TY - JOUR
T1 - Tissue-specific expression of Cas9 has no impact on whole-body metabolism in four transgenic mouse lines
AU - Bond, Simon T.
AU - Zhuang, Aowen
AU - Yang, Christine
AU - Gould, Eleanor A.M.
AU - Sikora, Tim
AU - Liu, Yingying
AU - Fu, Ying
AU - Watt, Kevin I.
AU - Tan, Yanie
AU - Kiriazis, Helen
AU - Lancaster, Graeme I.
AU - Gregorevic, Paul
AU - Henstridge, Darren C.
AU - McMullen, Julie R.
AU - Meikle, Peter J.
AU - Calkin, Anna C.
AU - Drew, Brian G.
N1 - Funding Information:
We acknowledge funding support from the Victorian State Government OIS program to Baker Heart & Diabetes Institute. These studies were supported by funding from the Baker Heine Trust through both the Obesity & Lipid and the Bioinformatics Programs, as well as the Bertalli mini-grant scheme at Baker. During the period of this study, some authors were supported by fellowship funding as follows: BGD and ACC, National Heart Foundation of Australia ( 101789 and 100067 , respectively); JRM and PG, NHMRC of Australia ( APP1078985 and APP1117835 , respectively). We thank members of the MMA, LMCD, Cardiac Hypertrophy, Metabolomics, and Hematopoiesis & Leukocyte Biology laboratories at BHDI for their contributions. We also acknowledge the use of the facilities and technical assistance of the Monash Histology Platform, Department of Anatomy and Developmental Biology, Monash University. Figure 1 A was created with Biorender ( Biorender.com ).
Funding Information:
We acknowledge funding support from the Victorian State Government OIS program to Baker Heart & Diabetes Institute. These studies were supported by funding from the Baker Heine Trust through both the Obesity & Lipid and the Bioinformatics Programs, as well as the Bertalli mini-grant scheme at Baker. During the period of this study, some authors were supported by fellowship funding as follows: BGD and ACC, National Heart Foundation of Australia (101789 and 100067, respectively); JRM and PG, NHMRC of Australia (APP1078985 and APP1117835, respectively). We thank members of the MMA, LMCD, Cardiac Hypertrophy, Metabolomics, and Hematopoiesis & Leukocyte Biology laboratories at BHDI for their contributions. We also acknowledge the use of the facilities and technical assistance of the Monash Histology Platform, Department of Anatomy and Developmental Biology, Monash University. Figure 1A was created with Biorender (Biorender.com).
Publisher Copyright:
© 2021 The Authors
PY - 2021/11
Y1 - 2021/11
N2 - Objective: CRISPR/Cas9 technology has revolutionized gene editing and fast tracked our capacity to manipulate genes of interest for the benefit of both research and therapeutic applications. Whilst many advances have, and continue to be made in this area, perhaps the most utilized technology to date has been the generation of knockout cells, tissues and animals. The advantages of this technology are many fold, however some questions still remain regarding the effects that long term expression of foreign proteins such as Cas9, have on mammalian cell function. Several studies have proposed that chronic overexpression of Cas9, with or without its accompanying guide RNAs, may have deleterious effects on cell function and health. This is of particular concern when applying this technology in vivo, where chronic expression of Cas9 in tissues of interest may promote disease-like phenotypes and thus confound the investigation of the effects of the gene of interest. Although these concerns remain valid, no study to our knowledge has yet to demonstrate this directly. Methods: In this study we used the lox-stop-lox (LSL) spCas9 ROSA26 transgenic (Tg) mouse line to generate four tissue-specific Cas9-Tg models that express Cas9 in the heart, liver, skeletal muscle or adipose tissue. We performed comprehensive phenotyping of these mice up to 20-weeks of age and subsequently performed molecular analysis of their organs. Results: We demonstrate that Cas9 expression in these tissues had no detrimental effect on whole body health of the animals, nor did it induce any tissue-specific effects on whole body energy metabolism, liver health, inflammation, fibrosis, heart function or muscle mass. Conclusions: Our data suggests that these models are suitable for studying the tissue specific effects of gene deletion using the LSL-Cas9-Tg model, and that phenotypes observed utilizing these models can be confidently interpreted as being gene specific, and not confounded by the chronic overexpression of Cas9.
AB - Objective: CRISPR/Cas9 technology has revolutionized gene editing and fast tracked our capacity to manipulate genes of interest for the benefit of both research and therapeutic applications. Whilst many advances have, and continue to be made in this area, perhaps the most utilized technology to date has been the generation of knockout cells, tissues and animals. The advantages of this technology are many fold, however some questions still remain regarding the effects that long term expression of foreign proteins such as Cas9, have on mammalian cell function. Several studies have proposed that chronic overexpression of Cas9, with or without its accompanying guide RNAs, may have deleterious effects on cell function and health. This is of particular concern when applying this technology in vivo, where chronic expression of Cas9 in tissues of interest may promote disease-like phenotypes and thus confound the investigation of the effects of the gene of interest. Although these concerns remain valid, no study to our knowledge has yet to demonstrate this directly. Methods: In this study we used the lox-stop-lox (LSL) spCas9 ROSA26 transgenic (Tg) mouse line to generate four tissue-specific Cas9-Tg models that express Cas9 in the heart, liver, skeletal muscle or adipose tissue. We performed comprehensive phenotyping of these mice up to 20-weeks of age and subsequently performed molecular analysis of their organs. Results: We demonstrate that Cas9 expression in these tissues had no detrimental effect on whole body health of the animals, nor did it induce any tissue-specific effects on whole body energy metabolism, liver health, inflammation, fibrosis, heart function or muscle mass. Conclusions: Our data suggests that these models are suitable for studying the tissue specific effects of gene deletion using the LSL-Cas9-Tg model, and that phenotypes observed utilizing these models can be confidently interpreted as being gene specific, and not confounded by the chronic overexpression of Cas9.
KW - Cas9
KW - CRISPR
KW - Metabolism
KW - Phenotype
KW - Tissue specific
KW - Transgenic mice
UR - http://www.scopus.com/inward/record.url?scp=85111990314&partnerID=8YFLogxK
U2 - 10.1016/j.molmet.2021.101292
DO - 10.1016/j.molmet.2021.101292
M3 - Article
C2 - 34246805
AN - SCOPUS:85111990314
SN - 2212-8778
VL - 53
JO - Molecular Metabolism
JF - Molecular Metabolism
M1 - 101292
ER -