1. Institute of Health and Biomedical Innovation
The CRISPR revolution
Thor Friis, PhD
Bone Group
Institute of Health and Biomedical Innovation
Kelvin Grove

2. Gene-editing – a brief history
Introducing site-specific genetic modification:
Site-directed mutagenesis
Molecular cloning, expression vectors
FLP recombinase/FRT
Cre recombinase
Lentivirus infections

3. Gene-editing nucleases – a brief history
Zinc-finger nucleases (ZFNs)
Tailor-made restriction enzymes
(Kim, Cha and Chandrasegaran, PNAS, 1999)

4. Gene-editing nucleases – a brief history
Transcription activator-like effector nucleases (TALENs)
DNA binding domain isolated from Xanthomonas plant pathogens.
(Zhang, et al., Nature Biotechnology, 2011)
“Method of the Year 2011”
(Nature Methods, 2012)
pnabio.com

5. CRISPR – a brief history
Clustered regularly interspaced short palindromic repeats (CRISPR).
Acquired immunity in bacteria and archea.
Short sequence repeats (SSRs) first discovered in prokaryotes in late 1980s.
(van der Oost et al., Trends Biochem Sci, 2009)
(van Belkum et al., MMBR, 1998)

6. CRISPR – a brief history
The CRISPR/Cas – proposed to act in a manner similar to RNAi in eukaryotes
(Al-Attar, S. BiolChem, 2011)
Relies on two main components:
A guide RNA
CRISPR associated protein (CAS)

7. The CRISPR locus in bacteria
spacers
tracrRNA
CAS
cas operon
CRISPR
Bacterial chromosome
Adapted from Sorek, Annual Review Biochemistry, 2013

8. CRISPR-associated protein (CAS)
Two components:
A helicase
Two endonuclease domains: HNH and RuvC
A single-guide RNA (sgRNA), tweaking natures most ancient immune system.

9. CRISPR/Cas system in Streptococcus pyogenes
PAM (NGG)
target DNA
Proto-spacer
crRNA 5’ 3’ 5’
spacer 3’
tracrRNA
Adapted from Jinek, Science, 2012

10. Programmable CRISPR/Cas9
PAM (NGG)
target DNA
Proto-spacer
Linker loop 5’
20 nt
A single-guide RNA (sgRNA), tweaking natures most ancient immune system.
spacer 3’
crRNA-tracrRNA chimera
(sgRNA)
Adapted from Jinek, Science, 2012

11. CRISPR/Cas9 gene-editing
Non-homologous
end joining
(NHEJ)
Homology- directed repair
(HDR)
Random insertions or deletions (indels)
Assisted recombination of nucleotides or insertions
Adapted from Duodna and Charpentier, Science, 2014

12. Pennisi, E. Science, August 2013

13. The CRISPR Craze
Genetics. 2013 Nov;195(3): Heritable custom genomic modifications in Caenorhabditis elegans via a CRISPR-Cas9 system.

14. The CRISPR Craze
Cell Rep. 2013 Jul 11;4(1): Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system.

15. The CRISPR Craze
Auer, T., et al. Genome Res. 2014
Genome Res. 2014 Jan;24(1): Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair.

16. The CRISPR Craze
The mutation of a second tyr gene target site also caused albinism.
Genesis. 2013 Dec;51(12):
Simple and efficient CRISPR/Cas9-mediated targeted mutagenesis in Xenopus tropicalis.

17. The CRISPR Craze
The thymus of IL2rg KO rabbits smaller than that from age-matched WT ones
Cell Regen (Lond). 2014 Sep 27;3(1):12.
Generation of multi-gene knockout rabbits using the Cas9/gRNA system.

18. The CRISPR Craze Nature 2014 Oct 16;514(7522):380-4
CRISPR-mediated direct mutation 
of cancer genes in the mouse liver.

19. The CRISPR Craze Scientific Reports 2016 Jul 15;6:29855.
Generation and evaluation of Myostatin knock-out rabbits and goats using CRISPR/Cas9 system.

20. The CRISPR Craze
Cell. 2014 Feb 13;156(4): Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos.

21. CRISPR – Conclusions
The use of CRISPR technology is still in its infancy.
A facile tool for reverse genetics  point mutations in promoters and open reading frames.
UQ has a facility specializing in CRISPR technology (Queensland Facility for Advanced Genome Editing)
Model organisms for skeletal disorders: zebrafish and mice