive criticism. In fact, he was fired from his position shortly after and sentenced to 3 years in jail.

   Why do you think He Jiankui's research sparked such a public outcry?

   Listen to students' responses. If students struggle with this question, you might bring up the following points: He violated the guidelines and regulations that have banned germline genome editing on human embryos. Germline mutations can be passed on to offspring, whereas somatic mutations cannot be inherited. This means that the changes Jiankui made in the twins' DNA will be passed on to future generations. The changes Jiankui made in the twins' DNA could potentially have ripple effects that we don't know of, yet. The full function of the gene that has been altered is not known. He might have accidentally changed other functions in the twins' bodies that might only become visible much later. The twins never had a chance to decide if they wanted their genome to be edited or not. Now they have to live with all the potential consequences for the rest of their lives.

   What do you think about Jiankui's experiment? Do you support his research or not? Why?

   Have students share their thoughts.

   Based on your discussions, what ethical concerns do you have about the CRISPR technology?

   Collect students' responses and make a list on the board of all the questions and concerns that the students mention. Some concerns that have been raised in the past are the following: Germline gene editing: This could have the potential to rewrite the gene pool for future generations. Stigmatization of genetic diseases: If genetic diseases could be cured with gene editing, will people that still carry such diseases be discriminated against? Safety standards: How safe is CRISPR? What if CRISPR accidentally cuts the wrong part of the DNA? There are still lots of questions about the accuracy and safety of CRISPR. Potential limits: Are there certain traits that are off-limits and should not be edited? Do we want so-called "designer babies"? Equal access to the technology: How can we make sure this technology is equally accessible to everyone?

5. Conclude the activity by telling students that all these issues are active questions that are currently discussed by the scientific community, regulators, and governments. Point out that the students are part of this society and will most likely live to see a future where gene editing plays a significant role in many different sectors (agriculture, environment, human health, etc.). Thus, it is important that they are informed about the capabilities of CRISPR and that they take part in these discussions!
6. Optional: Recommend that students watch the movie Gattaca as one science-fiction example of what a future society with human gene editing might look like. You can also provide an article for students to read if they are interested: Experiments that led to the first gene-edited babies: the ethical failings and the urgent need for better governance.

Assess

You can use this quiz to assess student learning after the activity:

• Online quiz, assignable in any LMS
• Quiz (pdf) and answer key (pdf)

Make Career Connections

Discussing or reading about these careers can help students make important connections between the in-class lesson and STEM job opportunities in the real world.

Microbiologist

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Career Profile

Microbiologists study the growth, structure, development, and general characteristics of bacteria and other microorganisms. It was microbiologists and molecular biologists who discovered the CRISPR/Cas9 system for the first time in bacteria and identified its role in bacterial immunity. Microbiologists use biotechnology and develop new molecular tools such as CRIPSR-Cas9 to advance knowledge of cells and human disease. Read more

Bioinformatics Scientist

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Career Profile

Bioinformatics scientists work at the intersection of biology, computer science, and information technology (IT). They create novel computational approaches and analytical tools that they can apply to the large amounts of data generated by biologists and geneticists through research. For example, they could develop tools that allow researchers to optimize the design of guide RNAs and allows to detect potential off-target sites in a given target region of a genome. Read more

Lesson Plan Variations

• Have students research the most current developments in gene editing and the CRISPR-Cas9 technology.
• Instead of making a stop-motion animation, have students create a cartoon or a flipbook that displays the molecular mechanism of CRISPR-Cas9.
• Focus more on the bioethical aspects of CRISPR. Simulate a podium discussion in which students take different roles; for example, a scientist, a politician, a CRISPR skeptic, a patient with a genetic disease, an ethicist, a religious figure, etc.
• Discuss what other applications CRISPR could have beyond gene editing. You can use the video "CRISPR: Gene Editing and Beyond" as a starting point.