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1. Whole genome analysis for 163 gRNAs in Cas9-edited mice reveals minimal off-target activity

Author

Peterson, Kevin A, Khalouei, Sam, Hanafi, Nour, Wood, Joshua A, Lanza, Denise G, Lintott, Lauri G, Willis, Brandon J, Seavitt, John R, Braun, Robert E, Dickinson, Mary E, White, Jacqueline K, Lloyd, KC Kent, Heaney, Jason D, Murray, Stephen A, Ramani, Arun, and Nutter, Lauryl MJ

Subjects
Gene Editing, Mice, Genome, Mutagenesis, Mutation, Human Genome, Genetics, Animals, 2.1 Biological and endogenous factors, CRISPR-Cas Systems, Aetiology, Biotechnology
Abstract

Genome editing with CRISPR-associated (Cas) proteins holds exceptional promise for "correcting" variants causing genetic disease. To realize this promise, off-target genomic changes cannot occur during the editing process. Here, we use whole genome sequencing to compare the genomes of 50 Cas9-edited founder mice to 28 untreated control mice to assess the occurrence of S. pyogenes Cas9-induced off-target mutagenesis. Computational analysis of whole-genome sequencing data detects 26 unique sequence variants at 23 predicted off-target sites for 18/163 guides used. While computationally detected variants are identified in 30% (15/50) of Cas9 gene-edited founder animals, only 38% (10/26) of the variants in 8/15 founders validate by Sanger sequencing. In vitro assays for Cas9 off-target activity identify only two unpredicted off-target sites present in genome sequencing data. In total, only 4.9% (8/163) of guides tested have detectable off-target activity, a rate of 0.2 Cas9 off-target mutations per founder analyzed. In comparison, we observe ~1,100 unique variants in each mouse regardless of genome exposure to Cas9 indicating off-target variants comprise a small fraction of genetic heterogeneity in Cas9-edited mice. These findings will inform future design and use of Cas9-edited animal models as well as provide context for evaluating off-target potential in genetically diverse patient populations.

Published
2023

2. A resource of targeted mutant mouse lines for 5,061 genes

Author

Birling, Marie-Christine, Yoshiki, Atsushi, Chiani, Francesco, Kaloff, Cornelia, Hörlein, Andreas, Teichmann, Sandy, Tasdemir, Adriane, Krause, Heidi, German, Dorota, Könitzer, Anne, Weber, Sarah, Beig, Joachim, McKay, Matthew, Chin, Hsian-Jean Genie, Bedigian, Richard, Dion, Stephanie, Kutny, Peter, Kelmenson, Jennifer, Perry, Emily, Nguyen-Bresinsky, Dong, Seluke, Audrie, Leach, Timothy, Perkins, Sara, Slater, Amanda, Christou, Skevoulla, Petit, Michaela, Urban, Rachel, Kales, Susan, DaCosta, Michael, McFarland, Michael, Palazola, Rick, Peterson, Kevin A, Svenson, Karen, Braun, Robert E, Taft, Robert, Codner, Gemma F, Rhue, Mark, Garay, Jose, Clary, Dave, Araiza, Renee, Grimsrud, Kristin, Bower, Lynette, Anchell, Nicole L, Jager, Kayla M, Young, Diana L, Dao, Phuong T, DeMayo, Francesco J, Gardiner, Wendy, Bell, Toni, Kenyon, Janet, Stewart, Michelle E, Lynch, Denise, Loeffler, Jorik, Caulder, Adam, Hillier, Rosie, Quwailid, Mohamed M, Zaman, Rumana, Dickinson, Mary E, Santos, Luis, Obata, Yuichi, Iwama, Mizuho, Nakata, Hatsumi, Hashimoto, Tomomi, Kadota, Masayo, Masuya, Hiroshi, Tanaka, Nobuhiko, Miura, Ikuo, Yamada, Ikuko, Doe, Brendan, Furuse, Tamio, Selloum, Mohammed, Jacquot, Sylvie, Ayadi, Abdel, Ali-Hadji, Dalila, Charles, Philippe, Le Marchand, Elise, El Amri, Amal, Kujath, Christelle, Fougerolle, Jean-Victor, Donahue, Leah Rae, Mellul, Peggy, Legeay, Sandrine, Vasseur, Laurent, Moro, Anne-Isabelle, Lorentz, Romain, Schaeffer, Laurence, Dreyer, Dominique, Erbs, Valérie, Eisenmann, Benjamin, Rossi, Giovanni, Fray, Martin D, Luppi, Laurence, Mertz, Annelyse, Jeanblanc, Amélie, Grau, Evelyn, Sinclair, Caroline, Brown, Ellen, Kundi, Helen, Madich, Alla, Woods, Mike, Pearson, Laila, Gambadoro, Alessia, Mayhew, Danielle, Griggs, Nicola, Houghton, Richard, Bussell, James, Ingle, Catherine, Valentini, Sara, Gleeson, Diane, Sethi, Debarati, Bayzetinova, Tanya, Burvill, Jonathan, Adams, David J, Gao, Xiang, Habib, Bishoy, Weavers, Lauren, Maswood, Ryea, Miklejewska, Evelina, Cook, Ross, Platte, Radka, Price, Stacey, Vyas, Sapna, Collinson, Adam, Hardy, Matt, Gertsenstein, Marina, Dalvi, Priya, Iyer, Vivek, West, Tony, Thomas, Mark, Mujica, Alejandro, Sins, Elodie, Barrett, Daniel, Dobbie, Michael, Grobler, Anne, Loots, Glaudina, Gomez-Segura, Alba, Hayeshi, Rose, Scholtz, Liezl-Marie, Bester, Cor, Pheiffer, Wihan, Venter, Kobus, Bosch, Fatima, Goodwin, Leslie O, Heaney, Jason D, Hérault, Yann, de Angelis, Martin Hrabě, Jiang, Si-Tse, Justice, Monica J, Kasparek, Petr, Ayabe, Shinya, King, Ruairidh E, Kühn, Ralf, Lee, Ho, Lee, Youngik, Liu, Zhiwei, Lloyd, K C Kent, Lorenzo, Isabel, Mallon, Ann-Marie, McKerlie, Colin, Meehan, Terrence F, Beaudet, Arthur L, Fuentes, Violeta Munoz, Newman, Stuart, Nutter, Lauryl M J, Oh, Goo Taeg, Pavlovic, Guillaume, Ramirez-Solis, Ramiro, Rosen, Barry, Ryder, Edward J, Santos, Luis A, Schick, Joel, Bottomley, Joanna, Seavitt, John R, Sedlacek, Radislav, Seisenberger, Claudia, Seong, Je Kyung, Skarnes, William C, Sorg, Tania, Steel, Karen P, Tamura, Masaru, Tocchini-Valentini, Glauco P, Wang, Chi-Kuang Leo, Bradley, Allan, Wardle-Jones, Hannah, Wattenhofer-Donzé, Marie, Wells, Sara, Wiles, Michael V, Willis, Brandon J, Wood, Joshua A, Wurst, Wolfgang, Xu, Ying, Consortium, International Mouse Phenotyping, Teboul, Lydia, Brown, Steve D M, Murray, Stephen A, Gallegos, Juan J, Green, Jennie R, Bohat, Ritu, Zimmel, Katie, Pereira, Monica, MacMaster, Suzanne, Tondat, Sandra, Wei, Linda, Carroll, Tracy, Bürger, Antje, Cabezas, Jorge, Fan-Lan, Qing, Jacob, Elsa, Creighton, Amie, Castellanos-Penton, Patricia, Danisment, Ozge, Clarke, Shannon, Joeng, Joanna, Kelly, Deborah, To, Christine, Bushell, Wendy, van Bruggen, Rebekah, Gailus-Durner, Valerie, Fuchs, Helmut, Marschall, Susan, Dunst, Stefanie, Romberger, Markus, Rey, Bernhard, Fessele, Sabine, Gormanns, Philipp, Friedel, Roland, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), and Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Cancer Research, Genotype, Knockout, International Cooperation, [SDV]Life Sciences [q-bio], Mutant, Mutagenesis (molecular biology technique), cytology [Mouse Embryonic Stem Cells], Mouse Mutant Strains, Biology, Genome, Medical and Health Sciences, Article, 03 medical and health sciences, Mice, 0302 clinical medicine, Research community, ddc:570, Genetics, Animals, Allele, Gene, Genetic Association Studies, 030304 developmental biology, Mice, Knockout, 0303 health sciences, Internet, metabolism [Mouse Embryonic Stem Cells], Information Dissemination, mutant mouse IMPC, Mouse Embryonic Stem Cells, Biological Sciences, Null allele, Embryonic stem cell, Phenotype, Mutagenesis, Montertondo Mouse Production, Technology Platforms, International Mouse Phenotyping Consortium, Function (biology), 030217 neurology & neurosurgery, Gene Deletion, Developmental Biology
Abstract

The International Mouse Phenotyping Consortium reports the generation of new mouse mutant strains for over 5,000 genes from targeted embryonic stem cells on the C57BL/6N genetic background. This includes 2,850 null alleles for which no equivalent mutant mouse line exists, 2,987 novel conditional-ready alleles, and 4,433 novel reporter alleles. This nearly triples the number of genes with reporter alleles and almost doubles the number of conditional alleles available to the scientific community. When combined with more than 30 years of community effort, the total mutant allele mouse resource covers more than half of the genome. The extensively validated collection is archived and distributed through public repositories, facilitating availability to the worldwide biomedical research community, and expanding our understanding of gene function and human disease.

Published
2021
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3. Generation of desminopathy in rats using CRISPR-Cas9

Author

Langer, Henning T, Mossakowski, Agata A, Willis, Brandon J, Grimsrud, Kristin N, Wood, Joshua A, Lloyd, Kevin CK, Zbinden-Foncea, Hermann, and Baar, Keith

Subjects
Male, Physiology, Precision medicine, Clinical Sciences, Injury, Human Movement and Sports Sciences, Muscular dystrophy, Rats, Desmin, Dystrophin, Mice, Muscular Diseases, Mutation, Genetics, Animals, 2.1 Biological and endogenous factors, CRISPR-Cas Systems, Exercise, Force transfer
Abstract

BackgroundDesminopathy is a clinically heterogeneous muscle disease caused by over 60 different mutations in desmin. The most common mutation with a clinical phenotype in humans is an exchange of arginine to proline at position 350 of desmin leading to p.R350P. We created the first CRISPR-Cas9 engineered rat model for a muscle disease by mirroring the R350P mutation in humans.MethodsUsing CRISPR-Cas9 technology, Des c.1045-1046 (AGG>CCG) was introduced into exon 6 of the rat genome causing p.R349P. The genotype of each animal was confirmed via quantitative PCR. Six male rats with a mutation in desmin (n=6) between the age of 120-150days and an equal number of wild type littermates (n=6) were used for experiments. Maximal plantar flexion force was measured in vivo and combined with the collection of muscle weights, immunoblotting, and histological analysis. In addition to the baseline phenotyping, we performed a synergist ablation study in the same animals.ResultsWe found a difference in the number of central nuclei between desmin mutants (1±0.4%) and wild type littermates (0.2±0.1%; P&nbsp

Published
2020

4. Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation

Author

Gurumurthy, Channabasavaiah B, O'Brien, Aidan R, Quadros, Rolen M, Adams, John, Alcaide, Pilar, Ayabe, Shinya, Ballard, Johnathan, Batra, Surinder K, Beauchamp, Marie-Claude, Becker, Kathleen A, Bernas, Guillaume, Brough, David, Carrillo-Salinas, Francisco, Chan, Wesley, Chen, Hanying, Dawson, Ruby, DeMambro, Victoria, D'Hont, Jinke, Dibb, Katharine M, Eudy, James D, Gan, Lin, Gao, Jing, Gonzales, Amy, Guntur, Anyonya R, Guo, Huiping, Harms, Donald W, Harrington, Anne, Hentges, Kathryn E, Humphreys, Neil, Imai, Shiho, Ishii, Hideshi, Iwama, Mizuho, Jonasch, Eric, Karolak, Michelle, Keavney, Bernard, Khin, Nay-Chi, Konno, Masamitsu, Kotani, Yuko, Kunihiro, Yayoi, Lakshmanan, Imayavaramban, Larochelle, Catherine, Lawrence, Catherine B, Li, Lin, Lindner, Volkhard, Liu, Xian-De, Lopez-Castejon, Gloria, Loudon, Andrew, Lowe, Jenna, Jerome-Majewska, Loydie A, Matsusaka, Taiji, Miura, Hiromi, Miyasaka, Yoshiki, Morpurgo, Benjamin, Motyl, Katherine, Nabeshima, Yo-Ichi, Nakade, Koji, Nakashiba, Toshiaki, Nakashima, Kenichi, Obata, Yuichi, Ogiwara, Sanae, Ouellet, Mariette, Oxburgh, Leif, Piltz, Sandra, Pinz, Ilka, Ponnusamy, Moorthy P, Ray, David, Redder, Ronald J, Rosen, Clifford J, Ross, Nikki, Ruhe, Mark T, Ryzhova, Larisa, Salvador, Ane M, Alam, Sabrina Shameen, Sedlacek, Radislav, Sharma, Karan, Smith, Chad, Staes, Katrien, Starrs, Lora, Sugiyama, Fumihiro, Takahashi, Satoru, Tanaka, Tomohiro, Trafford, Andrew W, Uno, Yoshihiro, Vanhoutte, Leen, Vanrockeghem, Frederique, Willis, Brandon J, Wright, Christian S, Yamauchi, Yuko, Yi, Xin, Yoshimi, Kazuto, Zhang, Xuesong, Zhang, Yu, Ohtsuka, Masato, Das, Satyabrata, Garry, Daniel J, Hochepied, Tino, Thomas, Paul, Parker-Thornburg, Jan, Adamson, Antony D, and Yoshiki, Atsushi

Subjects
Male, Homology-directed repair, Microinjections, Mouse, Methyl-CpG-Binding Protein 2, Bioinformatics, Knockout, Mice, CRISPR-Associated Protein 9, Oligonucleotide, Information and Computing Sciences, Machine learning, Genetics, Animals, Alleles, Prevention, Floxed allele, Reproducibility of Results, Statistical, Biological Sciences, Long single-stranded DNA, Conditional knockout mouse, Reproducibility, Blastocyst, Regression Analysis, Female, Transgenesis, CRISPR-Cas Systems, CRISPR-Cas9, Factor Analysis, Environmental Sciences, Biotechnology
Abstract

BackgroundCRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as "two-donor floxing" method).ResultsWe re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach.ConclusionWe propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis, an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.

Published
2019

5. Efficient mouse genome engineering by CRISPR-EZ (CRISPR RNP Electroporation of Zygotes) technology

Author

Modzelewski, Andrew J., Chen, Sean, Willis, Brandon J., Lloyd, K C Kent, Wood, Joshua A., and He, Lin

Subjects
Gene Editing, Mice, Inbred C57BL, Mice, Knockout, Mice, Electroporation, Microinjections, Zygote, CRISPR-Associated Protein 9, Animals, Clustered Regularly Interspaced Short Palindromic Repeats, Article
Abstract

CRISPR/Cas9 technology has transformed mouse genome editing with unprecedented precision, efficiency, and ease; however, the current practice of microinjecting CRISPR reagents into pronuclear-stage embryos remains rate-limiting. We thus developed CRISRP-EZ (CRISPR RNP Electroporation of Zygotes), an electroporation-based technology that outperforms pronuclear and cytoplasmic microinjection in efficiency, simplicity, cost, and throughput. In C57BL/6J and C57BL/6N mouse strains, CRISPR-EZ achieves 100% delivery of Cas9/sgRNA ribonucleoproteins (RNPs), facilitating indel mutations (insertions or deletions), exon deletions, point mutations, and small insertions. In a side-by-side comparison in the high-throughput KnockOut Mouse Project (KOMP) pipeline, CRISPR-EZ consistently outperformed microinjection. Here, we provide an optimized protocol covering single guide RNA (sgRNA) synthesis, embryo collection, RNP electroporation, mouse generation, and genotyping strategies. Using CRISPR-EZ, a graduate-level researcher with basic embryo manipulation skills can obtain genetically modified mice in 6 weeks. Altogether, CRISPR-EZ is a simple, economic, efficient, and high-throughput technology that is potentially applicable to other mammalian species.

Published
2018

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