Kansas College and Career Ready Standards

Science

• HS-LS1-1. Construct an explanation based on evidence for how the structure of DNA determines the structure of proteins which carry out the essential functions of life through systems of specialized cells.
• HS-LS2-7. Design, evaluate, and refine a solution for reducing the impacts of human activities on the environment and biodiversity.
• HS-LS3-1. Ask questions to clarify relationships about the role of DNA and chromosomes in coding the instructions for characteristic traits passed from parents to offspring.
• HS-LS3-2 . Make and defend a claim based on evidence that inheritable genetic variations may result
  from (1) new genetic combinations through meiosis, (2) viable errors occurring during replication, and/or
  (3) mutations caused by environmental factors.

Learning Objectives

• Understand the structure of DNA and its role in genetic inheritance.
• Comprehend how DNA encodes traits that are passed across generations.
• Understand the components of the CRISPR-Cas system.
• Understand that CRISPR can be used to edit specific genes in living organisms.
• Understand that editing genes can induce positive traits in crops.

Materials

• Kansas Corn: Genetic Cut and Paste PowerPoint presentation (available at kansascornstem.com)
• 37 degree C heat source, e.g., thermocycler, incubator, or water bath
• micropipette (20-200 mL) for dispensing reagents
• Distilled Water

For each student group
Prelab

• CRISPR model handout (p 40-42 MiniPCR Chopped)
• Scissors
• Transparent tape or copy of digital Chopped Prelab (google doc)

Lab

• Electrophoresis Gel box
• micropipette (2-20 mL)
• micropipette tips (at least 24/group)
• gel cup
• buffer
• Disposable laboratory gloves
• Protective eyewear
• Permanent marker
• Cup to dispose of tips
• Crushed ice

Safety Considerations

Keep lab area clean and free of unnecessary equipment. Students should wear protective eyewear and gloves throughout the lab.

Procedures for Instruction

• Length of Time for Preparation: 30 minutes to thaw reagents and dilute the Cas9 enzyme.
• Length of Time for Classroom Teaching: Two 45-50 minute class periods or one 90-minute class period.

Preparation Procedure

• Located on page 7-10 of “Chopped” MiniPCR Teacher’s Guide.

Background Information

CRISPR-Cas systems are naturally found in bacteria and other prokaryotes as a form of protection.  These systems defend these simple organisms from bacteriophage, viruses that attack bacteria, by identifying sequences of viral DNA and cutting these strands before the bacteria can utilize this viral DNA to create protein which effectively prevents the virus from reproducing.  This action is very similar to restriction enzymes that perform a similar function. Restriction Enzymes cut any DNA that matches their short individual sequences.  We often utilize these naturally occurring restriction enzymes to cut DNA sequences at targeted locations in the lab but we have been limited by the sequences found in nature. CRISPR-Cas systems are different in a very important way.  They use guide RNA to find specific viral DNA sequences. Honing in like a sniper on a target. This allows the same nuclease enzyme to target different sequences and adapt more quickly to changes in the virus.  In 2012 scientists programmed CRISPR RNA to target DNA sequences they wanted to change.  This approach enables scientists to cut genes that they want to disable or even cut a specific gene out while pasting in a different gene to replace it.  Effectively turning on, turning off, or replacing genes at the discretion of the desired traits.  This ability to edit genes has many potential applications in treating diseases such as sickle cell disease, cystic fibrosis, and types of cancer.  This technology also shows promise in the development of crops.  Waxy Corn has a different starch composition that is desirable for certain cooking and industrial applications.  This difference is due to mutations disabling a gene called Waxy1.  These Waxy1 alleles have arisen naturally as well as being induced using chemicals and radiation.  Using CRISPR scientists precisely deleted the waxy1 gene in high yielding corn lines.  This process produced waxy hybrids much more quickly than conventional methods and the yields were dramatically higher.  This gene editing tool has enormous potential in the medical field as well as the agricultural and industrial setting and has the potential to confer a public perception benefit.  Currently, food products containing gene edited crops do not require genetically modified labeling eliminating some controversy surrounding such varieties.

More information located on page 12-15 of “Chopped” MiniPCR Teacher’s Guide.

Available for download at https://www.minipcr.com/products/minipcr-learning-labs/crispr/

Classroom Discussion

In the prelab activity, located on pages 17-20, the DNA sequence that will be edited is written out in A, G, T, and C format in a google document. Students will look for the target sequence for each of the guide RNA strands and determine the location where the Cas-9 enzyme will cut the strand. The length of the fragments produced can then be predicted and compared with the fragment lengths found in the gel during the lab analysis. There are two options for this activity depending on preference and technology available. Yeast and anaerobic respiration

• Pre-Lab Paper Model- Located on pages 40-42. The paper model involves cutting the sequence from
  paper and taping it into one piece. The entire sequence isn’t written out but the number of bases left out
  in the strands are labeled. Students search for the complementary sequence in the parts that are written
  out. Once the location of the cut is found they can count the typed bases in each fragment and add the
  number counted to the number given that was not typed. This number will be the predicted fragment
  length.
• Digital Prelab- available as a google document. Can be found in the PowerPoint presentation or at
  https://links.minipcr.com/chopped_prelab This version of the prelab is quicker and requires less counting than the paper model but does require a device that can open and edit a google doc. Students can open and edit a copy of the google doc by clicking the link above. This version uses the character count and search capability to find the target sequence and count the letters representing bases in the fragment
  before and after the Cas-9 edit. This activity can be performed together as a class using a projector. There are detailed instructions and demonstrations in the PowerPoint.
• Prelab questions located on pages 27-29 of Chopped Students Guide.

Follow lab guide 21-26 miniPCR Chopped

Teacher Resources

Page 44-54 of Instructors Guide includes sections on differentiation, trouble shooting, expected results, etc. this will assist you as you conduct the lab.

Lab Analysis

Did the fragments on the gel match the prediction?

Reflection and Conclusion

Pages 34-36 miniPCR Chopped Students Guide

Science and Agriculture Careers

To learn more about agriculture careers, visit agexplorer.com.  You can also find career profiles at kscorn.com/careerconnections.

Sources

• https://bitesizebio.com/47927/history-crispr/
• https://bioengineeringcommunity.nature.com/posts/61316-using-crispr-to-develop-superior-cornhybrids

Disclaimer

Any educator electing to perform demonstrations is expected to follow NSTA Minimum Safety Practices and Regulations for Demonstrations, Experiments, and Workshops, which are available at http://static.nsta.org/pdfs/MinimumSafetyPracticesAndRegulations.pdf, as well as all school policies and rules and all state and federal laws, regulations, codes and professional standards. Educators are responsible for abiding appropriate legal standards and better professional practices under a duty of care to make laboratories and demonstrations in and out of the classroom as safe as possible. If in doubt, do not perform the demonstrations.