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1. @miniPCRfb.com/miniPCR@miniPCRCHOPPED!Using CRISPR/Cas9 to cut DNA

2. Today’s labIn this lab, you will take a close-up look at the molecular machinery that makes CRISPR/Cas such a powerful genome editing tool!

3. Genome editingChanging a specific sequence of DNA inside a living cell is called genome editing Genome editing has many applications such as investigating how genes work or correcting mutations that cause disease Until recently, genome editing was very difficult to perform and could only be carried out in a few select organisms.

4. CRISPR/Cas revolutionized genome editinghttps://www.nobelprize.org/prizes/chemistry/2020/press-release/Easily programmable: Scientists can target virtually any DNA sequence Highly specific: Unlikely to target more than one region in a genome.Widely adaptable: Can theoretically be used to target DNA in any organism.Emmanuelle Charpentier and Jennifer Doudna won the 2020 Nobel Prize for their work on CRISPR/Cas

5. How CRISPR/Cas worksThe CRISPR/Cas system has 2 main components:Cas protein: a nuclease that cuts DNA – Cas9 is most commonly usedGuide RNA: a small RNA that binds to Cas9 and controls where Cas9 will cut the DNA Cas9 protein (red)Guide RNA(yellow)

6. The Cas9 nuclease cuts DNANucleases are enzymes that cut DNA or RNACas9 nucleases cut through both strands of DNACas nucleases are useful because they can be directed to cut specific DNA sequences Cas9 protein (red)

7. A guide RNA controls where Cas9 cutsGuide RNAs (gRNA) have two parts:Scaffold – the folded loop that binds to the Cas9Spacer – the section that finds and binds to the target DNA sequenceSpacerScaffold

8. Cas9 and gRNA bind to form a complex<Click to play>

9. Cas9 and gRNA bind to form a complexgRNA is aligned to DNA sequence

10. If all 20 bases are complementary, Cas9 nuclease cuts both strands of target DNAgRNA is aligned to DNA sequence

11. DNA not cutDNA cut

12. The power of CRISPR/CasProgrammable - scientists design custom guide RNAs, allowing them to target Cas9 to different DNA sequencesSpecific - Cas9 should only cut the DNA when the guide RNA binds to a complementary sequence DNA cut

13. CHOPPED! Using CRISPR/Cas to cut DNAThe goal of today’s lab is to use CRIPSR/Cas9 to target and cut a DNA sample in a tube (in vitro)We’ll use two different gRNAs to investigate how the Cas9 nuclease can be programmed to specifically target a DNA sequence—just by changing the guide RNA.

14. Experiment overviewStep 1: Make DNA cutting predictions based on gRNA sequencesStep 2: Form Cas9/gRNA molecular complexes and mix with DNAStep 3: Analyze cut DNA using gel electrophoresisDNAgRNACas9gRNADNA

15. First , let’s predict where the gRNAs will targetWe will be targeting a DNA sample that is 3,142 base pairs (bp) longWe will be using two different gRNAs, each targeting a different 20 bp section in the DNA sampleWhen the gRNA is aligned with the complementary sequence in the DNA sample, the Cas9 will cut the DNA into two fragments

16. Paper model for predicting DNA fragmentsA paper model of the DNA sample and the two gRNAs is provided in your lab guideUse the paper model to find where the gRNAs are complementary to the DNA sample This will tell you where the Cas9 will cut the DNA

17. Paper model for predicting DNA fragmentsAfter determining where the gRNAs will target and guide the Cas9 to cut, we will calculate the size of the two resulting fragments.Base pair where DNA is cutFragment 2Fragment 1bp 1bp 3142

18. Next, let’s set up our CRISPR/Cas9 reactionsCas9DNADNADNADNAgRNA1Cas9gRNA2Cas9

19. Protocol: part ALabel tubesMix Cas9, gRNA, reaction buffer, and water as directed by the lab protocolIncubate 10 minutes at room temperature

20. Protocol: part BAdd DNA sample as directed by the lab protocolIncubate 15 minutes at 37 °C

21. Protocol: part CAdd Proteinase K solution as directed by the lab protocol

22. Option A: Stop here for the dayStore your samples in the freezer.In the next class period, samples can be thawed and used immediately for gel electrophoresis. Note: If freezing your sample overnight, there is no need to incubate tubes at 37 °C after adding proteinase K as indicated in Option B.Protocol: part C

23. Protocol: part COption B: Run your samples on the gel in the same class periodIncubate your samples at 37 °C for at least 10 minutes. Before running your gel.Longer incubation times (up to 20 minutes) may result in clearer gel results.

24. Analyze DNA fragments with gel electrophoresis Load your samples on a 1% agarose gel with DNA stainLane 1: 10 μl of Fast DNA Ladder 3Lane 2: 15 μl of Tube ALane 3: 15 μl of Tube BLane 4: 15 μl of Tube CLane 5: 15 μl of Tube DConduct electrophoresis until you observe clear separation between the bands in your gel.

25. Interpreting our resultsThe Cas9 nuclease cut at specific points in the DNA sample depending on which gRNA guided itDid these match up with your predictions?

26. Why are there three bands?Due to many potential reasons, the reactions may not be complete and not all the DNA sample in the tube will be cut by the Cas9The uncut DNA will show up at ~3142 bp (the same as your negative controls)The dimmer the uncut band, the more complete your reaction

27. ConclusionsHow did todays lab demonstrate that CRISPR/Cas9 is highly specific?How did todays lab demonstrate that the Cas9 enzyme is programmable?Why were tubes A and B important?Which component of the Cas9/gRNA complex is responsible for directing where the DNA will be cut? How do you know?

28. What happens after cutting?Today’s lab only cut the DNA, but that alone doesn’t change the DNAIn an organism, after the DNA is cut, the cell’s repair mechanisms will typically repair the double strand breakScientists use these repair mechanisms to introduce changes to the DNA

29. CRISPR/Cas resource library Free to downloadComprehensive background and study questions http://minipcr.com/CRISPR