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Lecture 6

BIOC14H3 Lecture Notes - Lecture 6: Homology Directed Repair, Cas9, Crispr


Department
Biological Sciences
Course Code
BIOC14H3
Professor
Patrick Mc Gowan
Lecture
6

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BIOC14 Lecture 6
Genome Editing using CRISPR/ CAS9
- Naturally occurring system in prokaryotes, that defends against Viral Genetic Material
- CRISPR Clustered Regularly Interspace Short Palindromic Repeats
o This is a stretch of DNA that contains short regions, where transcription of these regions will make RNA that
serve as guides (Short-guide RNAS or sgRNAS) to recognize viral DNA segments.
o CAS9: The crispr associated enzyme 9”, which is an endonuclease that binds to short-guide RNA/ sgRNA, and
will assist in catalyzing the double-stranded break, in foreign viral DNA, disabling them
o sgRNA can be engineered to recognize any gene or DNA Sequence to cause double stranded break.
o The double stranded break can
A) Induce a mutation
B) Insert a piece of DNA that can be used as a transgene
- Note: With this method, you can introduce many sgRNA that target different genes. This method can be used in a
multiplex way. For example, fibroblasts cells can become other types of cells using this system. Researchers know a
few genes involved in making fibroblast becoming neurons, so by editing the fibroblast gene, they could turn the cell
into a neuron
- Major Advantage: because CRISPR involves RNAse, its simple, and you could do it in a multiplex way, to edit multiple
genes. Since we can affect multiple genes, this is a way that we can modify quantitative traits since they are
determined by multiple genes.
- CRISPR/ CAS9 Process
1) Scientist will create a genetic sequence known as the guide RNA (sgRNA), that matches a piece of DNA that
they want to modify it is a complementary sequence
2) The sequence is injected to a cell/zygote along a protein known as CAS9, which acts like a pair of scissors that
cut the DNA
3) CAS9 will recognize a PAM Sequence Protospacer Adjacent Motif, which will induce a double-stranded cut. It
is a location where CAS9 can edit.
4) The sgRNA will complementarily bind to the target DNA, forming a DNA-RNA Hybrid.
5) CAS 9 will induce a double stranded break, and cut it out. Once their job is complete, the guide RNA and CAS9
leave the scene
6) Two things can happen
A) Cells will try to repair the double stranded break using Non-Homologous End Joining (NEHJ). NHEJ is
imperfect and will created indels: insertions/deletions (2-30BPs)
This will lead to frame shifts mutations depending on the sequences that are left over. This can lead
to a premature stop codon which will ultimately lose gene function
B) We can use Homology Directed Repair (HDR). We can engineer DNA fragments with homology arms
so complementary base pairing could occur to insert that piece of DNA.
Using vector designed homology to regions surrounding the sgRNA target sites.
Currently, this technique is being used for repairing known human mutations.
Ethical concerns because you can modify a lot of things, like baby eye colour. This technique is
banned to be used on human zygotes. (although you can modify human embryos)
Genetic Dissection of Neural Circuits
- Insufficient to look at a single gene, or a single neuron type to study the brain.
- Must look at gene in context/ in it’s population of neurons that it is connected.
- Neural activity and connections will then be used to understand behaviour
Neural Circuit
- Neurons are organized into circuits that process information
- Circuits: Interconnected neurons forming a functional unit (billions of neurons with trillions of synaptic connections)
o Researchers can study Simple organisms, which tend to have fewer/ individual neurons (like C-Elegans)
o You can also focus on a group of neurons of the same type in Vertebrates

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- The analysis of cell types and their connectivity allows to determine the wiring of the circuits allows you to study
the properties of a neural circuit.
- Experiments involving the loss/gain of function allows scientist to determine how a specific neuronal type will
contribute to a circuit.
Studying the effects of GENES in NEURONS
- Broadly, to study the effects of genes in neurons:
o First need to identify the neurons expressing the gene of interest. (Which neurons are expressing which genes?)
o Measure their activity (electrically or chemically)
o Look at their connection pattern (wiring of the circuit), determine the circuit.
o Activity is modulated to understand the effects (ie on behaviour)
Identifying neurons expressing the Gene of interest RNA in situ Hybridization
- Method for localizing and detecting specific mRNA sequences in preserved tissues
- Uses RNA Probe (Riboprobe) that is complementary to the target gene sequences Complementary sequence
hybridizes to the target DNA sequence
o The probe is labelled such that it can be detected via radioisotopes, fluorescent dye, antigen- to be detected
by an antibody.
o Colorimetric Assay: Look at the colour of different dyes, to figure out how much RNA is present. Using this, you
can figure out where there is more/less expression of a particular gene
- There are 2 ways to create a Riboprobe
o A) From Plasmid: Insert an RNA species of interest into the plasmid, and use this to generate multiple copies of
this mRNA. You need to create antisense riboprobes, that are complementary to the gene of interest, while the
sense riboprobes are a control for non specific binding of that sequence. Using the promoter regions, you can
drive the transcription of that gene
o B) Can use a PCR Template, from a DNA sequence that you generate in the lab. Process is the same as above,
you use a promoter sequence to transcribe the RNA.
- With in-situ hybridization you can use multiple different tags, to figure different expression.
Allen Brain Atlas
- Paul Allen funded the Allen Institute of Brain Science. He created a Digital Atlas of Gene Expression patterns in the
mouse and in humans. (Microarray and MRI data is also available for human in addition to ISH)
- They used a systematic approach to do in situ hybridization for gene expression. (Think about the fact that gene
expression changes spatially and temporally in a lifetime, so they figured out a robotic way/ computers, to perform
this a lot quicker)
- They generated Genepaint map that shows the expression of a gene and how it changes across a lifetime. They
take brain slices and generate an image, of the expression of a gene.
Genes to Neural Phenotype
- Neurons expressing the genes of interest must be identified
- Measure their chemical and electrical physiological activity
- Determine their connection pattern with other neurons in the circuit
- Modulate the activity of neuron to see it’s effect.
- Note: It is critical to target specific neural populations without affecting others.
Genetic Access to specific neuron populations
- The question we ask; how do we deliver specific genetically encodable molecules to one specific neural population?
- 8 Different approaches
o Transgenic approaches using promoters/enhancer
Use specific promoter or enhancer that is only expressed in one population of neurons
o Transgenic approaches using BACs
Insert large pieces of DNA, that have ALL genomic regulatory elements within them
o Knock-in/out

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o Recombinase-mediated approach
Use the CRE with LOX P system, to induce alteration within genes that are only expressed within a neuron
of interest
o Virus mediated approachF
Use a virus, and hijack the ability to insert itself within a gene of interest, and express the gene that is
only going to be functional in a neuron of interest.
o CRISPR/Cas9 approach
Transgenic Approaches Using Promoters and Enhancers
- A gene regulatory element includes the promoter regions and enhancer regions of the gene.
- Enhancers are regions that drive transcription changes, that are located far from transcription start site. They can be
50kB away from the gene. They can be located in the
o 5’ Upstream region of the gene
o 3’ Untranslated Region
o Within the intron of the gene
- Promoters are closer to the start site, and will also regulate transcription.
- Both promoters and enhancers provide the opportunity for TF to bind, and enhance or surpass transcription
- If we know that a certain gene is only expressed in one population, we can use a promoter only found in those
genes, to drive the transcription of the target gene that we are introducing
- This method works only for
- Steps
o 1) Isolate the promoter/enhancer sequence of the gene of interest
o 2) Use the promoter/ enhancer to express transgene (The promoter will be specific to a specific neural cell type,
and will drive the transcription of that gene)
o 3) Via Pronuclear injection or electroporation, integrate your construct within the genome
Electroporation: involved blasting the cell with genomic DNA, which will be taken up through the lipid
membrane as a function of changing the electrical current, and opening pores within the cell.
- Limitation
o This method involved the transfer of only small portions of DNA (8kB of sequence). This could encompass the
promoter of a gene, but regulatory elements involve enhancers as well, which are far from the transcription
start site.
Therefore, it is possible that you may not capture all the regulatory elements that will drive transcription
o All relevant genetic elements will/ may not be included
o Random Integration in the genome will influence the expression patterns.
Transgenic Approaches using BACs
- BACs are DNA Vectors that can carry large fragments of DNA (400kB)
- Cloning in BACs allows to isolate a Clone, that has a complete set of promoter/ enhancer elements of a gene
- Using a BAC clone, we can insert a complete set of genes known as a DNA Cassette, which contain all the promoter
and enhancer elements for a given gene, along with your transgene of interest.
- Limitation
o Does not mediate against random integration.
- Steps
o 1) Identify a BAC clone, that contains your target gene
o 2) Insert your DNA Cassette, which has all your regulatory elements + your
transgene
o 3) Inject via Microinjection into a fertilize oocyte, and implant the oocyte
into your pseudo pregnant female, to create your offspring mice.
- Uses of BACS
o Can be used as a Receptor Genes (Such as GFP) to identify specific neural
types, which are showing a specific gene of interest
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