Using CRISPR-Cas9 to Construct Knockout Mutants in DNA-Repair Genes in Arabidopsis thaliana
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
- [00:00:28.000]First of all, plant
mitochondria are very large
- [00:00:32.280]in comparison to most
- [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
- [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
- [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
- [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
- [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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