BIOLOGY 1A Lecture Notes - Lecture 26: Zinc Finger Nuclease, Jennifer Doudna, Genome Editing
GENOME EDITING
In vivo: Overcome deficiency by delivering a vector to transfer the editing reagent into organ to restore genetics
•
Ex vivo: manipulating cells in culture and genetically modifying them
Use induced pluripotent stem cells to genome edit and differentiate into modified cells that can be reinfused
○
Isolate hematopoeitic stem cells (HSCs) that are multipotent and reinfuse
○
•
Human genome: 3000 megabases
What tool do we use to alter mutation specifically?
○
•
Maria Jasin
Experiment: I-Scel
If you make a break in the human genome, (DSB), the dna repair machinery will repair it. You can hijack the machinery to direct specific
modifications. Used an enzyme called I-Scel1 which can find DNA specific where to introduce a DSB. Sce1 is very big, not found in genome
sequence. So if you put this into human cells, and integrate it only once into genome, it will make a cut only there and nowhere else. Maria
demonstrated this will be repaired that will allow you to modify DNA around the cut site. We need to make nucleases that are simpler than
Sce1 to make specific cuts.
§
○
•
Aron Klug and Carl Pabo- zinc finger proteins
Look at differences in proteins, one finger in ZF makes contact to 3 bases. A different finger would make contact to a different 3 bases.
○
You can use different fingers to direct specificity in the genome
○
•
Dana Carrol- Zinc finger nuclease (ZFN)
You can engineer a ZFN to make a cut
○
You can target every site in the human genome.
○
•
Jennifer Doudna: use an RNA guided mechanism
•
Vocabulary:
DSB: double strand break
Xrays, UV light and replication errors can result in Base-excision repairs, Nucleotide-excision repair, recombinational repair, mismatch repairs
○
•
Nuclease: enzyme engineered to induce a DSB
•
ZFN: zinc finger nuclease
ZFNs recognize the DNA backbone using alpha helicises that fit. Into the major groove of DNA and wraps around with helix
○
1 module: 3 bp
○
•
TALEN: transcription activator like effector nuclease
Alpha helices that forms a spiral around the DNA and makes contact w it
○
1 module: 1 bp
○
Plant pathogens use these modules to infect plants
○
15-20 bp long
○
•
Cas9: CRISPR-associated gene number •
CRISPR: clustered regularly interspaced short palindromic repeats
Has an RNA that helps recognize sequences of DNA and there is a protein scaffold that guides the RNA to make contact with the opening of DNA. The
DNA will be nicked at two positions
○
1 base: 1 bp
○
Protein Cas9 will nick the DNA
○
•
CRISPR in 30 seconds:
Host cell invented a mech to fight bateriophages. It uses the DNA and chops it into pieces and bacteria will remember the phage because it used the
invading nucleic acids and chopped it up. This will lead to production of 2 RNAs- the CRSPR RNA which has specificity of DNA integrated and the
tracer RNA that can recognize the CRISPR RNA with proteins from this locus. The nuclease is made by one protein (cas9) and it will constantly hover
the bacteria waiting for another bacteriophage to come in and will cut it.
○
•
DSB-R: double strand break repair•
NHEJ: non-homologous end joining
Cell puts two ends back together and ligates it.
○
Then the nuclease that recognizes the sides of the break, it will come back and cut again and the cell will put it back together
Every now and then the cell makes a mistake and inserts or deletes DNA by the break, the site has now changed and nuclease cannot cut again
because it does not recognize it.
§
○
•
KO: knockout
Make random insertions and deletions and therefore disrupting the function.
○
•
HR(HDR): homology-directed repair
Use sister chromatid information to deal with DSB and repair based on the other template
○
•
Donor: DNA used to repair a nuclease-induced DSB by HDR
Deliver a piece of DNA that has homology
§
•
Knockin: the introduction of new genetic information
Use principle of isolating cells from kids with immune deficiency from a gene on X chromosome
Generated ZNF that would cut at the mutation. Delivered a repair template. And cured the genetic deficiency
§
○
•
TI: targeted•
Can l integration
Allows u to integrate sequence into locus
○
Can hijack any locus to express proteins
○
•
The TOOLBOX:
The toolbox of genome editing is largely nuclease-type-agnostic
We don’t care how we make the double strand break there's many ways to do it
○
Use nucleases to do gene disruptions
Can insert DNA (integrate coding sequence that will generate GDP protein to report on gene activity of a specific locus)
§
Apply 2 nucleases you can cut out big chunks of DNA
Can infer why this deleted sequence was important□
100,000 bp can be deleted□
§
○
Homology Directed Repair
Gene correction
§
Targeted gene addition
§
Transgene stacking
§
○
•
Multiplexing: doing more than one gene edit in the same cell, or multiple different edits on different cells at the same time
Helpful if you wanna investigate many phenotypes at once
○
•
Lecture 26-3/23
Sunday, April 8, 2018
4:36 PM
GENOME EDITING
In vivo: Overcome deficiency by delivering a vector to transfer the editing reagent into organ to restore genetics
•
Ex vivo: manipulating cells in culture and genetically modifying them
Use induced pluripotent stem cells to genome edit and differentiate into modified cells that can be reinfused
○
Isolate hematopoeitic stem cells (HSCs) that are multipotent and reinfuse
○
•
Human genome: 3000 megabases
What tool do we use to alter mutation specifically?
○
•
Maria Jasin
Experiment: I-Scel
If you make a break in the human genome, (DSB), the dna repair machinery will repair it. You can hijack the machinery to direct specific
modifications. Used an enzyme called I-Scel1 which can find DNA specific where to introduce a DSB. Sce1 is very big, not found in genome
sequence. So if you put this into human cells, and integrate it only once into genome, it will make a cut only there and nowhere else. Maria
demonstrated this will be repaired that will allow you to modify DNA around the cut site. We need to make nucleases that are simpler than
Sce1 to make specific cuts.
§
○
•
Aron Klug and Carl Pabo- zinc finger proteins
Look at differences in proteins, one finger in ZF makes contact to 3 bases. A different finger would make contact to a different 3 bases.
○
You can use different fingers to direct specificity in the genome
○
•
Dana Carrol- Zinc finger nuclease (ZFN)
You can engineer a ZFN to make a cut
○
You can target every site in the human genome.
○
•
Jennifer Doudna: use an RNA guided mechanism
•
Vocabulary:
DSB: double strand break
Xrays, UV light and replication errors can result in Base-excision repairs, Nucleotide-excision repair, recombinational repair, mismatch repairs
○
•
Nuclease: enzyme engineered to induce a DSB
•
ZFN: zinc finger nuclease
ZFNs recognize the DNA backbone using alpha helicises that fit. Into the major groove of DNA and wraps around with helix
○
1 module: 3 bp
○
•
TALEN: transcription activator like effector nuclease
Alpha helices that forms a spiral around the DNA and makes contact w it
○
1 module: 1 bp
○
Plant pathogens use these modules to infect plants
○
15-20 bp long
○
•
Cas9: CRISPR-associated gene number •
CRISPR: clustered regularly interspaced short palindromic repeats
Has an RNA that helps recognize sequences of DNA and there is a protein scaffold that guides the RNA to make contact with the opening of DNA. The
DNA will be nicked at two positions
○
1 base: 1 bp
○
Protein Cas9 will nick the DNA
○
•
CRISPR in 30 seconds:
Host cell invented a mech to fight bateriophages. It uses the DNA and chops it into pieces and bacteria will remember the phage because it used the
invading nucleic acids and chopped it up. This will lead to production of 2 RNAs- the CRSPR RNA which has specificity of DNA integrated and the
tracer RNA that can recognize the CRISPR RNA with proteins from this locus. The nuclease is made by one protein (cas9) and it will constantly hover
the bacteria waiting for another bacteriophage to come in and will cut it.
○
•
DSB-R: double strand break repair•
NHEJ: non-homologous end joining
Cell puts two ends back together and ligates it.
○
Then the nuclease that recognizes the sides of the break, it will come back and cut again and the cell will put it back together
Every now and then the cell makes a mistake and inserts or deletes DNA by the break, the site has now changed and nuclease cannot cut again
because it does not recognize it.
§
○
•
KO: knockout
Make random insertions and deletions and therefore disrupting the function.
○
•
HR(HDR): homology-directed repair
Use sister chromatid information to deal with DSB and repair based on the other template
○
•
Donor: DNA used to repair a nuclease-induced DSB by HDR
Deliver a piece of DNA that has homology
§
•
Knockin: the introduction of new genetic information
Use principle of isolating cells from kids with immune deficiency from a gene on X chromosome
Generated ZNF that would cut at the mutation. Delivered a repair template. And cured the genetic deficiency
§
○
•
TI: targeted•
Can l integration
Allows u to integrate sequence into locus
○
Can hijack any locus to express proteins
○
•
The TOOLBOX:
The toolbox of genome editing is largely nuclease-type-agnostic
We don’t care how we make the double strand break there's many ways to do it
○
Use nucleases to do gene disruptions
Can insert DNA (integrate coding sequence that will generate GDP protein to report on gene activity of a specific locus)
§
Apply 2 nucleases you can cut out big chunks of DNA
Can infer why this deleted sequence was important□
100,000 bp can be deleted□
§
○
Homology Directed Repair
Gene correction
§
Targeted gene addition
§
Transgene stacking
§
○
•
Multiplexing: doing more than one gene edit in the same cell, or multiple different edits on different cells at the same time
Helpful if you wanna investigate many phenotypes at once
○
•
Lecture 26-3/23
Sunday, April 8, 2018
4:36 PM
Document Summary
In vivo: overcome deficiency by delivering a vector to transfer the editing reagent into or. Ex vivo: manipulating cells in culture and genetically modifying them. Use induced pluripotent stem cells to genome edit and differentiate into modified c. Isolate hematopoeitic stem cells (hscs) that are multipotent and reinfuse. If you make a break in the human genome, (dsb), the dna repair machinery w modifications. Used an enzyme called i-scel1 which can find dna specific whe sequence. So if you put this into human cells, and integrate it only once into g demonstrated this will be repaired that will allow you to modify dna around. Aron klug and carl pabo- zinc finger proteins. Look at differences in proteins, one finger in zf makes contact to 3 bases. You can use different fingers to direct specificity in the genome. You can engineer a zfn to make a cut. You can target every site in the human genome.
