Use of Gene Editing Technologies in Rodents. Carlisle P. Landel, Ph.D.
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1 Use of Gene Editing Technologies in Rodents Carlisle P. Landel, Ph.D.
2 The Mouse as A Model Mammal Small, easy to maintain, fecund Well understood genetics Similarity to humans >90% Availability of inbred strains Highly amenable to genome modification Thousands of strains of genetically modified mouse strains exist Banks of ES cells with mutations in almost all mouse genes exist
3 Milestones in Mouse Genetics 1903 Mendel s laws verified in mammals using mouse coat colors 1920 s Genetic basis for cancer: tumor susceptible mouse strains 1930 s Extrachromosomal transmission of tumors (virus, MMTV) 1940 s Histocompatibility antigens mapped (8 linkage groups) Effect of uterine environment on birth defects established Mouse mutants used to model human congenital defects 1950 s Biological effects of radiation and chemical mutagens 1980 Transgenic mouse technology: pronuclear injection 1984 Interspecies homologies in linkages evident 1986 Embryonic stem cell technology: blastocyst injection 1990 s ENU mutagenesis projects 2000 >10,000 genes, >25,000 markers in mouse 2003 Mouse genome draft sequence available 2007 Knock-Out Mouse Project (KOMP) 2009 Targeted Nucleases come online commercially
4 The Rat as a Model Larger More expensive to maintain, slightly longer generation time More material per animal Preferred for toxicological and behavioral studies Some genetic phenotypes more closely mimic the human condition compared to mice Availability of genome modification was traditionally much more limited than with mice Initial transgenesis attempts were inefficient No ES cells until 2009
5 ES Cell Mediated Targeted (Obtain/produce ES Cells) Mutagenesis (the bad old days) Manipulate ES cells in vitro Isolate targeted clones Timeline: 4 8 weeks Insert targeted clones into blastocysts Transfer injected blasts to foster mothers Screen for chimeric offspring carrying targeted mutation Timeline: 4 8 weeks Mate to produce heterozygous germline founders Timeline: 8 weeks Mate to produce homozygous mutant animals Timeline: 8 weeks Total Timeline To Homozygous Mutants: 8 months minimum
6 The Targeted Nuclease Family ZFNs First applied to rats in 2009 and to mice in 2010 Disadvantages: expensive, not so easy to construct, limited target availability, onerous IP reach through TALENs (XTNs) Rodent applications began in 2011 Easier to make than ZFNs, can theoretically target any sequence CRISPR/CAS9 Rodent applications began in 2013 Extremely easy to engineer, amenable to multiplex targeting More prone to off target mutagenesis, somewhat targetrestricted
7 Double Strand Break Repair Non Homologous End Joining Homologous Recombination Repair Error Prone Introduces small deletions/insertions Error Free 7
8 Targeted Nuclease Mutagenesis DNE DNE DNE coding sequence coding seekwence DNA break coding se quence codingquence mutagenic repair by NHEJ cargo plasmid DNA cargo DNA break homologous recombination DNA break coding se ekwence coding sequence plasmid DNA coding sequence homologous recombination
9 Targeted Nuclease Mediated Mutagenesis Process Treat 1 cell embryos with reagent Transfer treated embryos to foster mothers Screen for mutant offspring Timeline: 6 8 weeks
10 Mouse Embryo Microinjection
11 Debut of CRISPR/CAS9
12 Results of Wang et al. CRISPR targeting is extremely efficient As high as 100% targeted pups and 95% homozygous targeting for some alleles/conditions 80% of pups from double gene targeting were double homozygotes
13 Implications of Wang et al. Gene targeting in rodents is now extremely easy Is ES cell mediated targeted mutagenesis obsolete? Simultaneous targeting of multiple genes promises tremendous savings of time and animals
14 Traditional Production of Lines Carrying Two Mutations 25% of animals 8 week process 6.25% of animals 8 week process
15 Traditional Production of Lines Carrying Three Mutations Mut 3 Mut % of animals 8 week process Mut 3 Mut 3 Mut 3 Mut 3 1.6% of animals 8 week process
16 Targeted Nuclease Mutagenesis Has Been Used To Generate: Knockout mutations, i.e., inactivate specific genes Knockin mutations, i.e., insert genetic material at a defined genomic site Single base changes at specific sites Small insertions of specific sequence, e.g. recombinase targets like loxp, etc. Transgene insertions at safe sites or under the control of endogenous promoters Larger scale deletions or insertions
17 Remaining Issues Off target mutagenesis System modification for increased fidelity In the animal model world, not such an issue: We ll just breed them out. Converting art to a routine methodology What defines a good target? Not all loci are equal. How do we bias the system indels vs insertions? What are the best reagents/conditions/practices for success?
18 Targeted Nucleases Have Revolutionized Mouse Genome Engineering No longer limited by ES cell availability Any strain can (potentially) be targeted Any (rodent) species can be targeted Timelines to model generation have been significantly shortened Animal use for model production has decreased
19 Questions? Carlisle P. Landel, Ph.D
Bart Williams, PhD Van Andel Research Center
A History of Genome Editing in the Laboratory Implications for Translational Applications Bart Williams, PhD Van Andel Research Center Introduction by Matthew Denenberg, MD DeVos Childrens Hospital Disclosures:
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