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INTRODUCTIONPART 1: CRISPR-Cas9 MechanismStep 1: TargetingStep 2: BindingStep 3: CleavingStep 4: DNA RepairPART 2: Inactivating Genes in ButterfliesPART 3: Designing a Guide RNAINTRODUCTIONScientists have determined the complete DNA sequences of the genomes for many organisms, including humans.By analyzing patterns in those sequences, they can estimate how many genes an organism has — humans, for example, have about 20,000. But sequence patterns alone don’t specifically show what each gene does. How canwe figure this out? In this activity, you will explore a tool that can be used to determine a gene’s function. You will then design your own version of this tool to examine genes that affect the colors and patterns on butterfly wings.CRISPR-Cas9, or CRISPR for short, is a biotechnology tool that can edit or inactivate specific genes. To learn more about the CRISPR-Cas9 system, you can explore the “How It Works” section of the CRISPR-Cas9 Mechanism and Applications Click & Learn.As shown in the class, the CRISPR-Cas9 tool uses a DNA-cutting enzyme, or nuclease, called Cas9. Cas9 was discovered in bacteria as a way for bacteria to fight off viruses. Scientists combine Cas9 with an RNA molecule called a guide RNA to form a Cas9-RNA complex. Part of the guide RNA matches a target DNA sequence within the gene that the scientists want to inactivate. You will now go through each step in the process of using CRISPR-Cas9 to inactivate a gene, using a sequence from an actual gene as an example.PART 1: CRISPR-Cas9 MechanismStep 1: TargetingFirst, the Cas9-RNA complex recognizes and binds to a three-nucleotide sequence called PAM, which stands for “proto-spacer adjacent motif.” PAM sequences are abundant throughout the genome and can occur on either strand of DNA. Every PAM sequence has the form 5'-NGG-3', where the “N” can be any DNA nucleotide (A, C, G, or T). 1. The partial gene (DNA) sequence below contains multiple PAM sequences. Highlight six PAM sequences in the top (5’ to 3’) strand.Once Cas9 binds to a PAM sequence, it unwinds the DNA. If the guide RNA matches the DNA sequence next to the PAM, the guide RNA will bind to the complementary DNA strand. If not, the DNA will zip back together and Cas9 will keep binding to other PAM sequences until it finds the matching target DNA.5'-GCACGGCGGAGCGGTTCTTGGCAGCGGCCGCACGATCTCGTTGCCGCCGG-3'3'-CGTGCCGCCTCGCCAAGAACCGTCGCCGGCGTGCTAGAGCAACGGCGG2. Below is a partial sequence of a guide RNA. The underlined section of the RNA is designed to match a specific target DNA sequence.Review the partial gene sequence reshown below. It contains a target DNA sequence that matches the guide RNA above. Highlight the one PAM sequence in the top (5’ to 3’) strand that is next to this target DNA sequence. (The sequence upstream, toward the 5’ end, of this PAM should match the underlined sequence in the guide RNA, which makes the opposite DNA strand complementary to the underlined sequence. Remember that U’s in RNA are equivalent to T’s in DNA.)Step 2: BindingOnce Cas9 binds to the correct PAM, the guide RNA binds to the complement of the target DNA sequence.3. Write down the guide RNA sequence that binds to the DNA, and the DNA sequence that it binds to (the complement of the target DNA). Label the 5' and 3' ends for both the RNA and DNA strands.Step 3: CleavingOnce the guide RNA binds to the complement of the target DNA sequence, it activates the nuclease activity (DNA-cutting ability) of the Cas9 enzyme. Cutting DNA is also called “cleaving.” Cas9 always cleaves both strands of DNA. It cleaves both the target DNA and its complement three nucleotides upstream (toward the 5’ end) of the PAM sequence.4. Rewrite the target DNA sequence and its complement below, indicating where Cas9 would cut both strands of DNA with a large space or vertical line (|).Step 4: DNA RepairAfter Cas9 cleaves the DNA, cellular enzymes will attempt to repair the break. Most of the time, these enzymes repair the DNA without errors. However, Cas9 will keep cutting the DNA at the same location until an error is made. 5. DNA repair errors include losing or inserting random nucleotides at the cut site. Explain how these changes might inactivate a gene.*****Return to main room for second part of lab*****5'-GGCGGAGCGGUUCUUGGCAGGUUUUAGAGCUAGAAAUAGC-3'5'-GCACGGCGGAGCGGTTCTTGGCAGCGGCCGCACGATCTCGTTGCCGCCGG-3'3'-CGTGCCGCCTCGCCAAGAACCGTCGCCGGCGTGCTAGAGCAACGGCGGPART 2: Inactivating Genes in ButterfliesRobert Reed, a biologist at Cornell University, wanted to identify genes that are important in butterfly wing patterns. He and his colleagues used CRISPR-Cas9 to inactivate different genes, then observed the effects on butterflies. Models of three genes they inactivated are shown in Figure 1.optix gene:spalt gene:Distal-less gene:Figure 1. Models of three genes involved in butterfly wing patterns: optix, spalt, and Distal-less. The shaded rectangles represent exons, and the horizontal lines between the rectangles represent introns. The black triangles above the rectanglespoint to the target DNA sequences, which match the guide RNAs made by the scientists. The arrows at the start of each gene represent the transcription start sites.1. What are exons and introns, and how do they differ?2. Figure 1 shows that the target DNA sequences are all in exons. Why might scientists want to target sequences in exons, rather than introns, when inactivating genes?PART 3: Designing a Guide RNAYou will now design your own guide RNA to inactivate a butterfly gene like Robert Reed did. Your goal is to knock out the optix gene in a species of butterfly called the painted lady (Vanessa cardui).Your guide RNA must match a target DNA sequence in the gene that you want to knock out. A partial sequence of an exon from the butterfly’s optix gene is shown below.1. Underline a 20-nucleotide target DNA sequence in the top (5’ to 3’) strand of the exon above. Remember that this sequence should be directly upstream (on the 5’ end) of a PAM sequence (5’-NGG-3’).2. Highlight the PAM sequence that is next to your target DNA sequence. This is where Cas9 will bind.3. Rewrite the target DNA sequence and its complement below, indicating where Cas9 would cut both strands of DNA with a large space or vertical line (|).4. Record the 20-nucleotide guide RNA sequence that matches your target DNA sequence. This sequence should not include the PAM.5. Robert Reed’s lab at Cornell University designed

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