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GENETICS: Lecture 21 (gene editing)- NOT DONE ENDED 26

Terms in this set (35)

what is used to cleave the DNA at a specific nucleotide at a specific chromosomal loci?
DNA endonuclease
what results from DNA endonuclease cut?
double stranded break (DSB)
what do DSB allow?
gene modification
two ways cells can fix DSB
non-homologous end joining (NHEJ) and homology-directed repair (HDR)
for NHEJ and HDR:
Nucleotide trimming
prone to error
uses DNA template
when does it occur
result?
NHEJ: yes,yes,no,before DNA replication, Gene knockdowns/outs

HDR: yes, no, yes, after DNA replication, gene knock-ins
DNA breaks during G2 phase, what repair mechanism is used?
homology directed repair
define gene knockdown/outs
taking out gene to see phenotypic result
you can figure out role of gene by doing this
what is gene knock in
inserting genes that werent there before
what happens when DNA fails to repair from double stranded break?
instability and incomplete replication in genome can lead to death, cancer, and mutations
describe pathway of NHEJ repair
proteins recognize double stranded break (PKcs, Ku70, Ku80)

proteins trim off jagged bits at spot of break to make blunt ends

DNA ligase glues the blunt ends back together
why is NHEJ more prone to error?
because it has to take out nucleotides to make the blunt ends
describe process of HDR
we have duplicated DNA and so we have sister chromatids (remember both are genetically identical)

One sister chromatid has DSB.

Nucleases come in a they trim off to make less jagged but we dont get blunt ends we get flaps of nucleotides hanging removed.

Rad51 protein attaches to one end and series of rad51 proteins that build up but on the nonbroken sister chromatid.

D loop is made b yopening nonbroken sister chromatid (done by stopping h-bonds between nucleotides) to do DNA replication and the broken strand can be repaired by replicating from the nonbroken strand that is identical so all nucleotides are kept non are lost because they are fully restored from D loop.

DNA ligase glues everything back together
what are the two types of DNA editing tools?
DNA binding domain like zinc fingers and TALENS
RNA based tools like crisper
how were ZFNs identified
african clawed frogs
how were TALENS identified
isolated from Xanthomonas
what is dimerising
when zinc fingers or TALENS complimentary base pair with each other which makes it pinch and then snaps the DNA
what is a negative part of ZFNS and TALENs
they target specific nucleotide sequences but if there is the same sequence somewhere else or multiple of the same they will cut at all spots with the nucleotide sequence.
They also use the same endonuclease (fok1) so you get off-target cleavege
what does CRISPR stand for?
Clustered Regularly Interspaced Short Palindromic Repeats
what are palindromic repeats?
palendrome unique spacer region in the CRISPR part of gene- they are the sequences used to help identify invader RNA
What is the original use of CRISPR

Describe how crRNA, tracrRNA, and Cas9 are transcribed/made

Describe how the crRNA-tracrRNA- and Cas9 complex cleaves the foreign DNA
natural defence mechanism in prokaryotes
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1. CRISPR sequences are transcribed into noncoding RNA which is processed into multiple crRNAs (sectioned according to palindromic repeats)

2. tracer gene is transcribed into tracrRNA
3. Cas 9 is transcribed
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1.tracrRNA binds to Cas9 and crRNA binds to tracRNA by base bairing
2. crRNA-tracrRNA-Cas9 complex cleaves the foreign DNA by targeting the site of base pairing of crRNA and target DNA
how is the foreign DNA cleaved
tracrRNA and crRNA form a complex with Cas endonucleases.

crRNA-tracrRNA-Cas complex cleave invading DNA into DSB to deactivate it
what is the crRNA-tracrRNA-Cas endonuclease complex represent? how?
scissors
binds to complementary DNA in foreign DNA which allows it to cut (not perfect complementary binding, they bond enough to cut)
How does CRISPR allow for immune memory in bacterial cells?

What do unique spacer sequences represent?
The bacteria can store the cleaved foreign DNA in its genome for future invader recognition. It incorporates it into its CRISPR region (made into more palendromic repeats)

Wanted criminal poster
What is single guide RNA

How is it used to make crRNA?
scientists engineered crRNA and tracrRNA to fuse together into single guide RNA

sgRNA insert their own target DNA into the sgRNA gene to make a unique crRNA and then Cas9 is used at endonuclease
why is Cas13 used instead of Cas9
Cas13 can transcribe RNA so we can transcribe mRNA instead of genome so it doesnt have to genetically modify organism but still allows us to see phenotypic effects

Cas9 targets DNA so modifications are permanent
what are the three ways to get recombinant DNA/CRISPR into cells?
particle bombardment
agrobacterium infiltration
agrobacterium mediated transformation
why is agrobacterium used for putting CRISP into cells?
agrobacterium is a natural pathogen of plant cells so it will invade and enter plant.
scientists have made a special crRNA, what is it made of and why is it better?
special crRNA is made of regular crRNA and tracrRNA that are fused together to make what is called sgRNA. sgRNA is the special crRNA and it allows scientists to insert their own target DNA into the sgRNA
what happens after Cas9 cuts the target DNA?(explain for both NHEJ and NDR)
NHEJ does its repair mechanisms by making blunt ends and ligase attaches the ends, it can result in deletions and/or insertions of DNA

HDR uses supplied DNA fragments as the template for DNA synthesis
define what the HNH domain, RuvC domain, and PAM parts of Cas9 are for?
HNH domain= cleaves the target DNA strand complementary to the crRNA
RuvC domain= cleaves the opposite strand of the target DNA
PAM= adjacent sequence for crRNA complementary pairing
which of the following use DNA or RNA:
Cas9
Cas12
Cas3
Cas13
Cas9- DNA
Cas12-DNA
Cas3-DNA
Cas13- RNA
what is the difference agrobacterium mediated transformation and agrobacterium infiltration
both make gene of interest into plasmid DNA and insert it into agrobacterium

mediated transformation: plant tissue and agrobacterium are co-cultured. Plant is then grown with the observation of phenotypic change

Infiltration: plant tissue is injected with agrobacterium and then grown with the observation of phenotypic change
desrcibe particle bombardment
gene of interest is made into a plasmid, plasmid is coated onto gold particles, plant tissue is bombarded with particles and plant is grown to observe phenotype
what parts of plasmid in agrobacterium are important
transfer DNA (T-DNA)- transfers to the plant and recombine into host genome
Ti plasmid- plasmid containing T-DNA
ended on slide 26
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