Institute of Health and Biomedical Innovation

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

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

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

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

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)

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)

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

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.

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

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

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

Pennisi, E. Science, August 2013

The CRISPR Craze Genetics. 2013 Nov;195(3):1181-5. Heritable custom genomic modifications in Caenorhabditis elegans via a CRISPR-Cas9 system.

The CRISPR Craze Cell Rep. 2013 Jul 11;4(1):220-8. Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system.

The CRISPR Craze Auer, T., et al. Genome Res. 2014 Genome Res. 2014 Jan;24(1):142-53. Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair.

The CRISPR Craze The mutation of a second tyr gene target site also caused albinism. Genesis. 2013 Dec;51(12):835-43. Simple and efficient CRISPR/Cas9-mediated targeted mutagenesis in Xenopus tropicalis.

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.

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

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

The CRISPR Craze Cell. 2014 Feb 13;156(4):836-43. Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos.

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