Hmb360 Lec 2 Week 1

Front 1

1) gene therapy involves manipulating _____ or ____ for ______

2) Name some strategies for gene therapy (general)

Back 1

1) DNA, RNA, Human disease treatment or prevention

2) -> rectifying (Corrections) genes

-> deleting genes

-> producing disabling mutations in pathogenic genomes

-> introduce therapeutic or protective somatic mutations

Front 2

1) Both fundamental genome editing techniques involve _______

2) Cas9 is a ________ nuclease

Back 2

1) nucleases

2) double

Front 3

1) Genome editing is _________ (targeted/general) with programmable __________, such as ________ and ________, which enable diverse genome manipulations in site-specific manner

Back 3

1) targeted, nucleases, zinc finger nucleases (ZF), transcription activator-like effector nucleases (TALENs)

Front 4

1) can use TALENS, ZF, or Cas9 for editing; ZF and TALENs also involve restriction enzymes called _____ that act as nucleases

Back 4

1) Fok1

Front 5

1) What's the difference of older gene editing techniques vs modern goals? whats the advantage?

Back 5

1) older techniques involved adding or inserting an exogenous DNA copy into cell nucleus; with new CRISPR tech we can target specific areas and delete/replace the targeted area. This is advantageous as we can avoid unwanted mutations

Front 6

1) name the old method of gene deletion

Back 6

1) Cre-LoxP

Front 7

TALENs (TAL Effector Nucleases) system vs CRISPR (RNA guided endonucleases) system

1) Both involve a stretch of ______

2) compare the nucleases

3) compare the DNA binding modules

4) resolution/specificity

Back 7

1) DNA

2) Fok1 in TALENs (requires dimerization for cleavage), that we need to genetically engineer; in CRISPR we have Cas9 protein, which are 2 separate nucleases

3) TALENs: TAL effector unit, each module recognizes 1 bp of target sequence)

CRISPR: Guide RNA that hybridizes to the target DNA (1:1 nucleotide base pairing)

4) Both have high resolution of target programmability, high specificity and low toxicity

Front 8

So why not TALENs instead of CRISPR? If using neurons the issues arises. Some of the points:

1) Their division

2) PSD property

3) Other structural properties

4) Result of global germline knockouts

5) What method could be useful so far?

Back 8

1) Theyre unique post-mytotic cells that are stuck in the G0 stage, so they're hard to target

2) They have dense network of PSD (Post synaptic density), making it difficult to use traditional targeted gene approaches using ES cell knockouts through homologus recombination

3) They have polarity, directionality, theyre not flat; cell has structural integrity

4) Sometime leads to lethality, or compensation; so not always effective

5) Conditional Cre-Lok, conditionally remove genes

Front 9

CRISPR Origin

1) Originally used by ________ as an _________ response, following __________ infection

2) Describe this process^

3) And now we use CRISPR by....

4) Advantage of CRISPR in neurons

Back 9

1) Bacteria, adaptive immune, viral or bacteriophage

2) Bacteria have their own nucleases, if they get infected they can chew apart the virus/phage that infected them because they have the reminisce/original immune response

3) using specific specific Cas9s using the short guide RNA segments to target specific genes.

4) we can in theory introduce genetic changes in the excitatory neuron regions

Front 10

1) How can we make CRISPR to be programmable, make it not target outside of the region that we want to change? What's the homing signal?

2) How do we get this CRISPR gene into the cell?

Back 10

1) The homing signal is a specific guide RNA 'sgRNA' (single guide RNA), we design this sgRNA, We read the DNA sequence we want to target then design the guide RNA.

2) Can use virus to deliver, use transfection technique or tag guide RNA with GFP to make sure its going into nucleus

Front 11

1) Name the 2 catalytic active nuclease domains of Cas9. What do they do?

Back 11

1) RuvC at top and HNH at bottom. Wherever guide RNA is binding, the HNH will cut the sequence, then in parallel, the RuvC nuclease will cut this as well.1

Front 12

1) The 2 segments of the sgRNA

Back 12

1) -> There is an element of the sgRNA that is derived from CRISPR RNA (crRNA) and trans-acting CRISPR RNA (tracrRNA).

-> We design the other complementary part that targets specific part of DNA

Front 13

1) Can we target any DNA sequence with CRISPR or is there a requirement ?

Back 13

1) The stretch of DNA has to contain a PAM (photospacer adjacent motif) sequence on the opposite strand. PAM allows the Cas9 nuclease to sit in the appropriate conformation.

Front 14

1) Describe a design advantage of the CRISPR system over the TALENs and the ZF system

2) Describe specificity advantage of CRISPR

Back 14

1) CRISPR only involves designing the complementary sgRNA while the nucleases stay the same; while in the other systems we need to form peptides/proteins to be able to target them, which is not easy and expensive.

2) We can only target 1 gene at a time with ZF or TALENs, while with CRISPR we can use multiplexing. We can insert multiple guide RNAs in the same reaction, target all 3 location in the DNA; can knockout 3 genes at same time. e.g. can target a gene that might compensate

Front 15

CRISPR Disadvantages vs TALENs

1) Targeting requirements

2) Specificity

3) Targeting Mitochondria

Back 15

1) need PAM sequence and guanine at 5' end; while for TALENs only need thymine at the 5' end. So we can target more genome sites with TALENs

2) TALENs thought to produce less off target effects in relative to CRISPR, due to heterodimeric construction of FOK1 nuclease in TALEN

3) TALEN can be used to target and cleave mutant mitochondrial DNA (That's involved in other disorders, e.g. psychological)

Front 16

RNAi tech

1) Briefly describe

2) Why forgotten?

3) CRISPR advantages

Back 16

1) RNA interference, in vivo manipulation of gene expression

2) effects are temporary, non-specific, limited to only transcribed genes (can't target e.g. enhancer regions)

3) can induce both gain and loss of function mutations, suffers from less off target effects

Front 17

CRISPR Considerations

1) GC (guanine cytosine content)

2) length

3) potential off-target effects

4) Some modes of introduction

Back 17

1) Generally around 50%, can be around 40-80%

2) 17-24 bp, avg of 20

3) 17-24 bp

4) nanoparticles, viruses, lentivirus, etc.

Front 18

1) Describe successful recent 2012 in vivo experiement

Back 18

1) SpCas 9 (bacterial isoform) has been applied in mice using hydrodynamic injection and adenovirus delivery in liver. Can in theory, inject directly in blood stream and it can target organs of interest in the periphery e.g. liver.

Front 19

Future goal is to enable SpCas9 use for editing cells in mammalian nervous system in vivo

1) What delivery method is favoured? why?

Back 19

1) adeno-associated virus (AAV), it is non-replicating. 1

Front 20

1) New Feng Zhang AAV

Back 20

1) Created built-in Cas9 system in AAV, making it easy to clone things around it; using sgRNA expression cassettes (AAV-SpGuide), can just replace it with your own guide RNA.

Front 21

1) Roger Nicoll 2014 paper

2) Feng Zheng paper after

Back 21

1) Removal of synaptic proteins using CRISPR

2) can deliver the method in living adult animal, target gene expression in vivo