2024 NSTA New Orleans • EDVOTEK® Workshops

EDVOTEK Workshops offered at the 2024 NSTA National Conference in New Orleans. Thursday & Friday Nov. 7-8. 1. Forensic Escape Room: Design Your Own Biotech Adventure 2. Code Breakers: Using CRISPR to Rewrite Genetics 3. The Sweet Laboratory: Exploring Food Science with Biotechnology 4. Put the M into STEM: Quantitative Techniques for Biotechnology 5. Heavy Metal: Investigating the Effects of Environmental Toxins on C. elegans 6. Introducing Your Students to CRISPR with Sickle Cell Gene Editing 7. Lion Family Reunion: Conservation Biology Genetics

EDVOTEK® WORKSHOPS • 2024 NSTA NEW ORLEANS

TABLE OF CONTENTS

WORKSHOP

PAGE

01 Forensic Escape Room: Design Your Own Biotech Adventure

02 Code Breakers: Using CRISPR to Rewrite Genetics

15 29 41 53 65 77

03 The Sweet Laboratory: Exploring Food Science with Biotechnology 04 Put the M into STEM: Quantitative Techniques for Biotechnology 05 Heavy Metal: Investigating the Effects of Environmental Toxins on C. elegans 06 Introducing Your Students to CRISPR with Sickle Cell Gene Editing

07 Lion Family Reunion: Conservation Biology Genetics

Check Out Our 2024-2025 Interactive Resource Guide!

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01 - Forensic Escape Room: Design Your Own Biotech Adventure

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01 - Forensic Escape Room: Design Your Own Biotech Adventure

Introduction Explore the world of forensic science with these fun and exciting escape room activities! Try forensic blood detection and agarose gel electrophoresis experiments, decipher clues, and solve puzzles. Learn to design your own escape room to have students unravel the evidence and free the innocent. Background Information Excerpts from EDVO-Kits 191 & 130 An abundance of material evidence can be left behind at the scene of a crime. This evidence can include blood on clothing, walls or floors, or even on the potential murder weapon. In some cases a few cells caught under the victim’s nails during a struggle can provide a wealth of information. Evidence can be obtained based on microscopic examination and biotechnological analysis, and then compared to a sample obtained from a person of interest who may have been at the site of the crime. Advances in molecular biology and genetics over the past 30 years have produced a variety of applications that have forever changed forensic science. Human tissue is made up of cells that contain DNA, which can be col- lected from evidence. When combined with the polymerase chain reaction (PCR) and DNA fingerprinting a very small amount of DNA from a biological sample can be analyzed. In many cases the crime can only be solved, and the criminals brought to justice, through the meticulous work of forensic scientists. DETECTION OF BLOOD SPATTERS: Presumptive & Confirmatory Tests The most common presumptive forensic blood test is the Kastle-Meyer test (Box 1). The Kastle-Meyer test uses a compound known as phenolphthalein (pr. fee-nawl-thal-een), which reacts with the iron carried by hemoglobin. First, presumptive blood is gathered on a cotton-tipped swab. The cellular membranes of cells on the swabs are then broken open (lysed) by applying a few drops of 95% ethanol. Phenolphthalin solution is then applied, followed quickly by hydrogen peroxide. If the cotton swab turns pink, it means that there was likely hemoglobin in the sample. BOX 1: Chemistry of the Kastle-Meyer Test The phenolphthalein (C 2 0H 16 O 4 ) used in the Kastle-Meyer test has been reduced, i.e. it has gained electrons, and is actually called phenolphthalin (C 2 0H 14 O 4 ) . The reaction in the Kastle-Meyer test is based on the reac- tion between the iron in hemoglobin and hydrogen peroxide (H 2 O 2 ). The iron in hemoglobin reduces (sup- plies electrons to) the H 2 O 2 , creating water (H 2 O). This reaction depletes the hemoglobin of electrons, which are in turn supplied by phenolphthalin. The oxidation, i.e. the release of electrons, of phenolphthalin turns it back into phenolphthalein, which has a characteristic pink color. Fe 4+ + C 2 0H 14 O 4 + H 2 O 2 → C 2 0H 16 O 4 + H 2 O + Fe 3+ Presumptive tests, such as the Kastle-Meyer, must be confirmed using a test that definitively detects blood. These are known as confirmatory tests. Confirmatory tests are often much more expensive and can take more time than presumptive tests. The most common confirmatory test for blood is the Rapid Stain Identification of Human Blood (RSID). The RSID works similarly to a pregnancy test. The sample is applied to the test strip, and antibodies that recognize blood proteins specifically bind to the sample. If the antibodies bind and the sample is positive for blood, a visible line is shown in the viewing window. Another confirmatory test for blood is ABO blood type testing. Testing for blood groups relies on the precipitation of an antigen-antibody complex, called agglutination. Only blood will produce this agglu- tination, which is why it is classified as a confirmatory blood test. In addition to being a confirmatory test, ABO blood typing is also a faster and more affordable identity test than other analysis techniques such as DNA fingerprinting. Indeed, forensic blood typing serves both as a confirmatory test and provides information about the suspect in the form of their blood type. Even though blood typing cannot point to a specific person as the criminal, it can point to a group of people that share the same blood type or eliminate suspects whose blood type does not match.

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01 - Forensic Escape Room: Design Your Own Biotech Adventure

BLOOD TYPING Blood typing is an immensely important clinical procedure. It is one of the first procedures performed during blood transfusions and surgery, and is also important in forensic science. Blood typing is an example of an agglutination assay, the precipitation of antigens on red blood cells and antibodies in the blood. When both components are present at a similar concentration they are in a state known as

equivalence. In an equivalent state, neither the antibody nor the antigen is in excess, and the antigen-antibody complex forms large networks that precipitate out of solution. Impor- tantly, the precipitate is easy to detect by eye, making agglutination assays both easy and cost-effective to perform. The most common blood typing system relates to the presence of the A and B antigens on the red blood cells. This system, known as the ABO blood types, produces four

Antigen on Red Blood Cells

Antibody in Blood

Percentage of Population

Blood Type

A B AB O

A B A & B O

anti-B anti-A none anti-A & anti-B

42% 10% 4% 44%

Figure 1: Types of Blood in the Population

possible blood types: A, B, AB, and O (Figure 4). Individuals with only A antigens will have type A blood, while someone with B antigens has type B blood. The A and B antigens are co-dominant, so a person can have both antigens on their red blood cells, leading to the AB blood type. If an individual has neither A nor B antigens they have type O blood. A person with type A blood will recognize red blood cells with the A antigen as “self”. However, if that person gets a blood transfusion with type B blood, the new red blood cells will be recognized by the immune system as foreign and will cause an immune response. Antibodies targeting the B antigens (anti-B antibodies) will bind to the B antigen on the transfused cells and agglutinate. In many cases, this severe immune response can be deadly. Therefore, it is important for hospitals and clinics to maintain records of patient blood types. The same reaction that can lead to severe immune responses in a patient is used for clinical and foren- sic blood typing experiments. For example, type B blood can be easily recognized by the agglutination between the anti-B antibodies and the B antigen. When something that is suspected to be blood is found at a crime scene, detectives will work quickly to secure the evidence and send it to a forensic lab for testing. In the lab, forensic scientists will perform presumptive and confirmatory tests for blood, potentially recommending additional testing such as DNA profiling. DNA FINGERPRINTING In humans, DNA is packaged into 23 pairs of chromosomes that are inherited from an individual’s bio- logical parents. Although most of this genetic material is identical in every person, small differences, or “polymorphisms”, in the DNA sequence occur throughout the genome. For example, the simplest differ - ence is a Single Nucleotide Polymorphism (or SNP). Changes in the number and location of restriction enzyme sites result in Restriction Fragment Length Polymorphisms (or RFLPs). Short repetitive stretches of DNA at specific locations in the genome can vary in number to produce STRs (Short Tandem Repeats) and VNTRs (Variable Number of Tandem Repeats). Although most polymorphisms occur in non-coding regions of DNA, those that disrupt a gene can result in disease. Medical diagnostic tests can identify specific polymorphisms associated with disease. Analyzing several different polymorphisms within a person’s genome generates a unique DNA “finger - print”. DNA fingerprints can allow us to distinguish one individual from another. Because polymorphisms are inherited, DNA fingerprints can also be used to determine paternity/maternity (and other familial relationships). The best-known application of DNA fingerprinting is in forensic science. DNA fingerprint - ing techniques are utilized to interpret blood, tissue, or fluid evidence collected at accidents and crime scenes. After DNA is extracted from these samples, forensic scientists can develop a DNA fingerprint. The DNA fingerprint from a crime scene can then be compared to the DNA fingerprints of different suspects. A match provides strong evidence that the suspect was present at the crime scene.

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01 - Forensic Escape Room: Design Your Own Biotech Adventure

Early fingerprinting analysis involved restriction digestion of the isolated DNA. Following electrophoresis of the digested sample, the DNA is transferred to a nylon membrane during a process known as Southern blotting. Sequence-specific DNA probes are used to visualize the membrane-bound DNA. If the DNA is not digested by the restriction enzyme, the probes will only hybridize to a single DNA segment. If a restriction site occurs within this sequence, the probe will hybridize with multiple bands of DNA. VNTRs are identified when a probe labels DNA at a dissimilar molecular weight. Although RFLP analysis is very precise, it is time-consuming and requires large amounts of DNA. To address these problems, forensic scientists use the polymerase chain reaction (PCR) to produce DNA fingerprints. PCR allows researchers to quickly create many copies of a specific region of DNA in vitro (summarized in Figure 1-1). This technique requires 500-fold less DNA than traditional RFLP analysis and it can be performed in an afternoon. To perform PCR, purified double-stranded DNA is mixed with primers (short synthetic DNA molecules that target DNA for amplification), a thermostable DNA polymerase (Taq) and nucleotides. The mixture is heated to 94°C to denature the DNA duplex (i.e., unzip it into single strands).Next, the sample is then cooled to 45°C - 60°C, allowing the primers to base pair with the target DNA sequence (called “annealing”). Lastly, the temperature is raised to 72°C, the optimal temperature at which Taq polymerase will extend the primer to synthesize a new strand of DNA. Each “PCR cycle” (denaturation, annealing, extension) doubles the amount of the target DNA in less than five minutes. In order to produce enough DNA for analysis, twenty to forty cycles may be required. To simplify this process, a specialized machine, called a “thermal cycler” or a “PCR machine”, was created to rapidly heat and cool the samples. ESCAPE ROOM SCENARIO Monica Homewood has been arrested as the main suspect in the murder of her husband, Bryce Home- wood. She has been questioned multiple times and continues to plead innocent to the crime. Monica’s lawyer is trying to prove her innocence and has convinced the Charleston Police Department to further investigate the crime scene. You are the best forensic analyst that money can buy in Charleston. Your job is to determine if the evidence collected at the crime scene contains blood, what blood type the blood evidence is, and to use DNA fingerprinting to determine who committed the crime. Will your forensics skill help prove Monica’s innocence? The following information and evidence are required for your forensic assessment of the crime.

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01 - Forensic Escape Room: Design Your Own Biotech Adventure

OFFICIAL POLICE REPORT

RESPONDING OFFICER: Caitlin Marlow

DETAILS OF EVENT: On Saturday night, police dispatch received a distressed phone call from a woman in her mid-30’s, claiming to have returned home to find her husband dead in their kitchen. Dispatch sent Caitlin Mar - low, the local police sheriff, to the crime scene in a small suburban condo in Charleston, South Caro - lina. Upon arriving at the condo, Sheriff Marlow found a deceased man with a traumatic head injury, including a deep laceration, in the kitchen. Marlow and her crime scene investigators collected the evidence from the scene to be analyzed by the crime scene team. Upon initial inspection of the scene, the following primary evidence was collect- ed: glass shards (from a broken iced tea pitcher) with what appears to be blood splatter on the kitchen table (Sample 1), a knife near the table with apparent blood on it (Sample 2), potential blood spatter on the hardwood floor near the kitchen table (Sample 3), and a tall glass with women’s lipstick on it. Later, the coroner was able to identify the body as Bryce Homewood. The cause of death was de - termined as a traumatic head injury. Glass shards in the head wound matched the broken glass from the iced tea pitcher found at the crime scene. A mysterious note with unintelligible text was found in the victim’s pocket. Marlow and her team of detectives questioned the neighbors in the condo complex. They deter- mined Monica Homewood, Deena Granville, and Alex Johnson to be the three main suspects of this crime. According to eyewitness reports, both Monica and Deena were seen at the scene of the crime one (1) hour before the call to the police dispatch. Neighbors also noted that a local real estate agent, Alex Johnson, was seen talking with Mr. Homewood at the condo the night before. THE VICTIM: • Bryce Homewood , aged 32, was a graphic designer for an advertising agency. He was married to Monica Homewood. He was known for his sharp wit and humor, though sometimes he veered into the realm of abuse and/or harassment when angry. SUSPECTS: • Monica Homewood , 33, works as a pediatric nurse at a local children's hospital. She was the distressed woman from the police dispatch phone call. She is also married to the victim. Most of the neighbors complained about their constant arguments. Their fights often disrupted the peaceful condo building. When questioned about her whereabouts during the crime, Monica claimed to have gone to an urgent care to take care of a deep wound she claims she obtained accidentally while cooking dinner with Bryce. It was confirmed by the local urgent care that Monica was treated for a knife wound. • Deena Granville , 34, is the neighbor and HOA president. She works as a marketing manager for a fashion retail company. She is known for being a stickler on the HOA rules and ever since the Home- woods moved in, there have only been fights between Bryce and Deena. Neighbors also noted that Bryce was also constantly fighting with Deena over excessive property management fees. The meeting notes from the HOA meetings prove that the two have a history of be- ing verbally aggressive towards each other. • Alex Johnson , 38 - Real Estate Agent. Alex Johnson has ongoing disputes with Bryce over a contest- ed condo sale. At the time of the crime, he claimed to be attending a local real estate conference which investigators have yet to confirm. Neighbors noted Alex had a strained relationship with the victim due to their frequent clashes over business deals, but he always maintained a professional demeanor in their interactions. SAMPLES PENDING FORENSIC ANALYSIS: Potential blood evidence found at the crime scene. Tests to be performed include Kastle-Meyer testing, Blood Group Typing and DNA Fingerprinting.

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01 - Forensic Escape Room: Design Your Own Biotech Adventure

Module I: Kastle-Maeyer Test

The first steps of your forensic analysis will use the Kastle-Meyer test as a presumptive test for blood. You will test the three crime scene samples to see if they are positive or negative for the presence of blood.

25 µL evidence

50 µL Phenolphthalin solution

50 µL Hydrogen peroxide solution

50 µL ethanol

1. Use a transfer pipet to ADD 1 drop (or 25 µL) of blood evidence to the swab. NOTE: Using more than one drop (25 µL) of evidence may affect the results.

2. Use a transfer pipet to ADD 2 drops (or 50 µL) of 95% ethanol to the swab. NOTE any color change. PLACE the pipet and remaining ethanol to the side for testing additional samples. 3. Use a new pipet to ADD 2 drops (or 50 µL) of the phenolphthalin solution to the swab. NOTE any color change. No color change is expected even if blood is present. PLACE the pipet and remaining phenolphthalin to the side for testing additional samples.

IMPORTANT For steps 2-4: When adding the detection reagents, hold the evidence swab at a steep angle, with the crime scene sample at the bottom.

4. Use a new pipet to ADD 2 drops (or 50 µL) of hydrogen peroxide solution “H2O2” to the swab. NOTE any color change. A pink color is expected after several seconds if blood is present. RECORD your results in the chart below.

REPEAT steps 1-4 for each blood evidence sample.

Crime Scene Sample #

Positive or Negative for Blood

CS1

CS2

CS3

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01 - Forensic Escape Room: Design Your Own Biotech Adventure

Module II: Blood Type Test

Now that some samples from the crime scene have been identified as blood using the Kastle-Meyer test, it is necessary to confirm the blood type of the samples. In this module, you will perform ABO testing on the blood samples from the crime scene. This will allow us to determine which crime scene blood samples are from the victim, and which samples were from the suspects. NOTE: Bryce is blood type O, Deena is blood type B, Alex is blood type A, and Monica is blood type B.

1. PLACE a microtiter plate piece as shown below. Using a permanent marker, label the plate. The column labels will include the four control blood types (A, B, AB, O) and any Module I crime scene samples that test positive for blood. The row labels will be Anti-A and Anti-B.

A B AB O

Anti A

Anti B

2. ADD 50 μL or two drops of each control blood type sample into each of the two corre - sponding wells. For example, control blood type A goes into the two wells under the letter “A”. Repeat the same procedure for the control blood types B, AB, O, and the two positive blood samples from the crime scene. Each well requires 50 μL. 3. Use a new pipette tip to ADD 50 μL or 2 drops of Anti-A serum into each of the wells in row #1. The same tip or transfer pipet can be used for all samples in row #1. 4. Use a new pipette tip to ADD 50 μL or 2 drops of Anti-B serum into each of the wells in row #2. The same tip or transfer pipet can be used for all samples in row #2. 5. Firmly HOLD the plate piece on the lab bench as shown. Very gently TAP the side of the plate with your finger near the positive crime scene samples to MIX for approximately 20- 30 seconds. Do this VERY CAREFULLY to avoid spilling any of the samples from the wells. PROCEED to the next step. 6. To visualize the results, PLACE the microtiter plate on a white light transilluminator, or use a bright flashlight held above the plate. COMPARE the crime scene evidence with the control blood samples. RECORD your results.

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01 - Forensic Escape Room: Design Your Own Biotech Adventure

Module III: DNA Fingerprinting After using blood typing to determine which samples are from the suspect, you must use DNA fingerprinting to find out who the blood came from. You send the suspects’ blood samples as well as the blood collected from the knife to an external DNA lab. Here they will extract DNA from each sample and amplify it by PCR. In this module, you will use gel electrophoresis to analyze the PCR samples obtained from the suspects and compare them to the blood found at the scene.

1:00

Concentrated buffer

Distilled water

Agarose

60°C

Flask

Caution! Flask will be HOT!

WAIT

POUR

60°C

ADD diluted SYBR Safe

60°C

CASTING THE AGAROSE GEL 1. DILUTE concentrated (50X) buffer with distilled water to create 1X buffer (see Table A).

REMINDER: This kit requires 0.8% agarose gels cast with at least 4 wells.

2. MIX agarose powder with 1X buffer in a 250 mL flask (see Table A). 3. DISSOLVE agarose powder by boiling the solution. MICROWAVE the solution on high for 1 minute. Carefully REMOVE the flask from the microwave and MIX by swirling the flask. Continue to HEAT the solution in 15-second bursts until the agarose is completely dissolved (the solution should be clear like water). 4. COOL agarose to 60 °C with careful swirling to promote even dissipation of heat. 5. While agarose is cooling, SEAL the ends of the gel-casting tray with the rubber end caps. PLACE the well template (comb) in the appropriate notch. 6. Before casting the gel, ADD diluted SYBR® Safe to the molten agarose and swirl to mix (see Table A). 7. POUR the cooled agarose solution into the prepared gel-casting tray. The gel should thoroughly solidify within 20 minutes. The gel will stiffen and become less transparent as it solidifies. 8. REMOVE end caps and comb.

Take particular care when re- moving the comb to prevent damage to the wells.

Individual 0.8% UltraSpec-Agarose™ with SYBR ® Stain

7 x 7 cm

0.6 mL

29.4 mL

0.24 g

30 mL

30 µL

10 x 7 cm*

0.9 mL

44.1 mL

0.36 g

45 mL

45 µL

14 x 7 cm

1.2 mL

58.8 mL

0.48 g

60 mL

60 µL

* Recommended gel volume for the EDGE™ Integrated Electrophoresis System.

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01 - Forensic Escape Room: Design Your Own Biotech Adventure

Module III: DNA Fingerprinting, continued

RUNNING THE GEL 9.

REMINDER: Before loading the samples, make sure the gel is properly oriented in the

PLACE gel (on the tray) into electrophoresis chamber. COVER the gel with 1X electrophoresis buffer (See Table B for recommended volumes). The gel should be completely submerged. 10. PUNCTURE the foil overlay of the QuickStrip™ with a pipet tip. LOAD the entire sample (35 µL) into the well as indicated by the Gel Loading table.

apparatus chamber.

11. PLACE safety cover. CHECK that the gel is properly oriented. Re- member, the DNA samples will migrate toward the positive (red) electrode. 12. CONNECT leads to the power source and PERFORM electrophoresis. (See Table C for time and voltage guidelines.) 13. After electrophoresis is complete, REMOVE the gel and casting tray from the electro- phoresis chamber.

GEL LOADING TABLE

VISUALIZING the SYBR® GEL SLIDE gel off the casting tray onto the viewing surface of the transilluminator. TURN the unit on. DNA should appear as bright green bands on a dark background. PHOTOGRAPH results.

SAMPLE A B C D

LANE 1 2 3 4

SAMPLE NAME DNA standard marker Crime scene sample Deena PCR Reaction Monica PCR Reaction

Time and Voltage Guidelines (0.8% Agarose Gel)

1x Electrophoresis Buffer (Chamber Buffer)

EDGE™

M12 & M36

Volts

Min/Max (minutes)

EDGE™

150 mL

3 mL

147 mL

150 125 100

10/20

20/35 30/45 40/60

M12

400 mL

8 mL

392 mL

N/A

M36

1000 mL

20 mL

980 mL

15/25

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01 - Forensic Escape Room: Design Your Own Biotech Adventure

Experimental Results and Analysis

MODULE I ANALYSIS

Negative Sample

Positive Sample

CS1 was negative and did not produce any color change after adding H 2 O 2 , while CS2 and CS3 turned bright pink and were posi- tive. Therefore, CS2 and CS3 should continue on for confirmatory testing.

MODULE II ANALYSIS

The crime scene sample CS2 is type O and crime scene sample CS3 is type B blood. Both Deena and Monica are type B. While blood typing can narrow down suspects, DNA testing would have to be

performed to conclusively say if the blood belongs to either of the two suspects. Therefore, PCR samples from crime scene sample CS3 should be DNA tested alongside PCR samples from Monica and Deena.

MODULE III ANALYSIS

The DNA standards in Lane 1 make it possible to measure the DNA bands obtained from the PCR reactions. The results of this analysis indicates an identical pattern in Lanes 2 and 4. This is strong evidence that the crime scene DNA matches the sample from Monica. In criminal investigations, several known variable regions in DNA are analyzed to match crime scene and suspect DNAs.

TUBE A B C D

LANE 1 2 3 4

SAMPLE NAME DNA standard marker Crime scene sample Deena PCR Reaction Monica PCR Reaction

MOLECULAR WEIGHTS 6751, 3652, 2827, 1568, 1118, 825, 630 3000, 1282 3000 3000, 1282

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01 - Forensic Escape Room: Design Your Own Biotech Adventure

Related Products Forensic Escape Room: Design Your Own Biotech Adventure For 10 groups. Explore the world of forensic science with this fun and exciting crime scene escape room! In this investigation, students decipher clues, solve puzzles, and unravel the evidence to free the innocent. Hands-on techniques include forensic blood detection, blood typing, and DNA fingerprinting. Comprehensive instructions on how to set up an escape room are included. Cat. #190 DNA Fingerprinting by PCR Amplification For 8 gels. Forensic DNA fingerprinting has become a universally accepted crime-fighting tool. Recent advances use the polymerase chain reaction (PCR) to amplify human DNA obtained from crime scenes. This experiment, based on a crime scene scenario, has an inquiry-based component. Cat. #130 Forensics Enhancement Techniques For 10 groups. Trace amounts of blood are often sufficient to identify the individual responsible for any number of crimes, including murder, burglary, or assault. Enhancement procedures can make a small stain of body fluid or tissue visible to the naked eye. In this experiment, students will act as detectives follow- ing the aftermath of a drug bust involving gang warfare over territory. Reagents that are routinely used as a first screen will be utilized to detect simulated blood and DNA. In addition, biological materials will be recovered from splatters, blood trajectory, and small droplets of simulated human materials. Cat. #194

Forensic Toxicology For 10 groups. In today’s forensic science labora- tory, toxicologists identify drugs and toxins in

samples collected from crime scenes, victims, and potential suspects. If present, the toxicologist also determines whether the drug or toxin contributed to a person’s behavioral changes or death. In this forensic science experiment, students will use the Enzyme Linked Immunosorbent Assay (ELISA) to analyze simulated crime scene samples for the presence of drugs. Cat. #195

Whose Fingerprints Were Left Behind? For 10 groups. After a crime has been committed, the evidence left behind can identify a potential culprit, although a single piece of evidence is not usually enough to convict someone. Even in this age of DNA, fingerprints and blood stains are still important at helping to identify a criminal. In this experiment your stu- dents will learn to detect and analyze fingerprints and then use these techniques to solve a classroom crime . Cat. #S-91

Details for all these products and MORE can be found on our website!

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01 - Forensic Escape Room: Design Your Own Biotech Adventure

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EDGE™ Integrated Electrophoresis System Runs one 10 x 7 cm gel Cat# 500

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DuoSource™ 100/150 V, for 1 or 2 Units Cat# 509

QuadraSource™ 10-300 V, for 1 or 4 Units Cat# 5010-Q

White Light LED Transilluminator Cat# 552

EDVOTEK® Variable Micropipette 5-50 µL Micropipette Cat. # 590

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Fixed Volume MiniPipette™ 35 µL MiniPipet™ Cat. # 587-2

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02 - Code Breakers: Using CRISPR to Rewrite Genetics

1155

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02 - Code Breakers: Using CRISPR to Rewrite Genetics

Introduction Unleash the power of gene editing with your students using REAL CRISPR-Cas technology to knock out colorful genes in E. coli. Identify successful knockout based on the cell color. Experi- ment by switching RNA templates and analyzing results, letting your students prove the specific - ity of CRISPR! Background Information CRISPR is a game-changing gene-editing tool that is catalyzing a wave of innovations across fields as diverse as biotechnology, medicine, agriculture, and energy. This system evolved in bacteria at the beginning of evolutionary history as a defense against viral attacks and was discovered by humans in the late 1980s. However, it would take scientists another 20 years to harness CRISPR as a precise and versatile gene editor. Today, CRISPR’s unmatched ability to rewrite the code of life has captured the imagination of re- searchers and the public alike. CRISPR technologies are used to cure diseases like cancer and sickle cell anemia, develop antibiotics, study complex genetic traits, understand evolutionary history, create drought-resistant crops, and develop new materials like bioplastics and biofuels. As CRISPR continues to advance its transformative potential will be limited only by our imagination and by the regulations we create. WHERE DOES THE NAME CRISPR COME FROM? CRISPR, short for Clustered Regularly Interspaced Short Palindromic Repeats describes uniquely patterned regions of bacterial DNA. An enzyme tightly linked to these CRISPR region was identified by scientists and called Cas for “CRISPR-associated proteins”. In bacteria and archaea, Cas enzymes cut and destroy foreign DNA, guided by the nucleotide sequences stored in CRISPR regions. Cas enzymes

are central to new DNA editing technologies but the CRISPR region itself is not. Never the less the CRISPR name has be- come synonymous with these technologies. EXPLORING THE CRISPR-CAS SYSTEM At the heart of CRISPR gene editing are two molecules: (1) an enzyme called Cas and (2) a

Figure 1: Bacterial CRISPR Region.

piece of RNA known as guide RNA (gRNA). These molecules collaborate to form a complex capable of identifying and cutting a specific DNA sequence within a genome, known as the target DNA. Following a cut, a cell’s intrinsic repair mechanisms mend the targeted DNA often with the help of additional DNA instructions, called template DNAs, that the experimenter provides. This repair process leads to changes in the DNA sequence that can induce a loss of function mutation, correct a mutation, or introduce a new trait in an organism. Cas proteins are endonucleases. These are enzymes that cut DNA by breaking the phosphodiester bonds between nucleotides. Some endonuclease cut DNA nonspecifically, but most cleave at specific nucleotide sequences called target sites. Cas enzymes fall into this latter category. They target specific sites within a DNA molecule and create a double-strand break. However, unlike most endonucle- ases (like restriction enzymes), which have a target DNA sequence built into their structure, CRISPR outsources its specificity to interchangeable molecules of guide RNA. This means that Cas enzymes can be programmed to cut any DNA region by creating guide RNA molecules that are complementary to the target’s sequence.

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02 - Code Breakers: Using CRISPR to Rewrite Genetics

Guide RNAs (gRNA), sometimes called single guide RNA (sgRNA),are the second key component for CRISPR gene editing. Serving as ‘molecular guides’, they direct the Cas enzymes to a specific target site within a genome. Guide RNA molecules consist of two functional parts. The first is a scaffolding sequence where the Cas enzyme binds. The second is an approximately 20-nucleotide sequence which determines where the DNA will be cut. Through base-pairing interactions, this nucleotide

sequence aligns with the target DNA and brings the Cas enzyme to the intended cut site. To ensure proper interactions between the gRNA - CAS complex and the target DNA, there’s an ad- ditional requirement: a nearby DNA sequence known as a protospacer adjacent motif, or PAM, sequence in the organism’s DNA. This is a sequence of two to six nucleotides found immediately downstream of the DNA sequence targeted

Figure 2: Target DNA and PAM site.

Cas9

gRNA

by the gRNA. PAM sequences allow the CAS enzyme to properly bind to, scan, and cut DNA. However, they also limit what DNA regions can be targeted. For example, the popular CRISPR enzyme Cas9 requires a 5′-NGG-3′ PAM sequence (where “N” can be any nucleotide base). This means that for a researcher using Cas9 the genomic locations that can be targeted are restricted to those containing a downstream 5′-NGG-3′ sequence. Luckily, there are a range of CAS enzymes each with its own unique PAM sequences. Multiple CAS nucleases have been identi- fied. Cas9 is a prominent example. Originally discovered in Streptococcus pyogenes, Cas9 quickly gained popularity in CRISPR gene ed- iting due to its high efficiency and accuracy. Other commonly used CRISPR endonucle- ases include Cas3, Cas12, Cas13, casino, SuperFI-Cas9, and Cs7-100.

Binding to target

Double-stranded cut

Figure 3: CRISPR targeting and digestion of DNA.

The target DNA represents the final critical component for the CRISPR experiment. This is

the specific sequence of DNA that researchers aim to modify. During a CRISPR experiment, the CAS- gRNA complex scans an organism’s DNA by unwinding short segments of DNA and comparing these sequences to the gRNA’s 20 bp binding sequence. When the complex reaches a region of DNA that is complimentary, it attaches. Once attached the Cas enzyme cuts the DNA. THE FORK IN THE ROAD: HOMOLOGOUS DIRECT REPAIR AND NON-HOMOLOGOUS END JOINING Once a CAS enzyme has cut through the double stranded DNA, the cell fixes the damaged nucleic acid chain using its intrinsic DNA repair pathways. Researchers design their CRISPR experiments so that the cell repairs the DNA in one of two ways: using non-homologous end joining (NHEJ) or homologous direct repair (HDR). These two repair mechanisms produce different results, allowing researchers to change the target DNA in distinct ways.

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02 - Code Breakers: Using CRISPR to Rewrite Genetics

NHEJ s a simple but error-prone process that occurs naturally in cells and is used in the lab for knock- out experiments. These are experiments that study the function of a gene by inactivating it with a loss of function mutation and then observing the results. When Cas cuts through the DNA backbone, the cell’s most direct means of repair is to join the two broken ends of DNA back together. However, this process usually results in small deletions or insertions at the cut site which cause frameshift mutations that disrupt the function of the targeted region. Unlike classical knockout methods, which required screening thousands of random mutations, CRISPR knockout experiments allow research- ers to design guide RNA that automatically targets their gene of interest, streamlining the process by eliminating one of the most laborious and time-intensive steps. HDR is also a naturally occurring process but more precise, controlled, and complex than NHEJ. It involves mending the double-stranded break using a donor or template DNA as a reference. To work properly, this template DNA must contain regions that match the target DNA sequences on both sides of the break. In cells, this is often the sister chromosome, which shares the same sequence. In CRISPR experiments, researchers develop a repair template that matches the genomic DNA around the cut site. However, in between these homologous beginning and ending regions, scientists can introduce specific mutations or new genes. As the cell repairs itself, these insertions are transcribed from the template to the target DNA allowing for specific and user-designed changes to an organ - ism’s genome.

Figure 4 - Repairing DNA Using HDR

CRISPR-Cas targeting DNA

Corrected DNA sequence

Homologous DNA sequences

Homology Directed Repair (HDR)

Repaired DNA

Figure 4: Repairing DNA Using HDR

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02 - Code Breakers: Using CRISPR to Rewrite Genetics

Experiment Summary THE CELL •

LyphoCells™ E. coli Purple: The host bacteria have been genetically engineered to pro- duce a purple pigment protein in the presence of IPTG. The genome has been modified to contain the gene for T7 polymerase under the control of the lac operon. The bacteria have also been transformed with a plasmid containing the Purple Chromoprotein under the control of the T7 promoter. When IPTG is added it de-represses (activates) the lac operon which allows the cell to synthesize T7 polymerase. This polymerase binds to the plasmid and turns on production of the purple protein.

THE PLASMIDS •

pCas9-PurpleGuide: This plasmid contains the Cas9 gene and a gRNA that targets the purple chromo- protein gene. It also contains a chloramphenicol resistance gene. pCas9-ControlGuide: This plasmid contains the Cas9 gene and a non-targeting guide RNA with a random sequence. This sequence was carefully checked against the E. coli purple genome and confirmed to be absent, ensuring that it won’t target any genomic sites during the experiment. The plasmid also contains a chloramphenicol resistance gene.

•

THE PLATES This experiment uses three additives to the plates: • IPTG (Isopropyl β-D-1-thiogalactopyranoside):

This compound induces the expression of genes controlled by the lac operon. It mimics the natural inducer of the lac operon, lactose, but unlike lactose, it is not metabolized by the cell, making it a more controlled and reliable inducer for gene expression. • Chloramphenicol: A popular antibiotic and selective agent in bacterial culture media. Effective against a range of bacteria by inhibiting their protein synthesis. • Ampicillin : A broad-spectrum antibiotic belonging to the penicillin group that works by inhibiting the synthesis of bacterial cell walls.

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02 - Code Breakers: Using CRISPR to Rewrite Genetics

Designing gRNA to Target LacZ

1. IDENTIFY and HIGHLIGHT five PAM sites in the target sequence. For this experiment, as - sume that you are using a Cas9 enzyme which uses an 5’-NGG-3’ PAM site. In this notation, the “N” can be any nucleotide. This means that the Cas9 will only bind to sequences imme- diately upstream (in the 5’ direction) of an AGG, TGG, CGG, or GGG sequence. NOTE: Since Cas9 can bind to either of the complementary DNA strands, remember to examine both for PAM sequences. 2. IDENTIFY and UNDERLINE the binding regions upstream of your highlighted PAM sites. In CRISPR, the guide RNA recognizes and binds to 20 nucleotides on the DNA strand opposite from the NGG PAM site. Binding always occurs upstream (in the 5’ direction) of the PAM sites. 3. Using base pairing rules, CREATE a guide sequence for the gRNA. This sequence should bind to the underlined region identified in step 2. For complimentary binding, Adenine (A) pairs with Thymine (T) and Cytosine (C) pairs with Guanine (G). Remember that for the bottom strand, a 5’ to 3’ direction means recording the sequence from right to left. 4. USING steps 1-3, DESIGN five candidate gRNAs for the LacZ Gene Sequence (0-200 bp). RECORD your answers in Table 1.

EXAMPLE SEQUENCE: 5’-TTCACTGCGTTCAGCAAAAAGTGAATTCTTGGTTACTGC 3’-AAGTGACGCAAGTCGTTTTTCACTTAAGAACCAATGACG

LacZ Gene Sequence (0-200 bp) 5’- ATGACCATGATTACGGATTCACTGGCCGTCGTTTTACAACGTCGTGACTG 3’- TACTGGTACTAATGCCTAAGTGACCGGCAGCAAAATGTTGCAGCACTGAC GGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTT CCTTTTGGGACCGCAATGGGTTGAATTAGCGGAACGTCGTGTAGGGGGAA TCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAA AGCGGTCGACCGCATTATCGCTTCTCCGGGCGTGGCTAGCGGGAAGGGAA CAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTTCCGGCACC - 3’ GTCAACGCGTCGGACTTACCGCTTACCGCGAAACGGACCAAAGGCCGTGG - 5’

Guide Sequence TABLE 1

PAM Sequence

gRNA Name Example gRNA 1 gRNA 2 gRNA 3 gRNA 4

TTCAGCAAAAAGTGAATTCT

TGG

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02 - Code Breakers: Using CRISPR to Rewrite Genetics

Experiment Procedure: Transformation

150 µL ice-cold CaCl 2

10 µL Plasmid (See table 2)

MIX

30-60 min @ 37°C

45 sec. @ 42°C

150 µL SOC to each tube.

Gently flick to mix.

NOTE: Keep tubes on ice as much as possible during this module. 1. COLLECT your two tubes of competent cells. LABEL them "1" and "2". ENSURE that the cells are kept on ice at all times. 2. ADD 150 µL ice cold CaCl 2 solution to each tube and MIX by gently pipetting up and down several times. 3. Using a new pipette tip for each tube, ADD 10 µL of the appropriate plasmids to each tube (see Table 2). MIX by gently pipetting up and down several times. 4. INCUBATE tubes on ice for 15 minutes. 5. Quickly PLACE the transformation tubes in a 42°C water bath for exactly 45 sec- onds. 6. Immediately RETURN the tubes to the ice bucket and INCUBATE for 2 minutes. 7. Using a new pipette tip for each tube, ADD 150 µL of SOC solution to each tube. MIX by gently flicking each tube. 8. PLACE tubes in a 37°C water bath and INCUBATE for 30-60 minutes to allow for recovery. continued

TABLE 2: Experiment Overview

Bacteria Strain

Plate

Plasmid

Experiment 1 2

E. coli Purple E. coli Purple

Amp/IPTG/Chloramphenicol Amp/IPTG/Chloramphenicol

pCas9-PurpleGuide “PG” pCas9-ControlGuide “CG”

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02 - Code Breakers: Using CRISPR to Rewrite Genetics

Experiment Procedure: Transformation

150 µL

REMOVE 250 µL

MIX

5000 rpm

ROTATE & SPREAD

SPREAD the cells

VISUALIZE and RECORD

48 hours @ 37°C

COVER

9. While cells are recovering, COLLECT and LABEL the bottom of two agar plates as indicated below. NOTE: Keep writing small and along the edge so you can easily see your colonies at the end. • 1 or "PG" for pCas9-PurpleGuide • 2 or "CG" for pCas9-ControlGuide 10. Following recovery, SPIN the tubes at 5000 rpm for 1 minute. 11. PLATE your two transformed cell lines by performing the following five steps one tube at a time. Use a new sterile pipet tip and loop for each tube/plate. (a) REMOVE 250 μL of the media. Work carefully during this step to making sure the cell pellet stays in place. (b) Gently RESUSPEND the pellet in the remaining media by pipetting up and down several times until no clumps are visible. (c) COLLECT 150 μL of the resuspension and PIPET into the center of the ap- propriate agar plate. (d) Use a loop to SPREAD cells evenly and thoroughly over the entire surface. (e) TURN the plate 90° and thoroughly SPREAD again. 12. COVER the plates with lids and INCUBATE at room temperature for 5 minutes to allow the liquid to be fully absorbed. 13. STACK the plates on top of one another and TAPE them together. INVERT plates (agar side on top) and PLACE in a 37°C bacterial incubation oven. INCUBATE for 48 hours. If you do not have an incubator, colonies will form at room tempera- ture in approximately 48-72 hours, but an incubator is HIGHLY recommended. 14. VISUALIZE the transformation and control plates and RECORD the number of colonies on the plate, the color(s) of the colonies (such as dark purple, light purple, white), and the number of colonies of each color. If the colors are faint, the plates can be left in the refrigerator (4°C) for 24-48 hours to for further color development.

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02 - Code Breakers: Using CRISPR to Rewrite Genetics

Experiment Results and Analysis

EXPERIMENT 1 Bacteria: E. coli Purple Introduced Plasmid: pCas9-PurpleGuide gRNA/CRISPR Target: purple chromoprotein gene Final Colony Color: White Did CRISPR occur? Yes. The colony color change from purple to white suggests that E. coli Purple went from producing a purple pig- ment to no longer producing a purple pigment. This suggests that the CRISPR-Cas9 system was able to disrupt the purple chromoprotein gene. Total number of colonies: 95

Number of dark purple colonies: 0 Number of light purple colonies: 0 Number of white colonies: 95

EXPERIMENT 2 Bacteria: E. coli Purple Introduced Plasmid: pCas9-ControlGuide gRNA/CRISPR Target: Random sequence not present in E. coli Purple Final Colony Color: Purple Did CRISPR occur? No. The colony color did not change. This suggests that this CRISPR-Cas9 system was not able to disrupt the purple chromoprotein gene responsible for the bacteria’s purple

appearance. This result also confirms that the color change observed in experiment 1 was not solely due to the transformation process (such as an unexpected by-product of intro- ducing the chloramphenicol resistance gene or another gene on the plasmid). Addition- ally, it demonstrates that Cas9 was unable to cut the purple chromoprotein gene without a specifically designed guide RNA. Total number of colonies: 91

Number of dark purple colonies: 91 Number of light purple colonies: 0 Number of white colonies: 0

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02 - Code Breakers: Using CRISPR to Rewrite Genetics

Experiment Results and Analysis