1. WHY IS EVERYONE CRAZY FOR CRISPR?

2. We have known about CRISPR for a while
Eric S. Lander, 2016, Cell, “The Heroes of CRISPR”

3. Only recently have we realized its potential for genome editing
Jennifer A. Doudna, and Emmanuelle Charpentier Science 2014;346:
Published by AAAS

4. Once we realized its potential for genome editing, research took off
Pubmed search “genome editing”
Pubmed search “CRISPR”

5. BUT WHAT IS CRISPR?

6. Bacteria can be infected by viruses too!
Viruses injecting their DNA into a bacterial cell
Elizabeth Pennisi Science 2013;341:
Published by AAAS

7. CRISPR is how bacteria defend themselves from viruses
The invading viral DNA is incorporated into the bacterial DNA (making the CRISPR array)
The portions of bacterial DNA that contain the virus are converted into RNA
This RNA that matches the virus is divided into pieces and joins together with a protein
Whenever this RNA/protein complex finds DNA that matches the viral sequence, it cut it up
Susan G. 2011, Nature, 471(7340):

8. Recognition of invading viral DNA
The system requires 3 components:
tracrRNA
Needed for the production of crRNA
crRNA
RNA derived from a previous viral infection
If the same virus infects the cell again, crRNA recognizes the invading DNA and binds to it
Cas9
Cuts the invading DNA into pieces
Jennifer A. Doudna, and Emmanuelle Charpentier Science 2014;346:
Published by AAAS

9. The crRNA and tracrRNA can be combined into one guide RNA
The system requires 3 components:
tracrRNA
Needed for the production of crRNA
crRNA
RNA derived from a previous viral infection
If the same virus infects the cell again, crRNA recognizes the invading DNA and binds to it
Cas9
Cuts the invading DNA into pieces
Published by AAAS

10. The crRNA and tracrRNA can be combined into one guide RNA
The system NOW requires 2 components:
Guide RNA
RNA derived from a previous viral infection
If the same virus infects the cell again, crRNA recognizes the invading DNA and binds to it
Cas9
Cuts the invading DNA into pieces
Published by AAAS

11. WHAT DOES THIS HAVE TO DO WITH GENOME EDITING?

12. Genome editing
A genetic engineering approach in which DNA is inserted, removed or replaced at a precise location within the genome. By changing the guide RNA, we can target Cas9 protein to any position in the genome, which will then be cut

13. When DNA is cut, it can have two fates
The cell attempt to rejoin the DNA ends, resulting in random mutations
The DNA ends are repaired by using a matching DNA sequence, if available, leading to precise changes

14. YEAH, BUT WHY IS CRISPR SO GREAT?

15. Edits DNA in the context of the chromosome
Advantages of CRISPR
Edits DNA in the context of the chromosome
It is relatively cheap and easy to design new guide RNAs, making genome editing technology much more accessible
It is highly efficient
The changes can be very precise
Can be used in many species, including those for which we have had very few other editing tools
If the target DNA appears more than once in the genome, all of those targets will be cut
Several different guide RNAs can be used for the introduction of multiple mutations at the same time.
Many different types of genome alterations are possible
Jiang et al. Nature Biotech, 2013

16. Many different types of genome alterations are possible
Edit genes:
Fix small defects in genes
Remove large portions of defective gene
Introduce new mutations
Mutate the genome at many positions to see the effects
Target genes rather than cutting them:
Increase amount of RNA produced from a gene
Decrease amount of RNA produced from a gene
Image genomic specific DNA locations in living cells
Jennifer A. Doudna, and Emmanuelle Charpentier Science 2014;346:
Published by AAAS

17. Future applications in biomedicine and biotechnology
Jennifer A. Doudna, and Emmanuelle Charpentier Science 2014;346:
Published by AAAS

18. Yeast Transformation

19. Steps in yeast transformation for chunk assembly
Create DNA that will repair the cut chromosome DNA
Transform into “competent” yeast cells
Competent yeast cells are ready to take up DNA
Select transformed cells
Those that have taken up the DNA

20. Transformation (Lithium acetated method)
Most common method since 1983
Unknown how DNA gets into the cell, moves into the nucleus and becomes maintained
Components of the reaction:
PEG (deposits DNA onto surface of yeast cell)
Carrier DNA (saturates nonspecific binding sites on yeast cell wall)
LiAc (increases permeability of the cell by (perhaps) shielding negative charges)
Heat shock (increases permeability of the cell wall)

21. Selecting transformed cells
We use a strain of yeast that contains a deletion in the URA3 gene
Without the URA3 gene, the yeast cells cannot make their own uracil
This strain cannot grow on SC-Ura media (media lacking uracil)
When plated on SC-Ura media, the cells will die
Chromosome
with deletion of
the URA3 gene
URA3 deletion

22. Selecting transformed cells
We are assembling our synthetic chunk DNAs in a plasmid vector that contains the URA3 gene
Yeast cells that become transformed and pick up a plasmid with the URA3 gene can make their own uracil
This strain can grow on SC-Ura media (media lacking uracil)
When plated on SC-Ura media, the cells will live and make colonies (after ~2 days at 30C)
Plasmid containing
chunk DNA and
the URA3 gene
URA3 gene
Chromosome
with deletion of
the URA3 gene
URA3 deletion

23. Other selectable markers in yeast
In addition to the URA3 gene, there are other commonly used selectable (auxotrophic) markers in yeast:
LYS2
LEU2
HIS3
TRP1
MET15
URA3 gene