Gonadal mosaicism mediated female-biased gender control in mice

In nature or during artificial breeding, evolutionary intervention or desirable trait selection could be achieved by means of gender control in an organism. Previously reported approaches including sex-sorted semen and genetic manipulation of a certain gene (Holden and Butler, 2018; Yosef et al., 2019). In mice, a recent study demonstrated a genetic system for biasing sex ratio through crossing two genetically engineered mouse lines (Yosef et al., 2019). The maternal line encodes functional Cas9 protein on an autosomal chromosome, whereas the paternal encodes guide RNAs on Y chromosome targeting vital genes. After fertilization, males carrying both Cas9 and guide RNAs were selfdestructed, resulting in a female biased sex ratio. However, the litter size is proximate half of normal size since half of the progeny are eliminated. Moreover, a reproductive reservoir of males and females must be maintained for crossing. Here, we established a mouse line carrying Hspa2 promoted Cas9 and constitutively expressed sgRNA targeting repetitive sequences on Y chromosome. In those mice, spermatogenic cells carrying Y chromosome were eliminated during spermatogenesis, resulting in female biased sex ratio in progenies whereas the total number was unaffected. The male-specific region of mouse Y chromosome (MSY) are composed of multiple DNA repeats (Cocquet et al., 2009). Sly, Ssty1, Ssty2 as multicopy genes with highly repetitive DNA sequences, are located at the long arm of mouse Y chromosome (MSYq) (Fig. 1A) (Toure et al., 2004). To target the repetitive sequence on Y chromosome via CRISPR/Cas9, firstly we designed three sgRNAs targeting each gene, with each sgRNA recognizing more than 100 copies of DNA on MSY (Fig. 1B). Meanwhile, male or female mouse embryonic stem cell (ESC) lines carrying constitutively expressed Cas9 were generated through blastocysts derivation, followed by Cas9-expressing lentivirus infection (Fig. S1A and S1B). Next, all the lentiviral sgRNAs targeting repetitive sequence on Y chromosome were packaged and infected into each male or female mESC line, respectively. Around 72 h after infection, cell viability was analyzed through cell counting. The result indicated that male mESCs, compared with female showed significantly decreased cell survival (Fig. 1C; Table S1). In addition, cell viability varied among different Y chromosome targeting sgRNAs, which may be associated with respective mutation efficiency. To prove that the variation of cell viability between male and female mESC lines were Y chromosome associated, we additionally designed 3 sgRNAs targeting repetitive sequences on autosome or X chromosome and 2 sgRNAs targeting a unique locus on Rosa26. For each sgRNA targeting autosome or X chromosome, there are more than 100 copies of target and none of them are essential genes or chromosome structure sequences related (Fig. 1B). When transduced these additional sgRNAs into male or female mESCs through lentivirus infection, we found that at 72 h post infection, both male and female mESCs carrying sgRNA targeting autosome or X chromosome died, while mESCs carrying sgRNA targeting Rosa26 proliferate normally, regardless of gender (Fig. 1C; Table S1). Then we identified the frequency of Y chromosome elimination in mESCs with Y chromosome targeting sgRNA infection through single cell expansion and genotyping. The Y-specific gene Sry is located on the short arm of mouse Y chromosome (MSYp) (Fig. 1A). Through genotyping, we observed that surviving cells lack of Sry were existed in all Y chromosome targeting groups (Fig. S1C). Further confirmation of complete Y chromosome elimination was performed by genotyping of other six Y chromosome genes, located on both short and long arm (Figs. 1A and S1D). Statistically, Y chromosome elimination occurred in all groups of surviving cells infected with different Y chromosome targeting sgRNAs, with a variable efficiency ranging from 40% to 100% according to our result (Fig. S1E). Overall, we demonstrate that CRISPR/Cas9 mediated multiple DNA double strand breaks (DSBs) induce cell death in mESCs (Fig. 1A). To illustrate the mechanism, we established male or female mESC lines carrying Cas9-T2A-mRFP transgene via PiggyBac transponson mediated insertion and single clone expansion (Fig. S2A). Cas9 expression could be indicated by mRFP fluorescence (Szymczak et al., 2004). In addition, based on the mutation efficiency of different sgRNAs, Ssty11, AutoXY-1, and Rosa26-1 were selected as Y chromosome, autosome or X chromosome, and Rosa26 targeting sgRNA for further study, respectively. Previously, it was reported that CRISPR/Cas9 mediated DNA DSBs induce


Dear Editor,
In nature or during artificial breeding, evolutionary intervention or desirable trait selection could be achieved by means of gender control in an organism. Previously reported approaches including sex-sorted semen and genetic manipulation of a certain gene (Holden and Butler, 2018;Yosef et al., 2019). In mice, a recent study demonstrated a genetic system for biasing sex ratio through crossing two genetically engineered mouse lines (Yosef et al., 2019). The maternal line encodes functional Cas9 protein on an autosomal chromosome, whereas the paternal encodes guide RNAs on Y chromosome targeting vital genes. After fertilization, males carrying both Cas9 and guide RNAs were selfdestructed, resulting in a female biased sex ratio. However, the litter size is proximate half of normal size since half of the progeny are eliminated. Moreover, a reproductive reservoir of males and females must be maintained for crossing. Here, we established a mouse line carrying Hspa2 promoted Cas9 and constitutively expressed sgRNA targeting repetitive sequences on Y chromosome. In those mice, spermatogenic cells carrying Y chromosome were eliminated during spermatogenesis, resulting in female biased sex ratio in progenies whereas the total number was unaffected.
The male-specific region of mouse Y chromosome (MSY) are composed of multiple DNA repeats (Cocquet et al., 2009). Sly, Ssty1, Ssty2 as multicopy genes with highly repetitive DNA sequences, are located at the long arm of mouse Y chromosome (MSYq) (Fig. 1A) (Toure et al., 2004). To target the repetitive sequence on Y chromosome via CRISPR/Cas9, firstly we designed three sgRNAs targeting each gene, with each sgRNA recognizing more than 100 copies of DNA on MSY (Fig. 1B). Meanwhile, male or female mouse embryonic stem cell (ESC) lines carrying constitutively expressed Cas9 were generated through blastocysts derivation, followed by Cas9-expressing lentivirus infection ( Fig. S1A and S1B). Next, all the lentiviral sgRNAs targeting repetitive sequence on Y chromosome were packaged and infected into each male or female mESC line, respectively. Around 72 h after infection, cell viability was analyzed through cell counting. The result indicated that male mESCs, compared with female showed significantly decreased cell survival ( Fig. 1C; Table S1). In addition, cell viability varied among different Y chromosome targeting sgRNAs, which may be associated with respective mutation efficiency. To prove that the variation of cell viability between male and female mESC lines were Y chromosome associated, we additionally designed 3 sgRNAs targeting repetitive sequences on autosome or X chromosome and 2 sgRNAs targeting a unique locus on Rosa26. For each sgRNA targeting autosome or X chromosome, there are more than 100 copies of target and none of them are essential genes or chromosome structure sequences related (Fig. 1B). When transduced these additional sgRNAs into male or female mESCs through lentivirus infection, we found that at 72 h post infection, both male and female mESCs carrying sgRNA targeting autosome or X chromosome died, while mESCs carrying sgRNA targeting Rosa26 proliferate normally, regardless of gender ( Fig. 1C; Table S1).
Then we identified the frequency of Y chromosome elimination in mESCs with Y chromosome targeting sgRNA infection through single cell expansion and genotyping. The Y-specific gene Sry is located on the short arm of mouse Y chromosome (MSYp) (Fig. 1A). Through genotyping, we observed that surviving cells lack of Sry were existed in all Y chromosome targeting groups (Fig. S1C). Further confirmation of complete Y chromosome elimination was performed by genotyping of other six Y chromosome genes, located on both short and long arm (Figs. 1A and S1D). Statistically, Y chromosome elimination occurred in all groups of surviving cells infected with different Y chromosome targeting sgRNAs, with a variable efficiency ranging from 40% to 100% according to our result (Fig. S1E). Overall, we demonstrate that CRISPR/Cas9 mediated multiple DNA double strand breaks (DSBs) induce cell death in mESCs (Fig. 1A).
To illustrate the mechanism, we established male or female mESC lines carrying Cas9-T2A-mRFP transgene via PiggyBac transponson mediated insertion and single clone expansion (Fig. S2A). Cas9 expression could be indicated by mRFP fluorescence (Szymczak et al., 2004). In addition, based on the mutation efficiency of different sgRNAs, Ssty1-1, AutoXY-1, and Rosa26-1 were selected as Y chromosome, autosome or X chromosome, and Rosa26 targeting sgRNA for further study, respectively. Previously, it was reported that CRISPR/Cas9 mediated DNA DSBs induce P53 dependent toxicity in human pluripotent stem cells or hematopoietic stem and progenitor cells (Ihry et al., 2018;Schiroli et al., 2019). Here, we hypothesized that CRISPR/ Cas9 mediated multiple DNA DSBs in mESCs activated DNA damage response and triggered P53 dependent cell death. To prove that, firstly the accumulation of 53BP1, a mediator in DNA damage signaling (Polo and Jackson, 2011), were detected in male or female Cas9 expressing mESC lines infected with Ssty1-1, AutoXY-1, or Rosa26-1 respectively at 24 h post infection. According to the result, increased 53BP1 signals were observed in Ssty1-1 infected male compared with female mESCs, reflecting higher DNA damage response burden (Figs. 1D and S3A). Meanwhile, Y chromosome targeting foci-bearing male mESCs showed more aggregated signals in comparison with autosome targeting ones, which presented more scattered spots ( Fig. S3A and S3B). For most Rosa26 targeting foci-bearing male mESCs, one or two 53BP1 foci were observed as expected (Figs. 1D and S3A).
Next, based on the male mESC line carrying Cas9-T2A-mRFP transgene, we generated two P53 knockout Cas9 expressing male mESC lines through CRISPR/Cas9 mediated transient transfection and single clone expansion (Fig. S2B). Western blot confirmed the complete P53 deficiency (Fig. S2C). Then, established P53 knockout Cas9 expressing male mESC lines were infected with Ssty1-1 and stained with 53BP1 antibody. Of note, according to 53BP1 signal, P53 deficiency did not impact the formation of DNA damage response foci induced by CRISPR/Cas9, which was consistent with previous research on human hematopoietic stem cells (Schiroli et al., 2019). Moreover, in contrast to single 53BP1 focus in P53 wild-type mESCs, double 53BP1 foci were observed in P53 knockout mESCs, implying mitigating effect of G 1 arrest ( Fig. S3A and S3C). Furthermore, cell proliferation was analyzed in wild-type female or P53 knockout male Cas9 expressing mESC lines infected with Ssty1-1, Ssty2-3, AutoXY-1, or Rosa26-1 at 0, 24, 48, 72 h post infection respectively ( Fig. S2D and S3D). Male mESCs infected with Y chromosome targeting sgRNAs presented significantly decreased cell proliferation and survival compared with female mESCs. Moreover, P53 deficiency partially rescued the impaired cell proliferation, indicating involvement of P53 in this process. For Cas9 expressing mESCs infected with AutoXY-1, both male and female mESCs died at 72 h post infection. However, in contrast to wild-type mESCs whose survival decreased with time, P53 deficient mESCs proliferated at the first 24 h and then all died in the following 48 h. In addition, male or female mESCs infected with Rosa26-1, wild-type or knockout P53 proliferated with no significant difference. Furthermore, we conducted high-throughput RNA-sequencing in wild-type male or female Cas9-expressing mESC lines infected with Ssty1-1, Ssty2-3, AutoXY-1, or Rosa26-1 at 24, 36 h post infection respectively. Venn diagram showed that differentially expressed genes (DEGs) between those mESC lines above (Fig. S4A). The results of high-throughput sequencing are plotted as a heat map (Fig. S4B). Subsequently, KEGG pathway analysis revealed that targeting repetitive sequence in mESCs resulted in significant variations of genes involved in P53 mediated cell death or apoptosis signaling pathway ( Fig. S4C and S4D). Overall, those confirmed that multiple DNA DSBs induced by CRISPR/Cas9 triggered DNA damage response and led to P53 dependent cell death in mESCs.
In summary, we provide a first proof of concept that repetitive sequence targeting, instead of particular genes through CRISPR/Cas9 could mediate female biased gender control in mice. Our research also paves the way for further study on evolutionary intervention as well as artificial breeding.

FOOTNOTES
We appreciate the support from Cell Biology Facility in Shanghai Institute of Biochemistry and Cell Biology as well as East China Normal University for cell sorting and imaging. Also, we thank, for the support, the Genome Tagging Project and Shanghai "Super MZB, DL, YC and YXW conceived and designed the project; MZB and GLL conducted mouse embryo and cell related experiments; YC, DL and QDW performed analyzed RNA-sequencing data, MZB, DL, YC and YXW analyzed the data and wrote the manuscript. JSL and YXW jointly supervised this project.
All the authors declare no competing interests.
All data are available in the main text or the supplementary materials.

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