Evidence map›Paper›PMID 31446895›Full record

ArticleGenome biology2019

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

Channabasavaiah B Gurumurthy, Aidan R O'Brien, Rolen M Quadros, John Adams, Pilar Alcaide, Shinya Ayabe, Johnathan Ballard, Surinder K Batra, Marie-Claude Beauchamp, Kathleen A Becker and 103 more

Abstract readMulticenter Study
PubMed Full text at PMC Publisher
In one paragraph

Article in Genome biology, 2019. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 52 papers.

0numbers the graph read from it
0cells of the map it votes in
52citing papers in PubMed
–field-weighted citation impact
1 · What the graph read from it

What it found

Each row is one number read from the abstract, on the scale the paper reported it, with its interval. Left of the dashed line favours the treatment, right favours the comparator. Under each row is the sentence it came from. New to these charts? A ten-minute tutorial.

The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.

2 · The registry

The trial behind it

Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.

Neither the registry nor the abstract names a trial number. If this is a trial report, that itself is worth knowing.

3 · Its place in the literature

Who cites it

52 citing papers in PubMed.

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4 · The record

Corrections and comments

5 · Who and what money

Authors and funding

113 authors.

Channabasavaiah B GurumurthyMouse Genome Engineering Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center, Omaha, NE, USA. cgurumurthy@unmc.edu.ORCID http://orcid.org/0000-0002-8022-4033
Aidan R O'BrienTransformational Bioinformatics, Health and Biosecurity Business Unit, CSIRO, North Ryde, Australia.
Rolen M QuadrosMouse Genome Engineering Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center, Omaha, NE, USA.
John AdamsTexas A&M Institute for Genomic Medicine (TIGM), Texas A&M University, College Station, TX, 77843, USA.
Pilar AlcaideDepartment of Immunology, Tufts University School of Medicine, Boston, USA.
Shinya AyabeRIKEN BioResource Research Center, Tsukuba, Ibaraki, 305-0074, Japan.
Johnathan BallardTexas A&M Institute for Genomic Medicine (TIGM), Texas A&M University, College Station, TX, 77843, USA.
Surinder K BatraDepartment of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA.
Marie-Claude BeauchampDepartments of Anatomy and Cell Biology, Human Genetics and Pediatrics, Research Institute McGill University Health Center (RI-MUHC), Montreal, Canada.
Kathleen A BeckerMaine Medical Center Research Institute (MMCRI), Scarborough, ME, USA.
Guillaume BernasTransgenesis and Animal Modeling Core Facility, Centre de Recherche du Centre Hospitalier Universitaire de Montreal (CRCHUM), Montreal, Canada.
David BroughDivision 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.
Francisco Carrillo-SalinasDepartment of Immunology, Tufts University School of Medicine, Boston, USA.
Wesley ChanDepartments of Anatomy and Cell Biology, Human Genetics and Pediatrics, Research Institute McGill University Health Center (RI-MUHC), Montreal, Canada.
Hanying ChenSchool of Medicine, Indiana University, Indianapolis, IN, 46202, USA.
Ruby DawsonSouth Australian Health & Medical Research Institute and Department of Medicine, University of Adelaide, Adelaide, Australia.
Victoria DeMambroMaine Medical Center Research Institute (MMCRI), Scarborough, ME, USA.
Jinke D'HontTransgenic Mouse Core Facility, VIB Center for Inflammation Research, Ghent, Belgium.
Katharine M DibbUnit of Cardiac Physiology, School of Medical Sciences, Manchester Academic Health Science Center, University of Manchester, Manchester, UK.
James D EudyHigh-Throughput DNA Sequencing and Genotyping Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center, Omaha, USA.
Lin GanUniversity of Rochester Medical Center, Rochester, NY, 14642, USA.
Jing GaoDepartment of Immunology and Infectious Disease, The John Curtin School of Medical Research, the Australian National University, Canberra, Australia.
Amy GonzalesTexas A&M Institute for Genomic Medicine (TIGM), Texas A&M University, College Station, TX, 77843, USA.
Anyonya R GunturMaine Medical Center Research Institute (MMCRI), Scarborough, ME, USA.
Huiping GuoTexas A&M Institute for Genomic Medicine (TIGM), Texas A&M University, College Station, TX, 77843, USA.
Donald W HarmsMouse Genome Engineering Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center, Omaha, NE, USA.
Anne HarringtonMaine Medical Center Research Institute (MMCRI), Scarborough, ME, USA.
Kathryn E HentgesDivision 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.
Neil HumphreysTransgenic Unit Core Facility, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK.
Shiho ImaiDepartment of Basic Medicine, Division of Basic Medical Science and Molecular Medicine, School of Medicine, Tokai University, 143, Shimokasuya, Isehara, Kanagawa, 259-1193, Japan.
Hideshi IshiiDepartment of Medical Data Science, Osaka University Graduate School of Medicine, Suita, Japan.
Mizuho IwamaRIKEN BioResource Research Center, Tsukuba, Ibaraki, 305-0074, Japan.
Eric JonaschThe University of Texas MD Anderson Cancer Center, Houston, TX, USA.
Michelle KarolakMaine Medical Center Research Institute (MMCRI), Scarborough, ME, USA.
Bernard KeavneyDivision 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.
Nay-Chi KhinDepartment of Immunology and Infectious Disease, The John Curtin School of Medical Research, the Australian National University, Canberra, Australia.
Masamitsu KonnoDepartment of Frontier Science for Cancer and Chemotherapy, Osaka University Graduate School of Medicine, Suita, Japan.
Yuko KotaniThe Institute of Experimental Animal Sciences, Osaka University Graduate School of Medicine, Suita, Japan.
Yayoi KunihiroThe Institute of Experimental Animal Sciences, Osaka University Graduate School of Medicine, Suita, Japan.
Imayavaramban LakshmananDepartment of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA.
Catherine LarochelleCentre de Recherche du Centre Hospitalier Universitaire de Montreal (CRCHUM), Montreal, Canada.
Catherine B LawrenceDivision 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.
Lin LiChildren's Research Institute Mouse Genome Engineering Core, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.
Volkhard LindnerMaine Medical Center Research Institute (MMCRI), Scarborough, ME, USA.
Xian-De LiuThe University of Texas MD Anderson Cancer Center, Houston, TX, USA.
Gloria Lopez-CastejonManchester Collaborative Centre for Inflammation Research (MCCIR), School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, UK.
Andrew LoudonCentre for Biological Timing, School of Medical Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK.
Jenna LoweDepartment of Immunology and Infectious Disease, The John Curtin School of Medical Research, the Australian National University, Canberra, Australia.
Loydie A Jerome-MajewskaDepartments of Anatomy and Cell Biology, Human Genetics and Pediatrics, Research Institute McGill University Health Center (RI-MUHC), Montreal, Canada.
Taiji MatsusakaDepartment of Basic Medicine, Division of Basic Medical Science and Molecular Medicine, School of Medicine, Tokai University, 143, Shimokasuya, Isehara, Kanagawa, 259-1193, Japan.
Hiromi MiuraCenter for Matrix Biology and Medicine, Graduate School of Medicine, Tokai University, Isehara, Kanagawa, 259-1193, Japan.
Yoshiki MiyasakaThe Institute of Experimental Animal Sciences, Osaka University Graduate School of Medicine, Suita, Japan.
Benjamin MorpurgoTexas A&M Institute for Genomic Medicine (TIGM), Texas A&M University, College Station, TX, 77843, USA.
Katherine MotylMaine Medical Center Research Institute (MMCRI), Scarborough, ME, USA.
Yo-Ichi NabeshimaLaboratory of Molecular Life Science, Foundation for Biomedical Research and Innovation, Kobe, Japan.
Koji NakadeRIKEN BioResource Research Center, Tsukuba, Ibaraki, 305-0074, Japan.
Toshiaki NakashibaRIKEN BioResource Research Center, Tsukuba, Ibaraki, 305-0074, Japan.
Kenichi NakashimaRIKEN BioResource Research Center, Tsukuba, Ibaraki, 305-0074, Japan.
Yuichi ObataRIKEN BioResource Research Center, Tsukuba, Ibaraki, 305-0074, Japan.
Sanae OgiwaraDepartment of Laboratory Animal Science, Support Center for Medical Research and Education, Tokai University, 143, Shimokasuya, Isehara, Kanagawa, 259-1193, Japan.
Mariette OuelletTransgenesis and Animal Modeling Core Facility, Centre de Recherche du Centre Hospitalier Universitaire de Montreal (CRCHUM), Montreal, Canada.
Leif OxburghMaine Medical Center Research Institute (MMCRI), Scarborough, ME, USA.
Sandra PiltzSouth Australian Health & Medical Research Institute and Department of Medicine, University of Adelaide, Adelaide, Australia.
Ilka PinzMaine Medical Center Research Institute (MMCRI), Scarborough, ME, USA.
Moorthy P PonnusamyDepartment of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA.
David RayOxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford, OX37LE, UK.
Ronald J RedderHigh-Throughput DNA Sequencing and Genotyping Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center, Omaha, USA.
Clifford J RosenMaine Medical Center Research Institute (MMCRI), Scarborough, ME, USA.
Nikki RossDepartment of Immunology and Infectious Disease, The John Curtin School of Medical Research, the Australian National University, Canberra, Australia.
Mark T RuheMouse Biology Program, University of California, Davis, USA.
Larisa RyzhovaMaine Medical Center Research Institute (MMCRI), Scarborough, ME, USA.
Ane M SalvadorDepartment of Immunology, Tufts University School of Medicine, Boston, USA.
Sabrina Shameen AlamDepartments of Anatomy and Cell Biology, Human Genetics and Pediatrics, Research Institute McGill University Health Center (RI-MUHC), Montreal, Canada.
Radislav SedlacekLaboratory of Transgenic Models of Diseases and Czech Centre for Phenogenomics, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic.
Karan SharmaCollege of Osteopathic Medicine, Marian University, Indianapolis, IN, 46222, USA.
Chad SmithThe University of Texas MD Anderson Cancer Center, Houston, TX, USA.
Katrien StaesTransgenic Mouse Core Facility, VIB Center for Inflammation Research, Ghent, Belgium.
Lora StarrsDepartment of Immunology and Infectious Disease, The John Curtin School of Medical Research, the Australian National University, Canberra, Australia.
Fumihiro SugiyamaLaboratory Animal Resource Center, University of Tsukuba, Tsukuba, Japan.
Satoru TakahashiLaboratory Animal Resource Center, University of Tsukuba, Tsukuba, Japan.
Tomohiro TanakaDepartment of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.
Andrew W TraffordUnit of Cardiac Physiology, School of Medical Sciences, Manchester Academic Health Science Center, University of Manchester, Manchester, UK.
Yoshihiro UnoThe Institute of Experimental Animal Sciences, Osaka University Graduate School of Medicine, Suita, Japan.
Leen VanhoutteTransgenic Mouse Core Facility, VIB Center for Inflammation Research, Ghent, Belgium.
Frederique VanrockeghemTransgenic Mouse Core Facility, VIB Center for Inflammation Research, Ghent, Belgium.
Brandon J WillisMouse Biology Program, University of California, Davis, USA.
Christian S WrightSchool of Health and Human Sciences, Department of Physical Therapy, Indiana University, Indianapolis, IN, 46202, USA.
Yuko YamauchiThe Institute of Experimental Animal Sciences, Osaka University Graduate School of Medicine, Suita, Japan.
Xin YiSchool of Health and Human Sciences, Department of Physical Therapy, Indiana University, Indianapolis, IN, 46202, USA.
Kazuto YoshimiThe Institute of Experimental Animal Sciences, Osaka University Graduate School of Medicine, Suita, Japan.
Xuesong ZhangThe University of Texas MD Anderson Cancer Center, Houston, TX, USA.
Yu ZhangChildren's Research Institute Mouse Genome Engineering Core, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.
Masato OhtsukaCenter for Matrix Biology and Medicine, Graduate School of Medicine, Tokai University, Isehara, Kanagawa, 259-1193, Japan.
Satyabrata DasLillehei Heart Institute Regenerative Medicine and Sciences Program, University of Minnesota, Minneapolis, MN, USA.
Daniel J GarryPaul and Sheila Wellstone Muscular Dystrophy Center, University of Minnesota, Minneapolis, MN, USA.
Tino HochepiedTransgenic Mouse Core Facility, VIB Center for Inflammation Research, Ghent, Belgium.
Paul ThomasSouth Australian Health & Medical Research Institute and Department of Medicine, University of Adelaide, Adelaide, Australia.
Jan Parker-ThornburgThe University of Texas MD Anderson Cancer Center, Houston, TX, USA.
Antony D AdamsonTransgenic Unit Core Facility, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK.
Atsushi YoshikiRIKEN BioResource Research Center, Tsukuba, Ibaraki, 305-0074, Japan.
Jean-Francois SchmouthTransgenesis and Animal Modeling Core Facility, Centre de Recherche du Centre Hospitalier Universitaire de Montreal (CRCHUM), Montreal, Canada.
Andrei GolovkoTexas A&M Institute for Genomic Medicine (TIGM), Texas A&M University, College Station, TX, 77843, USA.
William R ThompsonSchool of Health and Human Sciences, Department of Physical Therapy, Indiana University, Indianapolis, IN, 46202, USA.
K C Kent LloydMouse Biology Program, University of California, Davis, USA.
Joshua A WoodMouse Biology Program, University of California, Davis, USA.
Mitra CowanMcGill Integrated Core for Animal Modeling (MICAM), Montreal, Canada.
Tomoji MashimoThe Institute of Experimental Animal Sciences, Osaka University Graduate School of Medicine, Suita, Japan.
Seiya MizunoLaboratory Animal Resource Center, University of Tsukuba, Tsukuba, Japan.
Hao ZhuChildren's Research Institute Mouse Genome Engineering Core, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.
Petr KasparekLaboratory of Transgenic Models of Diseases and Czech Centre for Phenogenomics, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic.
Lucy LiawMaine Medical Center Research Institute (MMCRI), Scarborough, ME, USA.
Joseph M MianoUniversity of Rochester Medical Center, Rochester, NY, 14642, USA.
Gaetan BurgioDepartment of Immunology and Infectious Disease, The John Curtin School of Medical Research, the Australian National University, Canberra, Australia. gaetan.burgio@anu.edu.au.ORCID http://orcid.org/0000-0002-7434-926X

Funding

Tumor Evolution and Metastasis ProgramP30CA016672 · NCI · UNIVERSITY OF TX MD ANDERSON CAN CTR · PI Fabien Delerue · 1985 to 2026
$290.8M
UNMC/EPPLEY CANCER CENTER SUPPORT GRANTP30CA036727 · NCI · UNIVERSITY OF NEBRASKA MEDICAL CENTER · PI Joann B. Sweasy · 1985 to 2026
$55.0M
Understanding Factors Influencing COVID-19 Testing and Vaccination in Immigrant Low-income and Homeless Populations and Testing Targeted InterventionsU54GM115516 · NIGMS · MAINEHEALTH · PI CLIFFORD JAMES ROSEN, Gary S. Stein · 2017 to 2026
$51.6M
KOMP2-Phase3 Production and Phenotyping by the DTCC Consortium TCP Transfer to UC DavisUM1OD023221 · OD · UNIVERSITY OF CALIFORNIA AT DAVIS · PI KC KENT LLOYD · 2016 to 2026
$45.7M
Indiana Clinical and Translational Sciences InstituteUL1TR001108 · NCATS · INDIANA UNIVERSITY INDIANAPOLIS · PI DENNE, SCOTT C., SHEKHAR, ANANTHA · 2013 to 2017
$23.3M
Project 3: MUC16-Mediated Metabolic Reprograming Induces PC MetastasisP01CA217798 · NCI · UNIVERSITY OF NEBRASKA MEDICAL CENTER · PI BATRA, SURINDER K. · 2018 to 2022
$8.1M
UNMC Histology/Imaging and Creighton University Imaging Core FacilityP30GM110768 · NIGMS · UNIVERSITY OF NEBRASKA MEDICAL CENTER · PI WALSH, EDWARD J · 2014 to 2018
$5.1M
The Molecular Biology of Neurosurgery SystemsP20GM103471 · NIGMS · UNIVERSITY OF NEBRASKA MEDICAL CENTER · PI SMITH, SHELLEY D · 2012 to 2013
$4.1M
Mechanisms of T cell activation in cardiac fibrosis and non-ischemic heart failureR01HL144477 · NHLBI · TUFTS UNIVERSITY BOSTON · PI ALCAIDE ALONSO, MARIA PILAR · 2019 to 2023
$3.2M
Osteocyte Mechanotransduction and the Gabapentin-Sensitive Matrix-Channel Tethering ComplexR01AR074473 · NIAMS · INDIANA UNIVERSITY INDIANAPOLIS · PI THOMPSON, WILLIAM ROY · 2018 to 2023
$2.8M
Transcriptional Control of Myocardin and the MYOCARDomeR01HL138987 · NHLBI · UNIVERSITY OF ROCHESTER · PI MIANO, JOSEPH M · 2018 to 2021
$2.3M
T Cell Mediated Immune Responses as a Regulator of Heart FailureR01HL123658 · NHLBI · TUFTS UNIVERSITY BOSTON · PI ALCAIDE ALONSO, MARIA PILAR · 2014 to 2018
$2.2M
Biotechnology and Biological Sciences Research Council BB/N015584/1British Heart Foundation CH/13/2/30154British Heart Foundation FS12-57British Heart Foundation FS/12/57/29717British Heart Foundation FS12/57/29717British Heart Foundation PG/12/89/29970British Heart Foundation RG/15/12/31616Medical Research Council MR/L010240/1Medical Research Council MR/M008908/1Medical Research Council MR/N029992/1Medical Research Council MR/P011853/1Medical Research Council MR/P023576/1Medical Research Council MR/P023576/2NCATS NIH HHS UL1 TR001108NCI NIH HHS P01 CA217798NCI NIH HHS P30 CA016672NCI NIH HHS P30 CA036727NCI NIH HHS R50 CA211121NHLBI NIH HHS R01 HL123658NHLBI NIH HHS R01 HL138987NHLBI NIH HHS R01 HL144477NIAMS NIH HHS K01 AR067858NIAMS NIH HHS R01 AR074473NIGMS NIH HHS P20GM103471NIGMS NIH HHS P30 GM110768NIGMS NIH HHS U54 GM115516NIH HHS HL 123658NIH HHS HL138987NIH HHS P01 CA217798NIH HHS P30CA16672NIH HHS UM1 OD023221NIH HHS UM1OD023221Wellcome Trust 097820/Z11/BWellcome Trust 104192/Z/14/ZWellcome Trust 105610/Z/14/ZWellcome Trust 107849/Z/15/ZWellcome Trust 107851/Z/15/Z
6 · The paper itself

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.

Indexed as

AllelesAnimalsBlastocystCRISPR-Associated Protein 9CRISPR-Cas SystemsFactor Analysis, StatisticalFemaleMaleMethyl-CpG-Binding Protein 2Mice, KnockoutMicroinjectionsRegression AnalysisReproducibility of ResultsCRISPR-Associated Protein 9Mecp2 protein, mouseMethyl-CpG-Binding Protein 2Conditional knockout mouseCRISPR-Cas9Floxed alleleHomology-directed repairLong single-stranded DNAMachine learningMouseOligonucleotideReproducibilityTransgenesis

Identifiers

PMID31446895
PMCPMC6709553

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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.