Module 9 Lab Notebook - Restriction Digest and Genotyping done

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Genetics lab notebook entry #10 Genotyping small indels via restriction digest Background Previously, we’ve selected sgRNAs for CRISPR mutagenesis in our target genes. This week we will carry out a digest using restriction enzymes to simulate Cas9-mediated cutting and also discuss the role of restriction digest in genotyping alleles with indels too small to easily observe on a gel. Restriction enzymes (also known as restriction endonucleases or, less commonly, restrictases) are a class of enzyme that recognize specific DNA sequences and cleaves DNA in a specific way. For instance, the enzyme EcoRI will always recognize the following sequence and cut it as indicated: 5’ … G AATTC … 3’ 3’ … CTTAA G … 5’ Many restriction enzymes result in the formation of “sticky ends,” which allow DNA strands to be reconnected by DNA ligase . This can be used to synthesize new DNA molecules from different component parts in a process known as molecular cloning. Restriction enzymes are commonly used to genotype CRISPR mutations or other small differences in DNA sequences, typically SNPs or small indels. Two alleles that differ by a SNP or small indel may be indistinguishable on a gel but the differences in their sequences may result in one fragment being cut by a restriction enzyme while the other is not. In this lab we will use restriction enzymes to cut our amplicons. Each enzyme has been chosen to cut near a possible sgRNA in each sequence (i.e., each enzyme is near a PAM) and you will be left to determine what possible sgRNA would result in a similar band pattern to the one you see on the gel. Digest 1. To begin, we must first digest our amplicons with the appropriate restriction enzymes. In 100ul tubes, set up the following reactions: Gene 1 digest Gene 2 digest 2µl enzyme Hpy99I BfaI 2µl buffer - - Up to 10µl PCR Gene 1 PCR Gene 2 PCR Fill to 20µl H 2 O Volume = 6 µl Volume =6 µl

Figure 1  MW - Ladder (See sizes on the left side of the image)  Lane 1 - Gene 1 Digest with Hpy99I  Lane 2 - Gene 2 Digest BfaI  Lane 3 - Gene 1 Control (No Digest Enzyme added)  Lane 4 - Gene 2 Control (No Digest Enzyme added)  MW - Ladder (See sizes on the left side of the image)  Lanes 5-8 Empty 2. Place the reactions in a thermal cycler with the following conditions: 1. 37 o C for 1 hour 2. 80 o C for 20 minutes 3. During this time, you may pour a gel as described in steps 1-5 of the gel electrophoresis lab manual. Alternatively, your TA may elect to complete this step for you. You may also move on to the virtual component of this lab while you wait.

4. When the gel is ready, add loading dye to your reactions and load the entirety of your reactions into the gel. If you still have PCR left over, run up to 10ul of undigested product as a control. A good pattern to load your gel in might be as follows: Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Ladder Digest 1 Control 1 Digest 2 Control 2 5. Run the gel at 150V for at least 30 minutes or until your bands are easily resolved. Take an image of your gel and include it below the appropriate prompt at the end of the lab manual. Remember to label each lane and write the approximate size next to each band. Virtual digest 1. Go to http://www.restrictionmapper.org/ to perform your virtual digest. Select your enzyme (e.g., Hpy99I for gene 1) and paste the corresponding amplicon below “paste sequence here.” Press “virtual digest” to run your digest. Record the fragment lengths in Table 1 at the end of the lab manual. Repeat these steps for gene 2. 2. Go to https://www.neb.com/ and look up the two enzymes we’ve used today—Hpy99I and BfaI. Record below their characteristic cutting sequences. Note that the DNA code includes letters beyond A, T, C and G. For instance “W” stands for “weakly” binding bases like A and T, while “S” stands for bases that form “strong” bonds like C and G. When you see one of these letters, it can mean any base in that category. Record the cut sites in Table 2 at the end of the lab manual. 3. On your gene models, highlight the corresponding cut sites and save as a new feature. Name these features after each enzyme. Take a screen cap of the graphical map and include it below the appropriate prompt at the end of the lab manual. Table 1: Record the expected fragment lengths for digestion of the Gene 1 and Gene 2 amplicons here. Fragment 1 length (bp) Fragment 2 length (if any) Gene 1 control 289 184 Gene 1 digest 293 180 Gene 2 control 270 193 Gene 2 digest 280 190

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1. Paste your labelled gel image below.

2. Do your results match your predictions from the virtual digest? If not, please explain the discrepancy in your results. The results do match the prediction the two fragment base pair lengths were very close to the predicted lengths. Table 2: Record the cut site for each enzyme here. Cut site Hpy99I CGACG, CGTCG BfaI CTAG 3. Take a screen cap of the graphical map with your highlighted cut sites and include it below.

4. Do the cut sites you’ve found for Hpy99I and BfaI match the sgRNAs you picked earlier? If not, what sgRNA might you choose in light of this new information? Write the sequence of the sgRNAs below. Yes the cut sites match and the length of the fragments match the predicted lengths. 5. Use the following information to answer a genotyping question. Say you are genotyping two animals at one of the loci we are studying. One animal carries a small indel (~1bp) due to CRISPR mutagenesis, while the other animal is wildtype. Before mutagenesis, you picked your sgRNA such that it contains a restriction site immediately 5’ of its PAM in the WT sequence. Upon digestion, one of the individuals digests at this cut site while the other does not. Which result, if any, indicates successful mutagenesis? Explain. The individual that did not digest or get cut signifies a successful mutagenesis because the indel shifted the sequence thus not allowing the enzyme to cut because the cut sequence was no longer there.

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