Knocking out Genes Required for DNA Repair in Plant Mitochondria using CRISPR-Cas9

David Campbell Author

08/04/2020 Added

24 Plays

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CRISPR-Cas9 was used to generate knockout mutants in genes involved in DNA repair in plant mitochondria

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[00:00:01.497]Hi everyone, my name is David Campbell
[00:00:04.112]and I'm a senior biochemistry student
[00:00:05.862]at the University of Nebraska Lincoln
[00:00:08.258]Over the summer, I've been working
[00:00:09.713]on a research project through
[00:00:11.142]the UCARE program.
[00:00:12.079]It's called "Knocking out genes required for
[00:00:14.286]DNA repair in plant mitochondria using
[00:00:15.982]CRISPR-Cas9".
[00:00:20.770]I work in the Christensen Lab, in the
[00:00:22.815]Beadle Center.
[00:00:24.019]I wanted to start of by telling you a bit
[00:00:25.999]about our research interests as a whole
[00:00:28.087]before I go into my specific project.
[00:00:30.787]We're interested in plant mitochondria.
[00:00:33.206]Specifically, how they maintain their genome.
[00:00:36.082]There are a number of structural features
[00:00:38.062]that seem to indicate that the way they
[00:00:39.642]do this is really unique.
[00:00:42.016]First, mitochondrial genomes are normally
[00:00:44.057]very small.
[00:00:45.277]You'll see that the human mitochondria,
[00:00:46.977]for instance contains around 16.5 thousand
[00:00:49.539]base pairs of DNA. Everything but the bare
[00:00:52.729]essentials tend to migrate to the nuclear
[00:00:55.243]genome over time.
[00:00:56.794]In terms of how many genes are involved,
[00:00:59.049]plant mitochondria tend to follow this rule.
[00:01:02.323]But, you can see that their sizes present
[00:01:04.818]a major discrepancy.
[00:01:06.134]There seems to be a lot of junk DNA inflating
[00:01:09.085]the size of the genome, while serving no
[00:01:11.297]obvious purpose.
[00:01:13.752]We're interested in what forces cause plant
[00:01:16.089]mitochondrial genomes to get this big,
[00:01:18.382]when a lot of the DNA doesn't seem to be
[00:01:20.372]doing anything.
[00:01:23.348]Part of the reason that genes tend to migrate
[00:01:25.598]to the nuclear genome is that mitochondria
[00:01:28.579]are a really bad place to store DNA.
[00:01:31.615]Through their metabolism, mitochondria
[00:01:34.328]produce a lot of high-energy molecules that
[00:01:36.971]can absolutely wreak havoc on the genome.
[00:01:40.525]Mutation rates tend to be higher in
[00:01:42.478]mitochondria because of this.
[00:01:44.697]In plant mitochondria, though, we observe
[00:01:46.831]lower mutation rates than either the
[00:01:48.933]nucleus or the chloroplasts.
[00:01:50.520]Instead, we see frequent recombination.
[00:01:54.072]You can see in this diagram how, though
[00:01:56.105]the sequence of DNA doesn't change,
[00:01:58.198]large sections of DNA can move around
[00:02:00.956]to create a different overall structure.
[00:02:06.686]For us, the most likely cause of these
[00:02:09.306]unique features seems to be plant mitochondria's
[00:02:12.646]DNA repair pathways. The use of different
[00:02:15.228]repair methods, like double strand break
[00:02:17.748]repair, for instance, could cause the low
[00:02:19.899]mutation rate and high recombination rate
[00:02:22.101]that we've observed.
[00:02:25.110]In order to study these repair pathways,
[00:02:27.094]it's necessary for us to have knockout mutants
[00:02:29.204]of the genes involved.
[00:02:30.839]Knockout mutants are organisms that have
[00:02:32.968]a small variation in a gene that stops them
[00:02:35.900]from performing their normal tasks.
[00:02:39.450]An msh1 knockout mutant wouldn't be able to
[00:02:41.519]produce the protein encoded by the gene,
[00:02:44.039]or it would produce a protein that is
[00:02:46.328]completely ineffective at doing what it
[00:02:48.559]normally does.
[00:02:49.963]This allows us to investigate what exactly
[00:02:52.022]the gene is doing inside the cell.
[00:02:54.759]Creating double mutants is also desirable,
[00:02:56.906]because it helps us discover more about
[00:02:58.600]the relationships between different genes
[00:03:00.835]and their functions.
[00:03:03.821]To create these knockout mutants, we used
[00:03:05.882]CRISPR-Cas9. This is a bacterial system
[00:03:09.022]that allows us to make cuts in DNA at very
[00:03:11.439]specific sites. The CRISPR-RNA or crRNA
[00:03:15.641]contains the target sequence which determines
[00:03:18.398]where we make the cut.
[00:03:19.655]This binds to the Cas9 protein through the tracrRNA
[00:03:22.966]and the Cas9 protein makes the cut.
[00:03:28.642]Once the cut is made, though, we don't
[00:03:30.380]for sure have a knockout. There are a number
[00:03:33.624]of different ways the DNA can come back
[00:03:35.814]together, including going back to the
[00:03:37.669]original arrangement.
[00:03:38.964]To prevent this, we used a special gRNA
[00:03:41.233]scheme that allows us to select for two
[00:03:43.820]targeting sites in the same plasmid.
[00:03:48.610]This slide shows the general workflow of
[00:03:50.813]the project. First the constructs are designed,
[00:03:53.962]assembled in E. coli, and transformed into
[00:03:56.403]Agrobacterium, which is a plant pathogen
[00:03:59.622]that specializes in getting DNA into
[00:04:01.551]plant cells.
[00:04:02.484]Then, we can select for successful
[00:04:05.127]transformants using BASTA, or glufosinate
[00:04:07.580]ammonium. This is a fairly common herbicide.
[00:04:11.770]We can confirm transformants, then, using
[00:04:13.866]PCR and gel electrophoresis.
[00:04:18.710]The DNA that we inserted into successful
[00:04:20.940]transformants contains a small piece which
[00:04:23.496]confers resistance to the herbicide BASTA,
[00:04:26.384]which would normally kill Arabidopsis plants
[00:04:29.251]By spraying potential transformants with
[00:04:31.531]BASTA, we can kill everything that doesn't
[00:04:33.747]have that DNA inserted.
[00:04:35.675]You can see from this tray that a
[00:04:37.270]relatively low number of seeds actually
[00:04:39.029]ended up being transformants. But, with a
[00:04:42.107]rate of about 0.5-1% expected, that's
[00:04:47.564]relatively normal.
[00:04:51.869]To confirm transformants, we used PCR and
[00:04:54.375]gel electrophoresis. These tools allow us
[00:04:57.211]to check if a specific sequence of DNA
[00:04:59.897]is present in a sample. Primers attach to
[00:05:03.350]specific sequences in the DNA and
[00:05:05.422]an enzyme called a polymerase makes a copy
[00:05:08.067]of everything between the primers.
[00:05:09.909]If the DNA we inserted is present, we'll see
[00:05:12.330]a band on the gel. The location of the band
[00:05:15.132]has to do with the size of the DNA that was
[00:05:17.108]copied. Larger fragments move slower, and
[00:05:20.206]are found closer to the top of the gel.
[00:05:22.380]The leftmost column is called a ladder,
[00:05:25.723]which shows us that the two bands are at
[00:05:28.154]about 1.9Kb in size, which is what we expect
[00:05:31.783]our plasmid to be.
[00:05:33.990]The band in lane 2 is one of our plant samples,
[00:05:37.085]and the band on the right is a positive
[00:05:39.168]control, a pure sample of the plasmid we
[00:05:41.339]inserted
[00:05:42.710]On the right, you'll see a potential
[00:05:46.930]transformant showing signs of the knockout
[00:05:49.220]mutation. Leaf-curling is a phenotype
[00:05:52.352]sometimes seen in msh1 knockouts, Lending
[00:05:55.559]further evidence that our procedure is
[00:05:57.543]actually working
[00:06:01.160]Based on this, it seems that CRISPR-Cas9
[00:06:03.727]was an effective way to generate knockout
[00:06:05.837]mutants in DNA repair genes in Arabidopsis.
[00:06:09.459]Continuing this research, we'll use this
[00:06:11.019]method to make more knockout mutants,
[00:06:12.931]including double mutants, so we can look at
[00:06:15.590]how these effect the mutation and
[00:06:17.294]recombination rates in plant mitochondria.
[00:06:20.483]Thank you for listening and have a great day!

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Knocking out Genes Required for DNA Repair in Plant Mitochondria using CRISPR-Cas9

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