Using CRISPR-Cas9 to Construct Knockout Mutants in DNA-Repair Genes in Arabidopsis thaliana

David Campbell Author

03/28/2021 Added

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Knockout mutants of DNA repair genes were generated in Arabidopsis thaliana. These mutants will be used to analyze the effect of double strand break repair on mutation and recombination

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[00:00:02.120]Hi, my name is David Campbell,
[00:00:03.910]and I'd like to tell you a little bit about my project using CRISPR-Cas9
[00:00:07.960]to construct Knock-Out mutants in DNA repair genes in Arabidopsis thaliana.
[00:00:12.920]This is a project completed under the direction of Dr.
[00:00:16.040]Alan Christensen and funded in part through the UCARE program.
[00:00:20.480]To start, I'd like to tell you a little bit about our background as a lab why
[00:00:24.360]we're interested in plant mitochondrial genetics.
[00:00:28.000]First of all, plant mitochondria are very large
[00:00:32.280]in comparison to most mitochondrial genomes.
[00:00:34.560]If you see this chart on the right
[00:00:36.000]and most of them range from 200 to 500 Kb with some exceptional species
[00:00:41.080]in the 6000 to 10000 Kb range, which is really exceptional considering
[00:00:46.800]that the human mitochondrial genome is about sixteen point five Kb.
[00:00:51.920]In addition, the mitochondrial mutation rate is very strange.
[00:00:57.040]We expect it to have a high mutation rate as a result of oxidative energy generation
[00:01:02.200]causing DNA damage, what we actually observed is a lower
[00:01:05.840]mutation rate in the mitochondria than either the nucleus or the chloroplast.
[00:01:10.280]In addition, we observe a high rate of recombination in plant mitochondria
[00:01:14.840]where the structure and order of genes is changing very rapidly.
[00:01:19.640]So the big idea in our lab is that the DNA
[00:01:22.280]maintenance and repair pathways are to blame for these phenomena.
[00:01:25.680]And specifically, we're looking at double strand break repair as a way to explain
[00:01:30.520]both the low mutation and the high recombination rates in plant mitochondria.
[00:01:36.080]So in the study we're investigating four genes; MSH1 and RecA3 are
[00:01:41.120]both known to be involved in mitochondrial recombinational surveillance.
[00:01:45.840]SUV3 is a gene that showed up
[00:01:48.400]on a mutant screen for a mutant unable to repair double strand breaks
[00:01:53.000]and RNAseH1B in yeast cells works with Suv3 to resolve DNA RNA hybrid's.
[00:02:01.960]So part of this is looking to see whether
[00:02:04.400]it performs those same functions in mitochondria.
[00:02:08.800]So we'd like to use knockout mutants
[00:02:11.680]of these genes to analyze mutational and recombination rates.
[00:02:16.400]The problem is that the TDNA mutants,
[00:02:19.080]which are widely available, have some drawbacks.
[00:02:21.840]First of all, it's very difficult to cross these genes into one another because
[00:02:25.920]they're very close together on chromosome three of the Arabidopsis nuclear genome
[00:02:30.080]and some of them tend to act more like knockdowns, the knockout.
[00:02:34.120]So to solve this,
[00:02:35.160]we're going to use CRISPR-Cas9 to generate those deletion mutants instead.
[00:02:40.000]This project takes part in about three different sections,
[00:02:44.000]construction of the plasmid, transformation, getting it into plants
[00:02:48.600]and then processing where we get the mutants that we want.
[00:02:52.440]So we're using CRISPR CAS9, which is in archaeal and bacterial system
[00:02:59.240]that is two pieces.
[00:03:01.840]There's a gRNA and the CAS9 protein.
[00:03:04.640]The gRNA finds the target sequence
[00:03:07.000]in the genome and the CAS9 does the cutting.
[00:03:10.080]And we can use one gRNA to cut at one site and make a double strand break.
[00:03:16.160]But if we use two, we get a better chance
[00:03:19.160]of getting that full knockout where the protein is completely unable to function.
[00:03:25.120]So this is a little bit more detailed look at our methods,
[00:03:27.520]starting with construction, so we are taking our gRNA sequence
[00:03:32.480]by getting into pEN-Chimera, our entry vector, and then transferring
[00:03:36.920]a piece of that into pMR333, which is our destination vector.
[00:03:41.320]So this is a diagram of our gRNA sequence.
[00:03:44.200]We have two targeting sites in Exon seven
[00:03:47.760]and Exon 10 of MSH1 flanked by tRNA processing sites.
[00:03:52.200]So this whole sequence is transcribed in plants and in the tRNA processing
[00:03:56.800]sites about to be cut up into the pieces that we want.
[00:04:01.240]This piece is transferred into pEN-chimera
[00:04:04.240]our entry vector in between the 2 bbs1 sites in the top right.
[00:04:08.680]It lands directly downstream of the U6-26.
[00:04:11.240]promoter, which is going to drive expression in Arabidopsis.
[00:04:15.320]Once we have this in there,
[00:04:16.760]we're going to verify with PCR and sequencing and then we're going
[00:04:20.680]to transfer the whole section between AttL1 and AttL2 into our destination vector.
[00:04:27.120]So this is what we get when we do that.
[00:04:30.000]And then again, we're transferring into our destination Vector pMR333.
[00:04:35.600]So this is our destination vector.
[00:04:37.440]It contains the Cas9 protein, as well as a promoter for that
[00:04:42.880]It contains a BASTA resistance gene, which allows us to select four plants that contain this plasmid.
[00:04:48.000]And it contains a GFP sequence. This is the Seed-coat Expressd GFP,
[00:04:54.080]which allows us to select for seeds which contain the CRISPR machinery.
[00:04:59.040]So the section from pEN-Chimera is going to be inserted between AttR1
[00:05:05.160]and AttR2, which we then verify using PCR and sequencing analysis.
[00:05:12.240]After we finish that,
[00:05:13.390]that's going to be the end of the construction step we move on to transformation where we
[00:05:17.640]get it into plants using Agrobacterium and floral dip.
[00:05:23.200]So once we do that, we have to select for the plants that have
[00:05:27.120]been successfully transformed using Basta selection.
[00:05:29.920]Again, that resistance gene in the plasmid makes it so that the plants that have
[00:05:35.440]that plasmid are able to survive contact with the herbicide.
[00:05:39.400]So you'll see before and after spraying, the effects are very noticeable.
[00:05:43.920]Most of the plants there are dead with one
[00:05:46.520]in the top right that is surviving fairly well.
[00:05:50.160]So when we looked at that Basta resistant plant using PCR and sequencing analysis,
[00:05:55.240]we noticed that it was indeed a successful transformant.
[00:06:00.040]So, again, in our post processing steps, we are
[00:06:04.680]processing potential mutants and verifying homozygotes.
[00:06:08.080]So we confirmed the transformation and deletion using PCR.
[00:06:12.400]We did sequence analysis of the CRISPR deletion sites
[00:06:15.800]And then we isolate deletion mutants with and without the CRISPR machinery
[00:06:20.600]So this is a diagram of the sequencing
[00:06:23.360]results from that, you'll see that we had target
[00:06:26.560]sites in Exon 7 and exon 10 of MSH1.
[00:06:30.240]And what we saw when we sequenced it is that there was a full deletion in between
[00:06:34.520]those two sites, which means that CRISPR did cut where we wanted to and we ended up
[00:06:39.640]with the deletion of a fairly large chunk of that gene.
[00:06:44.280]Once we had a deletion mutant, we had to
[00:06:48.400]Verify that we had
[00:06:51.200]CRISPR-free and CRISPR-machinery containing knockout mutants,
[00:06:54.800]so the way we did that was using that Seedcoat Express GFP that I mentioned.
[00:07:00.240]So we collect seeds from the deletion
[00:07:02.360]mutant and we looked at those seeds under a fluorescent microscope.
[00:07:06.200]This allows us to see which ones contain that plasmid.
[00:07:09.720]So we sort them into GFP negative and GFP positive seeds.
[00:07:14.760]GFP negative seeds are CRISPR free deletion means those are done ready to go.
[00:07:20.160]The ones that are glowing green have at least one copy of the CRISPR plasmid.
[00:07:26.880]So we have to grow those up again
[00:07:28.960]and look at their progeny to see whether they were homozygous or heterozygous.
[00:07:33.120]And that way we can isolate homozygous transgene deletion mutants.
[00:07:40.720]And once we do that,
[00:07:41.960]we've completed the processing step of this process using the MSH1 gene,
[00:07:49.480]and that's the end of it.
[00:07:50.520]So in conclusion, we were able to successfully isolate MSH1
[00:07:55.960]deletion mutants our processing of those deletion mutants
[00:08:00.360]yielded homozygous mutants with and without the CRISPR machinery.
[00:08:04.680]In the future of this project, we would like to isolate more of these
[00:08:08.600]mutants and use them to construct double mutants,
[00:08:12.720]so that we can look at mutational and recombination effects of these genes.
[00:08:17.920]So that was my UCARE project for this year. I hope you enjoyed.
[00:08:22.960]Thank you.

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Using CRISPR-Cas9 to Construct Knockout Mutants in DNA-Repair Genes in Arabidopsis thaliana

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