![\"figure](\"https:\/\/platohealth.ai\/wp-content\/uploads\/2024\/06\/investigating-the-potential-of-x-chromosome-shredding-for-mouse-genetic-biocontrol-scientific-reports.png\")
<\/a><\/div>\nElimination of the X chromosome and creation of DNA double strand break in mouse ES cells by CRISPR\/Cas9-mediated gene editing. (a<\/b>) Schematic of X-B, X-C, and X-D target sites and their copies on the mouse X chromosome and representative brightfield microscopy images of mouse ES cells 24 h, 48 h, 72 h, and 144 h after transfection and puromycin selection. Co-transfection with a GFP-expression plasmid was performed to assess transfection efficiency (top). mES cells grow as dome shaped colonies which are apparent at the 144h timepoint. Scale bar\u2009=\u2009100 \u00b5M. (b<\/b>) Cell survival percentage at 144 h post-transfection of X-shredder gRNAs (n\u2009=\u20093; Bars show mean\u2009\u00b1\u2009SD, one way ANOVA with Sidak\u2019s multiple comparison test). (c<\/b>) Representative immunofluorescence images of mouse ES cells 24 h after transfection. Scale bar\u2009=\u200910 \u00b5M. (d<\/b>) Average numbers of GFP\/gamma-H2AX double positive cells (n\u2009=\u20093). Bars show mean\u2009\u00b1\u2009SD, one way ANOVA with Sidak\u2019s multiple comparison test.<\/p>\n<\/div>\n<\/div>\n To assess X-shredding activity in vivo, we employed a \u2018split drive\u2019 system. gRNA- and Cas9-expressing mouse lines were generated separately and then crossed, resulting in ubiquitous gRNA and male germline-specific Cas9 expression. All transgenes were randomly integrated into the genome using pronuclear injection. We established Cas9 expression lines using the previously validated germline promoter sequences of Stra8<\/i>, Ccna1<\/i>, and Prm1<\/i>15<\/a>,16<\/a>,17<\/a>,18<\/a>,19<\/a><\/sup> which are active in pre-meiotic, meiotic (leptotene-zygotene) and post-meiotic spermatocytes, respectively. Cas9 was linked to an EGFP reporter using a P2A self-cleaving peptide (Supplementary Fig. 2<\/a>a;6<\/a><\/sup>). X-B, X-C, and X-D gRNA sequences were driven by the U6 promoter. A CMV mCherry fluorescent reporter cassette was also included to facilitate identification of transgene carriers (Supplementary Fig. 2<\/a>b). We generated single lines for X-B and X-D which contained 40 and 10 transgene copies, respectively (Fig. 2<\/a>a). Two independent X-C lines, X-C-1 and X-C-2 were generated, both of which carried eight copies of the transgene (Fig. 2<\/a>a). All lines were positive for mCherry fluorescence as determined by ear skin biopsies, although, X-D mice had much lower expression compared with the other lines (Fig. 2<\/a>b).<\/p>\n Characterisation of X shredder-mCherry and germline promoter-Cas9 transgenic mice. (a<\/b>) Transgene copy number of hemizygous X-B (n\u2009=\u20092), X-C-1 (n\u2009=\u20093), X-C-2 (n\u2009=\u20093) (two founder lines), and X-D (n\u2009=\u200910) mice. Bars show mean\u2009\u00b1\u2009SD. (b<\/b>) Fluorescence imaging performed on ear skin punch biopsies for the transgenic mouse lines described in A. showing representative mCherry signal (red) with a WT mCherry negative control. n<\/i>\u2009=\u2009\u2009>\u200930 per genotype. Scale bar\u2009=\u2009200 \u00b5M. (c<\/b>) Expression of Cas9 RNA in testis isolated from Ccna1<\/i>-, Prm1<\/i>-, and Stra8<\/i>-Cas9 transgenic mouse lines. Expression is normalised to eEF2<\/i> and WT testis indicates background Cas9 detection in this assay. n<\/i>\u2009=\u20091 per genotype. (d<\/b>) Representative IF of Cas9-GFP expression (green) in the testis of Ccna1<\/i>-Cas9-GFP transgenic mice. GFP signal was amplified by staining with an anti-GFP antibody while DAPI nuclear staining (blue) shows tubule structures. n\u2009=\u20092 per genotype, mice 8\u201324 weeks of age. Scale bar\u2009=\u2009100 \u00b5M.<\/p>\n<\/div>\n<\/div>\n RNA harvested from the testis of Cas9-EGFP transgenic mouse lines was used for qRT-PCR to determine Cas9<\/i> expression levels. The Ccna1<\/i>-Cas9-EGFP and Prm1<\/i>-Cas9-EGFP lines expressed high levels of Cas9<\/i> mRNA while lower transgene expression was detected in Stra8<\/i>-Cas9-EGFP testes (Fig. 2<\/a>c). However, we could only detect EGFP fluorescence in testis sections of Ccna1<\/i>-Cas9-EGFP mice, where it localised to the tubules and was expressed in late spermatocytes (D and m) to elongating spermatids (1\u201314 of spermiogenic cycle) (Fig. 2<\/a>d, Supplementary Fig. 2<\/a>C). We therefore focussed mainly on the Ccna1<\/i>-Cas9 line for in vivo experiments.<\/p>\n We generated double transgenic progeny expressing an X-shredding gRNA together with Cas9 by mating X-B, X-C (two lines, X-C-1 and X-C-2), or X-D lines with Ccna1<\/i>-Cas9-EGFP. The weight of Ccna1<\/i>Tg<\/sup>; X-BTg<\/sup> testis were comparable to wild type (WT), while both Ccna1<\/i>Tg<\/sup>; X-CTg<\/sup> lines and Ccna1<\/i>Tg<\/sup>; X-DTg<\/sup> mice had significantly smaller testis (Fig. 3<\/a>a). Histological analysis showed relatively normal tubule structure in Ccna1<\/i>Tg<\/sup>; X-BTg<\/sup> mice while both Ccna1<\/i>Tg<\/sup>; X-CTg<\/sup> lines and Ccna1<\/i>Tg<\/sup>; X-DTg<\/sup> mice exhibited disrupted tubule architecture (Fig. 3<\/a>b). Two-dimensional area and perimeter of Ccna1<\/i>Tg<\/sup>; X-CTg<\/sup> and Ccna1<\/i>Tg<\/sup>; X-DTg<\/sup> testes were also significantly decreased compared with WT (Fig. 3<\/a>c and d, respectively). Tubules either lacking or with few spermatogenic cells were common in Ccna1<\/i>Tg<\/sup>; X-CTg<\/sup> and Ccna1<\/i>Tg<\/sup>; X-DTg<\/sup> testes (Fig. 3<\/a>b, e, f), as were the presence of tubule vacuoles. All double transgenic lines showed significantly reduced sperm concentrations and dramatically reduced sperm motility fitness compared with WT (Fig. 3<\/a>g, h).<\/p>\n Functional assessment of Ccna1<\/i>-Cas9Tg<\/sup>; X-shredderTg<\/sup> double transgenic mice. (a<\/b>) WT, Ccna1<\/i>Tg<\/sup>; X-BTg<\/sup>, Ccna1<\/i>Tg<\/sup>; X-CTg<\/sup>, and Ccna1<\/i>Tg<\/sup>; X-DTg<\/sup> ex-breeder males were sacrificed, testis removed and weighed on a fine balance. (b<\/b>) H&E staining of representative testis sections from mice used in A. showing disrupted tubule architecture in Ccna1<\/i>Tg<\/sup>; X-CTg<\/sup> and Ccna1<\/i>Tg<\/sup>; X-DTg<\/sup> mice. Arrows indicate empty tubules. Arrow heads indicate vacuoles. Asterisks indicate areas with an abundance of Leydig cells. Scale bar\u2009=\u2009250 \u00b5M. Quantification of two-dimensional area (c<\/b>) and perimeter size (d<\/b>) from sections. (e<\/b>) Enumeration of the frequency of tubules containing spermatogenic cells. (f<\/b>) Quantification of the frequency of empty tubules. (g<\/b>) Sperm isolated from the epididymis were enumerated using a Sperm Class Analyser. (h<\/b>) Sperm motility was measured according to WHO 4 metrics. Mean motility values from mice shown. (i<\/b>) X chromosome dosage was assessed by qPCR using sperm DNA isolated from WT, Ccna1<\/i>Tg<\/sup>; X-BTg<\/sup>, Ccna1<\/i>Tg<\/sup>; X-CTg<\/sup>, and Ccna1<\/i>Tg<\/sup>; X-DTg<\/sup> mice. Male mouse DNA containing one X chromosome was used as the reference. Mean\u2009\u00b1\u2009SD of triplicates for each mouse is shown. (a<\/b>) and (c\u2013h<\/b>) n\u2009=\u20093 for WT, Ccna1<\/i>Tg<\/sup>; X-BTg<\/sup>, Ccna1<\/i>Tg<\/sup>; X-CTg<\/sup>, and n\u2009=\u20094 Ccna1<\/i>Tg<\/sup>; X-DTg<\/sup>. Bars show mean\u2009\u00b1\u2009SD. Mice 21\u201367 weeks of age. (a<\/b>), (c\u2013i<\/b>) show X-C-1Tg<\/sup> and X-C-2Tg<\/sup> data combined. Bars show mean\u2009\u00b1\u2009SD, one way ANOVA with Sidak\u2019s multiple comparison test.<\/p>\n<\/div>\n<\/div>\n To investigate if disproportionate X chromosome sperm loss was occurring in vivo, we isolated sperm from the vas deferens and cauda epididymis of WT and double transgenic male mice and measured X chromosome dosage by qPCR using three genes spanning the X chromosome (Rpgr<\/i>, DMD<\/i>, and Tlr7<\/i>). Interestingly, Ccna1<\/i>Tg<\/sup>; X-DTg<\/sup> male 3 and Ccna1<\/i>Tg<\/sup>; X-CTg<\/sup> male 3 had lower gene dosage at all three loci (Fig. 3<\/a>i). The former was particularly striking with only\u2009~\u200960% X chromosome dosage detected. Ccna1<\/i>Tg<\/sup>; X-DTg<\/sup> males 1, 2 and 4 also had lower dosage across 2 of the 3 loci. In contrast, Ccna1<\/i>Tg<\/sup>; X-BTg<\/sup> males had normal X chromosome dosage. These data suggest that disproportionate X chromosome loss is occurring in vivo when the X-C and X-D gRNA and Cas9 are expressed in the male germline, although the effect is variable and incompletely penetrant.<\/p>\n To investigate male offspring bias, we mated Ccna1<\/i>-Cas9Tg<\/sup>; X-shredder-gRNATg<\/sup> males with WT females and assessed the proportion of male and female progeny (Fig. 4<\/a>a). As negative controls, we also mated genetically equivalent littermate transgenic females with wild type males (spermatogenesis promoters should not be active in females). Progeny from Ccna1<\/i>Tg<\/sup>; X-BTg<\/sup> and Ccna1<\/i>Tg<\/sup>; X-CTg<\/sup> sires from both X-CTg<\/sup> lines did not show significant deviation from a 50:50 male:female ratio (Fig. 4<\/a>b, c). Similarly, Cas9 expression driven by Prm1<\/i> or Stra8<\/i> did not significantly alter the male:female ratio when used in combination with the both X-CTg<\/sup> lines (Fig. 4<\/a>e, f). Interestingly, Ccna1<\/i>Tg<\/sup>; X-DTg<\/sup> males sired an excess of male progeny (152 males versus 122 females) but this failed to reach statistical significance (P<\/i>\u2009=\u20090.0699; Fig. 4<\/a>d). To further assess male bias, we performed in vitro fertilisation using sperm isolated from a Ccna1<\/i>Tg<\/sup>; X-DTg<\/sup> male. Following generation of zygotes, blastocysts were allowed to develop, DNA was extracted, and the ratio of male to female blastocysts was enumerated. No significant difference in the male:female ratio was observed, confirming that significant loss of X-bearing sperm was not generally occurring in double transgenic males (Supplementary Fig. 3<\/a>a, b).<\/p>\nX-shredder transgenic mouse generation<\/h3>\n
![\"figure](\"https:\/\/platohealth.ai\/wp-content\/uploads\/2024\/06\/investigating-the-potential-of-x-chromosome-shredding-for-mouse-genetic-biocontrol-scientific-reports-1.png\")
<\/a><\/div>\nCharacterisation of gRNA; Cas9 double transgenic mice<\/h3>\n
![\"figure](\"https:\/\/platohealth.ai\/wp-content\/uploads\/2024\/06\/investigating-the-potential-of-x-chromosome-shredding-for-mouse-genetic-biocontrol-scientific-reports-2.png\")
<\/a><\/div>\nBiased generation of male offspring is not observed in experimental matings<\/h3>\n
![\"figure](\"https:\/\/platohealth.ai\/wp-content\/uploads\/2024\/06\/investigating-the-potential-of-x-chromosome-shredding-for-mouse-genetic-biocontrol-scientific-reports-3.png\")
<\/a><\/div>\n