Replicating human disease in rodents: the good and the bad of CRISPR/Cas9 genome editing Guillaume Pavlovic, PhD Head of Unit at PHENOMIN ‐ Institut Clinique de la Souris email: [email protected] CMC Strategy Forum Europe linkedIn: http://bit.ly/2VMLoJ9 Understanding Human disease with rodents: three challenges phenotypes of protein Some failure of mice and other coding genes in Mouse model organisms studies to be replicated or translated to humans Multiple phenotypes No known Knowledge Translation phenotype on disease from rodent One genes to Human phenotype Probability of success Reproducibility of preclinical studies https://www.mousephenotype.org/ 50% of preclinical data are irreproducible CRISPR/Cas9 genome editing in rodents THE GOOD What new possibilities does CRISPR open to better mimic human diseases ? – The genetic context (background) is not adapted – The designed mutation does not mimic the Human pathology With CRISPR/Cas9 new types of mutations can be easily engineered THE BAD There is more than off targets ! Understanding CRISPR/Cas9 genome editing to reduce lack of reproducibility – You are facing experimental variability, poor experimental design or bad reproducibility why do the results discovered using in vivo models sometimes fail to translate to human disease? Plenty of literature showing that the inbred genetic background has an effect on the phenotype Cerebellar phenotype of En1Hd/Hd mutants on 129/Sv and C57BL/6J backgrounds. deletion of the cerebellum 129/Sv—En1Hd/Hd C57BL/6J—En1Hd/Hd Bilovocky et al. J. Neurosci. 2003 why do the results discovered using in vivo models sometimes fail to translate to human disease? Plenty of literature showing that the inbred genetic background has an effect on the phenotype genetic background limits generalizability of genotype-phenotype relationships Sittig et al. demonstrated low generalizability of mouse null allele phenotypes across a panel of F1 genetic backgrounds; It suggests that the use of single strains is a barrier to robust characterization of genotype‐phenotype relationships. Sittig et al. Neuron. 2016 Are you working on C57BL/6 or on C57BL/6 background ? The extensive use of only few mouse strains like C57BL/6 cannot mimic the outbred diversity of human beings In mice, C57BL/6 lines (congenic or coisogenic) represent 68% of the >28 000 lines available in MGI Most of the phenotyping analyses were done in one of these genetic contexts, mixed backgrounds or 129* models were backcrossed to C57BL/6. *129 mice are a complex collection of various backgrounds (Simpson et al., 1997) C57BL/6 lines represent 68% of the >28 000 lines available (MGI extract, February 2017) Gene editing in animal disease model NOD/ShiLtJ CRISPR/Cas9‐mediated gene editing for 10 genes delivered by microinjection Gene X CRISPR/Cas9 NOD/ShiLtJ Gene X KO Qin et al. Genetics 2015 NOD/ShiLtJ: Non Obese diabetic, polygenic model for type 1 diabetes Manipulation of the Ts65Dn minichromosome Ts65Dn mice carry a small extra chromosome derived from a reciprocal translocation (mouse chr16 and 17) Partial trisomy model Ts65Dn Ts65Dn mice are trisomic for about two‐thirds of the genes orthologous to human chromosome 21. Chr17 Ts65Dn are well‐characterized and highly relevant model for studying Down Syndrome with neural cognitive deficits and behavioral abnormalities. Chr16 Manipulation of the Ts65Dn minichromosome Partial trisomy model Ts65Dn 70 Mb are not related to Chr17 Down Syndrome disease Chr16 Manipulation of the Ts65Dn minichromosome Partial trisomy model Ts65Dn CRISPR /Cas9 Chr17 CRISPR /Cas9 Chr16 Manipulation of the Ts65Dn minichromosome The Ts65Dn males show a decreased fertility We combined in vitro fertilization with CRISPR/Cas9 injection in eggs. B6C3H xTs65Dn Understanding human structural variations leading to diseases . Structural variants (SVs) are large genomic alterations that involve segments of DNA greater than one kb (Freeman et al., 2006) . Copy number variants (CNVs) are a subfamily of SV that correspond only to deletions or duplications and do not include inversions or translocations (Freeman et al., 2006) . One of the most common cause in morbidity and mortality in human population . e.g. Down syndrome affecting 1 out of 800 births . SVs likely play a major role in very various diseases not only restricted to neuronal disorders (Conrad et al., 2010; Fanciulli et al., 2007; McCarroll and Altshuler, 2007; Wu and Hurst, 2016) . More than >60 000 SVs were discovered in human (Huddleston and Eichler, 2016) . The ones that are pathological SVs are mostly not known . Chromosomalrearrangementshaveacentralroleinthepathogenesis of human cancers (Taki & Taniwaki, 2006) Generation of Cbs structural variant rat model CRISPR/Cas9 injection in Sprague Dawley fertilized oocytes 4 gRNA used – 37.2 kb region 2 gRNAs 2 gRNAs 37.2 kb CRISPR/Cas9 for structural variant models ‐ Our results CRISPR CRISPR gene(s) Wild‐type Deletion Duplication (DUP) gene(s) gene(s) Inversion (INV) gene(s) DUP + INV gene(s) gene(s) INV + DUP INV gene(s) gene(s) INV + DUP gene(s) gene(s) NHEJ gene(s) CRISPR/Cas9 make easy generating more complex mutations Understanding the human structural variations that leads to disease target DNA deletion – Working with a large panel of SV mutations both in mice and rat – Read our paper Efficient and rapid generation of large genomic variants in rats and mice using CRISMERE ‐ Birling et al. 2017 Sci Rep. http://bit.ly/2Jlwxip Humanization of large locus in mice Replacement of ApoE mouse gene by ApoE2, 3 and 4 human alleles including Tomm40 Humanization of large locus in mice Replacement of ApoE mouse gene by ApoE2, 3 and 4 human alleles including Tomm40 Full replacement of a 37 kb sequence achieved in C57BL/6N ES cells using Neo/Hygro + CRISPR/Cas9 selection – 4 variants achieved Human sequence 37.7 kb Mouse locus Mouse locus ~29 kb CRISPR/Cas9 genome editing can improve replicating Human disease Generation of complex humanization in mouse and rat Achieving structural variants models Genetic context can be easily changed Working in a large panel of species Read our review Modeling human disease in rodents by CRISPR/Cas9 genome editing. Birling et al. 2017 Mamm Genome. http://bit.ly/2WtsHYh Understating CRISPR/Cas9 genome editing to reduce reproducibility issues CRISPR/Cas9 is a (targeted) mutagen DNA The only function of the CRISPR/Cas9 system is to create a double strand break at a chosen DNA sequence CRISPR/Cas9 targeted double strand break (dsb) CRISPR/Cas9 is a (targeted) mutagen DNA The only function of the CRISPR/Cas9 system is to create a double strand break at a chosen DNA sequence CRISPR/Cas9 targeted double strand break (dsb) The cell repairs the double strand break as it can NHEJ HR Gene disruption Correction from by small the other insertions or chromosome deletions template The causes of unwanted mutations with CRISPR/Cas9 CRISPR/Cas9 is like bromide ethidium it is a mutagen … a targeted mutagen but still a mutagen The causes of unwanted mutations with CRISPR/Cas9 CRISPR/Cas9 off‐target: – Lack of specificity of the system that cut DNA sequence that are similar to the target Cell reparation off‐target: – Dsb are boosting cell reparation – Addition of CRISPR/Cas9 will favour unwanted (unanticipated) DNA reparation Cell reparation on‐target: – Dsb are boosting cell reparation – Do I really have the model I think? CRISPR/Cas9 off‐target ‐ Lack of specificity of the system that cut DNA sequence that are similar to the target Off‐target are very frequent Off‐target happens but are rare event Cells In vivo ‐mice Plasmid construct – constitutive expression mRNA or protein Cas9 – transient expression + IMPC data (Iyer et al. 2015) CRISPR/Cas9 off‐target With a good experimental design (CRISPR/Cas9 expressed as mRNA / protein for transient gene editing), off‐target level may be less important than natural genetic drift Generating a SNP model DNA + CRISPR/Cas9 targeted double strand break NHEJ HR Cell reparation off‐targets Single strand or double strand DNA used as template for recombination can integrate randomly in the genome Cell reparation off‐targets Our data show that ssODN used to achieve knock‐in mutations can integrate at random in the genome >15% F0 animals have integrated random ssODN – > 100 animals screened from 5 projects Cell reparation off‐targets Frequency of random integration of ssDNA need to be evaluated Solutions – Random integration mutation can be removed by crossing with wt animals – qPCR screening for SNP (shortssDNA) or CKO / knock‐in (longssDNA) need to be performed Cell reparation on‐target Reparation of the double strand break can lead to unexpected events CRISPR/Cas9 mediate double strand‐ break may induce chromosomal rearrangements Lessons learned with CRISMERE CRISPR CRISPR Target • The use of 2 or more sgRNAs may result in reparation of DNA leading to complex rearrangements like duplications, inversions or deletions. • By consequence those mutations should be anticipated. • Such rearrangements are frequent and have been observed for several loci both in mouse and rats. Translocations with CRISPR/Cas9 Two sgRNA in different genes = Two double strand breaks = 2 knock-out genes + Chromosomal Rearrangements Sequences rearrangements Yin et al. proof of concepts of in vivo excision of HIV‐1 provirus in mouse Yin et al., 2017 In Vivo Excision of HIV-1 Provirus by saCas9 and Multiplex Single-Guide RNAs in Animal Models Cell reparation on‐targets CRISPR/Cas9 boost homologous recombination mechanisms HDR of a complex construct using CRISPR/cas