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21 Cards in this Set

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1) gene therapy involves manipulating _____ or ____ for ______


2) Name some strategies for gene therapy (general)

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

1) Both fundamental genome editing techniques involve _______


2) Cas9 is a ________ nuclease

1) nucleases


2) double

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



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





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

1) Fok1

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

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

1) name the old method of gene deletion



1) Cre-LoxP

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

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

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?

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

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

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

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?

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

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

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

1) The 2 segments of the sgRNA

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

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

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.

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


2) Describe specificity advantage of CRISPR

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

CRISPR Disadvantages vs TALENs


1) Targeting requirements


2) Specificity


3) Targeting Mitochondria

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)

RNAi tech


1) Briefly describe


2) Why forgotten?


3) CRISPR advantages

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

CRISPR Considerations


1) GC (guanine cytosine content)


2) length


3) potential off-target effects




4) Some modes of introduction

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.

1) Describe successful recent 2012 in vivo experiement

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.

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


1) What delivery method is favoured? why?

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

1) New Feng Zhang AAV

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.

1) Roger Nicoll 2014 paper


2) Feng Zheng paper after

1) Removal of synaptic proteins using CRISPR


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