Skip to content

Latest commit

 

History

History
161 lines (110 loc) · 4.44 KB

crispr.md

File metadata and controls

161 lines (110 loc) · 4.44 KB

Introduction

  • Introduction of Krzysztof

    • why is it us talking about this :-)

  • What is CRISPR

    • Abbreviation?
    • What is it? "CRISPR, is a family of DNA sequences in bacteria that contains snippets of DNA from viruses that have attacked the bacterium"
    • What was the breakthrough?
    • Why is it important?

Definitions of some terms

Even if this might make it a somewhat boring start of the episode, I think we have to introduce a few concepts to our listeners for the rest of the discussion to work. I think the following should suffice:

  • Gene
  • DNA
  • RNA
  • Enzymes
  • Nuclease

What is Genome Editing

  • Replace a part of the genome with other genes

    • in order to repair destroyed ones
    • or to plug in different ones

  • Targeted Genomic Cleavage, double stranded break

    • Homing Device
    • Endonuclease

  • Gene Editing on Living cells seems to be new? Practical, because no breeding of generations and generations in order to change genome.

  • Does (irrational) fear of GMOs carry over to CRISPR/Cas9 editing?

  • Do legal restrictions on GMOs apply to CRISPR edited organisms?

The natural occuring thing

  • CRISPR is actually a naturally-occurring, ancient defense mechanism found in a wide range of bacteria -> explain

  • CRISPR: One DNA sequence would be repeated over and over again, with unique sequences in between the repeats -> why?

  • Cas9

    • CRISPR-associated proteins
    • which can precisely snip DNA and slice invading viruses.

How to do Genome Editing with CRISPR/Cas

  • Genome Editing is not new, right?

    • How was it done before?
    • How is CRISPR/Cas better? (More precise, targets more genes (up to 20))

  • Guide RNA

  • Cutting DNA alone is not enough, because the cell might repair it randomly. So we have to supply the spare.

  • Simple: a transfection (compared to what earlier?)

  • How does it work, specifically

  • Different usage scenarios:

    • homologous repair . gene knockout . gene tagging . site-specific mutogenesis
    • non-homologous end-joining repair . mutations . deletions insertions . knockouts

  • Promotor study, reporter gene

  • How does the actual technical lab process work? "All biologists have to do is feed Cas9 the right sequence, called a guide RNA, and boom, you can cut and paste bits of DNA sequence into the genome wherever you want. All you have to do is design a target sequence using an online tool and order the guide RNA to match."

    "Occasionally, though, the enzyme still cuts in the wrong place, which is one of the stumbling blocks for wider use, especially in the clinic."

    • More on accuracy of CRISPR: How about reports on multiple non-targeted (undesirable) changes? Up to thousands? Was that completely rebutted?

    • How do you quality control that the editing went well?

    • how reliable are the editing processes?

    • is this a "on average" thing?

  • Target many genes at once.

  • Compare to other approaches

    • zinc finger nuclease (ZFN)
    • transcription activator-like effector nucleases (TALENs)

Specific CRISPR applications

  • It is currently a research tool or "industry ready?

    • how would it be used in research?

  • https://www.heise.de/tp/features/Genmanipulierte-menschliche-Embryonen-Zweifel-am-Durchbruch-3848037.html - nice summary about (technical, but also ethical) issues.

  • Research tool "mouse editing"?

    • What is a knock-out mouse, how did
    • How did you create them before,
    • how do you do it with CRISPR?

  • The prob that many genes are involved in many things is still there, right?

  • Applications

    • from creating monkeys with targeted mutations
    • to preventing HIV infection in human cells.
    • potential to cure any genetic disease.
    • And yes, it might even lead to designer babies.

  • Also works in Stem Cells, so ...

  • Why is it that CRISPR can be applied to all organisms, not just the typical model ones?

How was it found

  • Talk a bit about the process of discovering it

    • over the years, timeline
    • recent breakthrough
    • seredipidous?

  • What has changed since it was discovered?

Wrap Up

  • CRISPR could become a major force in ecology and conservation,

  • Ethical issues?