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3. 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
Biological Sciences, Genetics, Biotechnology, Prevention, Alleles, Animals, Blastocyst, CRISPR-Associated Protein 9, CRISPR-Cas Systems, Factor Analysis, Statistical, Female, Male, Methyl-CpG-Binding Protein 2, Mice, Knockout, Microinjections, Regression Analysis, Reproducibility of Results, CRISPR-Cas9, Mouse, Transgenesis, Homology-directed repair, Conditional knockout mouse, Floxed allele, Oligonucleotide, Long single-stranded DNA, Machine learning, Reproducibility, Environmental Sciences, Information and Computing Sciences, Bioinformatics
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. Reprogramming human T cell function and specificity with non-viral genome targeting.

Author

Roth, Theodore L, Puig-Saus, Cristina, Yu, Ruby, Shifrut, Eric, Carnevale, Julia, Li, P Jonathan, Hiatt, Joseph, Saco, Justin, Krystofinski, Paige, Li, Han, Tobin, Victoria, Nguyen, David N, Lee, Michael R, Putnam, Amy L, Ferris, Andrea L, Chen, Jeff W, Schickel, Jean-Nicolas, Pellerin, Laurence, Carmody, David, Alkorta-Aranburu, Gorka, Del Gaudio, Daniela, Matsumoto, Hiroyuki, Morell, Montse, Mao, Ying, Cho, Min, Quadros, Rolen M, Gurumurthy, Channabasavaiah B, Smith, Baz, Haugwitz, Michael, Hughes, Stephen H, Weissman, Jonathan S, Schumann, Kathrin, Esensten, Jonathan H, May, Andrew P, Ashworth, Alan, Kupfer, Gary M, Greeley, Siri Atma W, Bacchetta, Rosa, Meffre, Eric, Roncarolo, Maria Grazia, Romberg, Neil, Herold, Kevan C, Ribas, Antoni, Leonetti, Manuel D, and Marson, Alexander

Subjects
T-Lymphocytes, Cells, Cultured, Animals, Humans, Mice, Receptors, Antigen, T-Cell, Protein Engineering, Neoplasm Transplantation, Autoimmunity, Genome, Human, Male, Interleukin-2 Receptor alpha Subunit, CRISPR-Cas Systems, Cellular Reprogramming, Gene Editing, Human Genome, Biotechnology, Genetics, Gene Therapy, 5.2 Cellular and gene therapies, 5.1 Pharmaceuticals, Inflammatory and immune system, Cancer, General Science & Technology
Abstract

Decades of work have aimed to genetically reprogram T cells for therapeutic purposes1,2 using recombinant viral vectors, which do not target transgenes to specific genomic sites3,4. The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair5,6. Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.

Published
2018

9. CD4+ effector T cells accelerate Alzheimer’s disease in mice

Author

Machhi, Jatin, Yeapuri, Pravin, Lu, Yaman, Foster, Emma, Chikhale, Rupesh, Herskovitz, Jonathan, Namminga, Krista L., Olson, Katherine E., Abdelmoaty, Mai Mohamed, Gao, Ju, Quadros, Rolen M., Kiyota, Tomomi, Jingjing, Liang, Kevadiya, Bhavesh D., Wang, Xinglong, Liu, Yutong, Poluektova, Larisa Y., Gurumurthy, Channabasavaiah B., Mosley, R. Lee, and Gendelman, Howard E.

Published
2021
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11. Response to correspondence on “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, Jr, 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, Eudy, James D., Gan, Lin, Gao, Jing, Gonzales, Amy, Guntur, Anyonya, 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-Majeweska, Loydie, 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, 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., Yoshiki, Atsushi, Schmouth, Jean-Francois, Golovko, Andrei, Thompson, William R., Lloyd, K. C. Kent, Wood, Joshua A., Cowan, Mitra, Mashimo, Tomoji, Mizuno, Seiya, Zhu, Hao, Kasparek, Petr, Liaw, Lucy, Miano, Joseph M., and Burgio, Gaetan

Published
2021
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12. Validation of Simple Sequence Length Polymorphism Regions of Commonly Used Mouse Strains for Marker Assisted Speed Congenics Screening

Author

Gurumurthy, Channabasavaiah B, Joshi, Poonam S, Kurz, Scott G, Ohtsuka, Masato, Quadros, Rolen M, Harms, Donald W, and Lloyd, KC Kent

Subjects
Biological Sciences, Genetics, Biotechnology
Abstract

Marker assisted speed congenics technique is commonly used to facilitate backcrossing of mouse strains in nearly half the time it normally takes otherwise. Traditionally, the technique is performed by analyzing PCR amplified regions of simple sequence length polymorphism (SSLP) markers between the recipient and donor strains: offspring with the highest number of markers showing the recipient genome across all chromosomes is chosen for the next generation. Although there are well-defined panels of SSLP makers established between certain pairs of mice strains, they are incomplete for most strains. The availability of well-established marker sets for speed congenic screens would enable the scientific community to transfer mutations across strain backgrounds. In this study, we tested the suitability of over 400 SSLP marker sets among 10 mouse strains commonly used for generating genetically engineered models. The panel of markers presented here can readily identify the specified strains and will be quite useful in marker assisted speed congenic screens. Moreover, unlike newer single nucleotide polymorphism (SNP) array methods which require sophisticated equipment, the SSLP markers panel described here only uses PCR and agarose gel electrophoresis of amplified products; therefore it can be performed in most research laboratories.

Published
2015

18. Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins

Author

Quadros, Rolen M., Miura, Hiromi, Harms, Donald W., Akatsuka, Hisako, Sato, Takehito, Aida, Tomomi, Redder, Ronald, Richardson, Guy P., Inagaki, Yutaka, Sakai, Daisuke, Buckley, Shannon M., Seshacharyulu, Parthasarathy, Batra, Surinder K., Behlke, Mark A., Zeiner, Sarah A., Jacobi, Ashley M., Izu, Yayoi, Thoreson, Wallace B., Urness, Lisa D., Mansour, Suzanne L., Ohtsuka, Masato, and Gurumurthy, Channabasavaiah B.

Published
2017
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19. Ecdysoneless Overexpression Drives Mammary Tumorigenesis through Upregulation of C-MYC and Glucose Metabolism

Author

Mohapatra, Bhopal C., primary, Mirza, Sameer, additional, Bele, Aditya, additional, Gurumurthy, Channabasavaiah B., additional, Raza, Mohsin, additional, Saleem, Irfana, additional, Storck, Matthew D., additional, Sarkar, Aniruddha, additional, Kollala, Sai Sundeep, additional, Shukla, Surendra K., additional, Southekal, Siddesh, additional, Wagner, Kay-Uwe, additional, Qiu, Fang, additional, Lele, Subodh M., additional, Alsaleem, Mansour A., additional, Rakha, Emad A., additional, Guda, Chittibabu, additional, Singh, Pankaj K., additional, Cardiff, Robert D., additional, Band, Hamid, additional, and Band, Vimla, additional

Published
2022
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20. Additional file 1 of CRISPR-KRISPR: a method to identify on-target and random insertion of donor DNAs and their characterization in knock-in mice

Author

Tanaka, Masayuki, Yokoyama, Keiko, Hayashi, Hideki, Isaki, Sanae, Kitatani, Kanae, Wang, Ting, Kawata, Hisako, Matsuzawa, Hideyuki, Gurumurthy, Channabasavaiah B., Miura, Hiromi, and Ohtsuka, Masato

Abstract

Additional file 1. This includes fourteen supplementary figures, four supplementary tables and supplementary text.

Published
2022
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22. Structure of Long-Range Direct and Indirect Spinocerebellar Pathways as Well as Local Spinal Circuits Mediating Proprioception

Author

Pop, Iliodora V., primary, Espinosa, Felipe, additional, Blevins, Cheasequah J., additional, Okafor, Portia C., additional, Ogujiofor, Osita W., additional, Goyal, Megan, additional, Mona, Bishakha, additional, Landy, Mark A., additional, Dean, Kevin M., additional, Gurumurthy, Channabasavaiah B., additional, and Lai, Helen C., additional

Published
2021
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23. Additional file 1 of CD4+ effector T cells accelerate Alzheimer���s disease in mice

Author

Machhi, Jatin, Yeapuri, Pravin, Lu, Yaman, Foster, Emma, Chikhale, Rupesh, Herskovitz, Jonathan, Namminga, Krista L., Olson, Katherine E., Abdelmoaty, Mai Mohamed, Gao, Ju, Quadros, Rolen M., Kiyota, Tomomi, Jingjing, Liang, Kevadiya, Bhavesh D., Wang, Xinglong, Liu, Yutong, Poluektova, Larisa Y., Gurumurthy, Channabasavaiah B., Mosley, R. Lee, and Gendelman, Howard E.

Abstract

Additional file 1: Figure S1. Development of monoclonal A��-Th1 and A��-Th17 cells. Figure S2. Modelling and explicit solvent molecular dynamics simulations. Figure S3. Adoptive transfer of A��-Teffs did not affect T cell frequency in APP/PS1 mice. Figure S4. Transcriptomic analysis of immune genes. Figure S5. Western blot images.

Published
2021
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24. Pharmacotherapeutics of SARS-CoV-2 Infections

Author

Kevadiya, Bhavesh D., Machhi, Jatin, Herskovitz, Jonathan, Oleynikov, Maxim D., Blomberg, Wilson R., Bajwa, Neha, Soni, Dhruvkumar, Das, Srijanee, Hasan, Mahmudul, Patel, Milankumar, Senan, Ahmed M., Gorantla, Santhi, McMillan, Jo Ellyn, Edagwa, Benson, Eisenberg, Robert, Gurumurthy, Channabasavaiah B., Reid, St Patrick M., Punyadeera, Chamindie, Chang, Linda, Gendelman, Howard E., Kevadiya, Bhavesh D., Machhi, Jatin, Herskovitz, Jonathan, Oleynikov, Maxim D., Blomberg, Wilson R., Bajwa, Neha, Soni, Dhruvkumar, Das, Srijanee, Hasan, Mahmudul, Patel, Milankumar, Senan, Ahmed M., Gorantla, Santhi, McMillan, Jo Ellyn, Edagwa, Benson, Eisenberg, Robert, Gurumurthy, Channabasavaiah B., Reid, St Patrick M., Punyadeera, Chamindie, Chang, Linda, and Gendelman, Howard E.

Abstract

The COVID-19 pandemic has affected more than 38 million people world-wide by person to person transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therapeutic and preventative strategies for SARS-CoV-2 remains a significant challenge. Within the past several months, effective treatment options have emerged and now include repurposed antivirals, corticosteroids and virus-specific antibodies. The latter has included convalescence plasma and monoclonal antibodies. Complete viral eradication will be achieved through an effective, safe and preventative vaccine. To now provide a comprehensive summary for each of the pharmacotherapeutics and preventative strategies being offered or soon to be developed for SARS-CoV-2. Graphical abstract: [Figure not available: see fulltext.].

Published
2021

25. Diagnostics for SARS-CoV-2 infections

Author

Kevadiya, Bhavesh D., Machhi, Jatin, Herskovitz, Jonathan, Oleynikov, Maxim D., Blomberg, Wilson R., Bajwa, Neha, Soni, Dhruvkumar, Das, Srijanee, Hasan, Mahmudul, Patel, Milankumar, Senan, Ahmed M., Gorantla, Santhi, Mcmillan, Joellyn, Edagwa, Benson, Eisenberg, Robert, Gurumurthy, Channabasavaiah B., Reid, St Patrick M., Punyadeera, Chamindie, Chang, Linda, Gendelman, Howard E., Kevadiya, Bhavesh D., Machhi, Jatin, Herskovitz, Jonathan, Oleynikov, Maxim D., Blomberg, Wilson R., Bajwa, Neha, Soni, Dhruvkumar, Das, Srijanee, Hasan, Mahmudul, Patel, Milankumar, Senan, Ahmed M., Gorantla, Santhi, Mcmillan, Joellyn, Edagwa, Benson, Eisenberg, Robert, Gurumurthy, Channabasavaiah B., Reid, St Patrick M., Punyadeera, Chamindie, Chang, Linda, and Gendelman, Howard E.

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread to nearly every corner of the globe, causing societal instability. The resultant coronavirus disease 2019 (COVID-19) leads to fever, sore throat, cough, chest and muscle pain, dyspnoea, confusion, anosmia, ageusia and headache. These can progress to life-threatening respiratory insufficiency, also affecting the heart, kidney, liver and nervous systems. The diagnosis of SARS-CoV-2 infection is often confused with that of influenza and seasonal upper respiratory tract viral infections. Due to available treatment strategies and required containments, rapid diagnosis is mandated. This Review brings clarity to the rapidly growing body of available and in-development diagnostic tests, including nanomaterial-based tools. It serves as a resource guide for scientists, physicians, students and the public at large.

Published
2021

27. A transgenic pig model expressing a CMV-ZsGreen1 reporter across an extensive array of tissues

Author

Desaulniers, Amy T, Cederberg, Rebecca A, Carreiro, Elizabeth P, Gurumurthy, Channabasavaiah B, White, Brett R, Desaulniers, Amy T, Cederberg, Rebecca A, Carreiro, Elizabeth P, Gurumurthy, Channabasavaiah B, and White, Brett R

Abstract

Since genetic engineering of pigs can benefit both biomedicine and agriculture, selecting a suitable gene promoter is critically important. The cytomegalovirus (CMV) promoter, which can robustly drive ubiquitous transgene expression, is commonly used at present, yet recent reports suggest tissue-specific activity in the pig. The objective of this study was to quantify ZsGreen1 protein (in lieu of CMV promoter activity) in tissues from pigs harboring a CMV-ZsGreen1 transgene with a single integration site. Tissue samples (n=35) were collected from neonatal hemizygous (n=3) and homozygous (n=3) piglets and ZsGreen1 abundance was determined via immunoblotting. ZsGreen1 was detected in all tissues, except hypothalamus, kidney cortex and oviduct. The expression patterns of homozygous and hemizygous piglets were similar (P>0.05). However, quantification revealed that ZsGreen1 protein levels were tissue-specific. Within neural/endocrine tissues, ZsGreen1 abundance was highest in the anterior pituitary gland, intermediate in the cerebellum and lowest in the cerebrum, spinal cord and posterior pituitary (P<0.05). In the digestive system, ZsGreen1 was more abundant in the salivary gland than esophagus, stomach, pancreas, duodenum, jejunum, ileum, spleen, colon, gallbladder and liver (P<0.05). Interestingly, ZsGreen1 amounts also differed within an organ (i.e., the right ventricle had 3-fold higher levels than the other heart chambers; P<0.05). These results provide useful information for the use of the CMV promoter to drive transgene expression in the pig. Moreover, this swine model represents a novel resource of ZsGreen1- labeled organs and a valuable tool to advance genome editing research.

Published
2020

32. Generating mouse models for biomedical research: technological advances

Author

Gurumurthy, Channabasavaiah B. and Lloyd, Kevin C. Kent

Subjects
Biomedical Research, Mouse, Computer science, Neuroscience (miscellaneous), Medicine (miscellaneous), Mutagenesis (molecular biology technique), lcsh:Medicine, Mouse Models, Computational biology, Genome, General Biochemistry, Genetics and Molecular Biology, At A Glance, Mice, Immunology and Microbiology (miscellaneous), Genome editing, RNA interference, lcsh:Pathology, Animals, CRISPR, Embryonic Stem Cells, Gene Editing, lcsh:R, Gene targeting, Disease Models, Animal, Mutagenesis, Genetically Engineered Mouse, RNA Interference, Homologous recombination, lcsh:RB1-214
Abstract

Over the past decade, new methods and procedures have been developed to generate genetically engineered mouse models of human disease. This At a Glance article highlights several recent technical advances in mouse genome manipulation that have transformed our ability to manipulate and study gene expression in the mouse. We discuss how conventional gene targeting by homologous recombination in embryonic stem cells has given way to more refined methods that enable allele-specific manipulation in zygotes. We also highlight advances in the use of programmable endonucleases that have greatly increased the feasibility and ease of editing the mouse genome. Together, these and other technologies provide researchers with the molecular tools to functionally annotate the mouse genome with greater fidelity and specificity, as well as to generate new mouse models using faster, simpler and less costly techniques., Summary: Newer molecular technologies to precisely and efficiently manipulate the mammalian genome are enabling the production of more scientifically valuable animal models.

Published
2019

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

Author

Gurumurthy, Channabasavaiah B., O'Brien, Aidan, Quadros, Rolen M., Adams Jr, John, Alcaide, Pilar, Ayabe, Shinya, Ballard, John, Batra, Surinder K., Beauchamp, Marie-Claude, Becker, Kathleen A., Bernas, Guillaume, Jing, Gao, Khin, Nay Chi, Lowe, Jenna, Ross, Nikki, Starrs, Lora, Burgio, Gaetan, Gurumurthy, Channabasavaiah B., O'Brien, Aidan, Quadros, Rolen M., Adams Jr, John, Alcaide, Pilar, Ayabe, Shinya, Ballard, John, Batra, Surinder K., Beauchamp, Marie-Claude, Becker, Kathleen A., Bernas, Guillaume, Jing, Gao, Khin, Nay Chi, Lowe, Jenna, Ross, Nikki, Starrs, Lora, and Burgio, Gaetan

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.

Published
2019

37. Slc26a9P2ACre: a new CRE driver to regulate gene expression in the otic placode lineage and other FGFR2b-dependent epithelia.

Author

Urness, Lisa D., Xiaofen Wang, Chaoying Li, Quadros, Rolen M., Harms, Donald W., Gurumurthy, Channabasavaiah B., and Mansour, Suzanne L.

Subjects
INNER ear, GENE expression, EPITHELIUM, EPITHELIAL cells, GENETIC regulation, GENETIC recombination, HAIR cells
Abstract

Pan-otic CRE drivers enable gene regulation throughout the otic placode lineage, comprising the inner ear epithelium and neurons. However, intersection of extra-otic gene-of-interest expression with the CRE lineage can compromise viability and impede auditory analyses. Furthermore, extant pan-otic CREs recombine in auditory and vestibular brain nuclei, making it difficult to ascribe resulting phenotypes solely to the inner ear. We have previously identified Slc26a9 as an otic placode-specific target of the FGFR2b ligands FGF3 and FGF10. We show here that Slc26a9 is otic specific through E10.5, but is not required for hearing. We targeted P2ACre to the Slc26a9 stop codon, generating Slc26a9P2ACre mice, and observed CRE activity throughout the otic epithelium and neurons, with little activity evident in the brain. Notably, recombination was detected in many FGFR2b ligand-dependent epithelia. We generated Fgf10 and Fgf8 conditional mutants, and activated an FGFR2b ligand trap from E17.5 to P3. In contrast to analogous mice generated with other panotic CREs, these were viable. Auditory thresholds were elevated in mutants, and correlated with cochlear epithelial cell losses. Thus, Slc26a9P2ACre provides a useful complement to existing pan-otic CRE drivers, particularly for postnatal analyses. [ABSTRACT FROM AUTHOR]

Published
2020
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38. Antiretroviral Drug Metabolism in Humanized PXR-CAR-CYP3A-NOG Mice

Author

McMillan, JoEllyn M., primary, Cobb, Denise A., additional, Lin, Zhiyi, additional, Banoub, Mary G., additional, Dagur, Raghubendra S., additional, Branch Woods, Amanda A., additional, Wang, Weimin, additional, Makarov, Edward, additional, Kocher, Ted, additional, Joshi, Poonam S., additional, Quadros, Rolen M., additional, Harms, Donald W., additional, Cohen, Samuel M., additional, Gendelman, Howard E., additional, Gurumurthy, Channabasavaiah B., additional, Gorantla, Santhi, additional, and Poluektova, Larisa Y., additional

Published
2018
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42. A Novel Interaction of Ecdysoneless (ECD) Protein with R2TP Complex Component RUVBL1 Is Required for the Functional Role of ECD in Cell Cycle Progression

Author

Mir, Riyaz A., primary, Bele, Aditya, additional, Mirza, Sameer, additional, Srivastava, Shashank, additional, Olou, Appolinaire A., additional, Ammons, Shalis A., additional, Kim, Jun Hyun, additional, Gurumurthy, Channabasavaiah B., additional, Qiu, Fang, additional, Band, Hamid, additional, and Band, Vimla, additional

Published
2016
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43. One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT)

Author

1000090372945, Ohtsuka, Masato, 1000090599523, Miura, Hiromi, 1000060312287, Mochida, Keiji, Hirose, Michiko, Hasegawa, Ayumi, 1000020194524, Ogura, Atsuo, 1000070272482, Mizutani, Ryuta, 1000010146706, Kimura, Minoru, 1000020444523, Isotani, Ayako, 1000020304066, Ikawa, Masahito, 1000030287099, Sato, Masahiro, Gurumurthy, Channabasavaiah B., 1000090372945, Ohtsuka, Masato, 1000090599523, Miura, Hiromi, 1000060312287, Mochida, Keiji, Hirose, Michiko, Hasegawa, Ayumi, 1000020194524, Ogura, Atsuo, 1000070272482, Mizutani, Ryuta, 1000010146706, Kimura, Minoru, 1000020444523, Isotani, Ayako, 1000020304066, Ikawa, Masahito, 1000030287099, Sato, Masahiro, and Gurumurthy, Channabasavaiah B.

Abstract

Ohtsuka, M., Miura, H., Mochida, K. et al. One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT). BMC Genomics 16, 274 (2015). https://doi.org/10.1186/s12864-015-1432-5, Background: The pronuclear injection (PI) is the simplest and widely used method to generate transgenic (Tg) mice. Unfortunately, PI-based Tg mice show uncertain transgene expression due to random transgene insertion in the genome, usually with multiple copies. Thus, typically at least three or more Tg lines are produced by injecting over 200 zygotes and the best line/s among them are selected through laborious screening steps. Recently, we developed technologies using Cre-loxP system that allow targeted insertion of single-copy transgene into a predetermined locus through PI. We termed the method as PI-based Targeted Transgenesis (PITT). A similar method using PhiC31-attP/B system was reported subsequently. Results: Here, we developed an improved-PITT (i-PITT) method by combining Cre-loxP, PhiC31-attP/B and FLP-FRT systems directly under C57BL/6N inbred strain, unlike the mixed strain used in previous reports. The targeted Tg efficiency in the i-PITT typically ranged from 10 to 30%, with 47 and 62% in two of the sessions, which is by-far the best Tg rate reported. Furthermore, the system could generate multiple Tg mice simultaneously. We demonstrate that injection of up to three different Tg cassettes in a single injection session into as less as 181 zygotes resulted in production of all three separate Tg DNA containing targeted Tg mice. Conclusions: The i-PITT system offers several advantages compared to previous methods: multiplexing capability (i-PITT is the only targeted-transgenic method that is proven to generate multiple different transgenic lines simultaneously), very high efficiency of targeted-transgenesis (up to 62%), significantly reduces animal numbers in mouse-transgenesis and the system is developed under C57BL/6N strain, the most commonly used pure genetic background. Further, the i-PITT system is freely accessible to scientific community.

Published
2015

47. One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT)

Author

Ohtsuka, Masato, primary, Miura, Hiromi, additional, Mochida, Keiji, additional, Hirose, Michiko, additional, Hasegawa, Ayumi, additional, Ogura, Atsuo, additional, Mizutani, Ryuta, additional, Kimura, Minoru, additional, Isotani, Ayako, additional, Ikawa, Masahito, additional, Sato, Masahiro, additional, and Gurumurthy, Channabasavaiah B, additional

Published
2015
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