Adams, John

Adamson, Antony D.

Alam, Sabrina Shameen

Alcaide, Pilar

Ayabe, Shinya

Ballard, Johnathan

Batra, Surinder K.

Beauchamp, Marie-Claude

Becker, Kathleen A.

Bernas, Guillaume

Brough, David

Burgio, Gaetan

Carrillo-Salinas, Francisco

Chan, Wesley

Chen, Hanying

Cowan, Mitra

D\u27Hont, Jinke

Das, Satyabrata

Dawson, Ruby

DeMambro, Victoria

Dibb, Katharine

Eudy, James D.

Gan, Lin

Gao, Jing

Garry, Daniel J.

Golovko, Andrei

Gonzales, Amy

Guntur, Anyonya

Guo, Huiping

Gurumurthy, Channabasavaiah B.

Harms, Donald W.

Harrington, Anne

Hentges, Kathryn E.

Hochepied, Tino

Humphreys, Neil

Imai, Shiho

Ishii, Hideshi

Iwama, Mizuho

Jerome-Majeweska, Loydie

Jonasch, Eric

Karolak, Michelle

Kasparek, Petr

Keavney, Bernard

Khin, Nay-Chi

Konno, Masamitsu

Kotani, Yuko

Kunihiro, Yayoi

Lakshmanan, Imayavaramban

Larochelle, Catherine

Lawrence, Catherine B.

Li, Lin

Liaw, Lucy

Lindner, Volkhard

Liu, Xian-De

Lloyd, K C Kent

Lopez-Castejon, Gloria

Loudon, Andrew

Lowe, Jenna

Mashimo, Tomoji

Matsusaka, Taiji

Miano, Joseph M.

Miura, Hiromi

Miyasaka, Yoshiki

Mizuno, Seiya

Morpurgo, Benjamin

Motyl, Katherine

Nabeshima, Yo-Ichi

Nakade, Koji

Nakashiba, Toshiaki

Nakashima, Kenichi

O\u27Brien, Aidan R.

Obata, Yuichi

Ogiwara, Sanae

Ohtsuka, Masato

Ouellet, Mariette

Oxburgh, Leif

Parker-Thornburg, Jan

Piltz, Sandra

Pinz, Ilka

Ponnusamy, Moorthy P.

Quadros, Rolen M.

Ray, David

Redder, Ronald J.

Rosen, Clifford J.

Ross, Nikki

Ruhe, Mark T.

Ryzhova, Larisa

Salvador, Ane M.

Schmouth, Jean-Francois

Sedlacek, Radislav

Sharma, Karan

Smith, Chad

Staes, Katrien

Starrs, Lora

Sugiyama, Fumihiro

Takahashi, Satoru

Tanaka, Tomohiro

Thomas, Paul

Thompson, William R.

Trafford, Andrew

English

Uno, Yoshihiro

Vanhoutte, Leen

Vanrockeghem, Frederique

Willis, Brandon J.

Wright, Christian S.

Yamauchi, Yuko

Yi, Xin

Yoshimi, Kazuto

Zhang, Xuesong

Zhang, Yu

Yoshiki, Atsushi

University of Nebraska Medical Center Research: DigitalCommons@UNMC

University of Nebraska Medical Center DigitalCommons@UNMC Journal Articles: Pharmacology & Experimental Neuroscience Pharmacology & Experimental Neuroscience 4-7-2021 Response to correspondence on "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 See next page for additional authors Follow this and additional works at: https://digitalcommons.unmc.edu/com_pen_articles  Part of the Medical Pharmacology Commons, and the Neurosciences Commons Authors 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, Guillaume Bernas, David Brough, Francisco Carrillo-Salinas, Wesley Chan, Hanying Chen, Ruby Dawson, Victoria DeMambro, Jinke D'Hont, Katharine Dibb, James D. Eudy, Lin Gan, Jing Gao, Amy Gonzales, Anyonya Guntur, Huiping Guo, Donald W. Harms, Anne Harrington, Kathryn E. Hentges, Neil Humphreys, Shiho Imai, Hideshi Ishii, Mizuho Iwama, Eric Jonasch, Michelle Karolak, Bernard Keavney, Nay-Chi Khin, Masamitsu Konno, Yuko Kotani, Yayoi Kunihiro, Imayavaramban Lakshmanan, Catherine Larochelle, Catherine B. Lawrence, Lin Li, Volkhard Lindner, Xian-De Liu, Gloria Lopez-Castejon, Andrew Loudon, Jenna Lowe, Loydie Jerome-Majeweska, Taiji Matsusaka, Hiromi Miura, Yoshiki Miyasaka, Benjamin Morpurgo, Katherine Motyl, Yo-Ichi Nabeshima, Koji Nakade, Toshiaki Nakashiba, Kenichi Nakashima, Yuichi Obata, Sanae Ogiwara, Mariette Ouellet, Leif Oxburgh, Sandra Piltz, Ilka Pinz, Moorthy P. Ponnusamy, David Ray, Ronald J. Redder, Clifford J. Rosen, Nikki Ross, Mark T. Ruhe, Larisa Ryzhova, Ane M. Salvador, Sabrina Shameen Alam, Radislav Sedlacek, Karan Sharma, Chad Smith, Katrien Staes, Lora Starrs, Fumihiro Sugiyama, Satoru Takahashi, Tomohiro Tanaka, Andrew Trafford, Yoshihiro Uno, Leen Vanhoutte, Frederique Vanrockeghem, Brandon J. Willis, Christian S. Wright, Yuko Yamauchi, Xin Yi, Kazuto Yoshimi, Xuesong Zhang, Yu Zhang, Masato Ohtsuka, Satyabrata Das, Daniel J. Garry, Tino Hochepied, Paul Thomas, Jan Parker-Thornburg, Antony D. Adamson, Atsushi Yoshiki, Jean-Francois Schmouth, Andrei Golovko, William R. Thompson, K C Kent Lloyd, Joshua A. Wood, Mitra Cowan, Tomoji Mashimo, Seiya Mizuno, Hao Zhu, Petr Kasparek, Lucy Liaw, Joseph M. Miano, and Gaetan Burgio CORRESPONDENCE Open AccessResponse to correspondence on“Reproducibility of CRISPR-Cas9 methodsfor generation of conditional mouse alleles:a multi-center evaluation”Channabasavaiah B. Gurumurthy1,2*, Aidan R. O’Brien3,4, Rolen M. Quadros1, John Adams Jr5, Pilar Alcaide6,Shinya Ayabe7, Johnathan Ballard5, Surinder K. Batra8, Marie-Claude Beauchamp9, Kathleen A. Becker10,Guillaume Bernas11, David Brough12, Francisco Carrillo-Salinas6, Wesley Chan9, Hanying Chen13, Ruby Dawson14,Victoria DeMambro10, Jinke D’Hont15,16, Katharine Dibb17, James D. Eudy18, Lin Gan19, Jing Gao4, Amy Gonzales5,Anyonya Guntur10, Huiping Guo5, Donald W. Harms1, Anne Harrington10, Kathryn E. Hentges20, Neil Humphreys21,Shiho Imai22, Hideshi Ishii23, Mizuho Iwama7, Eric Jonasch24, Michelle Karolak10, Bernard Keavney25, Nay-Chi Khin4,Masamitsu Konno26, Yuko Kotani27, Yayoi Kunihiro27, Imayavaramban Lakshmanan8, Catherine Larochelle28,Catherine B. Lawrence12, Lin Li29, Volkhard Lindner10, Xian-De Liu24, Gloria Lopez-Castejon30, Andrew Loudon31,Jenna Lowe4, Loydie Jerome-Majeweska9, Taiji Matsusaka22, Hiromi Miura32,33, Yoshiki Miyasaka27, Benjamin Morpurgo5,Katherine Motyl10, Yo-ichi Nabeshima34, Koji Nakade7, Toshiaki Nakashiba7, Kenichi Nakashima7, Yuichi Obata7,Sanae Ogiwara35, Mariette Ouellet11, Leif Oxburgh10, Sandra Piltz14, Ilka Pinz10, Moorthy P. Ponnusamy8, David Ray36,Ronald J. Redder18, Clifford J. Rosen10, Nikki Ross4, Mark T. Ruhe37, Larisa Ryzhova10, Ane M. Salvador6,Sabrina Shameen Alam9, Radislav Sedlacek38, Karan Sharma39, Chad Smith24, Katrien Staes15,16, Lora Starrs4,Fumihiro Sugiyama40, Satoru Takahashi40, Tomohiro Tanaka41, Andrew Trafford17, Yoshihiro Uno27, Leen Vanhoutte15,16,Frederique Vanrockeghem15,16, Brandon J. Willis37, Christian S. Wright42, Yuko Yamauchi27, Xin Yi42, Kazuto Yoshimi27,Xuesong Zhang24, Yu Zhang29, Masato Ohtsuka32,33, Satyabrata Das43, Daniel J. Garry43,44, Tino Hochepied15,16,Paul Thomas14, Jan Parker-Thornburg24, Antony D. Adamson21, Atsushi Yoshiki7, Jean-Francois Schmouth11,Andrei Golovko5, William R. Thompson42, K. C. Kent Lloyd37,45, Joshua A. Wood37, Mitra Cowan46, Tomoji Mashimo27,Seiya Mizuno40, Hao Zhu29, Petr Kasparek38, Lucy Liaw10, Joseph M. Miano19 and Gaetan Burgio4*This reply refers to the commentavailable at https://doi.org/10.1186/s13059-021-02312-3.* Correspondence: cgurumurthy@unmc.edu; gaetan.burgio@anu.edu.au1Mouse Genome Engineering CoreFacility, Vice Chancellor for ResearchOffice, University of NebraskaMedical Center, Omaha, NE, USA4Department of Immunology andInfectious Disease, the John CurtinSchool of Medical Research, TheAustralian National University,Canberra, AustraliaFull list of author information isavailable at the end of the article© The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, whichpermits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit tothe original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. Theimages or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwisein a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is notpermitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyrightholder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public DomainDedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unlessotherwise stated in a credit line to the data.Gurumurthy et al. Genome Biology           (2021) 22:99 https://doi.org/10.1186/s13059-021-02320-3We would like to thank the Yang et al. (2013) authors for their comments and debateon optimal methods for mouse transgenesis. The two Jaenisch laboratory studies pub-lished in Cell in 2013 were ground-breaking, demonstrating for the first time proof ofprinciple CRISPR mediated gene editing in the mouse zygote to generate knockout andconditional alleles, and caused much excitement in the transgenic mouse community.However, over several years and in many laboratories, the reality did not match theexcitement when it came to generating conditional alleles in a single step. While it istrue that the 2-guides 2-oligo approach can work in certain circumstances (members ofour own consortium reported some success with this method), the efficiencies reportedin Yang et al. (2013) do not bear out across multiple gene targets. Indeed, as the com-ments from Yang et al. point out, they themselves have performed further reproducibil-ity experiments on the Mecp2 locus (their point #1) and these unpublished results failto reproduce the 16% efficiency from their original publication. And our study is notthe first time concerns have been raised as to the efficiency of the 2-guides 2-oligomethod, with anecdotal reports from others in the transgenic community (Science;2016. doi:https://doi.org/10.1126/science.aal0334 [doi.org]), which stated that “Whatwas disappointing is none of us could reproduce at the efficiencies reported by Jaenisch.… It works at 1% or 2% at JAX and a lot of projects are failing. It’s really not proven tobe a robust method.” And the Yang et al. group’s response was that “The paper reportedwhat we found,” Jaenisch says. “Now, we see there are issues”.In regard to their point #2, regrettably, details of concentrations of reagents usedwere not reported in Yang et al. (2013). The authors, in this correspondence, now statethat they had provided concentrations in their other report [ref #4], but this reference(#4) does not describe generation of conditional alleles, and therefore, the experimentalconditions of this paper (ref #4) cannot be extrapolated for generating the conditionalalleles. Also, because such critical details were unavailable in the Yang et al. paper (ref#1), some of us had contacted the authors asking for tips on how to get their methodto work, but we received no response. This oversight of the authors (in failing to de-scribe the concentrations in the original paper) indeed allowed us to assess efficienciesusing a range of conditions on many loci, both lower and greater than the now revealedconditions from Yang et al. (2013), and using different delivery platforms (microinjec-tion, electroporation), the results of which further confirm that the Yang et al. methodis not efficient as it was originally reported. We suggest the reader to refer to the exten-sive data in our additional file 1 (supplementary data file) where we show that the widerange of reagent concentrations does not affect the efficiency of the Yang et al. method.Furthermore, Hatada’s group (Horii et al.) attempted to reproduce the Mecp2 experi-ments, and they reported either very low efficiency or very high toxicity when the con-centration of reagents was in the higher range. See Table 1 in Horii et al.; theconcentration of 50/12/100 produced only 2% efficiency whereas the concentration100/24/200 led to the death of nearly 90% of embryos, and the authors were unable todetermine the method’s efficiency at this higher concentration.In regard to their point #3, the authors speculate that Piezo-driven zygote injectionmay contribute to the difference of success rates. It would be necessary to examine thisspeculation by comparing the efficiencies of Piezo-driven and pronuclear injectionmethods side-by-side for a few loci. Because efficiencies at different genomic loci oftenGurumurthy et al. Genome Biology           (2021) 22:99 Page 2 of 4vary highly (which the Yang et al. authors state in their paragraph below point #3), itwould be ideal to gather such side-by-side data for at least 6 to 10 loci or more to en-sure reproducibility. Otherwise, the assumption remains speculative.Further, the authors in the paragraph below their point #3 suggest that their originalmethod may not be efficient on other loci by stating that “it would be premature for sci-entific community to assume that their method would work on other loci” indicating thattheir study was too underpowered for routine use in core facilities. We discussed thisspecific point (underpower) in our paper: we suggest the reader to refer to the discus-sion section of our paper from the sentence that reads ‘While many published methodsare reproducible (as evidenced by their wide usage), the research community often en-counters issues in reproducing some published methods.’Lastly, our observations call into question the robustness of the approach and its suit-ability for widespread use. Additionally, we evaluated alternative methods in parallel toreport improved efficiencies across several gene targets using one-donor methods. It isvital we hold published methodologies to the highest possible standards, especially inthe field of mouse transgenesis, where widespread adoption of low efficiency geneticmanipulation strategies can have ethical consequences on the number of animals usedin research. Science in general currently has a reproducibility crisis (https://www.na-ture.com/collections/prbfkwmwvz [nature.com]), and it is our responsibility as scien-tists that published methods are robustly tested and that the results from higher-powered analyses, which can at times be contradictory, are themselves published.Author details1Mouse Genome Engineering Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center,Omaha, NE, USA. 2Developmental Neuroscience, Munroe Meyer Institute for Genetics and Rehabilitation, University ofNebraska Medical Center, Omaha, NE, USA. 3Transformational Bioinformatics, Health and Biosecurity Business Unit,CSIRO, Sydney, Australia. 4Department of Immunology and Infectious Disease, the John Curtin School of MedicalResearch, The Australian National University, Canberra, Australia. 5Texas A&M Institute for Genomic Medicine (TIGM),Texas A&M University, College Station, TX 77843, USA. 6Department of Immunology, Tufts University School ofMedicine, Boston, USA. 7RIKEN BioResource Research Center, Tsukuba, Ibaraki 305-0074, Japan. 8Department ofBiochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA. 9Departments ofAnatomy and Cell Biology, Human Genetics and Pediatrics, Research Institute McGill University Health Center(RI-MUHC), Montreal, Canada. 10Maine Medical Center Research Institute (MMCRI), Scarborough, ME, USA.11Transgenesis and Animal Modeling Core Facility, Centre de Recherche du Centre Hospitalier Universitaire deMontreal (CRCHUM), Montreal, Canada. 12Division of Neuroscience and Experimental Psychology, School of BiologicalSciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University ofManchester, AV Hill Building, Oxford Road, Manchester M13 9PT, UK. 13School of Medicine, Indiana University,Indianapolis, IN 46202, USA. 14South Australian Health & Medical Research Institute and Department of Medicine,University of Adelaide, Adelaide, Australia. 15Transgenic mouse core facility, VIB Center for Inflammation Research,Ghent, Belgium. 16Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium. 17Unit of CardiacPhysiology, School of Medical Sciences, Manchester Academic Health Science Center, University of Manchester,Manchester, UK. 18High-Throughput DNA Sequencing and Genotyping Core Facility, Vice Chancellor for ResearchOffice, University of Nebraska Medical Center, Omaha, USA. 19University of Rochester Medical Center, Rochester, NY14642, USA. 20Division of Evolution and Genomic Sciences, School of Biological Sciences, Faculty of Biology, Medicineand Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK. 21Transgenic Unitcore facility, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK. 22Department of BasicMedicine, Division of Basic Medical Science and Molecular Medicine, School of Medicine, Tokai University, 143,Shimokasuya, Isehara, Kanagawa 259-1193, Japan. 23Department of Medical Data Science, Osaka University GraduateSchool of Medicine, Suita, Japan. 24The University of Texas, MD Anderson Cancer Center, Houston, TX, USA. 25Divisionof Cardiovascular Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, The University ofManchester AND Manchester Heart Centre, Manchester University NHS Foundation Trust, Manchester Academic HealthScience Centre, Manchester, UK. 26Department of Frontier Science for Cancer and Chemotherapy, Osaka UniversityGraduate School of Medicine, Osaka, Japan. 27The Institute of Experimental Animal Sciences, Osaka University GraduateSchool of Medicine, Osaka, Japan. 28Centre de Recherche du Centre Hospitalier Universitaire de Montreal (CRCHUM),Montreal, Canada. 29Children’s Research Institute Mouse Genome Engineering Core, University of Texas SouthwesternMedical Center, Dallas, TX 75390, USA. 30Manchester Collaborative Centre for Inflammation Research (MCCIR), School ofBiological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, UK. 31Centrefor Biological Timing, School of Medical Sciences, Faculty of Biology, Medicine and Health, University of Manchester,Manchester, UK. 32Center for Matrix Biology and Medicine, Graduate School of Medicine, Tokai University, Isehara,Kanagawa 259-1193, Japan. 33Department of Molecular Life Science, Division of Basic Medical Science and MolecularGurumurthy et al. Genome Biology           (2021) 22:99 Page 3 of 4Medicine, School of Medicine, Tokai University, 143, Shimokasuya, Isehara, Kanagawa 259-1193, Japan. 34Laboratory ofMolecular Life Science, Foundation for Biomedical Research and Innovation, Kobe, Japan. 35Department of LaboratoryAnimal Science, Support Center for Medical Research and Education, Tokai University, 143, Shimokasuya, Isehara,Kanagawa 259-1193, Japan. 36Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, OxfordOX37LE, UK. 37Mouse Biology Program, University of California, Davis, USA. 38Laboratory of Transgenic Models ofDiseases and Czech Centre for Phenogenomics, Institute of Molecular Genetics of the Czech Academy of Sciences,Prague, Czech Republic. 39College of Osteopathic Medicine, Marian University, Indianapolis, IN 46222, USA. 40LaboratoryAnimal Resource Center, University of Tsukuba, Tsukuba, Japan. 41Department of Gastroenterology and Metabolism,Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan. 42Department of Physical Therapy, Schoolof Health and Human Sciences, Indiana University, Indianapolis, IN 46202, USA. 43Lillehei Heart Institute RegenerativeMedicine and Sciences Program, University of Minnesota, Minneapolis, MN, USA. 44Paul and Sheila Wellstone MuscularDystrophy Center, University of Minnesota, Minneapolis, MN, USA. 45Department of Surgery, School of Medicine,University of California, Davis, Davis, USA. 46McGill Integrated Core for Animal Modeling (MICAM), Montreal, Canada.Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Gurumurthy et al. Genome Biology           (2021) 22:99 Page 4 of 4

Response to correspondence on  Reproducibility of CRISPR-Cas9 Methods for Generation of Conditional Mouse Alleles: A Multi-Center Evaluation

O'Brien, Aidan R.

Adams, John, Jr.

D'Hont, Jinke

Nabeshima, Yo-ichi

Vanrockeghem, Frédérique

Ghent University Academic Bibliography

CORRESPONDENCE Open AccessResponse to correspondence on“Reproducibility of CRISPR-Cas9 methodsfor generation of conditional mouse alleles:a multi-center evaluation”Channabasavaiah B. Gurumurthy1,2*, Aidan R. O’Brien3,4, Rolen M. Quadros1, John Adams Jr5, Pilar Alcaide6,Shinya Ayabe7, Johnathan Ballard5, Surinder K. Batra8, Marie-Claude Beauchamp9, Kathleen A. Becker10,Guillaume Bernas11, David Brough12, Francisco Carrillo-Salinas6, Wesley Chan9, Hanying Chen13, Ruby Dawson14,Victoria DeMambro10, Jinke D’Hont15,16, Katharine Dibb17, James D. Eudy18, Lin Gan19, Jing Gao4, Amy Gonzales5,Anyonya Guntur10, Huiping Guo5, Donald W. Harms1, Anne Harrington10, Kathryn E. Hentges20, Neil Humphreys21,Shiho Imai22, Hideshi Ishii23, Mizuho Iwama7, Eric Jonasch24, Michelle Karolak10, Bernard Keavney25, Nay-Chi Khin4,Masamitsu Konno26, Yuko Kotani27, Yayoi Kunihiro27, Imayavaramban Lakshmanan8, Catherine Larochelle28,Catherine B. Lawrence12, Lin Li29, Volkhard Lindner10, Xian-De Liu24, Gloria Lopez-Castejon30, Andrew Loudon31,Jenna Lowe4, Loydie Jerome-Majeweska9, Taiji Matsusaka22, Hiromi Miura32,33, Yoshiki Miyasaka27, Benjamin Morpurgo5,Katherine Motyl10, Yo-ichi Nabeshima34, Koji Nakade7, Toshiaki Nakashiba7, Kenichi Nakashima7, Yuichi Obata7,Sanae Ogiwara35, Mariette Ouellet11, Leif Oxburgh10, Sandra Piltz14, Ilka Pinz10, Moorthy P. Ponnusamy8, David Ray36,Ronald J. Redder18, Clifford J. Rosen10, Nikki Ross4, Mark T. Ruhe37, Larisa Ryzhova10, Ane M. Salvador6,Sabrina Shameen Alam9, Radislav Sedlacek38, Karan Sharma39, Chad Smith24, Katrien Staes15,16, Lora Starrs4,Fumihiro Sugiyama40, Satoru Takahashi40, Tomohiro Tanaka41, Andrew Trafford17, Yoshihiro Uno27, Leen Vanhoutte15,16,Frederique Vanrockeghem15,16, Brandon J. Willis37, Christian S. Wright42, Yuko Yamauchi27, Xin Yi42, Kazuto Yoshimi27,Xuesong Zhang24, Yu Zhang29, Masato Ohtsuka32,33, Satyabrata Das43, Daniel J. Garry43,44, Tino Hochepied15,16,Paul Thomas14, Jan Parker-Thornburg24, Antony D. Adamson21, Atsushi Yoshiki7, Jean-Francois Schmouth11,Andrei Golovko5, William R. Thompson42, K. C. Kent Lloyd37,45, Joshua A. Wood37, Mitra Cowan46, Tomoji Mashimo27,Seiya Mizuno40, Hao Zhu29, Petr Kasparek38, Lucy Liaw10, Joseph M. Miano19 and Gaetan Burgio4*This reply refers to the commentavailable at https://doi.org/10.1186/s13059-021-02312-3.* Correspondence: cgurumurthy@unmc.edu; gaetan.burgio@anu.edu.au1Mouse Genome Engineering CoreFacility, Vice Chancellor for ResearchOffice, University of NebraskaMedical Center, Omaha, NE, USA4Department of Immunology andInfectious Disease, the John CurtinSchool of Medical Research, TheAustralian National University,Canberra, AustraliaFull list of author information isavailable at the end of the article© The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, whichpermits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit tothe original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. Theimages or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwisein a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is notpermitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyrightholder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public DomainDedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unlessotherwise stated in a credit line to the data.Gurumurthy et al. Genome Biology           (2021) 22:99 https://doi.org/10.1186/s13059-021-02320-3We would like to thank the Yang et al. (2013) authors for their comments and debateon optimal methods for mouse transgenesis. The two Jaenisch laboratory studies pub-lished in Cell in 2013 were ground-breaking, demonstrating for the first time proof ofprinciple CRISPR mediated gene editing in the mouse zygote to generate knockout andconditional alleles, and caused much excitement in the transgenic mouse community.However, over several years and in many laboratories, the reality did not match theexcitement when it came to generating conditional alleles in a single step. While it istrue that the 2-guides 2-oligo approach can work in certain circumstances (members ofour own consortium reported some success with this method), the efficiencies reportedin Yang et al. (2013) do not bear out across multiple gene targets. Indeed, as the com-ments from Yang et al. point out, they themselves have performed further reproducibil-ity experiments on the Mecp2 locus (their point #1) and these unpublished results failto reproduce the 16% efficiency from their original publication. And our study is notthe first time concerns have been raised as to the efficiency of the 2-guides 2-oligomethod, with anecdotal reports from others in the transgenic community (Science;2016. doi:https://doi.org/10.1126/science.aal0334 [doi.org]), which stated that “Whatwas disappointing is none of us could reproduce at the efficiencies reported by Jaenisch.… It works at 1% or 2% at JAX and a lot of projects are failing. It’s really not proven tobe a robust method.” And the Yang et al. group’s response was that “The paper reportedwhat we found,” Jaenisch says. “Now, we see there are issues”.In regard to their point #2, regrettably, details of concentrations of reagents usedwere not reported in Yang et al. (2013). The authors, in this correspondence, now statethat they had provided concentrations in their other report [ref #4], but this reference(#4) does not describe generation of conditional alleles, and therefore, the experimentalconditions of this paper (ref #4) cannot be extrapolated for generating the conditionalalleles. Also, because such critical details were unavailable in the Yang et al. paper (ref#1), some of us had contacted the authors asking for tips on how to get their methodto work, but we received no response. This oversight of the authors (in failing to de-scribe the concentrations in the original paper) indeed allowed us to assess efficienciesusing a range of conditions on many loci, both lower and greater than the now revealedconditions from Yang et al. (2013), and using different delivery platforms (microinjec-tion, electroporation), the results of which further confirm that the Yang et al. methodis not efficient as it was originally reported. We suggest the reader to refer to the exten-sive data in our additional file 1 (supplementary data file) where we show that the widerange of reagent concentrations does not affect the efficiency of the Yang et al. method.Furthermore, Hatada’s group (Horii et al.) attempted to reproduce the Mecp2 experi-ments, and they reported either very low efficiency or very high toxicity when the con-centration of reagents was in the higher range. See Table 1 in Horii et al.; theconcentration of 50/12/100 produced only 2% efficiency whereas the concentration100/24/200 led to the death of nearly 90% of embryos, and the authors were unable todetermine the method’s efficiency at this higher concentration.In regard to their point #3, the authors speculate that Piezo-driven zygote injectionmay contribute to the difference of success rates. It would be necessary to examine thisspeculation by comparing the efficiencies of Piezo-driven and pronuclear injectionmethods side-by-side for a few loci. Because efficiencies at different genomic loci oftenGurumurthy et al. Genome Biology           (2021) 22:99 Page 2 of 4vary highly (which the Yang et al. authors state in their paragraph below point #3), itwould be ideal to gather such side-by-side data for at least 6 to 10 loci or more to en-sure reproducibility. Otherwise, the assumption remains speculative.Further, the authors in the paragraph below their point #3 suggest that their originalmethod may not be efficient on other loci by stating that “it would be premature for sci-entific community to assume that their method would work on other loci” indicating thattheir study was too underpowered for routine use in core facilities. We discussed thisspecific point (underpower) in our paper: we suggest the reader to refer to the discus-sion section of our paper from the sentence that reads ‘While many published methodsare reproducible (as evidenced by their wide usage), the research community often en-counters issues in reproducing some published methods.’Lastly, our observations call into question the robustness of the approach and its suit-ability for widespread use. Additionally, we evaluated alternative methods in parallel toreport improved efficiencies across several gene targets using one-donor methods. It isvital we hold published methodologies to the highest possible standards, especially inthe field of mouse transgenesis, where widespread adoption of low efficiency geneticmanipulation strategies can have ethical consequences on the number of animals usedin research. Science in general currently has a reproducibility crisis (https://www.na-ture.com/collections/prbfkwmwvz [nature.com]), and it is our responsibility as scien-tists that published methods are robustly tested and that the results from higher-powered analyses, which can at times be contradictory, are themselves published.Author details1Mouse Genome Engineering Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center,Omaha, NE, USA. 2Developmental Neuroscience, Munroe Meyer Institute for Genetics and Rehabilitation, University ofNebraska Medical Center, Omaha, NE, USA. 3Transformational Bioinformatics, Health and Biosecurity Business Unit,CSIRO, Sydney, Australia. 4Department of Immunology and Infectious Disease, the John Curtin School of MedicalResearch, The Australian National University, Canberra, Australia. 5Texas A&M Institute for Genomic Medicine (TIGM),Texas A&M University, College Station, TX 77843, USA. 6Department of Immunology, Tufts University School ofMedicine, Boston, USA. 7RIKEN BioResource Research Center, Tsukuba, Ibaraki 305-0074, Japan. 8Department ofBiochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA. 9Departments ofAnatomy and Cell Biology, Human Genetics and Pediatrics, Research Institute McGill University Health Center(RI-MUHC), Montreal, Canada. 10Maine Medical Center Research Institute (MMCRI), Scarborough, ME, USA.11Transgenesis and Animal Modeling Core Facility, Centre de Recherche du Centre Hospitalier Universitaire deMontreal (CRCHUM), Montreal, Canada. 12Division of Neuroscience and Experimental Psychology, School of BiologicalSciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University ofManchester, AV Hill Building, Oxford Road, Manchester M13 9PT, UK. 13School of Medicine, Indiana University,Indianapolis, IN 46202, USA. 14South Australian Health & Medical Research Institute and Department of Medicine,University of Adelaide, Adelaide, Australia. 15Transgenic mouse core facility, VIB Center for Inflammation Research,Ghent, Belgium. 16Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium. 17Unit of CardiacPhysiology, School of Medical Sciences, Manchester Academic Health Science Center, University of Manchester,Manchester, UK. 18High-Throughput DNA Sequencing and Genotyping Core Facility, Vice Chancellor for ResearchOffice, University of Nebraska Medical Center, Omaha, USA. 19University of Rochester Medical Center, Rochester, NY14642, USA. 20Division of Evolution and Genomic Sciences, School of Biological Sciences, Faculty of Biology, Medicineand Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK. 21Transgenic Unitcore facility, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK. 22Department of BasicMedicine, Division of Basic Medical Science and Molecular Medicine, School of Medicine, Tokai University, 143,Shimokasuya, Isehara, Kanagawa 259-1193, Japan. 23Department of Medical Data Science, Osaka University GraduateSchool of Medicine, Suita, Japan. 24The University of Texas, MD Anderson Cancer Center, Houston, TX, USA. 25Divisionof Cardiovascular Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, The University ofManchester AND Manchester Heart Centre, Manchester University NHS Foundation Trust, Manchester Academic HealthScience Centre, Manchester, UK. 26Department of Frontier Science for Cancer and Chemotherapy, Osaka UniversityGraduate School of Medicine, Osaka, Japan. 27The Institute of Experimental Animal Sciences, Osaka University GraduateSchool of Medicine, Osaka, Japan. 28Centre de Recherche du Centre Hospitalier Universitaire de Montreal (CRCHUM),Montreal, Canada. 29Children’s Research Institute Mouse Genome Engineering Core, University of Texas SouthwesternMedical Center, Dallas, TX 75390, USA. 30Manchester Collaborative Centre for Inflammation Research (MCCIR), School ofBiological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, UK. 31Centrefor Biological Timing, School of Medical Sciences, Faculty of Biology, Medicine and Health, University of Manchester,Manchester, UK. 32Center for Matrix Biology and Medicine, Graduate School of Medicine, Tokai University, Isehara,Kanagawa 259-1193, Japan. 33Department of Molecular Life Science, Division of Basic Medical Science and MolecularGurumurthy et al. Genome Biology           (2021) 22:99 Page 3 of 4Medicine, School of Medicine, Tokai University, 143, Shimokasuya, Isehara, Kanagawa 259-1193, Japan. 34Laboratory ofMolecular Life Science, Foundation for Biomedical Research and Innovation, Kobe, Japan. 35Department of LaboratoryAnimal Science, Support Center for Medical Research and Education, Tokai University, 143, Shimokasuya, Isehara,Kanagawa 259-1193, Japan. 36Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, OxfordOX37LE, UK. 37Mouse Biology Program, University of California, Davis, USA. 38Laboratory of Transgenic Models ofDiseases and Czech Centre for Phenogenomics, Institute of Molecular Genetics of the Czech Academy of Sciences,Prague, Czech Republic. 39College of Osteopathic Medicine, Marian University, Indianapolis, IN 46222, USA. 40LaboratoryAnimal Resource Center, University of Tsukuba, Tsukuba, Japan. 41Department of Gastroenterology and Metabolism,Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan. 42Department of Physical Therapy, Schoolof Health and Human Sciences, Indiana University, Indianapolis, IN 46202, USA. 43Lillehei Heart Institute RegenerativeMedicine and Sciences Program, University of Minnesota, Minneapolis, MN, USA. 44Paul and Sheila Wellstone MuscularDystrophy Center, University of Minnesota, Minneapolis, MN, USA. 45Department of Surgery, School of Medicine,University of California, Davis, Davis, USA. 46McGill Integrated Core for Animal Modeling (MICAM), Montreal, Canada.Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Gurumurthy et al. Genome Biology           (2021) 22:99 Page 4 of 4

Response to correspondence on 'Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles : a multi-center evaluation'

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Response to correspondence on “Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation”

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Response to correspondence on "Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles:a multi-center evaluation"

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Response to correspondence on Reproducibility of CRISPR-Cas9 Methods for Generation of Conditional Mouse Alleles: A Multi-Center Evaluation

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