Genome Technology & Applica2ons – genome edi2ng: CRISPR-Cas
Introduc)on
Group of technologies that can make targeted changes in the DNA of a living cell or organism. It can be
used to add or remove DNA in the genome or to alter its sequence.
• Tagging of “protein of interest” with e.g. GFP, venus…
– You can put a fluorescent tag on the protein that you are interested in, and in that way
you visualize it directly under the microscope
– E.g. venus: yellow fluorescent protein, you can add it to your DNA sequence and then
your cell will make from itself this fluorescent protein
• To create mutant mice/cells to study a disease or biological process
– E.g. Alzheimer’s disease: there are some mechanisms known that cause this disease,
but a lot is not known à researchers are interested in making new models to study
the disease further and you can do it by adding/introducing for example a mutaOon
that is founded in humans in mice
• To remove muta+ons in pa+ents to cure a disease (genome ediOng is mostly known for this)
à The changes are sOll under control of the endogenous promotor in the cell. Everything stays in the
normal physiological circumstances.
QuesOon 1: true or false
You want to study the effects of a mutaOon by comparing the behavior of cells expressing wildtype
(normal) and mutant (disease) protein. Genome ediOng is always the preferred strategy over
transfecOon/transducOon of wildtype and mutant overexpression constructs.
à statement is both
• TRUE: Biological point of view à genome ediOng is the best approach
Expressed at physiological levels and Ome points
— no artefacts aYributed to excessive expression levels (e.g. aggregaOon)
— wild type/mutant cells should “in theory” similarly express protein of interest à there
can be some variability in the transfecOon efficiency just by random effect. If you
repeat this mulOple Omes, then these differences will average out (= not an issue). But
if you only do it once or twice, this will give you a difference at the phenotype level of
your cell.
— No confounding effect of endogenous wild type protein expression à you have for
example a cell with a wild type and you add the mutant protein, you will look at what
was present already, and not only at the effects of the mutant only ßà using CRSIPR-
Cas or genome ediOng: you will not have a confounding effect of the wild type
• FALSE: Technical point of view à genome ediOng is the best approach; it depends on the
availability of anObodies
SubopOmal sensiOvity of tools for subsequent funcOonal analyses
— Some anObodies cannot detect low protein levels
— Lack of anObody specificity in some cases
o E.g. you edit your mutant in endogenous cell, using genome ediOng, then you
will have normal levels of your mutant protein à you want to know where it
is expressed: in the cytoskeleton? In a different locaOon where you don’t want
it to be? à you use an anObody for immunochemistry or western blodng to
see what the expression levels are. If the anObodies are not sensiOve enough,
they will not be able to detect these baseline levels that are present in the cell.
o If the anObodies that are commercially available are improving, than this
doesn’t maYer anymore à but currently the sensiOvity and specificity is not
opOmal
, How does it work?
• It is based on the concept of eukaryoOc DNA repair à if you add a double stranded break to
your DNA, then there are two ways to repair this
— 1: based on the present of a repair template, e.g. homologous chromosome à you
will have homologous recombinaOon (HR)
If you give the cells a template with a mutaOon in there, you will insert that point
mutaOon in your cells = Knock-in
— 2: non-homologous end joining (NHEJ) = this is what happens most à the breaks will
just be ligated with each other again, but this method is error prone, which means you
can get the introducOon of some nucleoOdes, removing of some nucleoOdes à results
in a knock-out or loss of funcOon allele because we can introduce frame shih
mutaOons for example
• First approach for genome ediOng based on this process à Homing endonucleases
(=meganucleases)
— RestricOon enzymes à you give this restricOon enzyme to your cell, it recognizes a
certain consensus sequence, it cuts there, and then you can go for either opOons à
knock-in or knock-out
— What is the limitaOon? à these restricOon enzymes, they are for example derived from
bacteria à you only have an endless number of endonucleases and only an endless
number of recogniOon sites that you can use
à so if there is not an endonuclease available that can cut on the specific locaOon that
you want, then there is no way that you can make that specific point mutaOon
BeYer alternaOves?
à E.g. zinc finger nucleases (ZNF)
• Not a biological approach à it is arOficial made in the lab (means you have more opOons + it
is more flexible)
— ArOficial endonucleases
• Custom DNA target à you can choose where you want it to cut
• 2 parts: DNA-binding part & nuclease domain of Fok1
— DNA-binding domains of transcripOon factors linked to nuclease domain of Fok1
o DNA-binding part is based on the fact that transcripOon factors bind to the
DNA = protein arm that recognizes a certain DNA sequence
o When it is bind, Fok1 will make the breaks
à The protein that recognizes the DNA will guide the enOre zinc finger nuclease to the
locaOon where you want to cut and when it is bound, Fok1 will cut, creaOng the break
à HR or NHEJ
Introduc)on
Group of technologies that can make targeted changes in the DNA of a living cell or organism. It can be
used to add or remove DNA in the genome or to alter its sequence.
• Tagging of “protein of interest” with e.g. GFP, venus…
– You can put a fluorescent tag on the protein that you are interested in, and in that way
you visualize it directly under the microscope
– E.g. venus: yellow fluorescent protein, you can add it to your DNA sequence and then
your cell will make from itself this fluorescent protein
• To create mutant mice/cells to study a disease or biological process
– E.g. Alzheimer’s disease: there are some mechanisms known that cause this disease,
but a lot is not known à researchers are interested in making new models to study
the disease further and you can do it by adding/introducing for example a mutaOon
that is founded in humans in mice
• To remove muta+ons in pa+ents to cure a disease (genome ediOng is mostly known for this)
à The changes are sOll under control of the endogenous promotor in the cell. Everything stays in the
normal physiological circumstances.
QuesOon 1: true or false
You want to study the effects of a mutaOon by comparing the behavior of cells expressing wildtype
(normal) and mutant (disease) protein. Genome ediOng is always the preferred strategy over
transfecOon/transducOon of wildtype and mutant overexpression constructs.
à statement is both
• TRUE: Biological point of view à genome ediOng is the best approach
Expressed at physiological levels and Ome points
— no artefacts aYributed to excessive expression levels (e.g. aggregaOon)
— wild type/mutant cells should “in theory” similarly express protein of interest à there
can be some variability in the transfecOon efficiency just by random effect. If you
repeat this mulOple Omes, then these differences will average out (= not an issue). But
if you only do it once or twice, this will give you a difference at the phenotype level of
your cell.
— No confounding effect of endogenous wild type protein expression à you have for
example a cell with a wild type and you add the mutant protein, you will look at what
was present already, and not only at the effects of the mutant only ßà using CRSIPR-
Cas or genome ediOng: you will not have a confounding effect of the wild type
• FALSE: Technical point of view à genome ediOng is the best approach; it depends on the
availability of anObodies
SubopOmal sensiOvity of tools for subsequent funcOonal analyses
— Some anObodies cannot detect low protein levels
— Lack of anObody specificity in some cases
o E.g. you edit your mutant in endogenous cell, using genome ediOng, then you
will have normal levels of your mutant protein à you want to know where it
is expressed: in the cytoskeleton? In a different locaOon where you don’t want
it to be? à you use an anObody for immunochemistry or western blodng to
see what the expression levels are. If the anObodies are not sensiOve enough,
they will not be able to detect these baseline levels that are present in the cell.
o If the anObodies that are commercially available are improving, than this
doesn’t maYer anymore à but currently the sensiOvity and specificity is not
opOmal
, How does it work?
• It is based on the concept of eukaryoOc DNA repair à if you add a double stranded break to
your DNA, then there are two ways to repair this
— 1: based on the present of a repair template, e.g. homologous chromosome à you
will have homologous recombinaOon (HR)
If you give the cells a template with a mutaOon in there, you will insert that point
mutaOon in your cells = Knock-in
— 2: non-homologous end joining (NHEJ) = this is what happens most à the breaks will
just be ligated with each other again, but this method is error prone, which means you
can get the introducOon of some nucleoOdes, removing of some nucleoOdes à results
in a knock-out or loss of funcOon allele because we can introduce frame shih
mutaOons for example
• First approach for genome ediOng based on this process à Homing endonucleases
(=meganucleases)
— RestricOon enzymes à you give this restricOon enzyme to your cell, it recognizes a
certain consensus sequence, it cuts there, and then you can go for either opOons à
knock-in or knock-out
— What is the limitaOon? à these restricOon enzymes, they are for example derived from
bacteria à you only have an endless number of endonucleases and only an endless
number of recogniOon sites that you can use
à so if there is not an endonuclease available that can cut on the specific locaOon that
you want, then there is no way that you can make that specific point mutaOon
BeYer alternaOves?
à E.g. zinc finger nucleases (ZNF)
• Not a biological approach à it is arOficial made in the lab (means you have more opOons + it
is more flexible)
— ArOficial endonucleases
• Custom DNA target à you can choose where you want it to cut
• 2 parts: DNA-binding part & nuclease domain of Fok1
— DNA-binding domains of transcripOon factors linked to nuclease domain of Fok1
o DNA-binding part is based on the fact that transcripOon factors bind to the
DNA = protein arm that recognizes a certain DNA sequence
o When it is bind, Fok1 will make the breaks
à The protein that recognizes the DNA will guide the enOre zinc finger nuclease to the
locaOon where you want to cut and when it is bound, Fok1 will cut, creaOng the break
à HR or NHEJ
