Detailed metabolic phenotyping of four tissue specific Cas9 transgenic mouse lines

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 by taking advantage of Cas9 function to promote indels in precise locations in the genome. Whilst the advantages of this technology are many fold, 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. Thus, in this study we used the lox-stop-lox (LSL) spCas9 ROSA26 transgenic (Tg) mouse line to generate four tissue-specific Cas9-Tg models with expression in the heart, liver, skeletal muscle and adipose tissue. We performed comprehensive phenotyping of these mice up to 20-weeks of age and subsequently performed molecular analysis of their organs. We demonstrated 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 energy metabolism, liver health, inflammation, fibrosis, heart function or muscle mass. Thus, 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.


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Since the discovery and proven utility of CRISPR/Cas9 based gene editing technologies, 51 there has been a proliferation of applications that take advantage of this ground-breaking 52 technology. Whilst the potential for this relatively simple but precise, genetic manipulation 53 tool is obvious, the speed at which the field is developing often means that subtle off-target 54 and deleterious effects of such an approach can be overlooked. Studies over the past 5 years 55 have demonstrated that each system requires important optimization to ensure accurate gene 56 editing, whilst minimizing off-target editing and potential toxicity induced by the 57 introduction of foreign genetic machinery (Broeders et al., 2020;Molla and Yang, 2019). 58 59 A major advantage of CRISPR based editing in the pre-clinical biomedical arena is the rapid 60 development of animal models that harbor global gene deletions or conditional targeting of 61 alleles. These models historically took 2-3 years to generate, where now a global deletion 62 model using CRISPR can take 3 months or less to generate (Singh et al., 2015). Moreover, 63 CRISPR overcomes the need to generate one mouse model per gene of interest, as is the case 64 with floxed alleles. Indeed, by over-expressing Cas9 globally in mice, or in a tissue specific 65 manner, one can generate a model where almost any gene can be manipulated simply by 66 introducing a guide RNA that targets your gene of interest. This flexibility of manipulation 67 has made it feasible to use one mouse model, or even an existing disease model, to study the 68 effect of manipulating one or many genes in combination. 69 70 One such model developed is the lox-STOP-lox spCas9-transgenic (LSL-spCas9Tg) mouse 71 (Platt et al., 2014). This model harbors the spCas9 gene at the ROSA26 locus, but is silenced 72 in the basal state by commonly applied repressor elements. By flanking the repressor or 73 6 performed some model specific phenotyping, including echocardiography of the MHC-alpha 114 model to analyze heart function. 115 116 At the completion of each study (mice up to approximately 20-22 weeks of age), tissues were 117 collected, processed and analyzed to first confirm that each model displayed the expected 118 expression profiles. Using qPCR analysis, we demonstrated that each model exhibited tissue 119 specific Cre-recombinase expression, with robust expression in the expected tissue, but no 120 detected expression in other tissues ( Figure 1B). Importantly, we also demonstrate using 121 qPCR that Cas9 expression was tissue specific, and that this expression was dependent on 122 both Cre-expression and the administration of tamoxifen in the inducible models. As a 123 secondary confirmation we also determined the abundance of GFP by qPCR, which is co-124 expressed from the same transgene cassette as Cas9, but is independently processed by the 125 ribosome (i.e. not tagged to Cas9). We demonstrated that both Cas9 and GFP exhibited the 126 expected tissue specific expression profiles including liver ( Figure 1C), heart ( Figure 1D), 127 muscle ( Figure 1E) and white adipose tissue (WAT, Figure 1F). The level of Cas9 induction 128 varied slightly across the lines, which is likely a reflection of local transcriptional machinery, 129 number of cells per unit of tissue, and the differential activity of Cre-recombinase in each 130 tissue. 131 132 Tissue Specific Expression of Cre-Recombinase, Cas9 and GFP does not Impact Animal 133

Body Weight or Tissue Weights. 134
Upon demonstrating that each model expressed Cas9 in a tissue specific manner, we next 135 sought to test previously raised concerns that chronic over-expression of "foreign" enzymes 136 such as Cre and Cas9 in metabolic tissues, might lead to phenotypic differences in animal 137 growth and development. A simple way of testing for toxicity or growth inhibition is to 7 compare body weight and individual tissue weights from each model at study end. We 139 demonstrate that body weights for each model were comparable between cohorts at the time 140 of cull, with no significant differences in body weight, whether they were expressing Cre, 141 Cas9, or if they had been treated with tamoxifen/oil (Figure 2A). Moreover, assessment of 142 liver, WAT, Muscle (Tibialis anterior; TA) and heart weights at the time of cull, 143 demonstrated no difference in the weight of any of these tissue between the various groups 144 within each model (Figures 2B-2E). Whilst it was important to demonstrate that there was no effect of Cas9 expression on gross 149 tissue weights or animal growth, we also sought to investigate whether tissue specific 150 pathways were being impacted by chronic over expression of Cas9 in each tissue. Therefore, 151 we performed a series of analyzes on each tissue to investigate these parameters, utilizing 152 qPCR, histology and functional assessment. 153

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In the liver specific Cas9 model (Alb-Cre), we used qPCR to analyze the expression of genes 155 that were representative of pathways that provided insight into the health and activity of the 156 liver. These included Col1a2 and Vim as markers of fibrosis, Chop for ER stress, Plin2 for 157 lipid storage and Tnfa and Il1b for inflammation ( Figure 3A). We demonstrated that none of 158 these genes were differentially expressed in mice with livers expressing Cas9 compared to 159 control livers, indicating that the expression of Cas9 in the liver was not impacting on liver 3C). We demonstrate that were no differences in the expression of any of these markers in 171 the left ventricle (LV) across the four groups of mice, implying that these pathways were not 172 altered by the expression of Cas9 (or Cre-recombinase). This was supported by data 173 demonstrating that the weight of the whole heart and the different regions of the heart from 174 these mice including; atria, LV and right ventricle (RV), were also not different between 175 groups ( Figure 3D). Consistently, the lung weights, spleen weight and kidney weight (which 176 are useful readouts of health in cardiac models) were all comparable across groups 177 demonstrating no peripheral effects of cardiac specific overexpression of Cas9. Lastly, we 178 assessed heart function in these mice using echocardiography. We demonstrated that at 179 comparable heart rates (HR), under anesthesia there were no differences in fractional 180 shortening (FS%) -a measure of systolic function, between the four groups ( Figure 3E). 181 Thus, collectively these data demonstrate that overexpression of Cas9 in cardiomyocytes has 182 no impact on heart health and function. 183 184 Specific phenotyping of the muscle specific Cas9 model (ACTA1-Cre) was also performed 185 using qPCR. We measured the expression of muscle specific genes that are known readouts 186 of muscle development and growth in the TA muscle. These included myogenic transcription 187 factors Myod, Myog and Mef2c, as well as the pro-fusion protein myomaker (Tmem8c) and 188 9 mature muscle marker Mck ( Figure 3F). As with our previous models, we demonstrated no 189 difference in the expression of these genes between the four groups of mice, indicating that 190 there were no major differences in the growth and function of adult skeletal muscle in the 191 presence of Cas9 expression. In support of this data, using EchoMRI we demonstrated that 192 there was no difference in lean muscle mass across the four groups, at any time point 193 throughout the study period ( Figure 3G). Finally, histological analyzes of TA muscle 194 sections using H&E staining, indicated that there were no major morphological differences in 195 the muscle structure between Cas9 positive and Cas9 negative mice ( Figure 3H). Given that many groups which study metabolism have an interest in glucose homeostasis and 215 how it relates to tissues such as liver, adipose, skeletal muscle and the heart, we sought to 216 determine if the expression of Cas9 in these tissue led to any changes in whole body glucose 217 handling. To investigate this, we performed fasting blood glucose measurements and oral 218 glucose tolerance tests on all groups and models within the final two weeks of the study 219 period. We demonstrated that there was no difference in fasting blood glucose levels between 220 Cas9 positive and Cas9 negative mice in each of the four tissue specific mouse models 221 ( Figures 4A, 4D, 4G and 4J). In order to test the glucose tolerance of these models, we 222 challenged each cohort with a standardized oral dose of glucose (2mg/kg lean mass), and 223 subsequently measured their blood glucose concentration over two hours in an oral glucose 224 tolerance test (oGTT). We demonstrated that all groups and models showed a peak glucose 225 concentration of approximately 18-20mmol/L at 15 minutes post glucose delivery, which 226 mostly returned to baseline by 60 minutes after delivery of the glucose bolus ( Figures 4B,  227 4E, 4H and 4K). We also demonstrated that there was no difference in the clearance of 228 glucose across any of the groups and in each of the models, indicating that there was no 229 difference in the glucose tolerance of these animals. This is further demonstrated 230 quantitatively by assessing the 90 minute cumulative area under the curve (AUC) for the 231 tolerance test (Figures 4C, 4F, 4I and 4L), confirming that there was no difference in 232 glucose tolerance between the groups in each tissue specific Cas9 model. 233 234 Collectively, the data presented above demonstrates that long term overexpression of Cas9 in 235 four different tissue specific models, does not lead to any effects on body weight, tissue 236 weight, the expression of markers of pathological pathways, or readouts of whole body 237 11 glucose metabolism. These findings provide an important foundation for future studies that 238 wish to use the LSL-Cas9 mouse model to study their gene of interest, and affords confidence 239 to researchers that metabolic phenotypes they measure are unlikely to be impacted by the 240 chronic over expression of Cas9 or Cre-recombinase in these models. 241 adipose and heart), as well as the constitutive albumin (liver) line. Importantly, we were 290 14 unable to detect any deleterious effects on metabolic pathways, morphology of tissues, body 291 composition or glucose tolerance in any lines over-expressing Cas9, supporting the notion 292 that there is no negative impact of chronic Cas9 expression in these tissues. Moreover, 293 echocardiography also demonstrated no impact on systolic heart function in cardiac-specific 294

Discussion
Cas9 expressing mice after 20 weeks of induction, providing evidence that heart function in 295 these mice was not affected by chronic Cas9 expression. 296

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In summary, we provide critical evidence that the metabolism and general health of four 298 different metabolic tissue specific mouse lines are unaffected by the chronic expression of 299 Cas9. These findings provide confidence for researchers moving forward, who wish to use 300 these Cas9 mouse models to manipulate the expression of genes in these particular tissues. 301 The minimal impact of Cas9 in these studies will likely reduce the need for future studies to 302 perform specific controls groups, reducing animal numbers and sparing expensive resources. 303 Our data will also provide confidence that observed phenotypes related to gene deletions in 304 these models in future studies, are likely to be specific to the gene of interest rather than 305 being related to the chronic over-expression of Cas9. Thus these findings provide an 306 important resource for the research community. All mice were bred and sourced through the ARA Precinct Animal Centre and randomly 331 allocated into their respective groups. For tamoxifen inducible models, they were treated as 332 follows. For ACTA1-Cre-ERT2 and AdipoQ-Cre-ERT2 models, mice were aged to 6-8 333 weeks old before being gavaged with either Tamoxifen (80mg/kg) in sunflower oil, or 334 sunflower oil alone, for 3 consecutive days. For the MHC-alpha-Cre-ERT2 model, mice were 335 IP injected once with 40mg/kg of Tamoxifen in sunflower oil, or sunflower oil alone. 336 Following tamoxifen treatment, mice were left to recover for 2 weeks, after which they were 337 maintained on a normal chow diet (Normal rodent chow, Specialty feeds, Australia) and 338 housed at 22°C on a 12hr light/dark cycle with access to food and water ad libitum with cages 339 changed weekly for 12 weeks. Cohorts of mice were subjected to EchoMRI and body weight 340 analysis throughout the study period. In the last two weeks of the study period, all animals 341 underwent oral glucose tolerance tests, whilst the MHC-alpha mice were also subjected to 342 cardiac function assessment via echocardiography. At the end of the study, mice were fasted 343 for 4-6 hours and then anesthetized with a lethal dose of ketamine/xylazine before blood and 344 tissues were collected, weighed and snap frozen for subsequent analysis. Liver and muscle were embedded cut side down in OCT before being frozen in a bath of 361 isopentane submerged in liquid nitrogen. After freezing, blocks were brought to -20°C and 362 5µm sections were cut using a Leica Cryostat. Sections were mounted and dried overnight at 363 room temperature before being fixed in Methanol. WAT samples were fixed in formalin and 364 mounted in Paraffin, before 5µm sections were cut on a Leica microtome. All sections were 365 stained with hematoxylin and eosin and slide images were captured using Olympus Slide 366 scanner VS120 (Olympus, Japan) and viewed in the supplied program (OlyVIA Build 13771, 367 Olympus, Japan).