Multicenter Study

. 2019 Aug 26;20(1):171.

doi: 10.1186/s13059-019-1776-2.

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

Channabasavaiah B Gurumurthy^{1

2}, Aidan R O'Brien^{3

4}, Rolen M Quadros⁵, John Adams Jr⁶, Pilar Alcaide⁷, Shinya Ayabe⁸, Johnathan Ballard⁶, Surinder K Batra⁹, Marie-Claude Beauchamp¹⁰, Kathleen A Becker¹¹, Guillaume Bernas¹², David Brough¹³, Francisco Carrillo-Salinas⁷, Wesley Chan¹⁰, Hanying Chen¹⁴, Ruby Dawson¹⁵, Victoria DeMambro¹¹, Jinke D'Hont^{16

17}, Katharine M Dibb¹⁸, James D Eudy¹⁹, Lin Gan²⁰, Jing Gao⁴, Amy Gonzales⁶, Anyonya R Guntur¹¹, Huiping Guo⁶, Donald W Harms⁵, Anne Harrington¹¹, Kathryn E Hentges²¹, Neil Humphreys²², Shiho Imai²³, Hideshi Ishii²⁴, Mizuho Iwama⁸, Eric Jonasch²⁵, Michelle Karolak¹¹, Bernard Keavney²⁶, Nay-Chi Khin⁴, Masamitsu Konno²⁷, Yuko Kotani²⁸, Yayoi Kunihiro²⁸, Imayavaramban Lakshmanan⁹, Catherine Larochelle²⁹, Catherine B Lawrence¹³, Lin Li³⁰, Volkhard Lindner¹¹, Xian-De Liu²⁵, Gloria Lopez-Castejon³¹, Andrew Loudon³², Jenna Lowe⁴, Loydie A Jerome-Majewska¹⁰, Taiji Matsusaka²³, Hiromi Miura^{33

34}, Yoshiki Miyasaka²⁸, Benjamin Morpurgo⁶, Katherine Motyl¹¹, Yo-Ichi Nabeshima³⁵, Koji Nakade⁸, Toshiaki Nakashiba⁸, Kenichi Nakashima⁸, Yuichi Obata⁸, Sanae Ogiwara³⁶, Mariette Ouellet¹², Leif Oxburgh^{11

37}, Sandra Piltz¹⁵, Ilka Pinz¹¹, Moorthy P Ponnusamy⁹, David Ray³⁸, Ronald J Redder¹⁹, Clifford J Rosen¹¹, Nikki Ross⁴, Mark T Ruhe³⁹, Larisa Ryzhova¹¹, Ane M Salvador⁷, Sabrina Shameen Alam¹⁰, Radislav Sedlacek⁴⁰, Karan Sharma⁴¹, Chad Smith²⁵, Katrien Staes^{16

17}, Lora Starrs⁴, Fumihiro Sugiyama⁴², Satoru Takahashi⁴², Tomohiro Tanaka⁴³, Andrew W Trafford¹⁸, Yoshihiro Uno²⁸, Leen Vanhoutte^{16

17}, Frederique Vanrockeghem^{16

17}, Brandon J Willis³⁹, Christian S Wright⁴⁴, Yuko Yamauchi²⁸, Xin Yi⁴⁴, Kazuto Yoshimi²⁸, Xuesong Zhang²⁵, Yu Zhang³⁰, Masato Ohtsuka^{33

34}, Satyabrata Das⁴⁵, Daniel J Garry^{46

47}, Tino Hochepied^{16

17}, Paul Thomas¹⁵, Jan Parker-Thornburg²⁵, Antony D Adamson²², Atsushi Yoshiki⁸, Jean-Francois Schmouth¹², Andrei Golovko⁶, William R Thompson⁴⁴, K C Kent Lloyd^{39

47}, Joshua A Wood³⁹, Mitra Cowan⁴⁸, Tomoji Mashimo²⁸, Seiya Mizuno⁴², Hao Zhu³⁰, Petr Kasparek⁴⁰, Lucy Liaw¹¹, Joseph M Miano²⁰, Gaetan Burgio⁴⁹

Affiliations

¹ Mouse Genome Engineering Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center, Omaha, NE, USA. cgurumurthy@unmc.edu.
² Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, USA. cgurumurthy@unmc.edu.
³ Transformational Bioinformatics, Health and Biosecurity Business Unit, CSIRO, North Ryde, Australia.
⁴ Department of Immunology and Infectious Disease, The John Curtin School of Medical Research, the Australian National University, Canberra, Australia.
⁵ Mouse Genome Engineering Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center, Omaha, NE, USA.
⁶ Texas A&M Institute for Genomic Medicine (TIGM), Texas A&M University, College Station, TX, 77843, USA.
⁷ Department of Immunology, Tufts University School of Medicine, Boston, USA.
⁸ RIKEN BioResource Research Center, Tsukuba, Ibaraki, 305-0074, Japan.
⁹ Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA.
¹⁰ Departments of Anatomy and Cell Biology, Human Genetics and Pediatrics, Research Institute McGill University Health Center (RI-MUHC), Montreal, Canada.
¹¹ Maine Medical Center Research Institute (MMCRI), Scarborough, ME, USA.
¹² Transgenesis and Animal Modeling Core Facility, Centre de Recherche du Centre Hospitalier Universitaire de Montreal (CRCHUM), Montreal, Canada.
¹³ 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.
¹⁴ School of Medicine, Indiana University, Indianapolis, IN, 46202, USA.
¹⁵ South Australian Health & Medical Research Institute and Department of Medicine, University of Adelaide, Adelaide, Australia.
¹⁶ Transgenic Mouse Core Facility, VIB Center for Inflammation Research, Ghent, Belgium.
¹⁷ Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.
¹⁸ Unit of Cardiac Physiology, School of Medical Sciences, Manchester Academic Health Science Center, University of Manchester, Manchester, UK.
¹⁹ High-Throughput DNA Sequencing and Genotyping Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center, Omaha, USA.
²⁰ University of Rochester Medical Center, Rochester, NY, 14642, USA.
²¹ 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.
²² Transgenic Unit Core Facility, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK.
²³ Department of Basic Medicine, Division of Basic Medical Science and Molecular Medicine, School of Medicine, Tokai University, 143, Shimokasuya, Isehara, Kanagawa, 259-1193, Japan.
²⁴ Department of Medical Data Science, Osaka University Graduate School of Medicine, Suita, Japan.
²⁵ The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
²⁶ 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.
²⁷ Department of Frontier Science for Cancer and Chemotherapy, Osaka University Graduate School of Medicine, Suita, Japan.
²⁸ The Institute of Experimental Animal Sciences, Osaka University Graduate School of Medicine, Suita, Japan.
²⁹ Centre de Recherche du Centre Hospitalier Universitaire de Montreal (CRCHUM), Montreal, Canada.
³⁰ Children's Research Institute Mouse Genome Engineering Core, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.
³¹ Manchester Collaborative Centre for Inflammation Research (MCCIR), School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, UK.
³² Centre for Biological Timing, School of Medical Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK.
³³ Center for Matrix Biology and Medicine, Graduate School of Medicine, Tokai University, Isehara, Kanagawa, 259-1193, Japan.
³⁴ 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.
³⁵ Laboratory of Molecular Life Science, Foundation for Biomedical Research and Innovation, Kobe, Japan.
³⁶ Department of Laboratory Animal Science, Support Center for Medical Research and Education, Tokai University, 143, Shimokasuya, Isehara, Kanagawa, 259-1193, Japan.
³⁷ Basic and Clinical Research, The Rogosin Institute, New York, USA.
³⁸ Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford, OX37LE, UK.
³⁹ Mouse Biology Program, University of California, Davis, USA.
⁴⁰ Laboratory of Transgenic Models of Diseases and Czech Centre for Phenogenomics, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic.
⁴¹ College of Osteopathic Medicine, Marian University, Indianapolis, IN, 46222, USA.
⁴² Laboratory Animal Resource Center, University of Tsukuba, Tsukuba, Japan.
⁴³ Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.
⁴⁴ School of Health and Human Sciences, Department of Physical Therapy, Indiana University, Indianapolis, IN, 46202, USA.
⁴⁵ Lillehei Heart Institute Regenerative Medicine and Sciences Program, University of Minnesota, Minneapolis, MN, USA.
⁴⁶ Paul and Sheila Wellstone Muscular Dystrophy Center, University of Minnesota, Minneapolis, MN, USA.
⁴⁷ Department of Surgery, School of Medicine, University of California, Davis, Davis, USA.
⁴⁸ McGill Integrated Core for Animal Modeling (MICAM), Montreal, Canada.
⁴⁹ Department of Immunology and Infectious Disease, The John Curtin School of Medical Research, the Australian National University, Canberra, Australia. gaetan.burgio@anu.edu.au.

PMID: 31446895
PMCID: PMC6709553
DOI: 10.1186/s13059-019-1776-2

Multicenter Study

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

Channabasavaiah B Gurumurthy et al. Genome Biol. 2019.

. 2019 Aug 26;20(1):171.

doi: 10.1186/s13059-019-1776-2.

Authors

Affiliations

¹ Mouse Genome Engineering Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center, Omaha, NE, USA. cgurumurthy@unmc.edu.
² Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, USA. cgurumurthy@unmc.edu.
³ Transformational Bioinformatics, Health and Biosecurity Business Unit, CSIRO, North Ryde, Australia.
⁴ Department of Immunology and Infectious Disease, The John Curtin School of Medical Research, the Australian National University, Canberra, Australia.
⁵ Mouse Genome Engineering Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center, Omaha, NE, USA.
⁶ Texas A&M Institute for Genomic Medicine (TIGM), Texas A&M University, College Station, TX, 77843, USA.
⁷ Department of Immunology, Tufts University School of Medicine, Boston, USA.
⁸ RIKEN BioResource Research Center, Tsukuba, Ibaraki, 305-0074, Japan.
⁹ Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA.
¹⁰ Departments of Anatomy and Cell Biology, Human Genetics and Pediatrics, Research Institute McGill University Health Center (RI-MUHC), Montreal, Canada.
¹¹ Maine Medical Center Research Institute (MMCRI), Scarborough, ME, USA.
¹² Transgenesis and Animal Modeling Core Facility, Centre de Recherche du Centre Hospitalier Universitaire de Montreal (CRCHUM), Montreal, Canada.
¹³ 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.
¹⁴ School of Medicine, Indiana University, Indianapolis, IN, 46202, USA.
¹⁵ South Australian Health & Medical Research Institute and Department of Medicine, University of Adelaide, Adelaide, Australia.
¹⁶ Transgenic Mouse Core Facility, VIB Center for Inflammation Research, Ghent, Belgium.
¹⁷ Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.
¹⁸ Unit of Cardiac Physiology, School of Medical Sciences, Manchester Academic Health Science Center, University of Manchester, Manchester, UK.
¹⁹ High-Throughput DNA Sequencing and Genotyping Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center, Omaha, USA.
²⁰ University of Rochester Medical Center, Rochester, NY, 14642, USA.
²¹ 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.
²² Transgenic Unit Core Facility, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK.
²³ Department of Basic Medicine, Division of Basic Medical Science and Molecular Medicine, School of Medicine, Tokai University, 143, Shimokasuya, Isehara, Kanagawa, 259-1193, Japan.
²⁴ Department of Medical Data Science, Osaka University Graduate School of Medicine, Suita, Japan.
²⁵ The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
²⁶ 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.
²⁷ Department of Frontier Science for Cancer and Chemotherapy, Osaka University Graduate School of Medicine, Suita, Japan.
²⁸ The Institute of Experimental Animal Sciences, Osaka University Graduate School of Medicine, Suita, Japan.
²⁹ Centre de Recherche du Centre Hospitalier Universitaire de Montreal (CRCHUM), Montreal, Canada.
³⁰ Children's Research Institute Mouse Genome Engineering Core, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.
³¹ Manchester Collaborative Centre for Inflammation Research (MCCIR), School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, UK.
³² Centre for Biological Timing, School of Medical Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK.
³³ Center for Matrix Biology and Medicine, Graduate School of Medicine, Tokai University, Isehara, Kanagawa, 259-1193, Japan.
³⁴ 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.
³⁵ Laboratory of Molecular Life Science, Foundation for Biomedical Research and Innovation, Kobe, Japan.
³⁶ Department of Laboratory Animal Science, Support Center for Medical Research and Education, Tokai University, 143, Shimokasuya, Isehara, Kanagawa, 259-1193, Japan.
³⁷ Basic and Clinical Research, The Rogosin Institute, New York, USA.
³⁸ Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford, OX37LE, UK.
³⁹ Mouse Biology Program, University of California, Davis, USA.
⁴⁰ Laboratory of Transgenic Models of Diseases and Czech Centre for Phenogenomics, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic.
⁴¹ College of Osteopathic Medicine, Marian University, Indianapolis, IN, 46222, USA.
⁴² Laboratory Animal Resource Center, University of Tsukuba, Tsukuba, Japan.
⁴³ Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.
⁴⁴ School of Health and Human Sciences, Department of Physical Therapy, Indiana University, Indianapolis, IN, 46202, USA.
⁴⁵ Lillehei Heart Institute Regenerative Medicine and Sciences Program, University of Minnesota, Minneapolis, MN, USA.
⁴⁶ Paul and Sheila Wellstone Muscular Dystrophy Center, University of Minnesota, Minneapolis, MN, USA.
⁴⁷ Department of Surgery, School of Medicine, University of California, Davis, Davis, USA.
⁴⁸ McGill Integrated Core for Animal Modeling (MICAM), Montreal, Canada.
⁴⁹ Department of Immunology and Infectious Disease, The John Curtin School of Medical Research, the Australian National University, Canberra, Australia. gaetan.burgio@anu.edu.au.

PMID: 31446895
PMCID: PMC6709553
DOI: 10.1186/s13059-019-1776-2

Abstract

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.

Keywords: CRISPR-Cas9; Conditional knockout mouse; Floxed allele; Homology-directed repair; Long single-stranded DNA; Machine learning; Mouse; Oligonucleotide; Reproducibility; Transgenesis.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

**Fig. 1**
Schematic of two-donor floxing method of creating cKO alleles. a Wild-type locus showing exons 3, 4, and 5 of a hypothetical gene where exon 4 is chosen as a target exon for inserting *LoxP* sites. Guides 1 and 2 target introns 3 and 4, respectively. b CRISPR components for the two-donor floxing method Cas9 source. c Delivery method of CRISPR components into zygotes (microinjection or electroporation). d The cKO allele showing target exon (#4) with flanking *LoxP* sites

**Fig. 2**
Quantitative assessment of the success of the two-donor floxing method. a Method of zygote injections (pronuclear, cytoplasmic, or both) for delivery of the CRISPR reagents used by reporting centers. Numbers indicate the percentage of the total zygotes microinjected or electroporated. b Form of the CRISPR reagents (mRNA, protein, or plasmid) delivered to the zygotes. Numbers indicate percentages. c Number of successfully edited alleles and correct *LoxP* insertions out of the total number of live-born pups from microinjected and transferred zygotes. Numbers indicate absolute numbers. d Types of editing observed among the live-born pups genotyped from a subsample from 25 loci. Numbers indicate absolute values

**Fig. 3**
Desired and undesired outcomes of the two-donor floxing method. **a-f** Wild-type locus showing exons 3, 4, and 5 of a hypothetical gene where exon 4 is chosen as a target exon for inserting *LoxP* sites. a Desired outcome showing a floxed allele. Overall occurrence was < 1%. b–f Various undesired outcomes including only one *LoxP* site insertion (b), only *indels* created at one or both sites (c), combination of *LoxP* insertion and *indels* (d), deletion between the two cleavage sites (e), and no *indel* or no insertion events (f)

See this image and copyright information in PMC

Comment in

Response to "Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation".
Yang H, Wang H, Jaenisch R. Yang H, et al. Genome Biol. 2021 Apr 7;22(1):98. doi: 10.1186/s13059-021-02312-3. Genome Biol. 2021. PMID: 33827646 Free PMC article. No abstract available.
Response to correspondence on "Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation".
Gurumurthy CB, O'Brien AR, Quadros RM, Adams J Jr, Alcaide P, Ayabe S, Ballard J, Batra SK, Beauchamp MC, Becker KA, Bernas G, Brough D, Carrillo-Salinas F, Chan W, Chen H, Dawson R, DeMambro V, D'Hont J, Dibb K, Eudy JD, Gan L, Gao J, Gonzales A, Guntur A, Guo H, Harms DW, Harrington A, Hentges KE, Humphreys N, Imai S, Ishii H, Iwama M, Jonasch E, Karolak M, Keavney B, Khin NC, Konno M, Kotani Y, Kunihiro Y, Lakshmanan I, Larochelle C, Lawrence CB, Li L, Lindner V, Liu XD, Lopez-Castejon G, Loudon A, Lowe J, Jerome-Majeweska L, Matsusaka T, Miura H, Miyasaka Y, Morpurgo B, Motyl K, Nabeshima YI, Nakade K, Nakashiba T, Nakashima K, Obata Y, Ogiwara S, Ouellet M, Oxburgh L, Piltz S, Pinz I, Ponnusamy MP, Ray D, Redder RJ, Rosen CJ, Ross N, Ruhe MT, Ryzhova L, Salvador AM, Alam SS, Sedlacek R, Sharma K, Smith C, Staes K, Starrs L, Sugiyama F, Takahashi S, Tanaka T, Trafford A, Uno Y, Vanhoutte L, Vanrockeghem F, Willis BJ, Wright CS, Yamauchi Y, Yi X, Yoshimi K, Zhang X, Zhang Y, Ohtsuka M, Das S, Garry DJ, Hochepied T, Thomas P, Parker-Thornburg J, Adamson AD, Yoshiki A, Schmouth JF, Golovko A, Thompson WR, Lloyd KCK, Wood JA, Cowan M, Mashimo T, Mizuno S, Zhu H, Kasparek P, Liaw L… See abstract for full author list ➔ Gurumurthy CB, et al. Genome Biol. 2021 Apr 7;22(1):99. doi: 10.1186/s13059-021-02320-3. Genome Biol. 2021. PMID: 33827648 Free PMC article. No abstract available.

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

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

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Abstract

Conflict of interest statement

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