US: 1-888-822-6642 | EU: +45 70 23 04 05 | [email protected] | TACONIC.COM ©Taconic Biosciences, Inc. All rights reserved. Contents of this publication may not be reproduced in any form without prior permission. PT1087-EK-EN-1709 Generation of Knockout, Knock-in, and Humanized Mouse Models Using the CRISPR/Cas9 Technology: Lessons Learned and Open Questions Steffen Güttler 1 , Alexander Klimke 1 , Petric Kuballa 1 , Gesa Glöckel 1 , Jeanette Keßler 1 , Heidrun Kern 1 , Kenneth Albrecht 2 , Adriano Flora 1 , Eleanor Kolossovski 2 , Jochen Welcker 1 1 Taconic Biosciences GmbH, Cologne, Germany; 2 Taconic Biosciences Inc., Rensselaer, NY The CRISPR/Cas9 genome editing system has established itself as a versatile technology for inducing precise genetic alterations in a number of different species, and has proved to have the potential to increase the efficiency and speed of producing genetically engineered models of human disease. At Taconic Biosciences, we use both an in vivo strategy utilizing one-cell embryos and a complementary in vitro strategy utilizing embryonic stem (ES) cells to generate both mouse and rat models with CRISPR/Cas9. We provide a broad data-set to illustrate our experiences using these two strategies within our production pipeline. The major advantages of in vivo gene editing using CRISPR/Cas-based methods are the significantly reduced time frame and effort involved in establishing new mouse or rat models and the ability to utilize almost any available mouse and rat strain. The relative simplicity of this method compared to ES cell-mediated approaches facilitates the generation of founder animals with reduced costs and effort, providing an attractive alternative to homologous recombination-based approaches. We have implemented CRISPR/Cas9 technology in vitro for the accelerated generation of knockout and simple knock-in (e.g. point mutations and small tags) mice and rats and successfully generated more than 200 models in the past three years. However, the genetic modifications that can be introduced in the genome by the in vivo approach are currently limited to relatively simple allelic configuration, such as single base substitutions, gene deletion, and insertion of short sequences. To overcome this limitation, we have combined the use of CRISPR/Cas9 technology with the advantages of utilizing large targeting constructs in ES cells. The combination of the two technologies allows for the generation of large and complex alleles with the precision and efficiency provided by the CRISPR/Cas system and, importantly, the humanization of specific loci in the mouse genome by gene replacement to create relevant model for preclinical drug testing. Advantages of Humanization by Genomic Replacement • Successful means that the humanized allele has an expression of a similar level (60% or more) of the endogenous mouse gene and the same pattern of expression as measured by RT-qPCR. • While Minigene Insertions and Open Reading Frame (ORF) Exchanges are easy to accomplish, the lack of regulatory elements within intronic regions often compromises the expression levels and patterns. • We recommend genomic replacements as these seem to resemble expression-levels and patterns most closely. Protocol for CRISPR-mediated Gene Editing in ES Cells Transfection of ES Cells to Introduce a Targeted Mutation via CRISPR/Cas9-mediated Gene Editing ES cells (e.g. C57BL/6NTac or BALB/c) were grown on a mitotically inactivated feeder layer comprised of mouse embryonic fibroblasts in ES cell culture medium. Cas9, the specific gRNAs, and the targeting vector were co-transfected into cells along with a plasmid for the expression of the respective selection cassette. One day post transfection the antibiotic was transiently added to the medium to select for transfected cells. ES cell clones were isolated as soon they show a distinct morphology and were analyzed by PCR in a primary screen for recombination at the 5' and 3' side. Homologous recombinant ES cell clones were expanded and frozen in liquid nitrogen after extensive molecular validation by PCR and southern blot analysis. Analysis of Off-target Effects • For all CRISPR embryo and CRISPR in ES cell projects, off-target predictions were performed according to the number and position of mis-matches within the gRNA sequence. • For 12 CRISPR in ES cell projects and 43 CRISPR in embryo projects, potential loci were analyzed by PCR and sequencing. • Within 505 loci of 31 ES cell clones and 310 loci of 555 G1-mice, only three off-target effects were detected: — The first off-target in embryo with two deviations in the seed and one deviation in the non-seed region. — The second off-target in ES cells with two deviations in the seed and one deviation in the non-seed region. — The third off-target in embryo with no deviation in the seed and two deviations in the non-seed region (only present in one out of three validated clones). • The off-target frequency with our setup is smaller than 0.4% and lower than the spontaneous mutation rate detected per generation or passage. Protocol for CRISPR-mediated Gene Editing in Embryo Pronuclear Injection After administration of hormones, superovulated C57BL/6NJ females were mated with C5BL/6NJ males. One-cell stage fertilized embryos were isolated from the oviducts at dpc 0.5. For microinjection, the one-cell stage embryos were placed in a drop of M2 medium under mineral oil. A microinjection pipette with an approximate internal diameter of 0.5 micrometer (at tip) was used to inject the mixed nucleotide preparation into the pronucleus of each embryo. For knockout projects, the mix contained two specific gRNA and the Cas9-protein. For knock-in projects, an oligonucleotide was injected together with the appropriate gRNA. After recovery, 25–35 injected one-cell stage embryos were transferred to one of the oviducts of 0.5 dpc, pseudo- pregnant NMRI females. Founder Analysis Founder animals were genotyped for presence of the deletion or the inserted point mutation (and inserted restriction site). PCR samples per founder were subcloned and up to four clones were analyzed by DNA-sequencing. Gene Knockout (KO) in Embryo Knock-in (KI) of a Point Mutation in Embryo Humanizations and Knock-in in ES cells 60 50 40 30 20 10 0 103 Injections (43 Projects) Pup Number 98 Injections (39 Projects) 40 30 20 10 0 Pup Number 103 Injections (43 Projects) 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Percentage of Total Genotyped Animals Percentage of Total Genotyped Animals 98 Injections (39 Projects) 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Heterozygous Targeted Clones Homozygous 17.8 kb (wt) 3.0 kb (hum) wt 1 2 3 4 5 12.2 kb (hum) 5.9 kb (control) Size of Deletion [kb] Percentage of Total Genotyped Animals 00 01 01 01 01 01 01 01 02 02 02 02 03 03 03 03 03 03 04 04 04 05 05 06 06 06 07 07 08 09 10 10 12 18 19 24 59 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 90 105 120 150 180 Time for Completion [Days] 120% 100% 80% 60% 40% 20% 0% Percentage of Projects Completed 90 105 120 150 180 Time for Completion [Days] 120% 100% 80% 60% 40% 20% 0% Percentage of Projects Completed Size of Insertion [nt] 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Percentage of Total Genotyped Animals 42 42 42 51 51 51 67 67 67 Number of Validated Clones Size of Insertion/Humanization [kb] 10 9 8 7 6 5 4 3 2 1 0 0.4 0.9 1 2.2 3.4 3.4 3.4 3.4 4.5 5 5.2 5.6 6.5 8.4 19 0 2 4 6 8 10 12 14 16 18 20 Size of Insertion/Humanization [kb] Percentage of Homologous Recombination 1.0% 0.8% 0.6% 0.4% 0.2% 0.0% Genomic Replacement Minigene Insertion ORF Exchange Humanizing Point Mutations Exon Swapping • CRISPR/Cas9 is a powerful tool that can speed up model generation from simple point mutations to large knock-ins and humanizations. • While we can predictably control the design of each model, the efficiency of CRISPR/Cas9 strongly depends on the selected gRNA. • Despite various prediction tool, the relation between gRNA-sequence and cleavage-efficiency remains largely unknown. • Off target effects are a controllable risk in our setup. CONCLUSION Improvements of procedures have led to higher reproducibility and overall efficiency. This resulted in a vast majority of projects completed in or ahead of the anticipated timeline. Deletion efficiency is independent of the size of the deletion Tag sequences of up to 67 nucleotides were inserted using long oligonucleotides Deletion efficiency is highly variable and dependent on gRNA sequence Homologous recombination efficiency is highly variable and dependent on the gRNA sequence • Homologous recombination efficiency is highly variable, depending on the gRNA sequence • We successfully inserted up to 45 kb of human sequence CRISPR-mediated homologous recombination in ES cells occurs on both alleles with a high frequency Mouse Genomic Locus Mouse Genomic Locus Targeted Allele (after CRISPR/Cas9-medicated Gene Editing) Mouse Genomic Locus Constitutive KI-PM Allele (after CRISPR/Cas9-medicated Gene Editing) Constitutive Humanized Allele (after CRISPR/Cas9-medicated Gene Editing) Homologous Recombination Rate of 15 Humanization and Knock-in Projects Success of Humanization Projects Completed at Taconic Biosciences CRISPR-mediated KI Approach: Percentage of Modifications in ES cells PCR Pre-screen Southern Blot Analysis Number of projects Genomic Replacement Minigene Insertion ORF Exchange Point Mutations Exon Swapping 45 40 35 30 25 20 15 10 5 0 Life Born Rates of 43 Knockout Projects Life Born Rates of 39 Knock-in Projects CRISPR-mediated KO Approach: Percentage of Modifications in Embryo CRISPR-mediated KI Approach: Percentage of Modifications in Embryo CRISPR-mediated KO Approach: Modifications in Embryo in Relation to Deletion Size CRISPR-mediated Tag-KI Approach: Percentage of Modifications in Embryo Percentage of Knockout Projects Completed within a Given Timeframe Percentage of Knock-in Projects Completed within a Given Timeframe wt % indel% del% wt % indel% del% validated heterozygous clone validated homozygous clone wt % indel% HDR% wt % indel% del % KO 2014/2015 KO 2016 KI 2014/2015 KI 2016 successful unsuccessful