BACKGROUND: CRISPR-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). RESULTS: We 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. CONCLUSION: We 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.

Basic and Clinical Research The Rogosin Institute New York USA

Center for Matrix Biology and Medicine Graduate School of Medicine Tokai University Isehara Kanagawa 259 1193 Japan

Centre de Recherche du Centre Hospitalier Universitaire de Montreal Montreal Canada

Centre for Biological Timing School of Medical Sciences Faculty of Biology Medicine and Health University of Manchester Manchester UK

Children's Research Institute Mouse Genome Engineering Core University of Texas Southwestern Medical Center Dallas TX 75390 USA

College of Osteopathic Medicine Marian University Indianapolis IN 46222 USA

Department of Basic Medicine Division of Basic Medical Science and Molecular Medicine School of Medicine Tokai University 143 Shimokasuya Isehara Kanagawa 259 1193 Japan

Department of Biochemistry and Molecular Biology University of Nebraska Medical Center Omaha NE USA

Department of Biomedical Molecular Biology Ghent University Ghent Belgium

Department of Frontier Science for Cancer and Chemotherapy Osaka University Graduate School of Medicine Suita Japan

Department of Gastroenterology and Metabolism Nagoya City University Graduate School of Medical Sciences Nagoya Japan

Department of Immunology and Infectious Disease The John Curtin School of Medical Research the Australian National University Canberra Australia

Department of Immunology Tufts University School of Medicine Boston USA

Department of Laboratory Animal Science Support Center for Medical Research and Education Tokai University 143 Shimokasuya Isehara Kanagawa 259 1193 Japan

Department of Medical Data Science Osaka University Graduate School of Medicine Suita Japan

Department of Molecular Life Science Division of Basic Medical Science and Molecular Medicine School of Medicine Tokai University 143 Shimokasuya Isehara Kanagawa 259 1193 Japan

Department of Pharmacology and Experimental Neuroscience University of Nebraska Medical Center Omaha NE USA

Department of Surgery School of Medicine University of California Davis Davis USA

Departments of Anatomy and Cell Biology Human Genetics and Pediatrics Research Institute McGill University Health Center Montreal Canada

Division of Cardiovascular Sciences School of Medical Sciences Faculty of Biology Medicine and Health The University of Manchester and Manchester Heart Centre Manchester University NHS Foundation Trust Manchester Academic Health Science Centre Manchester UK

Division of Evolution and Genomic Sciences School of Biological Sciences Faculty of Biology Medicine and Health Manchester Academic Health Science Centre University of Manchester Manchester UK

Division of Neuroscience and Experimental Psychology School of Biological Sciences Faculty of Biology Medicine and Health Manchester Academic Health Science Centre University of Manchester AV Hill Building Oxford Road Manchester M13 9PT UK

High Throughput DNA Sequencing and Genotyping Core Facility Vice Chancellor for Research Office University of Nebraska Medical Center Omaha USA

Laboratory Animal Resource Center University of Tsukuba Tsukuba Japan

Laboratory of Molecular Life Science Foundation for Biomedical Research and Innovation Kobe Japan

Laboratory of Transgenic Models of Diseases and Czech Centre for Phenogenomics Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czech Republic

Lillehei Heart Institute Regenerative Medicine and Sciences Program University of Minnesota Minneapolis MN USA

Maine Medical Center Research Institute Scarborough ME USA

Manchester Collaborative Centre for Inflammation Research School of Biological Sciences Faculty of Biology Medicine and Health The University of Manchester Manchester UK

McGill Integrated Core for Animal Modeling Montreal Canada

Mouse Biology Program University of California Davis USA

Mouse Genome Engineering Core Facility Vice Chancellor for Research Office University of Nebraska Medical Center Omaha NE USA

Oxford Centre for Diabetes Endocrinology and Metabolism University of Oxford Oxford OX37LE UK

Paul and Sheila Wellstone Muscular Dystrophy Center University of Minnesota Minneapolis MN USA

RIKEN BioResource Research Center Tsukuba Ibaraki 305 0074 Japan

School of Health and Human Sciences Department of Physical Therapy Indiana University Indianapolis IN 46202 USA

School of Medicine Indiana University Indianapolis IN 46202 USA

South Australian Health and Medical Research Institute and Department of Medicine University of Adelaide Adelaide Australia

Texas A and M Institute for Genomic Medicine Texas A and M University College Station TX 77843 USA

The Institute of Experimental Animal Sciences Osaka University Graduate School of Medicine Suita Japan

The University of Texas MD Anderson Cancer Center Houston TX USA

Transformational Bioinformatics Health and Biosecurity Business Unit CSIRO North Ryde Australia

Transgenesis and Animal Modeling Core Facility Centre de Recherche du Centre Hospitalier Universitaire de Montreal Montreal Canada

Transgenic Mouse Core Facility VIB Center for Inflammation Research Ghent Belgium

Transgenic Unit Core Facility Faculty of Biology Medicine and Health University of Manchester Manchester UK

Unit of Cardiac Physiology School of Medical Sciences Manchester Academic Health Science Center University of Manchester Manchester UK

University of Rochester Medical Center Rochester NY 14642 USA

Genome Biol. 2021 Apr 7;22(1):98. doi: 10.1186/s13059-021-02312-3. PubMed

Genome Biol. 2021 Apr 7;22(1):99. doi: 10.1186/s13059-021-02320-3. PubMed

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