Artificial complexes for tethering chromatids or chromosomes

Abstract

During aging, egg cells exhibit a gradual loss of the cohesin complex. This loss of cohesin ultimately leads to premature separation of sister chromatids and aneuploidy. The inventors have manipulated an artificial aggregation system, which is a complex for chromatid or chromosome tethering, to reduce, for example, premature separation of sister chromatids associated with aging in oocytes. The artificial aggregation system is a complex that tethers chromosomes or chromatids, thereby reducing, for example, the risk of aneuploidy. The complex comprises (I) one or more first proteins and (II) one or more second proteins, where each of the (I) first and (II) second proteins comprises (i) a chromatin-binding component, (ii) a protein-binding region at the N-terminus of the chromatin-binding component, and (iii) a protein-binding region at the C-terminus of the chromatin-binding component. The invention also includes a nucleic acid molecule encoding the complex. Furthermore, several in vitro methods relating to the complex or the nucleic acid molecule encoding the complex also form part of the invention.

Description

[Technical Field]

[0001] Women become infertile with age. In particular, fertility begins to decline after around age 35. 1,3 The decline in fertility is caused by an increase in aneuploidy of a woman's eggs, which is related to age. Immature eggs are stored in the ovaries from birth and can therefore be decades old by the time of fertilization. The number of aneuploid eggs increases dramatically as a woman approaches her 40s. 1,4,5,10,11 Aneuploidy occurs after fertilization, when cells transition from the egg to the embryo (Figure 1a). In most cases, aneuploid embryos cannot implant in the uterus or develop to the expected delivery date. If there are too many aneuploid eggs, the woman can no longer become pregnant. High levels of aneuploidy in the eggs of women in their late 30s and 40s also explain why IVF treatment in this age group often fails despite successful egg retrieval.

[0002] The chromosomes of an egg cell are composed of individual chromatids linked together by cohesin complexes. Cohesin provides chromosome cohesion, which is crucial for the precise separation of chromosomes throughout meiotic cell division. 12-15 During aging, oocytes show a gradual loss of the cohesin complex (upper left panel, Figure 1a). 6,8,9,12-15 The loss of cohesin ultimately leads to the early separation of sister chromatids. 1,10,12-14,16-19 Once separated, sister chromatids randomly separate during both the first and second meiotic cell divisions (meiosis I and II), resulting in aneuploidy (upper right panel, Figure 1a). To the best of our knowledge, there is currently no method to prevent premature separation (PSSC) of sister chromatids by retaining cohesin on the sister chromatids, or to reverse its loss.

[0003] International Publication No. 2009 / 030932(A1) describes a method for reducing non-segregation and aneuploidy of chromosomes during meiosis in mammals by administering an effective dose of cdh1 to gametes. 68International Publication No. WO 2017 / 192847 (A1) describes a methodology for reducing FSH and a method of using an activin disruptor for preventing and / or treating fertilization disorders of eggs. 69 Chinese Patent Application Publication No. CN 115656509 (A) discloses ERK1 / 2 protein as a new biomarker for chromosomal aneuploidy in oocytes, where the biomarker is ERK1 / 2 protein in oocytes. 70 .

[0004] As a result, there are no available treatments for age-related infertility, and many women over 35 years old are unable to conceive.

Summary of the Invention

[0005] To address this need, the inventors manipulated an artificial aggregation system, which is a complex for chromatid or chromosome tethering, to reduce age-related PSSC in eggs (lower panel, Figure 1a). The components of the complex and the overall structure of the complex are shown in Figure 1b.

[0006] To demonstrate, for example, that an artificial agglutination system tethers sister chromatids, since human oocytes are difficult to obtain, the inventors mainly used mouse oocytes as a model of PSSC (Figs. 1c, 1d). Based on mouse data and data obtained from human somatic cells, the present invention further demonstrates that the artificial agglutination system functions in human eggs (see Examples 1 and 2). The technical advantage of the present invention is that the artificial agglutination system is a complex that tethers chromosomes or chromatids, such as sister chromatids, for example, so that the risk of aneuploidy is reduced. The complex includes (I) one or more first proteins and (II) one or more second proteins, and the (I) first and (II) second proteins each include (i) a chromatin-binding component, (ii) a protein-binding region (PBN region) that is the N-terminus of the chromatin-binding component, and (iii) a protein-binding region (PBC region) that is the C-terminus of the chromatin-binding component, as further defined in the claims. The overall arrangement is shown in Fig. 1b. The complex tethers chromosomes or chromatids by binding of the chromosome-binding component to the chromosome or chromatid and by binding of the protein-binding region of the first protein to the protein-binding region of the second protein. Further, the complex reversibly tethers chromosomes and chromatids. In other words, the tethering is releasable, thereby enabling the separation of chromosomes or chromatids, for example, during anaphase I or II of meiosis.

[0007] The present invention also encompasses nucleic acid molecules encoding a complex as further defined in the claims. Further, several in vitro methods related to the complex or the nucleic acid molecule encoding the complex also form part of the present invention and are further defined in the claims. Complexes or nucleic acid molecules for use in reproductive medicine are also claimed. In particular, assisted reproductive medicine is preferred. Various aspects of the present invention defined in the independent claims are incorporated herein by reference. Preferred embodiments are included in the dependent claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] [Figure 1A]This is an engineered cohesination system for preventing chromosomal errors in oocytes. (A) A schematic diagram of the engineered cohesination system on metaphase chromosomes of human oocytes. Cohesin gradually dissociates from oocyte chromosomes during aging, causing premature separation of sister chromatids (PSSCs) (upper panel, left) and aneuploidy (meiosis II, upper panel, center)3,49. Untreated secondary oocytes with PSSCs result in aneuploid embryos with two chromosomes shown in gray and aneuploid second polar bodies without chromosomes shown in gray (upper panel, right). The engineered cohesin system restores cohesination (black) to the centromere region, preventing PSSCs (lower panel, left) and aneuploidy (lower panel, center), and resulting in euploid embryos (lower panel, right). (B) An overall diagram of the present invention. The schematic diagram on the left shows the overall arrangement of (I) the first protein and (II) the second protein, each containing (i) a chromatin-binding component, (ii) a protein-binding region (PBN region) at the N-terminus of the chromatin-binding component, and (iii) a protein-binding region (PBC region) at the C-terminus of the chromatin-binding component. The schematic diagram in the center shows that the PBN of the first protein and the PBC of the second protein can bind, and the PBC of the first protein and the PBN of the second protein can bind. Alternatively, the PBN regions of the first and second proteins can bind to each other, and the PBC regions of the first and second proteins can bind to each other. The binding may be covalent or non-covalent. The panel on the right of the schematic diagram shows three exemplary methods for releasing the binding. The first is (a) cleavage of the complex via a protease cleavage site located between the chromatin-binding component and the protein-binding region. The second is (b) degradation of the complex, in which the first and / or second protein of the complex further includes a ubiquitination site. The third is (c) disruption of the binding between the PBN region and the PBC region of the first and second proteins, the disruption of which is mediated by chemically induced proximity. (C) Chromosomes (shown in gray) were correctly aligned with respect to the central region of the meiotic spindle (shown in white) in metaphase II mouse oocytes (left panel), or had PSSCs (right panel).In boxes I and II, isolated chromatids marked by a single kinetochore (shown in white) represent PSSCs, and in box III, paired sister chromatids are aligned in the central region. Scale bars are 2 μm. (D) Frequency of PSSCs in oocytes obtained from young mice (open bars) and mice aged 13, 17, and 20 months (filled bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars are 95% CI, 1 degree of freedom. (E) Models of TALhesin components A and B. DNA-binding TAL effectors (shown in dark gray) are adjacent to either SpyTags (component A) or Spy-Catcher (component B) (see Figure 2A for further details). Dashed lines represent linker regions L1 and L2. The C-terminal domain of TAL is not shown. Based on pdb, these are 3 UGM50 and 4 MLI51. (F) TALhesin[mmMajSat]A / B is localized to the chromosome in metaphase I (left panel) and the spindle (white) in metaphase II (right panel). Scale bar is 2 μm. (G) Enlarged view of the chromosome enclosed in the rectangle in Figure 1f. TALhesin[mmMajSat] is localized to the centromere region of the chromosome in the spindle (top) during mouse meiosis I and the spindle (bottom) during meiosis II. Scale bar is 2 μm. (H) Mobility of TALhesin[mmMajSat] chromosome foci (shown in dark gray) compared to TAL[mmMajSat] effector alone (light gray), as measured by FRAP. Bars are SD from the mean, with 30 repeats for each condition. (I) Measurement of the interval between kinetochores between sister chromatids of metaphase II chromosome in mouse oocytes injected with the indicated amount of TALhesin[mmMajSat]A / B mRNA. Black squares indicate the mean, and lines indicate the median for each condition. Statistical values ​​were measured by Bonferroni-corrected two-sample t-test (equal variances not assumed). n is the number of sister kinetochore pairs analyzed, 2391. [Figure 1B]This is an engineered cohesination system for preventing chromosomal errors in oocytes. (A) A schematic diagram of the engineered cohesination system on metaphase chromosomes of human oocytes. Cohesin gradually dissociates from oocyte chromosomes during aging, causing premature separation of sister chromatids (PSSCs) (upper panel, left) and aneuploidy (meiosis II, upper panel, center)3,49. Untreated secondary oocytes with PSSCs result in aneuploid embryos with two chromosomes shown in gray and aneuploid second polar bodies without chromosomes shown in gray (upper panel, right). The engineered cohesin system restores cohesination (black) to the centromere region, preventing PSSCs (lower panel, left) and aneuploidy (lower panel, center), and resulting in euploid embryos (lower panel, right). (B) An overall diagram of the present invention. The schematic diagram on the left shows the overall arrangement of (I) the first protein and (II) the second protein, each containing (i) a chromatin-binding component, (ii) a protein-binding region (PBN region) at the N-terminus of the chromatin-binding component, and (iii) a protein-binding region (PBC region) at the C-terminus of the chromatin-binding component. The schematic diagram in the center shows that the PBN of the first protein and the PBC of the second protein can bind, and the PBC of the first protein and the PBN of the second protein can bind. Alternatively, the PBN regions of the first and second proteins can bind to each other, and the PBC regions of the first and second proteins can bind to each other. The binding may be covalent or non-covalent. The panel on the right of the schematic diagram shows three exemplary methods for releasing the binding. The first is (a) cleavage of the complex via a protease cleavage site located between the chromatin-binding component and the protein-binding region. The second is (b) degradation of the complex, in which the first and / or second protein of the complex further includes a ubiquitination site. The third is (c) disruption of the binding between the PBN region and the PBC region of the first and second proteins, the disruption of which is mediated by chemically induced proximity. (C) Chromosomes (shown in gray) were correctly aligned with respect to the central region of the meiotic spindle (shown in white) in metaphase II mouse oocytes (left panel), or had PSSCs (right panel).In boxes I and II, isolated chromatids marked by a single kinetochore (shown in white) represent PSSCs, and in box III, paired sister chromatids are aligned in the central region. Scale bars are 2 μm. (D) Frequency of PSSCs in oocytes obtained from young mice (open bars) and mice aged 13, 17, and 20 months (filled bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars are 95% CI, 1 degree of freedom. (E) Models of TALhesin components A and B. DNA-binding TAL effectors (shown in dark gray) are adjacent to either SpyTags (component A) or Spy-Catcher (component B) (see Figure 2A for further details). Dashed lines represent linker regions L1 and L2. The C-terminal domain of TAL is not shown. Based on pdb, these are 3 UGM50 and 4 MLI51. (F) TALhesin[mmMajSat]A / B is localized to the chromosome in metaphase I (left panel) and the spindle (white) in metaphase II (right panel). Scale bar is 2 μm. (G) Enlarged view of the chromosome enclosed in the rectangle in Figure 1f. TALhesin[mmMajSat] is localized to the centromere region of the chromosome in the spindle (top) during mouse meiosis I and the spindle (bottom) during meiosis II. Scale bar is 2 μm. (H) Mobility of TALhesin[mmMajSat] chromosome foci (shown in dark gray) compared to TAL[mmMajSat] effector alone (light gray), as measured by FRAP. Bars are SD from the mean, with 30 repeats for each condition. (I) Measurement of the interval between kinetochores between sister chromatids of metaphase II chromosome in mouse oocytes injected with the indicated amount of TALhesin[mmMajSat]A / B mRNA. Black squares indicate the mean, and lines indicate the median for each condition. Statistical values ​​were measured by Bonferroni-corrected two-sample t-test (equal variances not assumed). n is the number of sister kinetochore pairs analyzed, 2391. [Figure 1C]This is an engineered cohesination system for preventing chromosomal errors in oocytes. (A) A schematic diagram of the engineered cohesination system on metaphase chromosomes of human oocytes. Cohesin gradually dissociates from oocyte chromosomes during aging, causing premature separation of sister chromatids (PSSCs) (upper panel, left) and aneuploidy (meiosis II, upper panel, center)3,49. Untreated secondary oocytes with PSSCs result in aneuploid embryos with two chromosomes shown in gray and aneuploid second polar bodies without chromosomes shown in gray (upper panel, right). The engineered cohesin system restores cohesination (black) to the centromere region, preventing PSSCs (lower panel, left) and aneuploidy (lower panel, center), and resulting in euploid embryos (lower panel, right). (B) An overall diagram of the present invention. The schematic diagram on the left shows the overall arrangement of (I) the first protein and (II) the second protein, each containing (i) a chromatin-binding component, (ii) a protein-binding region (PBN region) at the N-terminus of the chromatin-binding component, and (iii) a protein-binding region (PBC region) at the C-terminus of the chromatin-binding component. The schematic diagram in the center shows that the PBN of the first protein and the PBC of the second protein can bind, and the PBC of the first protein and the PBN of the second protein can bind. Alternatively, the PBN regions of the first and second proteins can bind to each other, and the PBC regions of the first and second proteins can bind to each other. The binding may be covalent or non-covalent. The panel on the right of the schematic diagram shows three exemplary methods for releasing the binding. The first is (a) cleavage of the complex via a protease cleavage site located between the chromatin-binding component and the protein-binding region. The second is (b) degradation of the complex, in which the first and / or second protein of the complex further includes a ubiquitination site. The third is (c) disruption of the binding between the PBN region and the PBC region of the first and second proteins, the disruption of which is mediated by chemically induced proximity. (C) Chromosomes (shown in gray) were correctly aligned with respect to the central region of the meiotic spindle (shown in white) in metaphase II mouse oocytes (left panel), or had PSSCs (right panel).In boxes I and II, isolated chromatids marked by a single kinetochore (shown in white) represent PSSCs, and in box III, paired sister chromatids are aligned in the central region. Scale bars are 2 μm. (D) Frequency of PSSCs in oocytes obtained from young mice (open bars) and mice aged 13, 17, and 20 months (filled bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars are 95% CI, 1 degree of freedom. (E) Models of TALhesin components A and B. DNA-binding TAL effectors (shown in dark gray) are adjacent to either SpyTags (component A) or Spy-Catcher (component B) (see Figure 2A for further details). Dashed lines represent linker regions L1 and L2. The C-terminal domain of TAL is not shown. Based on pdb, these are 3 UGM50 and 4 MLI51. (F) TALhesin[mmMajSat]A / B is localized to the chromosome in metaphase I (left panel) and the spindle (white) in metaphase II (right panel). Scale bar is 2 μm. (G) Enlarged view of the chromosome enclosed in the rectangle in Figure 1f. TALhesin[mmMajSat] is localized to the centromere region of the chromosome in the spindle (top) during mouse meiosis I and the spindle (bottom) during meiosis II. Scale bar is 2 μm. (H) Mobility of TALhesin[mmMajSat] chromosome foci (shown in dark gray) compared to TAL[mmMajSat] effector alone (light gray), as measured by FRAP. Bars are SD from the mean, with 30 repeats for each condition. (I) Measurement of the interval between kinetochores between sister chromatids of metaphase II chromosome in mouse oocytes injected with the indicated amount of TALhesin[mmMajSat]A / B mRNA. Black squares indicate the mean, and lines indicate the median for each condition. Statistical values ​​were measured by Bonferroni-corrected two-sample t-test (equal variances not assumed). n is the number of sister kinetochore pairs analyzed, 2391. [Figure 1D]This is an engineered cohesination system for preventing chromosomal errors in oocytes. (A) A schematic diagram of the engineered cohesination system on metaphase chromosomes of human oocytes. Cohesin gradually dissociates from oocyte chromosomes during aging, causing premature separation of sister chromatids (PSSCs) (upper panel, left) and aneuploidy (meiosis II, upper panel, center)3,49. Untreated secondary oocytes with PSSCs result in aneuploid embryos with two chromosomes shown in gray and aneuploid second polar bodies without chromosomes shown in gray (upper panel, right). The engineered cohesin system restores cohesination (black) to the centromere region, preventing PSSCs (lower panel, left) and aneuploidy (lower panel, center), and resulting in euploid embryos (lower panel, right). (B) An overall diagram of the present invention. The schematic diagram on the left shows the overall arrangement of (I) the first protein and (II) the second protein, each containing (i) a chromatin-binding component, (ii) a protein-binding region (PBN region) at the N-terminus of the chromatin-binding component, and (iii) a protein-binding region (PBC region) at the C-terminus of the chromatin-binding component. The schematic diagram in the center shows that the PBN of the first protein and the PBC of the second protein can bind, and the PBC of the first protein and the PBN of the second protein can bind. Alternatively, the PBN regions of the first and second proteins can bind to each other, and the PBC regions of the first and second proteins can bind to each other. The binding may be covalent or non-covalent. The panel on the right of the schematic diagram shows three exemplary methods for releasing the binding. The first is (a) cleavage of the complex via a protease cleavage site located between the chromatin-binding component and the protein-binding region. The second is (b) degradation of the complex, in which the first and / or second protein of the complex further includes a ubiquitination site. The third is (c) disruption of the binding between the PBN region and the PBC region of the first and second proteins, the disruption of which is mediated by chemically induced proximity. (C) Chromosomes (shown in gray) were correctly aligned with respect to the central region of the meiotic spindle (shown in white) in metaphase II mouse oocytes (left panel), or had PSSCs (right panel).In boxes I and II, isolated chromatids marked by a single kinetochore (shown in white) represent PSSCs, and in box III, paired sister chromatids are aligned in the central region. Scale bars are 2 μm. (D) Frequency of PSSCs in oocytes obtained from young mice (open bars) and mice aged 13, 17, and 20 months (filled bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars are 95% CI, 1 degree of freedom. (E) Models of TALhesin components A and B. DNA-binding TAL effectors (shown in dark gray) are adjacent to either SpyTags (component A) or Spy-Catcher (component B) (see Figure 2A for further details). Dashed lines represent linker regions L1 and L2. The C-terminal domain of TAL is not shown. Based on pdb, these are 3 UGM50 and 4 MLI51. (F) TALhesin[mmMajSat]A / B is localized to the chromosome in metaphase I (left panel) and the spindle (white) in metaphase II (right panel). Scale bar is 2 μm. (G) Enlarged view of the chromosome enclosed in the rectangle in Figure 1f. TALhesin[mmMajSat] is localized to the centromere region of the chromosome in the spindle (top) during mouse meiosis I and the spindle (bottom) during meiosis II. Scale bar is 2 μm. (H) Mobility of TALhesin[mmMajSat] chromosome foci (shown in dark gray) compared to TAL[mmMajSat] effector alone (light gray), as measured by FRAP. Bars are SD from the mean, with 30 repeats for each condition. (I) Measurement of the interval between kinetochores between sister chromatids of metaphase II chromosome in mouse oocytes injected with the indicated amount of TALhesin[mmMajSat]A / B mRNA. Black squares indicate the mean, and lines indicate the median for each condition. Statistical values ​​were measured by Bonferroni-corrected two-sample t-test (equal variances not assumed). n is the number of sister kinetochore pairs analyzed, 2391. [Figure 1E]This is an engineered cohesination system for preventing chromosomal errors in oocytes. (A) A schematic diagram of the engineered cohesination system on metaphase chromosomes of human oocytes. Cohesin gradually dissociates from oocyte chromosomes during aging, causing premature separation of sister chromatids (PSSCs) (upper panel, left) and aneuploidy (meiosis II, upper panel, center)3,49. Untreated secondary oocytes with PSSCs result in aneuploid embryos with two chromosomes shown in gray and aneuploid second polar bodies without chromosomes shown in gray (upper panel, right). The engineered cohesin system restores cohesination (black) to the centromere region, preventing PSSCs (lower panel, left) and aneuploidy (lower panel, center), and resulting in euploid embryos (lower panel, right). (B) An overall diagram of the present invention. The schematic diagram on the left shows the overall arrangement of (I) the first protein and (II) the second protein, each containing (i) a chromatin-binding component, (ii) a protein-binding region (PBN region) at the N-terminus of the chromatin-binding component, and (iii) a protein-binding region (PBC region) at the C-terminus of the chromatin-binding component. The schematic diagram in the center shows that the PBN of the first protein and the PBC of the second protein can bind, and the PBC of the first protein and the PBN of the second protein can bind. Alternatively, the PBN regions of the first and second proteins can bind to each other, and the PBC regions of the first and second proteins can bind to each other. The binding may be covalent or non-covalent. The panel on the right of the schematic diagram shows three exemplary methods for releasing the binding. The first is (a) cleavage of the complex via a protease cleavage site located between the chromatin-binding component and the protein-binding region. The second is (b) degradation of the complex, in which the first and / or second protein of the complex further includes a ubiquitination site. The third is (c) disruption of the binding between the PBN region and the PBC region of the first and second proteins, the disruption of which is mediated by chemically induced proximity. (C) Chromosomes (shown in gray) were correctly aligned with respect to the central region of the meiotic spindle (shown in white) in metaphase II mouse oocytes (left panel), or had PSSCs (right panel).In boxes I and II, isolated chromatids marked by a single kinetochore (shown in white) represent PSSCs, and in box III, paired sister chromatids are aligned in the central region. Scale bars are 2 μm. (D) Frequency of PSSCs in oocytes obtained from young mice (open bars) and mice aged 13, 17, and 20 months (filled bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars are 95% CI, 1 degree of freedom. (E) Models of TALhesin components A and B. DNA-binding TAL effectors (shown in dark gray) are adjacent to either SpyTags (component A) or Spy-Catcher (component B) (see Figure 2A for further details). Dashed lines represent linker regions L1 and L2. The C-terminal domain of TAL is not shown. Based on pdb, these are 3 UGM50 and 4 MLI51. (F) TALhesin[mmMajSat]A / B is localized to the chromosome in metaphase I (left panel) and the spindle (white) in metaphase II (right panel). Scale bar is 2 μm. (G) Enlarged view of the chromosome enclosed in the rectangle in Figure 1f. TALhesin[mmMajSat] is localized to the centromere region of the chromosome in the spindle (top) during mouse meiosis I and the spindle (bottom) during meiosis II. Scale bar is 2 μm. (H) Mobility of TALhesin[mmMajSat] chromosome foci (shown in dark gray) compared to TAL[mmMajSat] effector alone (light gray), as measured by FRAP. Bars are SD from the mean, with 30 repeats for each condition. (I) Measurement of the interval between kinetochores between sister chromatids of metaphase II chromosome in mouse oocytes injected with the indicated amount of TALhesin[mmMajSat]A / B mRNA. Black squares indicate the mean, and lines indicate the median for each condition. Statistical values ​​were measured by Bonferroni-corrected two-sample t-test (equal variances not assumed). n is the number of sister kinetochore pairs analyzed, 2391. [Figure 1F]This is an engineered cohesination system for preventing chromosomal errors in oocytes. (A) A schematic diagram of the engineered cohesination system on metaphase chromosomes of human oocytes. Cohesin gradually dissociates from oocyte chromosomes during aging, causing premature separation of sister chromatids (PSSCs) (upper panel, left) and aneuploidy (meiosis II, upper panel, center)3,49. Untreated secondary oocytes with PSSCs result in aneuploid embryos with two chromosomes shown in gray and aneuploid second polar bodies without chromosomes shown in gray (upper panel, right). The engineered cohesin system restores cohesination (black) to the centromere region, preventing PSSCs (lower panel, left) and aneuploidy (lower panel, center), and resulting in euploid embryos (lower panel, right). (B) An overall diagram of the present invention. The schematic diagram on the left shows the overall arrangement of (I) the first protein and (II) the second protein, each containing (i) a chromatin-binding component, (ii) a protein-binding region (PBN region) at the N-terminus of the chromatin-binding component, and (iii) a protein-binding region (PBC region) at the C-terminus of the chromatin-binding component. The schematic diagram in the center shows that the PBN of the first protein and the PBC of the second protein can bind, and the PBC of the first protein and the PBN of the second protein can bind. Alternatively, the PBN regions of the first and second proteins can bind to each other, and the PBC regions of the first and second proteins can bind to each other. The binding may be covalent or non-covalent. The panel on the right of the schematic diagram shows three exemplary methods for releasing the binding. The first is (a) cleavage of the complex via a protease cleavage site located between the chromatin-binding component and the protein-binding region. The second is (b) degradation of the complex, in which the first and / or second protein of the complex further includes a ubiquitination site. The third is (c) disruption of the binding between the PBN region and the PBC region of the first and second proteins, the disruption of which is mediated by chemically induced proximity. (C) Chromosomes (shown in gray) were correctly aligned with respect to the central region of the meiotic spindle (shown in white) in metaphase II mouse oocytes (left panel), or had PSSCs (right panel).In boxes I and II, isolated chromatids marked by a single kinetochore (shown in white) represent PSSCs, and in box III, paired sister chromatids are aligned in the central region. Scale bars are 2 μm. (D) Frequency of PSSCs in oocytes obtained from young mice (open bars) and mice aged 13, 17, and 20 months (filled bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars are 95% CI, 1 degree of freedom. (E) Models of TALhesin components A and B. DNA-binding TAL effectors (shown in dark gray) are adjacent to either SpyTags (component A) or Spy-Catcher (component B) (see Figure 2A for further details). Dashed lines represent linker regions L1 and L2. The C-terminal domain of TAL is not shown. Based on pdb, these are 3 UGM50 and 4 MLI51. (F) TALhesin[mmMajSat]A / B is localized to the chromosome in metaphase I (left panel) and the spindle (white) in metaphase II (right panel). Scale bar is 2 μm. (G) Enlarged view of the chromosome enclosed in the rectangle in Figure 1f. TALhesin[mmMajSat] is localized to the centromere region of the chromosome in the spindle (top) during mouse meiosis I and the spindle (bottom) during meiosis II. Scale bar is 2 μm. (H) Mobility of TALhesin[mmMajSat] chromosome foci (shown in dark gray) compared to TAL[mmMajSat] effector alone (light gray), as measured by FRAP. Bars are SD from the mean, with 30 repeats for each condition. (I) Measurement of the interval between kinetochores between sister chromatids of metaphase II chromosome in mouse oocytes injected with the indicated amount of TALhesin[mmMajSat]A / B mRNA. Black squares indicate the mean, and lines indicate the median for each condition. Statistical values ​​were measured by Bonferroni-corrected two-sample t-test (equal variances not assumed). n is the number of sister kinetochore pairs analyzed, 2391. [Figure 1G]This is an engineered cohesination system for preventing chromosomal errors in oocytes. (A) A schematic diagram of the engineered cohesination system on metaphase chromosomes of human oocytes. Cohesin gradually dissociates from oocyte chromosomes during aging, causing premature separation of sister chromatids (PSSCs) (upper panel, left) and aneuploidy (meiosis II, upper panel, center)3,49. Untreated secondary oocytes with PSSCs result in aneuploid embryos with two chromosomes shown in gray and aneuploid second polar bodies without chromosomes shown in gray (upper panel, right). The engineered cohesin system restores cohesination (black) to the centromere region, preventing PSSCs (lower panel, left) and aneuploidy (lower panel, center), and resulting in euploid embryos (lower panel, right). (B) An overall diagram of the present invention. The schematic diagram on the left shows the overall arrangement of (I) the first protein and (II) the second protein, each containing (i) a chromatin-binding component, (ii) a protein-binding region (PBN region) at the N-terminus of the chromatin-binding component, and (iii) a protein-binding region (PBC region) at the C-terminus of the chromatin-binding component. The schematic diagram in the center shows that the PBN of the first protein and the PBC of the second protein can bind, and the PBC of the first protein and the PBN of the second protein can bind. Alternatively, the PBN regions of the first and second proteins can bind to each other, and the PBC regions of the first and second proteins can bind to each other. The binding may be covalent or non-covalent. The panel on the right of the schematic diagram shows three exemplary methods for releasing the binding. The first is (a) cleavage of the complex via a protease cleavage site located between the chromatin-binding component and the protein-binding region. The second is (b) degradation of the complex, in which the first and / or second protein of the complex further includes a ubiquitination site. The third is (c) disruption of the binding between the PBN region and the PBC region of the first and second proteins, the disruption of which is mediated by chemically induced proximity. (C) Chromosomes (shown in gray) were correctly aligned with respect to the central region of the meiotic spindle (shown in white) in metaphase II mouse oocytes (left panel), or had PSSCs (right panel).In boxes I and II, isolated chromatids marked by a single kinetochore (shown in white) represent PSSCs, and in box III, paired sister chromatids are aligned in the central region. Scale bars are 2 μm. (D) Frequency of PSSCs in oocytes obtained from young mice (open bars) and mice aged 13, 17, and 20 months (filled bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars are 95% CI, 1 degree of freedom. (E) Models of TALhesin components A and B. DNA-binding TAL effectors (shown in dark gray) are adjacent to either SpyTags (component A) or Spy-Catcher (component B) (see Figure 2A for further details). Dashed lines represent linker regions L1 and L2. The C-terminal domain of TAL is not shown. Based on pdb, these are 3 UGM50 and 4 MLI51. (F) TALhesin[mmMajSat]A / B is localized to the chromosome in metaphase I (left panel) and the spindle (white) in metaphase II (right panel). Scale bar is 2 μm. (G) Enlarged view of the chromosome enclosed in the rectangle in Figure 1f. TALhesin[mmMajSat] is localized to the centromere region of the chromosome in the spindle (top) during mouse meiosis I and the spindle (bottom) during meiosis II. Scale bar is 2 μm. (H) Mobility of TALhesin[mmMajSat] chromosome foci (shown in dark gray) compared to TAL[mmMajSat] effector alone (light gray), as measured by FRAP. Bars are SD from the mean, with 30 repeats for each condition. (I) Measurement of the interval between kinetochores between sister chromatids of metaphase II chromosome in mouse oocytes injected with the indicated amount of TALhesin[mmMajSat]A / B mRNA. Black squares indicate the mean, and lines indicate the median for each condition. Statistical values ​​were measured by Bonferroni-corrected two-sample t-test (equal variances not assumed). n is the number of sister kinetochore pairs analyzed, 2391. [Figure 1H]This is an engineered cohesination system for preventing chromosomal errors in oocytes. (A) A schematic diagram of the engineered cohesination system on metaphase chromosomes of human oocytes. Cohesin gradually dissociates from oocyte chromosomes during aging, causing premature separation of sister chromatids (PSSCs) (upper panel, left) and aneuploidy (meiosis II, upper panel, center)3,49. Untreated secondary oocytes with PSSCs result in aneuploid embryos with two chromosomes shown in gray and aneuploid second polar bodies without chromosomes shown in gray (upper panel, right). The engineered cohesin system restores cohesination (black) to the centromere region, preventing PSSCs (lower panel, left) and aneuploidy (lower panel, center), and resulting in euploid embryos (lower panel, right). (B) An overall diagram of the present invention. The schematic diagram on the left shows the overall arrangement of (I) the first protein and (II) the second protein, each containing (i) a chromatin-binding component, (ii) a protein-binding region (PBN region) at the N-terminus of the chromatin-binding component, and (iii) a protein-binding region (PBC region) at the C-terminus of the chromatin-binding component. The schematic diagram in the center shows that the PBN of the first protein and the PBC of the second protein can bind, and the PBC of the first protein and the PBN of the second protein can bind. Alternatively, the PBN regions of the first and second proteins can bind to each other, and the PBC regions of the first and second proteins can bind to each other. The binding may be covalent or non-covalent. The panel on the right of the schematic diagram shows three exemplary methods for releasing the binding. The first is (a) cleavage of the complex via a protease cleavage site located between the chromatin-binding component and the protein-binding region. The second is (b) degradation of the complex, in which the first and / or second protein of the complex further includes a ubiquitination site. The third is (c) disruption of the binding between the PBN region and the PBC region of the first and second proteins, the disruption of which is mediated by chemically induced proximity. (C) Chromosomes (shown in gray) were correctly aligned with respect to the central region of the meiotic spindle (shown in white) in metaphase II mouse oocytes (left panel), or had PSSCs (right panel).In boxes I and II, isolated chromatids marked by a single kinetochore (shown in white) represent PSSCs, and in box III, paired sister chromatids are aligned in the central region. Scale bars are 2 μm. (D) Frequency of PSSCs in oocytes obtained from young mice (open bars) and mice aged 13, 17, and 20 months (filled bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars are 95% CI, 1 degree of freedom. (E) Models of TALhesin components A and B. DNA-binding TAL effectors (shown in dark gray) are adjacent to either SpyTags (component A) or Spy-Catcher (component B) (see Figure 2A for further details). Dashed lines represent linker regions L1 and L2. The C-terminal domain of TAL is not shown. Based on pdb, these are 3 UGM50 and 4 MLI51. (F) TALhesin[mmMajSat]A / B is localized to the chromosome in metaphase I (left panel) and the spindle (white) in metaphase II (right panel). Scale bar is 2 μm. (G) Enlarged view of the chromosome enclosed in the rectangle in Figure 1f. TALhesin[mmMajSat] is localized to the centromere region of the chromosome in the spindle (top) during mouse meiosis I and the spindle (bottom) during meiosis II. Scale bar is 2 μm. (H) Mobility of TALhesin[mmMajSat] chromosome foci (shown in dark gray) compared to TAL[mmMajSat] effector alone (light gray), as measured by FRAP. Bars are SD from the mean, with 30 repeats for each condition. (I) Measurement of the interval between kinetochores between sister chromatids of metaphase II chromosome in mouse oocytes injected with the indicated amount of TALhesin[mmMajSat]A / B mRNA. Black squares indicate the mean, and lines indicate the median for each condition. Statistical values ​​were measured by Bonferroni-corrected two-sample t-test (equal variances not assumed). n is the number of sister kinetochore pairs analyzed, 2391. [Figure 1I]This is an engineered cohesination system for preventing chromosomal errors in oocytes. (A) A schematic diagram of the engineered cohesination system on metaphase chromosomes of human oocytes. Cohesin gradually dissociates from oocyte chromosomes during aging, causing premature separation of sister chromatids (PSSCs) (upper panel, left) and aneuploidy (meiosis II, upper panel, center)3,49. Untreated secondary oocytes with PSSCs result in aneuploid embryos with two chromosomes shown in gray and aneuploid second polar bodies without chromosomes shown in gray (upper panel, right). The engineered cohesin system restores cohesination (black) to the centromere region, preventing PSSCs (lower panel, left) and aneuploidy (lower panel, center), and resulting in euploid embryos (lower panel, right). (B) An overall diagram of the present invention. The schematic diagram on the left shows the overall arrangement of (I) the first protein and (II) the second protein, each containing (i) a chromatin-binding component, (ii) a protein-binding region (PBN region) at the N-terminus of the chromatin-binding component, and (iii) a protein-binding region (PBC region) at the C-terminus of the chromatin-binding component. The schematic diagram in the center shows that the PBN of the first protein and the PBC of the second protein can bind, and the PBC of the first protein and the PBN of the second protein can bind. Alternatively, the PBN regions of the first and second proteins can bind to each other, and the PBC regions of the first and second proteins can bind to each other. The binding may be covalent or non-covalent. The panel on the right of the schematic diagram shows three exemplary methods for releasing the binding. The first is (a) cleavage of the complex via a protease cleavage site located between the chromatin-binding component and the protein-binding region. The second is (b) degradation of the complex, in which the first and / or second protein of the complex further includes a ubiquitination site. The third is (c) disruption of the binding between the PBN region and the PBC region of the first and second proteins, the disruption of which is mediated by chemically induced proximity. (C) Chromosomes (shown in gray) were correctly aligned with respect to the central region of the meiotic spindle (shown in white) in metaphase II mouse oocytes (left panel), or had PSSCs (right panel).In boxes I and II, isolated chromatids marked by a single kinetochore (shown in white) represent PSSCs, and in box III, paired sister chromatids are aligned in the central region. Scale bars are 2 μm. (D) Frequency of PSSCs in oocytes obtained from young mice (open bars) and mice aged 13, 17, and 20 months (filled bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars are 95% CI, 1 degree of freedom. (E) Models of TALhesin components A and B. DNA-binding TAL effectors (shown in dark gray) are adjacent to either SpyTags (component A) or Spy-Catcher (component B) (see Figure 2A for further details). Dashed lines represent linker regions L1 and L2. The C-terminal domain of TAL is not shown. Based on pdb, these are 3 UGM50 and 4 MLI51. (F) TALhesin[mmMajSat]A / B is localized to the chromosome in metaphase I (left panel) and the spindle (white) in metaphase II (right panel). Scale bar is 2 μm. (G) Enlarged view of the chromosome enclosed in the rectangle in Figure 1f. TALhesin[mmMajSat] is localized to the centromere region of the chromosome in the spindle (top) during mouse meiosis I and the spindle (bottom) during meiosis II. Scale bar is 2 μm. (H) Mobility of TALhesin[mmMajSat] chromosome foci (shown in dark gray) compared to TAL[mmMajSat] effector alone (light gray), as measured by FRAP. Bars are SD from the mean, with 30 repeats for each condition. (I) Measurement of the interval between kinetochores between sister chromatids of metaphase II chromosome in mouse oocytes injected with the indicated amount of TALhesin[mmMajSat]A / B mRNA. Black squares indicate the mean, and lines indicate the median for each condition. Statistical values ​​were measured by Bonferroni-corrected two-sample t-test (equal variances not assumed). n is the number of sister kinetochore pairs analyzed, 2391.

[0009] [Figure 2A]High expression of the artificial complex (TALhesin) induces errors in meiosis I and II.

[0010] (A) Schematic diagram of the domain arrangement of TALhesin[mmMajSat] targeting mouse centromere repeats (not drawn to scale). Fluorescent proteins, mCherry or mClover, are incorporated into the L2 linker region. (B) Titration of TALhesin[mmMajSat]A / B mRNA analyzed by 1% agarose gel electrophoresis. Domain composition and expected size of each transcript are shown next to annotated images of nucleotides (nt). (C) TALhesin[mmMajSat]A / B localizes to chromosomes after NEBD. Chromosomes are gray; TALhesin is dark gray (hrs: time after NEBD). Scale bar is 5 μm. (D) Duration of mouse meiosis I from NEBD to polar body protrusion in oocytes microinjected with TALhesin[mmMajSat]-B only (white bars) or oocytes microinjected with increased amounts of TALhesin[mmMajSat]A / B mRNA (amol, attomol, Hrs, hours). Black squares indicate the mean, and lines indicate the median for each condition. Bars are SD from the mean. n is the number of cells analyzed. P-values ​​were measured by Student's t-test. (E) Metaphase II spindles of two exemplary mouse oocytes expressing large amounts (0.5 amol) of TALhesin[mmMajSat] mRNA. Chromosome clustering (box I) and fractured kinetochores (white dots) (box II) are shown in each example. (F) Enlarged view of the chromosomes enclosed in the square in Figure 2e, showing chromosome clustering and fractured kinetochores. (EF) represents TALhesin, dark gray, kinetochore, microtubule, and chromosome. The scale bar is 2 μm. (G) This is a quantification of error detected by live microscopy of meiosis I in mouse oocytes expressing gradually increasing amounts of TALhesin[mmMajSat]A / B mRNA. Low-quality chromosomes are shown as open bars. Chromosomes in the oocyte that remain attached to the polar body and are being tracked are shown as gray bars. Clustered chromosomes are shown as filled bars. [Figure 2B]High expression of the artificial complex (TALhesin) induces errors in meiosis I and II. (A) Schematic diagram of the domain arrangement of TALhesin[mmMajSat] targeting mouse centromere repeats (not drawn to scale). Fluorescent proteins, mCherry or mClover, are incorporated into the L2 linker region. (B) Titration of TALhesin[mmMajSat]A / B mRNA analyzed by 1% agarose gel electrophoresis. Domain composition and expected size of each transcript are shown next to annotated images of nucleotides (nt). (C) TALhesin[mmMajSat]A / B localizes to chromosomes after NEBD. Chromosomes are gray; TALhesin is dark gray (hrs: time after NEBD). Scale bar is 5 μm. (D) Duration of mouse meiosis I from NEBD to polar body protrusion in oocytes microinjected with TALhesin[mmMajSat]-B only (white bars) or oocytes microinjected with increased amounts of TALhesin[mmMajSat]A / B mRNA (amol, attomol, Hrs, hours). Black squares indicate the mean, and lines indicate the median for each condition. Bars are SD from the mean. n is the number of cells analyzed. P-values ​​were measured by Student's t-test. (E) Metaphase II spindles of two exemplary mouse oocytes expressing large amounts (0.5 amol) of TALhesin[mmMajSat] mRNA. Chromosome clustering (box I) and fractured kinetochores (white dots) (box II) are shown in each example. (F) Enlarged view of the chromosomes enclosed in the square in Figure 2e, showing chromosome clustering and fractured kinetochores. (EF) represents TALhesin, dark gray, kinetochore, microtubule, and chromosome. The scale bar is 2 μm. (G) This is a quantification of error detected by live microscopy of meiosis I in mouse oocytes expressing gradually increasing amounts of TALhesin[mmMajSat]A / B mRNA. Low-quality chromosomes are shown as open bars. Chromosomes in the oocyte that remain attached to the polar body and are being tracked are shown as gray bars. Clustered chromosomes are shown as filled bars. [Figure 2C] High expression of the artificial complex (TALhesin) induces errors in meiosis I and II. (A) Schematic diagram of the domain arrangement of TALhesin[mmMajSat] targeting mouse centromere repeats (not drawn to scale). Fluorescent proteins, mCherry or mClover, are incorporated into the L2 linker region. (B) Titration of TALhesin[mmMajSat]A / B mRNA analyzed by 1% agarose gel electrophoresis. Domain composition and expected size of each transcript are shown next to annotated images of nucleotides (nt). (C) TALhesin[mmMajSat]A / B localizes to chromosomes after NEBD. Chromosomes are gray; TALhesin is dark gray (hrs: time after NEBD). Scale bar is 5 μm. (D) Duration of mouse meiosis I from NEBD to polar body protrusion in oocytes microinjected with TALhesin[mmMajSat]-B only (white bars) or oocytes microinjected with increased amounts of TALhesin[mmMajSat]A / B mRNA (amol, attomol, Hrs, hours). Black squares indicate the mean, and lines indicate the median for each condition. Bars are SD from the mean. n is the number of cells analyzed. P-values ​​were measured by Student's t-test. (E) Metaphase II spindles of two exemplary mouse oocytes expressing large amounts (0.5 amol) of TALhesin[mmMajSat] mRNA. Chromosome clustering (box I) and fractured kinetochores (white dots) (box II) are shown in each example. (F) Enlarged view of the chromosomes enclosed in the square in Figure 2e, showing chromosome clustering and fractured kinetochores. (EF) represents TALhesin, dark gray, kinetochore, microtubule, and chromosome. The scale bar is 2 μm. (G) This is a quantification of error detected by live microscopy of meiosis I in mouse oocytes expressing gradually increasing amounts of TALhesin[mmMajSat]A / B mRNA. Low-quality chromosomes are shown as open bars. Chromosomes in the oocyte that remain attached to the polar body and are being tracked are shown as gray bars. Clustered chromosomes are shown as filled bars. [Figure 2D] High expression of the artificial complex (TALhesin) induces errors in meiosis I and II. (A) Schematic diagram of the domain arrangement of TALhesin[mmMajSat] targeting mouse centromere repeats (not drawn to scale). Fluorescent proteins, mCherry or mClover, are incorporated into the L2 linker region. (B) Titration of TALhesin[mmMajSat]A / B mRNA analyzed by 1% agarose gel electrophoresis. Domain composition and expected size of each transcript are shown next to annotated images of nucleotides (nt). (C) TALhesin[mmMajSat]A / B localizes to chromosomes after NEBD. Chromosomes are gray; TALhesin is dark gray (hrs: time after NEBD). Scale bar is 5 μm. (D) Duration of mouse meiosis I from NEBD to polar body protrusion in oocytes microinjected with TALhesin[mmMajSat]-B only (white bars) or oocytes microinjected with increased amounts of TALhesin[mmMajSat]A / B mRNA (amol, attomol, Hrs, hours). Black squares indicate the mean, and lines indicate the median for each condition. Bars are SD from the mean. n is the number of cells analyzed. P-values ​​were measured by Student's t-test. (E) Metaphase II spindles of two exemplary mouse oocytes expressing large amounts (0.5 amol) of TALhesin[mmMajSat] mRNA. Chromosome clustering (box I) and fractured kinetochores (white dots) (box II) are shown in each example. (F) Enlarged view of the chromosomes enclosed in the square in Figure 2e, showing chromosome clustering and fractured kinetochores. (EF) represents TALhesin, dark gray, kinetochore, microtubule, and chromosome. The scale bar is 2 μm. (G) This is a quantification of error detected by live microscopy of meiosis I in mouse oocytes expressing gradually increasing amounts of TALhesin[mmMajSat]A / B mRNA. Low-quality chromosomes are shown as open bars. Chromosomes in the oocyte that remain attached to the polar body and are being tracked are shown as gray bars. Clustered chromosomes are shown as filled bars. [Figure 2E]High expression of the artificial complex (TALhesin) induces errors in meiosis I and II. (A) Schematic diagram of the domain arrangement of TALhesin[mmMajSat] targeting mouse centromere repeats (not drawn to scale). Fluorescent proteins, mCherry or mClover, are incorporated into the L2 linker region. (B) Titration of TALhesin[mmMajSat]A / B mRNA analyzed by 1% agarose gel electrophoresis. Domain composition and expected size of each transcript are shown next to annotated images of nucleotides (nt). (C) TALhesin[mmMajSat]A / B localizes to chromosomes after NEBD. Chromosomes are gray; TALhesin is dark gray (hrs: time after NEBD). Scale bar is 5 μm. (D) Duration of mouse meiosis I from NEBD to polar body protrusion in oocytes microinjected with TALhesin[mmMajSat]-B only (white bars) or oocytes microinjected with increased amounts of TALhesin[mmMajSat]A / B mRNA (amol, attomol, Hrs, hours). Black squares indicate the mean, and lines indicate the median for each condition. Bars are SD from the mean. n is the number of cells analyzed. P-values ​​were measured by Student's t-test. (E) Metaphase II spindles of two exemplary mouse oocytes expressing large amounts (0.5 amol) of TALhesin[mmMajSat] mRNA. Chromosome clustering (box I) and fractured kinetochores (white dots) (box II) are shown in each example. (F) Enlarged view of the chromosomes enclosed in the square in Figure 2e, showing chromosome clustering and fractured kinetochores. (EF) represents TALhesin, dark gray, kinetochore, microtubule, and chromosome. The scale bar is 2 μm. (G) This is a quantification of error detected by live microscopy of meiosis I in mouse oocytes expressing gradually increasing amounts of TALhesin[mmMajSat]A / B mRNA. Low-quality chromosomes are shown as open bars. Chromosomes in the oocyte that remain attached to the polar body and are being tracked are shown as gray bars. Clustered chromosomes are shown as filled bars. [Figure 2F] High expression of the artificial complex (TALhesin) induces errors in meiosis I and II. (A) Schematic diagram of the domain arrangement of TALhesin[mmMajSat] targeting mouse centromere repeats (not drawn to scale). Fluorescent proteins, mCherry or mClover, are incorporated into the L2 linker region. (B) Titration of TALhesin[mmMajSat]A / B mRNA analyzed by 1% agarose gel electrophoresis. Domain composition and expected size of each transcript are shown next to annotated images of nucleotides (nt). (C) TALhesin[mmMajSat]A / B localizes to chromosomes after NEBD. Chromosomes are gray; TALhesin is dark gray (hrs: time after NEBD). Scale bar is 5 μm. (D) Duration of mouse meiosis I from NEBD to polar body protrusion in oocytes microinjected with TALhesin[mmMajSat]-B only (white bars) or oocytes microinjected with increased amounts of TALhesin[mmMajSat]A / B mRNA (amol, attomol, Hrs, hours). Black squares indicate the mean, and lines indicate the median for each condition. Bars are SD from the mean. n is the number of cells analyzed. P-values ​​were measured by Student's t-test. (E) Metaphase II spindles of two exemplary mouse oocytes expressing large amounts (0.5 amol) of TALhesin[mmMajSat] mRNA. Chromosome clustering (box I) and fractured kinetochores (white dots) (box II) are shown in each example. (F) Enlarged view of the chromosomes enclosed in the square in Figure 2e, showing chromosome clustering and fractured kinetochores. (EF) represents TALhesin, dark gray, kinetochore, microtubule, and chromosome. The scale bar is 2 μm. (G) This is a quantification of error detected by live microscopy of meiosis I in mouse oocytes expressing gradually increasing amounts of TALhesin[mmMajSat]A / B mRNA. Low-quality chromosomes are shown as open bars. Chromosomes in the oocyte that remain attached to the polar body and are being tracked are shown as gray bars. Clustered chromosomes are shown as filled bars. [Figure 2G] High expression of the artificial complex (TALhesin) induces errors in meiosis I and II. (A) Schematic diagram of the domain arrangement of TALhesin[mmMajSat] targeting mouse centromere repeats (not drawn to scale). Fluorescent proteins, mCherry or mClover, are incorporated into the L2 linker region. (B) Titration of TALhesin[mmMajSat]A / B mRNA analyzed by 1% agarose gel electrophoresis. Domain composition and expected size of each transcript are shown next to annotated images of nucleotides (nt). (C) TALhesin[mmMajSat]A / B localizes to chromosomes after NEBD. Chromosomes are gray; TALhesin is dark gray (hrs: time after NEBD). Scale bar is 5 μm. (D) Duration of mouse meiosis I from NEBD to polar body protrusion in oocytes microinjected with TALhesin[mmMajSat]-B only (white bars) or oocytes microinjected with increased amounts of TALhesin[mmMajSat]A / B mRNA (amol, attomol, Hrs, hours). Black squares indicate the mean, and lines indicate the median for each condition. Bars are SD from the mean. n is the number of cells analyzed. P-values ​​were measured by Student's t-test. (E) Metaphase II spindles of two exemplary mouse oocytes expressing large amounts (0.5 amol) of TALhesin[mmMajSat] mRNA. Chromosome clustering (box I) and fractured kinetochores (white dots) (box II) are shown in each example. (F) Enlarged view of the chromosomes enclosed in the square in Figure 2e, showing chromosome clustering and fractured kinetochores. (EF) represents TALhesin, dark gray, kinetochore, microtubule, and chromosome. The scale bar is 2 μm. (G) This is a quantification of error detected by live microscopy of meiosis I in mouse oocytes expressing gradually increasing amounts of TALhesin[mmMajSat]A / B mRNA. Low-quality chromosomes are shown as open bars. Chromosomes in the oocyte that remain attached to the polar body and are being tracked are shown as gray bars. Clustered chromosomes are shown as filled bars.

[0011] [Figure 3A] The artificial complex (TALhesin) induces aggregation in mouse chromosomes.

[0012] (A) Schematic diagram of the results of depletion of Rec8 by trim-away in Figure 3b. (B) Depletion of Rec8 by trim-away in young mouse oocytes injected with only 2 amol of TALhesin[mmMajSat]-B component (upper panel). Depletion of Rec8 by trim-away in oocytes injected with 2 amol of TALhesin[mmMajSat]A / B (lower panel) (min is minutes). Min is minutes. Scale bar is 5 μm. (C) Schematic diagram of the results of depletion of TALhesin[mmMajSat] protein by trim-away in Figure 3d. (D) Depletion of TALhesin[mmMajSat] by anti-GFP trim-away (upper panel), or subsequent depletion of Rec8 (lower panel). Dashed lines indicate polar bodies in the field of view. hrs is hours. Scale bar is 5 μm. (E) Schematic diagram of the chromosomal error reduction assay in mouse oocytes. (F) Example of a meiotic II spindle from aged mouse oocytes used for analysis. Automated spot detection of 40 kinetochores (white spheres) is performed in each example. PSSCs (center panel), indicated by asterisks, are those in which more than two errors are detected. TALhesin[mmMajSat]A / B was injected with 0.05 amol mRNA (right panel). Microtubules are white. Scale bar is 3 μm. (G) Frequency of PSSCs in untreated oocytes from young mice (white bars), untreated oocytes from aged mice (filled bars), and aged oocytes treated with TALhesin[mmMajSat]A / B mRNA (gray bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars are 95% CI, 1 degree of freedom. (H) Reduction in PSSCs in aged mouse oocytes microinjected with TALhesin[mmMajSat] mRNA compared to untreated young and aged oocytes. (I) Percentage of oocytes from aged mice treated with TALhesin[mmMajSat] having 0 (white bar), 1 (light gray bar), 2 (dark gray bar), or more than 2 (filled bar) PSSC errors compared to untreated young and aged oocytes.The number of oocytes analyzed is shown below for each condition. (J) Increase in error-free oocytes from aged mice treated with TALhesin[mmMajSat]mRNA compared to untreated oocytes from young and aged mice. [Figure 3B]The artificial complex (TALhesin) induces condensation in mouse chromosomes. (A) Schematic diagram of the results of depletion of Rec8 by trim-away in Figure 3b. (B) Depletion of Rec8 by trim-away in young mouse oocytes injected with only 2 amol of TALhesin[mmMajSat]-B component (upper panel). Depletion of Rec8 by trim-away in oocytes injected with 2 amol of TALhesin[mmMajSat]A / B (lower panel) (min is minutes). Min is minutes. Scale bar is 5 μm. (C) Schematic diagram of the results of depletion of TALhesin[mmMajSat] protein by trim-away in Figure 3d. (D) Depletion of TALhesin[mmMajSat] by anti-GFP trim-away (upper panel), or subsequent depletion of Rec8 (lower panel). Dashed lines indicate polar bodies in the field of view. hrs is hours. The scale bar is 5 μm. (E) Schematic diagram of the chromosomal error reduction assay in mouse oocytes. (F) Example of a meiotic II spindle from aged mouse oocytes used for analysis. Automated spot detection of 40 kinetochores (white spheres) is performed in each example. PSSCs (center panel), indicated by asterisks, are those in which more than two errors are detected. TALhesin[mmMajSat]A / B was injected with 0.05 amol mRNA (right panel). Microtubules are white. The scale bar is 3 μm. (G) Frequency of PSSCs in untreated oocytes from young mice (blank bars), untreated oocytes from aged mice (filled bars), and aged oocytes treated with TALhesin[mmMajSat]A / B mRNA (gray bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars represent 95% CI, 1 degree of freedom. (H) Reduction in PSSCs in aged mouse oocytes microinjected with TALhesin[mmMajSat]mRNA compared to untreated young and aged oocytes.(I) Percentage of oocytes from aged mice treated with TALhesin[mmMajSat] that have 0 (blank bar), 1 (light gray bar), 2 (dark gray bar), or more than 2 (filled bar) PSSC errors, compared to untreated young and aged oocytes. The number of oocytes analyzed is shown below for each condition. (J) Increase in error-free oocytes from aged mice treated with TALhesin[mmMajSat] mRNA, compared to untreated oocytes from young and aged mice. [Figure 3C]The artificial complex (TALhesin) induces condensation in mouse chromosomes. (A) Schematic diagram of the results of depletion of Rec8 by trim-away in Figure 3b. (B) Depletion of Rec8 by trim-away in young mouse oocytes injected with only 2 amol of TALhesin[mmMajSat]-B component (upper panel). Depletion of Rec8 by trim-away in oocytes injected with 2 amol of TALhesin[mmMajSat]A / B (lower panel) (min is minutes). Min is minutes. Scale bar is 5 μm. (C) Schematic diagram of the results of depletion of TALhesin[mmMajSat] protein by trim-away in Figure 3d. (D) Depletion of TALhesin[mmMajSat] by anti-GFP trim-away (upper panel), or subsequent depletion of Rec8 (lower panel). Dashed lines indicate polar bodies in the field of view. hrs is hours. The scale bar is 5 μm. (E) Schematic diagram of the chromosomal error reduction assay in mouse oocytes. (F) Example of a meiotic II spindle from aged mouse oocytes used for analysis. Automated spot detection of 40 kinetochores (white spheres) is performed in each example. PSSCs (center panel), indicated by asterisks, are those in which more than two errors are detected. TALhesin[mmMajSat]A / B was injected with 0.05 amol mRNA (right panel). Microtubules are white. The scale bar is 3 μm. (G) Frequency of PSSCs in untreated oocytes from young mice (blank bars), untreated oocytes from aged mice (filled bars), and aged oocytes treated with TALhesin[mmMajSat]A / B mRNA (gray bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars represent 95% CI, 1 degree of freedom. (H) Reduction in PSSCs in aged mouse oocytes microinjected with TALhesin[mmMajSat]mRNA compared to untreated young and aged oocytes.(I) Percentage of oocytes from aged mice treated with TALhesin[mmMajSat] that have 0 (blank bar), 1 (light gray bar), 2 (dark gray bar), or more than 2 (filled bar) PSSC errors, compared to untreated young and aged oocytes. The number of oocytes analyzed is shown below for each condition. (J) Increase in error-free oocytes from aged mice treated with TALhesin[mmMajSat] mRNA, compared to untreated oocytes from young and aged mice. [Figure 3D]The artificial complex (TALhesin) induces condensation in mouse chromosomes. (A) Schematic diagram of the results of depletion of Rec8 by trim-away in Figure 3b. (B) Depletion of Rec8 by trim-away in young mouse oocytes injected with only 2 amol of TALhesin[mmMajSat]-B component (upper panel). Depletion of Rec8 by trim-away in oocytes injected with 2 amol of TALhesin[mmMajSat]A / B (lower panel) (min is minutes). Min is minutes. Scale bar is 5 μm. (C) Schematic diagram of the results of depletion of TALhesin[mmMajSat] protein by trim-away in Figure 3d. (D) Depletion of TALhesin[mmMajSat] by anti-GFP trim-away (upper panel), or subsequent depletion of Rec8 (lower panel). Dashed lines indicate polar bodies in the field of view. hrs is hours. The scale bar is 5 μm. (E) Schematic diagram of the chromosomal error reduction assay in mouse oocytes. (F) Example of a meiotic II spindle from aged mouse oocytes used for analysis. Automated spot detection of 40 kinetochores (white spheres) is performed in each example. PSSCs (center panel), indicated by asterisks, are those in which more than two errors are detected. TALhesin[mmMajSat]A / B was injected with 0.05 amol mRNA (right panel). Microtubules are white. The scale bar is 3 μm. (G) Frequency of PSSCs in untreated oocytes from young mice (blank bars), untreated oocytes from aged mice (filled bars), and aged oocytes treated with TALhesin[mmMajSat]A / B mRNA (gray bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars represent 95% CI, 1 degree of freedom. (H) Reduction in PSSCs in aged mouse oocytes microinjected with TALhesin[mmMajSat]mRNA compared to untreated young and aged oocytes.(I) Percentage of oocytes from aged mice treated with TALhesin[mmMajSat] that have 0 (blank bar), 1 (light gray bar), 2 (dark gray bar), or more than 2 (filled bar) PSSC errors, compared to untreated young and aged oocytes. The number of oocytes analyzed is shown below for each condition. (J) Increase in error-free oocytes from aged mice treated with TALhesin[mmMajSat] mRNA, compared to untreated oocytes from young and aged mice. [Figure 3E]The artificial complex (TALhesin) induces condensation in mouse chromosomes. (A) Schematic diagram of the results of depletion of Rec8 by trim-away in Figure 3b. (B) Depletion of Rec8 by trim-away in young mouse oocytes injected with only 2 amol of TALhesin[mmMajSat]-B component (upper panel). Depletion of Rec8 by trim-away in oocytes injected with 2 amol of TALhesin[mmMajSat]A / B (lower panel) (min is minutes). Min is minutes. Scale bar is 5 μm. (C) Schematic diagram of the results of depletion of TALhesin[mmMajSat] protein by trim-away in Figure 3d. (D) Depletion of TALhesin[mmMajSat] by anti-GFP trim-away (upper panel), or subsequent depletion of Rec8 (lower panel). Dashed lines indicate polar bodies in the field of view. hrs is hours. The scale bar is 5 μm. (E) Schematic diagram of the chromosomal error reduction assay in mouse oocytes. (F) Example of a meiotic II spindle from aged mouse oocytes used for analysis. Automated spot detection of 40 kinetochores (white spheres) is performed in each example. PSSCs (center panel), indicated by asterisks, are those in which more than two errors are detected. TALhesin[mmMajSat]A / B was injected with 0.05 amol mRNA (right panel). Microtubules are white. The scale bar is 3 μm. (G) Frequency of PSSCs in untreated oocytes from young mice (blank bars), untreated oocytes from aged mice (filled bars), and aged oocytes treated with TALhesin[mmMajSat]A / B mRNA (gray bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars represent 95% CI, 1 degree of freedom. (H) Reduction in PSSCs in aged mouse oocytes microinjected with TALhesin[mmMajSat]mRNA compared to untreated young and aged oocytes.(I) Percentage of oocytes from aged mice treated with TALhesin[mmMajSat] that have 0 (blank bar), 1 (light gray bar), 2 (dark gray bar), or more than 2 (filled bar) PSSC errors, compared to untreated young and aged oocytes. The number of oocytes analyzed is shown below for each condition. (J) Increase in error-free oocytes from aged mice treated with TALhesin[mmMajSat] mRNA, compared to untreated oocytes from young and aged mice. [Figure 3F]The artificial complex (TALhesin) induces condensation in mouse chromosomes. (A) Schematic diagram of the results of depletion of Rec8 by trim-away in Figure 3b. (B) Depletion of Rec8 by trim-away in young mouse oocytes injected with only 2 amol of TALhesin[mmMajSat]-B component (upper panel). Depletion of Rec8 by trim-away in oocytes injected with 2 amol of TALhesin[mmMajSat]A / B (lower panel) (min is minutes). Min is minutes. Scale bar is 5 μm. (C) Schematic diagram of the results of depletion of TALhesin[mmMajSat] protein by trim-away in Figure 3d. (D) Depletion of TALhesin[mmMajSat] by anti-GFP trim-away (upper panel), or subsequent depletion of Rec8 (lower panel). Dashed lines indicate polar bodies in the field of view. hrs is hours. The scale bar is 5 μm. (E) Schematic diagram of the chromosomal error reduction assay in mouse oocytes. (F) Example of a meiotic II spindle from aged mouse oocytes used for analysis. Automated spot detection of 40 kinetochores (white spheres) is performed in each example. PSSCs (center panel), indicated by asterisks, are those in which more than two errors are detected. TALhesin[mmMajSat]A / B was injected with 0.05 amol mRNA (right panel). Microtubules are white. The scale bar is 3 μm. (G) Frequency of PSSCs in untreated oocytes from young mice (blank bars), untreated oocytes from aged mice (filled bars), and aged oocytes treated with TALhesin[mmMajSat]A / B mRNA (gray bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars represent 95% CI, 1 degree of freedom. (H) Reduction in PSSCs in aged mouse oocytes microinjected with TALhesin[mmMajSat]mRNA compared to untreated young and aged oocytes.(I) Percentage of oocytes from aged mice treated with TALhesin[mmMajSat] that have 0 (blank bar), 1 (light gray bar), 2 (dark gray bar), or more than 2 (filled bar) PSSC errors, compared to untreated young and aged oocytes. The number of oocytes analyzed is shown below for each condition. (J) Increase in error-free oocytes from aged mice treated with TALhesin[mmMajSat] mRNA, compared to untreated oocytes from young and aged mice. [Figure 3G]The artificial complex (TALhesin) induces condensation in mouse chromosomes. (A) Schematic diagram of the results of depletion of Rec8 by trim-away in Figure 3b. (B) Depletion of Rec8 by trim-away in young mouse oocytes injected with only 2 amol of TALhesin[mmMajSat]-B component (upper panel). Depletion of Rec8 by trim-away in oocytes injected with 2 amol of TALhesin[mmMajSat]A / B (lower panel) (min is minutes). Min is minutes. Scale bar is 5 μm. (C) Schematic diagram of the results of depletion of TALhesin[mmMajSat] protein by trim-away in Figure 3d. (D) Depletion of TALhesin[mmMajSat] by anti-GFP trim-away (upper panel), or subsequent depletion of Rec8 (lower panel). Dashed lines indicate polar bodies in the field of view. hrs is hours. The scale bar is 5 μm. (E) Schematic diagram of the chromosomal error reduction assay in mouse oocytes. (F) Example of a meiotic II spindle from aged mouse oocytes used for analysis. Automated spot detection of 40 kinetochores (white spheres) is performed in each example. PSSCs (center panel), indicated by asterisks, are those in which more than two errors are detected. TALhesin[mmMajSat]A / B was injected with 0.05 amol mRNA (right panel). Microtubules are white. The scale bar is 3 μm. (G) Frequency of PSSCs in untreated oocytes from young mice (blank bars), untreated oocytes from aged mice (filled bars), and aged oocytes treated with TALhesin[mmMajSat]A / B mRNA (gray bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars represent 95% CI, 1 degree of freedom. (H) Reduction in PSSCs in aged mouse oocytes microinjected with TALhesin[mmMajSat]mRNA compared to untreated young and aged oocytes.(I) Percentage of oocytes from aged mice treated with TALhesin[mmMajSat] that have 0 (blank bar), 1 (light gray bar), 2 (dark gray bar), or more than 2 (filled bar) PSSC errors, compared to untreated young and aged oocytes. The number of oocytes analyzed is shown below for each condition. (J) Increase in error-free oocytes from aged mice treated with TALhesin[mmMajSat] mRNA, compared to untreated oocytes from young and aged mice. [Figure 3H]The artificial complex (TALhesin) induces condensation in mouse chromosomes. (A) Schematic diagram of the results of depletion of Rec8 by trim-away in Figure 3b. (B) Depletion of Rec8 by trim-away in young mouse oocytes injected with only 2 amol of TALhesin[mmMajSat]-B component (upper panel). Depletion of Rec8 by trim-away in oocytes injected with 2 amol of TALhesin[mmMajSat]A / B (lower panel) (min is minutes). Min is minutes. Scale bar is 5 μm. (C) Schematic diagram of the results of depletion of TALhesin[mmMajSat] protein by trim-away in Figure 3d. (D) Depletion of TALhesin[mmMajSat] by anti-GFP trim-away (upper panel), or subsequent depletion of Rec8 (lower panel). Dashed lines indicate polar bodies in the field of view. hrs is hours. The scale bar is 5 μm. (E) Schematic diagram of the chromosomal error reduction assay in mouse oocytes. (F) Example of a meiotic II spindle from aged mouse oocytes used for analysis. Automated spot detection of 40 kinetochores (white spheres) is performed in each example. PSSCs (center panel), indicated by asterisks, are those in which more than two errors are detected. TALhesin[mmMajSat]A / B was injected with 0.05 amol mRNA (right panel). Microtubules are white. The scale bar is 3 μm. (G) Frequency of PSSCs in untreated oocytes from young mice (blank bars), untreated oocytes from aged mice (filled bars), and aged oocytes treated with TALhesin[mmMajSat]A / B mRNA (gray bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars represent 95% CI, 1 degree of freedom. (H) Reduction in PSSCs in aged mouse oocytes microinjected with TALhesin[mmMajSat]mRNA compared to untreated young and aged oocytes.(I) Percentage of oocytes from aged mice treated with TALhesin[mmMajSat] that have 0 (blank bar), 1 (light gray bar), 2 (dark gray bar), or more than 2 (filled bar) PSSC errors, compared to untreated young and aged oocytes. The number of oocytes analyzed is shown below for each condition. (J) Increase in error-free oocytes from aged mice treated with TALhesin[mmMajSat] mRNA, compared to untreated oocytes from young and aged mice. [Figure 3I]The artificial complex (TALhesin) induces condensation in mouse chromosomes. (A) Schematic diagram of the results of depletion of Rec8 by trim-away in Figure 3b. (B) Depletion of Rec8 by trim-away in young mouse oocytes injected with only 2 amol of TALhesin[mmMajSat]-B component (upper panel). Depletion of Rec8 by trim-away in oocytes injected with 2 amol of TALhesin[mmMajSat]A / B (lower panel) (min is minutes). Min is minutes. Scale bar is 5 μm. (C) Schematic diagram of the results of depletion of TALhesin[mmMajSat] protein by trim-away in Figure 3d. (D) Depletion of TALhesin[mmMajSat] by anti-GFP trim-away (upper panel), or subsequent depletion of Rec8 (lower panel). Dashed lines indicate polar bodies in the field of view. hrs is hours. The scale bar is 5 μm. (E) Schematic diagram of the chromosomal error reduction assay in mouse oocytes. (F) Example of a meiotic II spindle from aged mouse oocytes used for analysis. Automated spot detection of 40 kinetochores (white spheres) is performed in each example. PSSCs (center panel), indicated by asterisks, are those in which more than two errors are detected. TALhesin[mmMajSat]A / B was injected with 0.05 amol mRNA (right panel). Microtubules are white. The scale bar is 3 μm. (G) Frequency of PSSCs in untreated oocytes from young mice (blank bars), untreated oocytes from aged mice (filled bars), and aged oocytes treated with TALhesin[mmMajSat]A / B mRNA (gray bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars represent 95% CI, 1 degree of freedom. (H) Reduction in PSSCs in aged mouse oocytes microinjected with TALhesin[mmMajSat]mRNA compared to untreated young and aged oocytes.(I) Percentage of oocytes from aged mice treated with TALhesin[mmMajSat] that have 0 (blank bar), 1 (light gray bar), 2 (dark gray bar), or more than 2 (filled bar) PSSC errors, compared to untreated young and aged oocytes. The number of oocytes analyzed is shown below for each condition. (J) Increase in error-free oocytes from aged mice treated with TALhesin[mmMajSat] mRNA, compared to untreated oocytes from young and aged mice. [Figure 3J]The artificial complex (TALhesin) induces condensation in mouse chromosomes. (A) Schematic diagram of the results of depletion of Rec8 by trim-away in Figure 3b. (B) Depletion of Rec8 by trim-away in young mouse oocytes injected with only 2 amol of TALhesin[mmMajSat]-B component (upper panel). Depletion of Rec8 by trim-away in oocytes injected with 2 amol of TALhesin[mmMajSat]A / B (lower panel) (min is minutes). Min is minutes. Scale bar is 5 μm. (C) Schematic diagram of the results of depletion of TALhesin[mmMajSat] protein by trim-away in Figure 3d. (D) Depletion of TALhesin[mmMajSat] by anti-GFP trim-away (upper panel), or subsequent depletion of Rec8 (lower panel). Dashed lines indicate polar bodies in the field of view. hrs is hours. The scale bar is 5 μm. (E) Schematic diagram of the chromosomal error reduction assay in mouse oocytes. (F) Example of a meiotic II spindle from aged mouse oocytes used for analysis. Automated spot detection of 40 kinetochores (white spheres) is performed in each example. PSSCs (center panel), indicated by asterisks, are those in which more than two errors are detected. TALhesin[mmMajSat]A / B was injected with 0.05 amol mRNA (right panel). Microtubules are white. The scale bar is 3 μm. (G) Frequency of PSSCs in untreated oocytes from young mice (blank bars), untreated oocytes from aged mice (filled bars), and aged oocytes treated with TALhesin[mmMajSat]A / B mRNA (gray bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars represent 95% CI, 1 degree of freedom. (H) Reduction in PSSCs in aged mouse oocytes microinjected with TALhesin[mmMajSat]mRNA compared to untreated young and aged oocytes.(I) Percentage of oocytes from aged mice treated with TALhesin[mmMajSat] that have 0 (blank bar), 1 (light gray bar), 2 (dark gray bar), or more than 2 (filled bar) PSSC errors, compared to untreated young and aged oocytes. The number of oocytes analyzed is shown below for each condition. (J) Increase in error-free oocytes from aged mice treated with TALhesin[mmMajSat] mRNA, compared to untreated oocytes from young and aged mice.

[0013] [Figure 4A] The artificial complex (TALhesin) reduces PSSC errors in aged mice.

[0014] (A) Dextran Texas Red was used as a tracer for antibody injection in the Trim-Away assay. Mouse oocytes injected with or not injected with different amounts of dextran Texas Red (219-43.8 femtograms) (far right). 43.8 femtograms were used as tracers for the anti-Rec 8 Trim-Away assay. Dashed lines indicate cell membranes. Scale bars are 10 μm. (B)-(E) Chromosome error reduction assays for 13-month-old mouse oocytes. (F)-(I) Chromosome error reduction assays for 20-month-old mouse oocytes. (B), (F) Frequency of PSSCs in 13-month-old (B) or 20-month-old (F) oocytes from young mice (open bars) and aged mice (filled bars), as well as oocytes treated with TALhesin[mmMajSat]A / B mRNA (gray bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars represent a 95% confidence interval and 1 degree of freedom. (C) and (G) show the reduction in PSSC in oocytes from 13-month-old (C) or 20-month-old (G) mice treated with TALhesin[mmMajSat]A / B mRNA, compared to untreated oocytes from young and aged mice. (D) and (H) show the percentage of oocytes from 13-month-old (C) or 20-month-old (H) mice treated with TALhesin[mmMajSat] that have a PSSC error of zero (white bar), 1 (light gray bar), 2 (dark gray bar), or more than 2 (filled bar), compared to untreated oocytes from young and aged mice. The number of oocytes analyzed is shown below each condition. (E) and (I) show the increase in error-free oocytes from 13-month-old (E) or 20-month-old (I) mice treated with TALhesin[mmMajSat] mRNA, compared to untreated oocytes from young and aged mice. (J) to (L) show the interval between sister chromatid kinetochores (iKt) in oocytes from young mice (white bars), TALhesin[mmMajSat]-treated (light gray bars), or untreated (dark gray bars), 13-month-old (J), 17-month-old (K), and 20-month-old (L) mice.Statistical values ​​were measured using a two-sample t-test (assuming no equal variances). Black squares indicate the mean, and lines indicate the median for each condition. "mos." represents months. Bars represent standard deviation (SD). [Figure 4B]The artificial complex (TALhesin) reduces PSSC errors in aged mice. (A) Dextran Texas Red was used as a tracer for antibody injection in the Trim-Away assay. Mouse oocytes injected with or not injected with different amounts of dextran Texas Red (219-43.8 femtograms) (far right). 43.8 femtograms were used as a tracer for the anti-Rec 8 Trim-Away assay. The dashed line indicates the cell membrane. The scale bar is 10 μm. (B)-(E) Chromosome error reduction assays for 13-month-old mouse oocytes. (F)-(I) Chromosome error reduction assays for 20-month-old mouse oocytes. (B) and (F) show the frequency of PSSCs in untreated oocytes from young mice (white bars) and aged mice (filled bars), as well as in oocytes from 13-month-old (B) or 20-month-old (F) mice treated with TALhesin[mmMajSat]A / B mRNA (gray bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars have a 95% confidence interval and 1 degree of freedom. (C) and (G) show the reduction in PSSCs in oocytes from 13-month-old (C) or 20-month-old (G) mice treated with TALhesin[mmMajSat]A / B mRNA compared to untreated oocytes from young and aged mice. (D) and (H) represent the percentage of oocytes from 13-month-old (C) or 20-month-old (H) mice treated with TALhesin[mmMajSat] that had a PSSC error of zero (white bar), 1 (light gray bar), 2 (dark gray bar), or more than 2 (filled bar), compared to untreated oocytes from young and aged mice. The number of oocytes analyzed is shown below for each condition. (E) and (I) represent the increase in error-free oocytes from 13-month-old (E) or 20-month-old (I) mice treated with TALhesin[mmMajSat] mRNA, compared to untreated oocytes from young and aged mice.(J)~(L) represents the interval (iKt) between sister chromatid kinetochores in oocytes from young mice (white bars), and mice treated with TALhesin[mmMajSat] (light gray bars), or untreated mice (dark gray bars), at 13 months (J), 17 months (K), and 20 months (L). Statistical values ​​were measured by a two-sample t-test (equal variances not assumed). Black squares indicate the mean, and lines indicate the median for each condition. "mos." represents months. Bars represent standard deviation (SD). [Figure 4C]The artificial complex (TALhesin) reduces PSSC errors in aged mice. (A) Dextran Texas Red was used as a tracer for antibody injection in the Trim-Away assay. Mouse oocytes injected with or not injected with different amounts of dextran Texas Red (219-43.8 femtograms) (far right). 43.8 femtograms were used as a tracer for the anti-Rec 8 Trim-Away assay. The dashed line indicates the cell membrane. The scale bar is 10 μm. (B)-(E) Chromosome error reduction assays for 13-month-old mouse oocytes. (F)-(I) Chromosome error reduction assays for 20-month-old mouse oocytes. (B) and (F) show the frequency of PSSCs in untreated oocytes from young mice (white bars) and aged mice (filled bars), as well as in oocytes from 13-month-old (B) or 20-month-old (F) mice treated with TALhesin[mmMajSat]A / B mRNA (gray bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars have a 95% confidence interval and 1 degree of freedom. (C) and (G) show the reduction in PSSCs in oocytes from 13-month-old (C) or 20-month-old (G) mice treated with TALhesin[mmMajSat]A / B mRNA compared to untreated oocytes from young and aged mice. (D) and (H) represent the percentage of oocytes from 13-month-old (C) or 20-month-old (H) mice treated with TALhesin[mmMajSat] that had a PSSC error of zero (white bar), 1 (light gray bar), 2 (dark gray bar), or more than 2 (filled bar), compared to untreated oocytes from young and aged mice. The number of oocytes analyzed is shown below for each condition. (E) and (I) represent the increase in error-free oocytes from 13-month-old (E) or 20-month-old (I) mice treated with TALhesin[mmMajSat] mRNA, compared to untreated oocytes from young and aged mice.(J)~(L) represents the interval (iKt) between sister chromatid kinetochores in oocytes from young mice (white bars), and mice treated with TALhesin[mmMajSat] (light gray bars), or untreated mice (dark gray bars), at 13 months (J), 17 months (K), and 20 months (L). Statistical values ​​were measured by a two-sample t-test (equal variances not assumed). Black squares indicate the mean, and lines indicate the median for each condition. "mos." represents months. Bars represent standard deviation (SD). [Figure 4D]The artificial complex (TALhesin) reduces PSSC errors in aged mice. (A) Dextran Texas Red was used as a tracer for antibody injection in the Trim-Away assay. Mouse oocytes injected with or not injected with different amounts of dextran Texas Red (219-43.8 femtograms) (far right). 43.8 femtograms were used as a tracer for the anti-Rec 8 Trim-Away assay. The dashed line indicates the cell membrane. The scale bar is 10 μm. (B)-(E) Chromosome error reduction assays for 13-month-old mouse oocytes. (F)-(I) Chromosome error reduction assays for 20-month-old mouse oocytes. (B) and (F) show the frequency of PSSCs in untreated oocytes from young mice (white bars) and aged mice (filled bars), as well as in oocytes from 13-month-old (B) or 20-month-old (F) mice treated with TALhesin[mmMajSat]A / B mRNA (gray bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars have a 95% confidence interval and 1 degree of freedom. (C) and (G) show the reduction in PSSCs in oocytes from 13-month-old (C) or 20-month-old (G) mice treated with TALhesin[mmMajSat]A / B mRNA compared to untreated oocytes from young and aged mice. (D) and (H) represent the percentage of oocytes from 13-month-old (C) or 20-month-old (H) mice treated with TALhesin[mmMajSat] that had a PSSC error of zero (white bar), 1 (light gray bar), 2 (dark gray bar), or more than 2 (filled bar), compared to untreated oocytes from young and aged mice. The number of oocytes analyzed is shown below for each condition. (E) and (I) represent the increase in error-free oocytes from 13-month-old (E) or 20-month-old (I) mice treated with TALhesin[mmMajSat] mRNA, compared to untreated oocytes from young and aged mice.(J)~(L) represents the interval (iKt) between sister chromatid kinetochores in oocytes from young mice (white bars), and mice treated with TALhesin[mmMajSat] (light gray bars), or untreated mice (dark gray bars), at 13 months (J), 17 months (K), and 20 months (L). Statistical values ​​were measured by a two-sample t-test (equal variances not assumed). Black squares indicate the mean, and lines indicate the median for each condition. "mos." represents months. Bars represent standard deviation (SD). [Figure 4E]The artificial complex (TALhesin) reduces PSSC errors in aged mice. (A) Dextran Texas Red was used as a tracer for antibody injection in the Trim-Away assay. Mouse oocytes injected with or not injected with different amounts of dextran Texas Red (219-43.8 femtograms) (far right). 43.8 femtograms were used as a tracer for the anti-Rec 8 Trim-Away assay. The dashed line indicates the cell membrane. The scale bar is 10 μm. (B)-(E) Chromosome error reduction assays for 13-month-old mouse oocytes. (F)-(I) Chromosome error reduction assays for 20-month-old mouse oocytes. (B) and (F) show the frequency of PSSCs in untreated oocytes from young mice (white bars) and aged mice (filled bars), as well as in oocytes from 13-month-old (B) or 20-month-old (F) mice treated with TALhesin[mmMajSat]A / B mRNA (gray bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars have a 95% confidence interval and 1 degree of freedom. (C) and (G) show the reduction in PSSCs in oocytes from 13-month-old (C) or 20-month-old (G) mice treated with TALhesin[mmMajSat]A / B mRNA compared to untreated oocytes from young and aged mice. (D) and (H) represent the percentage of oocytes from 13-month-old (C) or 20-month-old (H) mice treated with TALhesin[mmMajSat] that had a PSSC error of zero (white bar), 1 (light gray bar), 2 (dark gray bar), or more than 2 (filled bar), compared to untreated oocytes from young and aged mice. The number of oocytes analyzed is shown below for each condition. (E) and (I) represent the increase in error-free oocytes from 13-month-old (E) or 20-month-old (I) mice treated with TALhesin[mmMajSat] mRNA, compared to untreated oocytes from young and aged mice.(J)~(L) represents the interval (iKt) between sister chromatid kinetochores in oocytes from young mice (white bars), and mice treated with TALhesin[mmMajSat] (light gray bars), or untreated mice (dark gray bars), at 13 months (J), 17 months (K), and 20 months (L). Statistical values ​​were measured by a two-sample t-test (equal variances not assumed). Black squares indicate the mean, and lines indicate the median for each condition. "mos." represents months. Bars represent standard deviation (SD). [Figure 4F]The artificial complex (TALhesin) reduces PSSC errors in aged mice. (A) Dextran Texas Red was used as a tracer for antibody injection in the Trim-Away assay. Mouse oocytes injected with or not injected with different amounts of dextran Texas Red (219-43.8 femtograms) (far right). 43.8 femtograms were used as a tracer for the anti-Rec 8 Trim-Away assay. The dashed line indicates the cell membrane. The scale bar is 10 μm. (B)-(E) Chromosome error reduction assays for 13-month-old mouse oocytes. (F)-(I) Chromosome error reduction assays for 20-month-old mouse oocytes. (B) and (F) show the frequency of PSSCs in untreated oocytes from young mice (white bars) and aged mice (filled bars), as well as in oocytes from 13-month-old (B) or 20-month-old (F) mice treated with TALhesin[mmMajSat]A / B mRNA (gray bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars have a 95% confidence interval and 1 degree of freedom. (C) and (G) show the reduction in PSSCs in oocytes from 13-month-old (C) or 20-month-old (G) mice treated with TALhesin[mmMajSat]A / B mRNA compared to untreated oocytes from young and aged mice. (D) and (H) represent the percentage of oocytes from 13-month-old (C) or 20-month-old (H) mice treated with TALhesin[mmMajSat] that had a PSSC error of zero (white bar), 1 (light gray bar), 2 (dark gray bar), or more than 2 (filled bar), compared to untreated oocytes from young and aged mice. The number of oocytes analyzed is shown below for each condition. (E) and (I) represent the increase in error-free oocytes from 13-month-old (E) or 20-month-old (I) mice treated with TALhesin[mmMajSat] mRNA, compared to untreated oocytes from young and aged mice.(J)~(L) represents the interval (iKt) between sister chromatid kinetochores in oocytes from young mice (white bars), and mice treated with TALhesin[mmMajSat] (light gray bars), or untreated mice (dark gray bars), at 13 months (J), 17 months (K), and 20 months (L). Statistical values ​​were measured by a two-sample t-test (equal variances not assumed). Black squares indicate the mean, and lines indicate the median for each condition. "mos." represents months. Bars represent standard deviation (SD). [Figure 4G]The artificial complex (TALhesin) reduces PSSC errors in aged mice. (A) Dextran Texas Red was used as a tracer for antibody injection in the Trim-Away assay. Mouse oocytes injected with or not injected with different amounts of dextran Texas Red (219-43.8 femtograms) (far right). 43.8 femtograms were used as a tracer for the anti-Rec 8 Trim-Away assay. The dashed line indicates the cell membrane. The scale bar is 10 μm. (B)-(E) Chromosome error reduction assays for 13-month-old mouse oocytes. (F)-(I) Chromosome error reduction assays for 20-month-old mouse oocytes. (B) and (F) show the frequency of PSSCs in untreated oocytes from young mice (white bars) and aged mice (filled bars), as well as in oocytes from 13-month-old (B) or 20-month-old (F) mice treated with TALhesin[mmMajSat]A / B mRNA (gray bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars have a 95% confidence interval and 1 degree of freedom. (C) and (G) show the reduction in PSSCs in oocytes from 13-month-old (C) or 20-month-old (G) mice treated with TALhesin[mmMajSat]A / B mRNA compared to untreated oocytes from young and aged mice. (D) and (H) represent the percentage of oocytes from 13-month-old (C) or 20-month-old (H) mice treated with TALhesin[mmMajSat] that had a PSSC error of zero (white bar), 1 (light gray bar), 2 (dark gray bar), or more than 2 (filled bar), compared to untreated oocytes from young and aged mice. The number of oocytes analyzed is shown below for each condition. (E) and (I) represent the increase in error-free oocytes from 13-month-old (E) or 20-month-old (I) mice treated with TALhesin[mmMajSat] mRNA, compared to untreated oocytes from young and aged mice.(J)~(L) represents the interval (iKt) between sister chromatid kinetochores in oocytes from young mice (white bars), and mice treated with TALhesin[mmMajSat] (light gray bars), or untreated mice (dark gray bars), at 13 months (J), 17 months (K), and 20 months (L). Statistical values ​​were measured by a two-sample t-test (equal variances not assumed). Black squares indicate the mean, and lines indicate the median for each condition. "mos." represents months. Bars represent standard deviation (SD). [Figure 4H]The artificial complex (TALhesin) reduces PSSC errors in aged mice. (A) Dextran Texas Red was used as a tracer for antibody injection in the Trim-Away assay. Mouse oocytes injected with or not injected with different amounts of dextran Texas Red (219-43.8 femtograms) (far right). 43.8 femtograms were used as a tracer for the anti-Rec 8 Trim-Away assay. The dashed line indicates the cell membrane. The scale bar is 10 μm. (B)-(E) Chromosome error reduction assays for 13-month-old mouse oocytes. (F)-(I) Chromosome error reduction assays for 20-month-old mouse oocytes. (B) and (F) show the frequency of PSSCs in untreated oocytes from young mice (white bars) and aged mice (filled bars), as well as in oocytes from 13-month-old (B) or 20-month-old (F) mice treated with TALhesin[mmMajSat]A / B mRNA (gray bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars have a 95% confidence interval and 1 degree of freedom. (C) and (G) show the reduction in PSSCs in oocytes from 13-month-old (C) or 20-month-old (G) mice treated with TALhesin[mmMajSat]A / B mRNA compared to untreated oocytes from young and aged mice. (D) and (H) represent the percentage of oocytes from 13-month-old (C) or 20-month-old (H) mice treated with TALhesin[mmMajSat] that had a PSSC error of zero (white bar), 1 (light gray bar), 2 (dark gray bar), or more than 2 (filled bar), compared to untreated oocytes from young and aged mice. The number of oocytes analyzed is shown below for each condition. (E) and (I) represent the increase in error-free oocytes from 13-month-old (E) or 20-month-old (I) mice treated with TALhesin[mmMajSat] mRNA, compared to untreated oocytes from young and aged mice.(J)~(L) represents the interval (iKt) between sister chromatid kinetochores in oocytes from young mice (white bars), and mice treated with TALhesin[mmMajSat] (light gray bars), or untreated mice (dark gray bars), at 13 months (J), 17 months (K), and 20 months (L). Statistical values ​​were measured by a two-sample t-test (equal variances not assumed). Black squares indicate the mean, and lines indicate the median for each condition. "mos." represents months. Bars represent standard deviation (SD). [Figure 4I]The artificial complex (TALhesin) reduces PSSC errors in aged mice. (A) Dextran Texas Red was used as a tracer for antibody injection in the Trim-Away assay. Mouse oocytes injected with or not injected with different amounts of dextran Texas Red (219-43.8 femtograms) (far right). 43.8 femtograms were used as a tracer for the anti-Rec 8 Trim-Away assay. The dashed line indicates the cell membrane. The scale bar is 10 μm. (B)-(E) Chromosome error reduction assays for 13-month-old mouse oocytes. (F)-(I) Chromosome error reduction assays for 20-month-old mouse oocytes. (B) and (F) show the frequency of PSSCs in untreated oocytes from young mice (white bars) and aged mice (filled bars), as well as in oocytes from 13-month-old (B) or 20-month-old (F) mice treated with TALhesin[mmMajSat]A / B mRNA (gray bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars have a 95% confidence interval and 1 degree of freedom. (C) and (G) show the reduction in PSSCs in oocytes from 13-month-old (C) or 20-month-old (G) mice treated with TALhesin[mmMajSat]A / B mRNA compared to untreated oocytes from young and aged mice. (D) and (H) represent the percentage of oocytes from 13-month-old (C) or 20-month-old (H) mice treated with TALhesin[mmMajSat] that had a PSSC error of zero (white bar), 1 (light gray bar), 2 (dark gray bar), or more than 2 (filled bar), compared to untreated oocytes from young and aged mice. The number of oocytes analyzed is shown below for each condition. (E) and (I) represent the increase in error-free oocytes from 13-month-old (E) or 20-month-old (I) mice treated with TALhesin[mmMajSat] mRNA, compared to untreated oocytes from young and aged mice.(J)~(L) represents the interval (iKt) between sister chromatid kinetochores in oocytes from young mice (white bars), and mice treated with TALhesin[mmMajSat] (light gray bars), or untreated mice (dark gray bars), at 13 months (J), 17 months (K), and 20 months (L). Statistical values ​​were measured by a two-sample t-test (equal variances not assumed). Black squares indicate the mean, and lines indicate the median for each condition. "mos." represents months. Bars represent standard deviation (SD). [Figure 4J]The artificial complex (TALhesin) reduces PSSC errors in aged mice. (A) Dextran Texas Red was used as a tracer for antibody injection in the Trim-Away assay. Mouse oocytes injected with or not injected with different amounts of dextran Texas Red (219-43.8 femtograms) (far right). 43.8 femtograms were used as a tracer for the anti-Rec 8 Trim-Away assay. The dashed line indicates the cell membrane. The scale bar is 10 μm. (B)-(E) Chromosome error reduction assays for 13-month-old mouse oocytes. (F)-(I) Chromosome error reduction assays for 20-month-old mouse oocytes. (B) and (F) show the frequency of PSSCs in untreated oocytes from young mice (white bars) and aged mice (filled bars), as well as in oocytes from 13-month-old (B) or 20-month-old (F) mice treated with TALhesin[mmMajSat]A / B mRNA (gray bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars have a 95% confidence interval and 1 degree of freedom. (C) and (G) show the reduction in PSSCs in oocytes from 13-month-old (C) or 20-month-old (G) mice treated with TALhesin[mmMajSat]A / B mRNA compared to untreated oocytes from young and aged mice. (D) and (H) represent the percentage of oocytes from 13-month-old (C) or 20-month-old (H) mice treated with TALhesin[mmMajSat] that had a PSSC error of zero (white bar), 1 (light gray bar), 2 (dark gray bar), or more than 2 (filled bar), compared to untreated oocytes from young and aged mice. The number of oocytes analyzed is shown below for each condition. (E) and (I) represent the increase in error-free oocytes from 13-month-old (E) or 20-month-old (I) mice treated with TALhesin[mmMajSat] mRNA, compared to untreated oocytes from young and aged mice.(J)~(L) represents the interval (iKt) between sister chromatid kinetochores in oocytes from young mice (white bars), and mice treated with TALhesin[mmMajSat] (light gray bars), or untreated mice (dark gray bars), at 13 months (J), 17 months (K), and 20 months (L). Statistical values ​​were measured by a two-sample t-test (equal variances not assumed). Black squares indicate the mean, and lines indicate the median for each condition. "mos." represents months. Bars represent standard deviation (SD). [Figure 4K]The artificial complex (TALhesin) reduces PSSC errors in aged mice. (A) Dextran Texas Red was used as a tracer for antibody injection in the Trim-Away assay. Mouse oocytes injected with or not injected with different amounts of dextran Texas Red (219-43.8 femtograms) (far right). 43.8 femtograms were used as a tracer for the anti-Rec 8 Trim-Away assay. The dashed line indicates the cell membrane. The scale bar is 10 μm. (B)-(E) Chromosome error reduction assays for 13-month-old mouse oocytes. (F)-(I) Chromosome error reduction assays for 20-month-old mouse oocytes. (B) and (F) show the frequency of PSSCs in untreated oocytes from young mice (white bars) and aged mice (filled bars), as well as in oocytes from 13-month-old (B) or 20-month-old (F) mice treated with TALhesin[mmMajSat]A / B mRNA (gray bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars have a 95% confidence interval and 1 degree of freedom. (C) and (G) show the reduction in PSSCs in oocytes from 13-month-old (C) or 20-month-old (G) mice treated with TALhesin[mmMajSat]A / B mRNA compared to untreated oocytes from young and aged mice. (D) and (H) represent the percentage of oocytes from 13-month-old (C) or 20-month-old (H) mice treated with TALhesin[mmMajSat] that had a PSSC error of zero (white bar), 1 (light gray bar), 2 (dark gray bar), or more than 2 (filled bar), compared to untreated oocytes from young and aged mice. The number of oocytes analyzed is shown below for each condition. (E) and (I) represent the increase in error-free oocytes from 13-month-old (E) or 20-month-old (I) mice treated with TALhesin[mmMajSat] mRNA, compared to untreated oocytes from young and aged mice.(J)~(L) represents the interval (iKt) between sister chromatid kinetochores in oocytes from young mice (white bars), and mice treated with TALhesin[mmMajSat] (light gray bars), or untreated mice (dark gray bars), at 13 months (J), 17 months (K), and 20 months (L). Statistical values ​​were measured by a two-sample t-test (equal variances not assumed). Black squares indicate the mean, and lines indicate the median for each condition. "mos." represents months. Bars represent standard deviation (SD). [Figure 4L]The artificial complex (TALhesin) reduces PSSC errors in aged mice. (A) Dextran Texas Red was used as a tracer for antibody injection in the Trim-Away assay. Mouse oocytes injected with or not injected with different amounts of dextran Texas Red (219-43.8 femtograms) (far right). 43.8 femtograms were used as a tracer for the anti-Rec 8 Trim-Away assay. The dashed line indicates the cell membrane. The scale bar is 10 μm. (B)-(E) Chromosome error reduction assays for 13-month-old mouse oocytes. (F)-(I) Chromosome error reduction assays for 20-month-old mouse oocytes. (B) and (F) show the frequency of PSSCs in untreated oocytes from young mice (white bars) and aged mice (filled bars), as well as in oocytes from 13-month-old (B) or 20-month-old (F) mice treated with TALhesin[mmMajSat]A / B mRNA (gray bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars have a 95% confidence interval and 1 degree of freedom. (C) and (G) show the reduction in PSSCs in oocytes from 13-month-old (C) or 20-month-old (G) mice treated with TALhesin[mmMajSat]A / B mRNA compared to untreated oocytes from young and aged mice. (D) and (H) represent the percentage of oocytes from 13-month-old (C) or 20-month-old (H) mice treated with TALhesin[mmMajSat] that had a PSSC error of zero (white bar), 1 (light gray bar), 2 (dark gray bar), or more than 2 (filled bar), compared to untreated oocytes from young and aged mice. The number of oocytes analyzed is shown below for each condition. (E) and (I) represent the increase in error-free oocytes from 13-month-old (E) or 20-month-old (I) mice treated with TALhesin[mmMajSat] mRNA, compared to untreated oocytes from young and aged mice.(J)~(L) represents the interval (iKt) between sister chromatid kinetochores in oocytes from young mice (white bars), and mice treated with TALhesin[mmMajSat] (light gray bars), or untreated mice (dark gray bars), at 13 months (J), 17 months (K), and 20 months (L). Statistical values ​​were measured by a two-sample t-test (equal variances not assumed). Black squares indicate the mean, and lines indicate the median for each condition. "mos." represents months. Bars represent standard deviation (SD).

[0015] [Figure 5A]The artificial complex (TALhesin) provides condensation to human chromosomes. (A) Representative images of mitotic HeLa cells lacking visible DNA crosslinks (upper panel), or representative images of mitotic HeLa cells with obvious DNA crosslinks (lower panel, Figure 5B). Arrows indicate TALhesin[hsCentro] focus on human chromosomes. Scale bar is 5 μm. (B) TALhesin[hsCentro] coexists with DNA crosslinking the nuclei of two daughter cells expressing TALhesin[hsCentro]A / B. Scale bar is 5 μm. (C) Quantification of daughter cells linked by DNA crosslinking in untransfected HeLa cells (white bars), HeLa cells transfected with TALhesin[hsCentro]-B only (gray bars), or HeLa cells transfected with TALhesin[hsCentro]A / B (filled bars). The number in parentheses indicates the number of daughter cell pairs analyzed for each condition. Statistical values ​​were measured by a two-sample t-test with Bonferroni correction (equal variances not assumed). Bars represent SEM. (D) Schematic diagram showing treatment of human oocytes with TALhesin[hsCentro]. (E) Localization of TALhesin[hsCentro] to human meiotic I chromosomes (upper panel) and meiotic II chromosomes (lower panel). Kinetochores, microtubules, and chromosomes. Scale bar is 5 μm. (F) Enlarged view of the chromosome enclosed in the rectangle in Figure 5e. TALhesin[hsCentro] is localized to the centromere region of human chromosomes in meiotic I oocytes (upper) and meiotic II oocytes (lower). Scale bar is 2 μm. (G) Mean fluorescence intensity (MFI) of normalized TALhesin[hsCentro] of chromosome pairs and prematurely separated sister chromatids (PSSCs) in human meiotic II oocytes. (H) Normalized TALhesin[hsCentro]MFI of intact human chromosomes and PSSCs grouped according to the interval between the smallest and largest kinetochores. For (G) and (H), the number of sister kinetochore pairs n analyzed is shown under each condition. The total number of cells analyzed is 24. Statistical values ​​were measured by a two-sample t-test (equal variances not assumed). mos. is in months.The bar is SD. [Figure 5B]The artificial complex (TALhesin) provides condensation to human chromosomes. (A) Representative images of mitotic HeLa cells lacking visible DNA crosslinks (upper panel), or representative images of mitotic HeLa cells with obvious DNA crosslinks (lower panel, Figure 5B). Arrows indicate TALhesin[hsCentro] focus on human chromosomes. Scale bar is 5 μm. (B) TALhesin[hsCentro] coexists with DNA crosslinking the nuclei of two daughter cells expressing TALhesin[hsCentro]A / B. Scale bar is 5 μm. (C) Quantification of daughter cells linked by DNA crosslinking in untransfected HeLa cells (white bars), HeLa cells transfected with TALhesin[hsCentro]-B only (gray bars), or HeLa cells transfected with TALhesin[hsCentro]A / B (filled bars). The number in parentheses indicates the number of daughter cell pairs analyzed for each condition. Statistical values ​​were measured by a two-sample t-test with Bonferroni correction (equal variances not assumed). Bars represent SEM. (D) Schematic diagram showing treatment of human oocytes with TALhesin[hsCentro]. (E) Localization of TALhesin[hsCentro] to human meiotic I chromosomes (upper panel) and meiotic II chromosomes (lower panel). Kinetochores, microtubules, and chromosomes. Scale bar is 5 μm. (F) Enlarged view of the chromosome enclosed in the rectangle in Figure 5e. TALhesin[hsCentro] is localized to the centromere region of human chromosomes in meiotic I oocytes (upper) and meiotic II oocytes (lower). Scale bar is 2 μm. (G) Mean fluorescence intensity (MFI) of normalized TALhesin[hsCentro] of chromosome pairs and prematurely separated sister chromatids (PSSCs) in human meiotic II oocytes. (H) Normalized TALhesin[hsCentro]MFI of intact human chromosomes and PSSCs grouped according to the interval between the smallest and largest kinetochores. For (G) and (H), the number of sister kinetochore pairs n analyzed is shown under each condition. The total number of cells analyzed is 24. Statistical values ​​were measured by a two-sample t-test (equal variances not assumed). mos. is in months.The bar is SD. [Figure 5C]The artificial complex (TALhesin) provides condensation to human chromosomes. (A) Representative images of mitotic HeLa cells lacking visible DNA crosslinks (upper panel), or representative images of mitotic HeLa cells with obvious DNA crosslinks (lower panel, Figure 5B). Arrows indicate TALhesin[hsCentro] focus on human chromosomes. Scale bar is 5 μm. (B) TALhesin[hsCentro] coexists with DNA crosslinking the nuclei of two daughter cells expressing TALhesin[hsCentro]A / B. Scale bar is 5 μm. (C) Quantification of daughter cells linked by DNA crosslinking in untransfected HeLa cells (white bars), HeLa cells transfected with TALhesin[hsCentro]-B only (gray bars), or HeLa cells transfected with TALhesin[hsCentro]A / B (filled bars). The number in parentheses indicates the number of daughter cell pairs analyzed for each condition. Statistical values ​​were measured by a two-sample t-test with Bonferroni correction (equal variances not assumed). Bars represent SEM. (D) Schematic diagram showing treatment of human oocytes with TALhesin[hsCentro]. (E) Localization of TALhesin[hsCentro] to human meiotic I chromosomes (upper panel) and meiotic II chromosomes (lower panel). Kinetochores, microtubules, and chromosomes. Scale bar is 5 μm. (F) Enlarged view of the chromosome enclosed in the rectangle in Figure 5e. TALhesin[hsCentro] is localized to the centromere region of human chromosomes in meiotic I oocytes (upper) and meiotic II oocytes (lower). Scale bar is 2 μm. (G) Mean fluorescence intensity (MFI) of normalized TALhesin[hsCentro] of chromosome pairs and prematurely separated sister chromatids (PSSCs) in human meiotic II oocytes. (H) Normalized TALhesin[hsCentro]MFI of intact human chromosomes and PSSCs grouped according to the interval between the smallest and largest kinetochores. For (G) and (H), the number of sister kinetochore pairs n analyzed is shown under each condition. The total number of cells analyzed is 24. Statistical values ​​were measured by a two-sample t-test (equal variances not assumed). mos. is in months.The bar is SD. [Figure 5D]The artificial complex (TALhesin) provides condensation to human chromosomes. (A) Representative images of mitotic HeLa cells lacking visible DNA crosslinks (upper panel), or representative images of mitotic HeLa cells with obvious DNA crosslinks (lower panel, Figure 5B). Arrows indicate TALhesin[hsCentro] focus on human chromosomes. Scale bar is 5 μm. (B) TALhesin[hsCentro] coexists with DNA crosslinking the nuclei of two daughter cells expressing TALhesin[hsCentro]A / B. Scale bar is 5 μm. (C) Quantification of daughter cells linked by DNA crosslinking in untransfected HeLa cells (white bars), HeLa cells transfected with TALhesin[hsCentro]-B only (gray bars), or HeLa cells transfected with TALhesin[hsCentro]A / B (filled bars). The number in parentheses indicates the number of daughter cell pairs analyzed for each condition. Statistical values ​​were measured by a two-sample t-test with Bonferroni correction (equal variances not assumed). Bars represent SEM. (D) Schematic diagram showing treatment of human oocytes with TALhesin[hsCentro]. (E) Localization of TALhesin[hsCentro] to human meiotic I chromosomes (upper panel) and meiotic II chromosomes (lower panel). Kinetochores, microtubules, and chromosomes. Scale bar is 5 μm. (F) Enlarged view of the chromosome enclosed in the rectangle in Figure 5e. TALhesin[hsCentro] is localized to the centromere region of human chromosomes in meiotic I oocytes (upper) and meiotic II oocytes (lower). Scale bar is 2 μm. (G) Mean fluorescence intensity (MFI) of normalized TALhesin[hsCentro] of chromosome pairs and prematurely separated sister chromatids (PSSCs) in human meiotic II oocytes. (H) Normalized TALhesin[hsCentro]MFI of intact human chromosomes and PSSCs grouped according to the interval between the smallest and largest kinetochores. For (G) and (H), the number of sister kinetochore pairs n analyzed is shown under each condition. The total number of cells analyzed is 24. Statistical values ​​were measured by a two-sample t-test (equal variances not assumed). mos. is in months.The bar is SD. [Figure 5E]The artificial complex (TALhesin) provides condensation to human chromosomes. (A) Representative images of mitotic HeLa cells lacking visible DNA crosslinks (upper panel), or representative images of mitotic HeLa cells with obvious DNA crosslinks (lower panel, Figure 5B). Arrows indicate TALhesin[hsCentro] focus on human chromosomes. Scale bar is 5 μm. (B) TALhesin[hsCentro] coexists with DNA crosslinking the nuclei of two daughter cells expressing TALhesin[hsCentro]A / B. Scale bar is 5 μm. (C) Quantification of daughter cells linked by DNA crosslinking in untransfected HeLa cells (white bars), HeLa cells transfected with TALhesin[hsCentro]-B only (gray bars), or HeLa cells transfected with TALhesin[hsCentro]A / B (filled bars). The number in parentheses indicates the number of daughter cell pairs analyzed for each condition. Statistical values ​​were measured by a two-sample t-test with Bonferroni correction (equal variances not assumed). Bars represent SEM. (D) Schematic diagram showing treatment of human oocytes with TALhesin[hsCentro]. (E) Localization of TALhesin[hsCentro] to human meiotic I chromosomes (upper panel) and meiotic II chromosomes (lower panel). Kinetochores, microtubules, and chromosomes. Scale bar is 5 μm. (F) Enlarged view of the chromosome enclosed in the rectangle in Figure 5e. TALhesin[hsCentro] is localized to the centromere region of human chromosomes in meiotic I oocytes (upper) and meiotic II oocytes (lower). Scale bar is 2 μm. (G) Mean fluorescence intensity (MFI) of normalized TALhesin[hsCentro] of chromosome pairs and prematurely separated sister chromatids (PSSCs) in human meiotic II oocytes. (H) Normalized TALhesin[hsCentro]MFI of intact human chromosomes and PSSCs grouped according to the interval between the smallest and largest kinetochores. For (G) and (H), the number of sister kinetochore pairs n analyzed is shown under each condition. The total number of cells analyzed is 24. Statistical values ​​were measured by a two-sample t-test (equal variances not assumed). mos. is in months.The bar is SD. [Figure 5F]The artificial complex (TALhesin) provides condensation to human chromosomes. (A) Representative images of mitotic HeLa cells lacking visible DNA crosslinks (upper panel), or representative images of mitotic HeLa cells with obvious DNA crosslinks (lower panel, Figure 5B). Arrows indicate TALhesin[hsCentro] focus on human chromosomes. Scale bar is 5 μm. (B) TALhesin[hsCentro] coexists with DNA crosslinking the nuclei of two daughter cells expressing TALhesin[hsCentro]A / B. Scale bar is 5 μm. (C) Quantification of daughter cells linked by DNA crosslinking in untransfected HeLa cells (white bars), HeLa cells transfected with TALhesin[hsCentro]-B only (gray bars), or HeLa cells transfected with TALhesin[hsCentro]A / B (filled bars). The number in parentheses indicates the number of daughter cell pairs analyzed for each condition. Statistical values ​​were measured by a two-sample t-test with Bonferroni correction (equal variances not assumed). Bars represent SEM. (D) Schematic diagram showing treatment of human oocytes with TALhesin[hsCentro]. (E) Localization of TALhesin[hsCentro] to human meiotic I chromosomes (upper panel) and meiotic II chromosomes (lower panel). Kinetochores, microtubules, and chromosomes. Scale bar is 5 μm. (F) Enlarged view of the chromosome enclosed in the rectangle in Figure 5e. TALhesin[hsCentro] is localized to the centromere region of human chromosomes in meiotic I oocytes (upper) and meiotic II oocytes (lower). Scale bar is 2 μm. (G) Mean fluorescence intensity (MFI) of normalized TALhesin[hsCentro] of chromosome pairs and prematurely separated sister chromatids (PSSCs) in human meiotic II oocytes. (H) Normalized TALhesin[hsCentro]MFI of intact human chromosomes and PSSCs grouped according to the interval between the smallest and largest kinetochores. For (G) and (H), the number of sister kinetochore pairs n analyzed is shown under each condition. The total number of cells analyzed is 24. Statistical values ​​were measured by a two-sample t-test (equal variances not assumed). mos. is in months.The bar is SD. [Figure 5G]The artificial complex (TALhesin) provides condensation to human chromosomes. (A) Representative images of mitotic HeLa cells lacking visible DNA crosslinks (upper panel), or representative images of mitotic HeLa cells with obvious DNA crosslinks (lower panel, Figure 5B). Arrows indicate TALhesin[hsCentro] focus on human chromosomes. Scale bar is 5 μm. (B) TALhesin[hsCentro] coexists with DNA crosslinking the nuclei of two daughter cells expressing TALhesin[hsCentro]A / B. Scale bar is 5 μm. (C) Quantification of daughter cells linked by DNA crosslinking in untransfected HeLa cells (white bars), HeLa cells transfected with TALhesin[hsCentro]-B only (gray bars), or HeLa cells transfected with TALhesin[hsCentro]A / B (filled bars). The number in parentheses indicates the number of daughter cell pairs analyzed for each condition. Statistical values ​​were measured by a two-sample t-test with Bonferroni correction (equal variances not assumed). Bars represent SEM. (D) Schematic diagram showing treatment of human oocytes with TALhesin[hsCentro]. (E) Localization of TALhesin[hsCentro] to human meiotic I chromosomes (upper panel) and meiotic II chromosomes (lower panel). Kinetochores, microtubules, and chromosomes. Scale bar is 5 μm. (F) Enlarged view of the chromosome enclosed in the rectangle in Figure 5e. TALhesin[hsCentro] is localized to the centromere region of human chromosomes in meiotic I oocytes (upper) and meiotic II oocytes (lower). Scale bar is 2 μm. (G) Mean fluorescence intensity (MFI) of normalized TALhesin[hsCentro] of chromosome pairs and prematurely separated sister chromatids (PSSCs) in human meiotic II oocytes. (H) Normalized TALhesin[hsCentro]MFI of intact human chromosomes and PSSCs grouped according to the interval between the smallest and largest kinetochores. For (G) and (H), the number of sister kinetochore pairs n analyzed is shown under each condition. The total number of cells analyzed is 24. Statistical values ​​were measured by a two-sample t-test (equal variances not assumed). mos. is in months.The bar is SD. [Figure 5H]The artificial complex (TALhesin) provides condensation to human chromosomes. (A) Representative images of mitotic HeLa cells lacking visible DNA crosslinks (upper panel), or representative images of mitotic HeLa cells with obvious DNA crosslinks (lower panel, Figure 5B). Arrows indicate TALhesin[hsCentro] focus on human chromosomes. Scale bar is 5 μm. (B) TALhesin[hsCentro] coexists with DNA crosslinking the nuclei of two daughter cells expressing TALhesin[hsCentro]A / B. Scale bar is 5 μm. (C) Quantification of daughter cells linked by DNA crosslinking in untransfected HeLa cells (white bars), HeLa cells transfected with TALhesin[hsCentro]-B only (gray bars), or HeLa cells transfected with TALhesin[hsCentro]A / B (filled bars). The number in parentheses indicates the number of daughter cell pairs analyzed for each condition. Statistical values ​​were measured by a two-sample t-test with Bonferroni correction (equal variances not assumed). Bars represent SEM. (D) Schematic diagram showing treatment of human oocytes with TALhesin[hsCentro]. (E) Localization of TALhesin[hsCentro] to human meiotic I chromosomes (upper panel) and meiotic II chromosomes (lower panel). Kinetochores, microtubules, and chromosomes. Scale bar is 5 μm. (F) Enlarged view of the chromosome enclosed in the rectangle in Figure 5e. TALhesin[hsCentro] is localized to the centromere region of human chromosomes in meiotic I oocytes (upper) and meiotic II oocytes (lower). Scale bar is 2 μm. (G) Mean fluorescence intensity (MFI) of normalized TALhesin[hsCentro] of chromosome pairs and prematurely separated sister chromatids (PSSCs) in human meiotic II oocytes. (H) Normalized TALhesin[hsCentro]MFI of intact human chromosomes and PSSCs grouped according to the interval between the smallest and largest kinetochores. For (G) and (H), the number of sister kinetochore pairs n analyzed is shown under each condition. The total number of cells analyzed is 24. Statistical values ​​were measured by a two-sample t-test (equal variances not assumed). mos. is in months.The bar is SD.

[0016] [Figure 6A] Artificial composite chromosomes (TALhesins) provide condensation to human chromosomes.

[0017] (A) Schematic diagram of the domain arrangement of TALhesin[hsCentro] with spy-catcher 003 targeting human centromere repeats used for expression in HeLa cells. (B) Schematic diagram of the domain arrangement of TALhesin[hsCentro] with spy-catcher 001 targeting human centromere repeats used for expression in human oocytes. (C) TALhesin[hsCentro]A / B (containing spy-catcher 001) mRNA used for human oocyte microinjection and analyzed by 1% agarose gel electrophoresis. Domain composition and expected size of each transcript are shown next to the annotated image with nucleotide (nt) values. (D) Age distribution of human oocyte donors. The number of cells from each age group is shown above each bar. (E) Normalized mean fluorescence intensity (MFI) of TALhesin[hsCentro] is inversely proportional to the normalized kinetochore spacing in treated human meiotic II oocytes. (F) In human meiotic II oocytes, chromosomes with high TALhesin[hsCentro]MFI have the lowest interkinetochore spacing. The n of sister kinetochore pairs is shown below for each condition. The total number of cells analyzed was 24. Statistical values ​​were measured by a two-sample t-test (equal variances not assumed). Bars represent SD. [Figure 6B]Artificial composite chromosomes (TALhesin) provide condensation to human chromosomes. (A) Schematic diagram of the domain arrangement of TALhesin[hsCentro] with spy-catcher 003 targeting human percentromere repeats used for expression in HeLa cells. (B) Schematic diagram of the domain arrangement of TALhesin[hsCentro] with spy-catcher 001 targeting human percentromere repeats used for expression in human oocytes. (C) TALhesin[hsCentro]A / B (containing spy-catcher 001) mRNA used for human oocyte microinjection and analyzed by 1% agarose gel electrophoresis. Domain composition and expected size of each transcript are shown next to the annotated image with nucleotide (nt) values. (D) Age distribution of human oocyte donors. The number of cells from each age group is shown above each bar. (E) The normalized mean fluorescence intensity (MFI) of TALhesin[hsCentro] is inversely proportional to the normalized interkinetochore spacing in treated human meiotic II oocytes. (F) In human meiotic II oocytes, chromosomes with high TALhesin[hsCentro] MFI have the lowest interkinetochore spacing. The n of sister kinetochore pairs is shown under each condition. The total number of cells analyzed was 24. Statistics were measured by a two-sample t-test (equal variances not assumed). Bars represent SD. [Figure 6C]Artificial composite chromosomes (TALhesin) provide condensation to human chromosomes. (A) Schematic diagram of the domain arrangement of TALhesin[hsCentro] with spy-catcher 003 targeting human percentromere repeats used for expression in HeLa cells. (B) Schematic diagram of the domain arrangement of TALhesin[hsCentro] with spy-catcher 001 targeting human percentromere repeats used for expression in human oocytes. (C) TALhesin[hsCentro]A / B (containing spy-catcher 001) mRNA used for human oocyte microinjection and analyzed by 1% agarose gel electrophoresis. Domain composition and expected size of each transcript are shown next to the annotated image with nucleotide (nt) values. (D) Age distribution of human oocyte donors. The number of cells from each age group is shown above each bar. (E) The normalized mean fluorescence intensity (MFI) of TALhesin[hsCentro] is inversely proportional to the normalized interkinetochore spacing in treated human meiotic II oocytes. (F) In human meiotic II oocytes, chromosomes with high TALhesin[hsCentro] MFI have the lowest interkinetochore spacing. The n of sister kinetochore pairs is shown under each condition. The total number of cells analyzed was 24. Statistics were measured by a two-sample t-test (equal variances not assumed). Bars represent SD. [Figure 6D]Artificial composite chromosomes (TALhesin) provide condensation to human chromosomes. (A) Schematic diagram of the domain arrangement of TALhesin[hsCentro] with spy-catcher 003 targeting human percentromere repeats used for expression in HeLa cells. (B) Schematic diagram of the domain arrangement of TALhesin[hsCentro] with spy-catcher 001 targeting human percentromere repeats used for expression in human oocytes. (C) TALhesin[hsCentro]A / B (containing spy-catcher 001) mRNA used for human oocyte microinjection and analyzed by 1% agarose gel electrophoresis. Domain composition and expected size of each transcript are shown next to the annotated image with nucleotide (nt) values. (D) Age distribution of human oocyte donors. The number of cells from each age group is shown above each bar. (E) The normalized mean fluorescence intensity (MFI) of TALhesin[hsCentro] is inversely proportional to the normalized interkinetochore spacing in treated human meiotic II oocytes. (F) In human meiotic II oocytes, chromosomes with high TALhesin[hsCentro] MFI have the lowest interkinetochore spacing. The n of sister kinetochore pairs is shown under each condition. The total number of cells analyzed was 24. Statistics were measured by a two-sample t-test (equal variances not assumed). Bars represent SD. [Figure 6E]Artificial composite chromosomes (TALhesin) provide condensation to human chromosomes. (A) Schematic diagram of the domain arrangement of TALhesin[hsCentro] with spy-catcher 003 targeting human percentromere repeats used for expression in HeLa cells. (B) Schematic diagram of the domain arrangement of TALhesin[hsCentro] with spy-catcher 001 targeting human percentromere repeats used for expression in human oocytes. (C) TALhesin[hsCentro]A / B (containing spy-catcher 001) mRNA used for human oocyte microinjection and analyzed by 1% agarose gel electrophoresis. Domain composition and expected size of each transcript are shown next to the annotated image with nucleotide (nt) values. (D) Age distribution of human oocyte donors. The number of cells from each age group is shown above each bar. (E) The normalized mean fluorescence intensity (MFI) of TALhesin[hsCentro] is inversely proportional to the normalized interkinetochore spacing in treated human meiotic II oocytes. (F) In human meiotic II oocytes, chromosomes with high TALhesin[hsCentro] MFI have the lowest interkinetochore spacing. The n of sister kinetochore pairs is shown under each condition. The total number of cells analyzed was 24. Statistics were measured by a two-sample t-test (equal variances not assumed). Bars represent SD. [Figure 6F]Artificial composite chromosomes (TALhesin) provide condensation to human chromosomes. (A) Schematic diagram of the domain arrangement of TALhesin[hsCentro] with spy-catcher 003 targeting human percentromere repeats used for expression in HeLa cells. (B) Schematic diagram of the domain arrangement of TALhesin[hsCentro] with spy-catcher 001 targeting human percentromere repeats used for expression in human oocytes. (C) TALhesin[hsCentro]A / B (containing spy-catcher 001) mRNA used for human oocyte microinjection and analyzed by 1% agarose gel electrophoresis. Domain composition and expected size of each transcript are shown next to the annotated image with nucleotide (nt) values. (D) Age distribution of human oocyte donors. The number of cells from each age group is shown above each bar. (E) The normalized mean fluorescence intensity (MFI) of TALhesin[hsCentro] is inversely proportional to the normalized interkinetochore spacing in treated human meiotic II oocytes. (F) In human meiotic II oocytes, chromosomes with high TALhesin[hsCentro] MFI have the lowest interkinetochore spacing. The n of sister kinetochore pairs is shown under each condition. The total number of cells analyzed was 24. Statistics were measured by a two-sample t-test (equal variances not assumed). Bars represent SD.

[0018] [Figure 7A] Separase-cleaved motifs release chromosome condensation mediated by artificial complexes.

[0019] (A) A schematic diagram (right, not drawn to scale) of the domain arrangement of TALhesin[mmMajSat](Rec8) with inactivation of TALhesin-mediated chromosome condensation (left) and mouse Rec8 fragments incorporated into two linker sites. (B)-(C) Live imaging of activated mice (B) or human oocytes (C) microinjected with mRNA encoding only TALhesin(Rec8)-B (top panel), mRNA encoding an uncleavable TALhesin (middle panel), or mRNA encoding a separase-cleavable TALhesin(Rec8) (bottom panel). The area enclosed by the rectangle is magnified below. Black arrows indicate non-segregation of chromosomes. White arrows indicate chromatid separation. TALhesins, chromosomes, min: minutes. Scale bar is 5 μm. (D)-(E) Duration (minutes) of anaphase II in activated mouse oocytes (D) or human oocytes (E) measured from the start to the completion of anaphase II. (F)~(G) Percentage of activated mouse oocytes (F) or human oocytes (G) in which chromosomes have not separated in late stage II. The atomol of injected mRNA is listed below for each condition. The number of cells analyzed for each condition is shown below. (H) Frequency of PSSCs in untreated oocytes from young mice (white bars), untreated oocytes from aged mice (filled bars), and aged oocytes treated with TALhesin[mmMajSat](Rec8)-A / B mRNA (gray bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars have a 95% confidence interval and 1 degree of freedom. [Figure 7B-01]Separase-cleavage motifs release chromosome condensation mediated by artificial complexes. (A) Inactivation of TALhesin-mediated chromosome condensation (left) and schematic diagram of the domain arrangement of TALhesin[mmMajSat](Rec8) with a mouse Rec8 fragment incorporated into two linker sites (right, not drawn to scale). (B)-(C) Live imaging of activated mice (B) or human oocytes (C) microinjected with mRNA encoding only TALhesin(Rec8)-B (top panel), mRNA encoding an uncleavable TALhesin (middle panel), or mRNA encoding a separase-cleavable TALhesin(Rec8) (bottom panel). The area enclosed by the rectangle is magnified below. Black arrows indicate non-segregation of chromosomes. White arrows indicate chromatid separation. TALhesins, chromosomes, min: minutes. Scale bar is 5 μm. (D)-(E) Duration (minutes) of late phase II in activated mouse oocytes (D) or human oocytes (E), measured from the start to completion of late phase II. (F)-(G) Percentage of activated mouse oocytes (F) or human oocytes (G) in which chromosomes were not separated during late phase II. The atomomorphs (amol) of injected mRNA are listed below for each condition. The number of cells analyzed for each condition is shown below. (H) Frequency of PSSCs in untreated oocytes from young mice (white bars), untreated oocytes from aged mice (filled bars), and aged oocytes treated with TALhesin[mmMajSat](Rec8)-A / B mRNA (gray bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars represent 95% confidence intervals and 1 degree of freedom. [Figure 7B-02]Separase-cleavage motifs release chromosome condensation mediated by artificial complexes. (A) Inactivation of TALhesin-mediated chromosome condensation (left) and schematic diagram of the domain arrangement of TALhesin[mmMajSat](Rec8) with a mouse Rec8 fragment incorporated into two linker sites (right, not drawn to scale). (B)-(C) Live imaging of activated mice (B) or human oocytes (C) microinjected with mRNA encoding only TALhesin(Rec8)-B (top panel), mRNA encoding an uncleavable TALhesin (middle panel), or mRNA encoding a separase-cleavable TALhesin(Rec8) (bottom panel). The area enclosed by the rectangle is magnified below. Black arrows indicate non-segregation of chromosomes. White arrows indicate chromatid separation. TALhesins, chromosomes, min: minutes. Scale bar is 5 μm. (D)-(E) Duration (minutes) of late phase II in activated mouse oocytes (D) or human oocytes (E), measured from the start to completion of late phase II. (F)-(G) Percentage of activated mouse oocytes (F) or human oocytes (G) in which chromosomes were not separated during late phase II. The atomomorphs (amol) of injected mRNA are listed below for each condition. The number of cells analyzed for each condition is shown below. (H) Frequency of PSSCs in untreated oocytes from young mice (white bars), untreated oocytes from aged mice (filled bars), and aged oocytes treated with TALhesin[mmMajSat](Rec8)-A / B mRNA (gray bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars represent 95% confidence intervals and 1 degree of freedom. [Figure 7B-03]Separase-cleavage motifs release chromosome condensation mediated by artificial complexes. (A) Inactivation of TALhesin-mediated chromosome condensation (left) and schematic diagram of the domain arrangement of TALhesin[mmMajSat](Rec8) with a mouse Rec8 fragment incorporated into two linker sites (right, not drawn to scale). (B)-(C) Live imaging of activated mice (B) or human oocytes (C) microinjected with mRNA encoding only TALhesin(Rec8)-B (top panel), mRNA encoding an uncleavable TALhesin (middle panel), or mRNA encoding a separase-cleavable TALhesin(Rec8) (bottom panel). The area enclosed by the rectangle is magnified below. Black arrows indicate non-segregation of chromosomes. White arrows indicate chromatid separation. TALhesins, chromosomes, min: minutes. Scale bar is 5 μm. (D)-(E) Duration (minutes) of late phase II in activated mouse oocytes (D) or human oocytes (E), measured from the start to completion of late phase II. (F)-(G) Percentage of activated mouse oocytes (F) or human oocytes (G) in which chromosomes were not separated during late phase II. The atomomorphs (amol) of injected mRNA are listed below for each condition. The number of cells analyzed for each condition is shown below. (H) Frequency of PSSCs in untreated oocytes from young mice (white bars), untreated oocytes from aged mice (filled bars), and aged oocytes treated with TALhesin[mmMajSat](Rec8)-A / B mRNA (gray bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars represent 95% confidence intervals and 1 degree of freedom. [Figure 7C-01]Separase-cleavage motifs release chromosome condensation mediated by artificial complexes. (A) Inactivation of TALhesin-mediated chromosome condensation (left) and schematic diagram of the domain arrangement of TALhesin[mmMajSat](Rec8) with a mouse Rec8 fragment incorporated into two linker sites (right, not drawn to scale). (B)-(C) Live imaging of activated mice (B) or human oocytes (C) microinjected with mRNA encoding only TALhesin(Rec8)-B (top panel), mRNA encoding an uncleavable TALhesin (middle panel), or mRNA encoding a separase-cleavable TALhesin(Rec8) (bottom panel). The area enclosed by the rectangle is magnified below. Black arrows indicate non-segregation of chromosomes. White arrows indicate chromatid separation. TALhesins, chromosomes, min: minutes. Scale bar is 5 μm. (D)-(E) Duration (minutes) of late phase II in activated mouse oocytes (D) or human oocytes (E), measured from the start to completion of late phase II. (F)-(G) Percentage of activated mouse oocytes (F) or human oocytes (G) in which chromosomes were not separated during late phase II. The atomomorphs (amol) of injected mRNA are listed below for each condition. The number of cells analyzed for each condition is shown below. (H) Frequency of PSSCs in untreated oocytes from young mice (white bars), untreated oocytes from aged mice (filled bars), and aged oocytes treated with TALhesin[mmMajSat](Rec8)-A / B mRNA (gray bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars represent 95% confidence intervals and 1 degree of freedom. [Figure 7C-02]Separase-cleavage motifs release chromosome condensation mediated by artificial complexes. (A) Inactivation of TALhesin-mediated chromosome condensation (left) and schematic diagram of the domain arrangement of TALhesin[mmMajSat](Rec8) with a mouse Rec8 fragment incorporated into two linker sites (right, not drawn to scale). (B)-(C) Live imaging of activated mice (B) or human oocytes (C) microinjected with mRNA encoding only TALhesin(Rec8)-B (top panel), mRNA encoding an uncleavable TALhesin (middle panel), or mRNA encoding a separase-cleavable TALhesin(Rec8) (bottom panel). The area enclosed by the rectangle is magnified below. Black arrows indicate non-segregation of chromosomes. White arrows indicate chromatid separation. TALhesins, chromosomes, min: minutes. Scale bar is 5 μm. (D)-(E) Duration (minutes) of late phase II in activated mouse oocytes (D) or human oocytes (E), measured from the start to completion of late phase II. (F)-(G) Percentage of activated mouse oocytes (F) or human oocytes (G) in which chromosomes were not separated during late phase II. The atomomorphs (amol) of injected mRNA are listed below for each condition. The number of cells analyzed for each condition is shown below. (H) Frequency of PSSCs in untreated oocytes from young mice (white bars), untreated oocytes from aged mice (filled bars), and aged oocytes treated with TALhesin[mmMajSat](Rec8)-A / B mRNA (gray bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars represent 95% confidence intervals and 1 degree of freedom. [Figure 7C-03]Separase-cleavage motifs release chromosome condensation mediated by artificial complexes. (A) Inactivation of TALhesin-mediated chromosome condensation (left) and schematic diagram of the domain arrangement of TALhesin[mmMajSat](Rec8) with a mouse Rec8 fragment incorporated into two linker sites (right, not drawn to scale). (B)-(C) Live imaging of activated mice (B) or human oocytes (C) microinjected with mRNA encoding only TALhesin(Rec8)-B (top panel), mRNA encoding an uncleavable TALhesin (middle panel), or mRNA encoding a separase-cleavable TALhesin(Rec8) (bottom panel). The area enclosed by the rectangle is magnified below. Black arrows indicate non-segregation of chromosomes. White arrows indicate chromatid separation. TALhesins, chromosomes, min: minutes. Scale bar is 5 μm. (D)-(E) Duration (minutes) of late phase II in activated mouse oocytes (D) or human oocytes (E), measured from the start to completion of late phase II. (F)-(G) Percentage of activated mouse oocytes (F) or human oocytes (G) in which chromosomes were not separated during late phase II. The atomomorphs (amol) of injected mRNA are listed below for each condition. The number of cells analyzed for each condition is shown below. (H) Frequency of PSSCs in untreated oocytes from young mice (white bars), untreated oocytes from aged mice (filled bars), and aged oocytes treated with TALhesin[mmMajSat](Rec8)-A / B mRNA (gray bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars represent 95% confidence intervals and 1 degree of freedom. [Figure 7D]Separase-cleavage motifs release chromosome condensation mediated by artificial complexes. (A) Inactivation of TALhesin-mediated chromosome condensation (left) and schematic diagram of the domain arrangement of TALhesin[mmMajSat](Rec8) with a mouse Rec8 fragment incorporated into two linker sites (right, not drawn to scale). (B)-(C) Live imaging of activated mice (B) or human oocytes (C) microinjected with mRNA encoding only TALhesin(Rec8)-B (top panel), mRNA encoding an uncleavable TALhesin (middle panel), or mRNA encoding a separase-cleavable TALhesin(Rec8) (bottom panel). The area enclosed by the rectangle is magnified below. Black arrows indicate non-segregation of chromosomes. White arrows indicate chromatid separation. TALhesins, chromosomes, min: minutes. Scale bar is 5 μm. (D)-(E) Duration (minutes) of late phase II in activated mouse oocytes (D) or human oocytes (E), measured from the start to completion of late phase II. (F)-(G) Percentage of activated mouse oocytes (F) or human oocytes (G) in which chromosomes were not separated during late phase II. The atomomorphs (amol) of injected mRNA are listed below for each condition. The number of cells analyzed for each condition is shown below. (H) Frequency of PSSCs in untreated oocytes from young mice (white bars), untreated oocytes from aged mice (filled bars), and aged oocytes treated with TALhesin[mmMajSat](Rec8)-A / B mRNA (gray bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars represent 95% confidence intervals and 1 degree of freedom. [Figure 7E]Separase-cleavage motifs release chromosome condensation mediated by artificial complexes. (A) Inactivation of TALhesin-mediated chromosome condensation (left) and schematic diagram of the domain arrangement of TALhesin[mmMajSat](Rec8) with a mouse Rec8 fragment incorporated into two linker sites (right, not drawn to scale). (B)-(C) Live imaging of activated mice (B) or human oocytes (C) microinjected with mRNA encoding only TALhesin(Rec8)-B (top panel), mRNA encoding an uncleavable TALhesin (middle panel), or mRNA encoding a separase-cleavable TALhesin(Rec8) (bottom panel). The area enclosed by the rectangle is magnified below. Black arrows indicate non-segregation of chromosomes. White arrows indicate chromatid separation. TALhesins, chromosomes, min: minutes. Scale bar is 5 μm. (D)-(E) Duration (minutes) of late phase II in activated mouse oocytes (D) or human oocytes (E), measured from the start to completion of late phase II. (F)-(G) Percentage of activated mouse oocytes (F) or human oocytes (G) in which chromosomes were not separated during late phase II. The atomomorphs (amol) of injected mRNA are listed below for each condition. The number of cells analyzed for each condition is shown below. (H) Frequency of PSSCs in untreated oocytes from young mice (white bars), untreated oocytes from aged mice (filled bars), and aged oocytes treated with TALhesin[mmMajSat](Rec8)-A / B mRNA (gray bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars represent 95% confidence intervals and 1 degree of freedom. [Figure 7F]Separase-cleavage motifs release chromosome condensation mediated by artificial complexes. (A) Inactivation of TALhesin-mediated chromosome condensation (left) and schematic diagram of the domain arrangement of TALhesin[mmMajSat](Rec8) with a mouse Rec8 fragment incorporated into two linker sites (right, not drawn to scale). (B)-(C) Live imaging of activated mice (B) or human oocytes (C) microinjected with mRNA encoding only TALhesin(Rec8)-B (top panel), mRNA encoding an uncleavable TALhesin (middle panel), or mRNA encoding a separase-cleavable TALhesin(Rec8) (bottom panel). The area enclosed by the rectangle is magnified below. Black arrows indicate non-segregation of chromosomes. White arrows indicate chromatid separation. TALhesins, chromosomes, min: minutes. Scale bar is 5 μm. (D)-(E) Duration (minutes) of late phase II in activated mouse oocytes (D) or human oocytes (E), measured from the start to completion of late phase II. (F)-(G) Percentage of activated mouse oocytes (F) or human oocytes (G) in which chromosomes were not separated during late phase II. The atomomorphs (amol) of injected mRNA are listed below for each condition. The number of cells analyzed for each condition is shown below. (H) Frequency of PSSCs in untreated oocytes from young mice (white bars), untreated oocytes from aged mice (filled bars), and aged oocytes treated with TALhesin[mmMajSat](Rec8)-A / B mRNA (gray bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars represent 95% confidence intervals and 1 degree of freedom. [Figure 7G]Separase-cleavage motifs release chromosome condensation mediated by artificial complexes. (A) Inactivation of TALhesin-mediated chromosome condensation (left) and schematic diagram of the domain arrangement of TALhesin[mmMajSat](Rec8) with a mouse Rec8 fragment incorporated into two linker sites (right, not drawn to scale). (B)-(C) Live imaging of activated mice (B) or human oocytes (C) microinjected with mRNA encoding only TALhesin(Rec8)-B (top panel), mRNA encoding an uncleavable TALhesin (middle panel), or mRNA encoding a separase-cleavable TALhesin(Rec8) (bottom panel). The area enclosed by the rectangle is magnified below. Black arrows indicate non-segregation of chromosomes. White arrows indicate chromatid separation. TALhesins, chromosomes, min: minutes. Scale bar is 5 μm. (D)-(E) Duration (minutes) of late phase II in activated mouse oocytes (D) or human oocytes (E), measured from the start to completion of late phase II. (F)-(G) Percentage of activated mouse oocytes (F) or human oocytes (G) in which chromosomes were not separated during late phase II. The atomomorphs (amol) of injected mRNA are listed below for each condition. The number of cells analyzed for each condition is shown below. (H) Frequency of PSSCs in untreated oocytes from young mice (white bars), untreated oocytes from aged mice (filled bars), and aged oocytes treated with TALhesin[mmMajSat](Rec8)-A / B mRNA (gray bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars represent 95% confidence intervals and 1 degree of freedom. [Figure 7H]Separase-cleavage motifs release chromosome condensation mediated by artificial complexes. (A) Inactivation of TALhesin-mediated chromosome condensation (left) and schematic diagram of the domain arrangement of TALhesin[mmMajSat](Rec8) with a mouse Rec8 fragment incorporated into two linker sites (right, not drawn to scale). (B)-(C) Live imaging of activated mice (B) or human oocytes (C) microinjected with mRNA encoding only TALhesin(Rec8)-B (top panel), mRNA encoding an uncleavable TALhesin (middle panel), or mRNA encoding a separase-cleavable TALhesin(Rec8) (bottom panel). The area enclosed by the rectangle is magnified below. Black arrows indicate non-segregation of chromosomes. White arrows indicate chromatid separation. TALhesins, chromosomes, min: minutes. Scale bar is 5 μm. (D)-(E) Duration (minutes) of late phase II in activated mouse oocytes (D) or human oocytes (E), measured from the start to completion of late phase II. (F)-(G) Percentage of activated mouse oocytes (F) or human oocytes (G) in which chromosomes were not separated during late phase II. The atomomorphs (amol) of injected mRNA are listed below for each condition. The number of cells analyzed for each condition is shown below. (H) Frequency of PSSCs in untreated oocytes from young mice (white bars), untreated oocytes from aged mice (filled bars), and aged oocytes treated with TALhesin[mmMajSat](Rec8)-A / B mRNA (gray bars). n is the number of chromosomes analyzed. Statistical significance is determined by Fisher's exact test. Bars represent 95% confidence intervals and 1 degree of freedom.

[0020] [Figure 8A] This involves incorporating the Rec8 motif into an artificial complex structure.

[0021] (A) Schematic diagram of the domain arrangement of TALhesin[mmMajSat](Rec8) targeting mouse centromere repeats (not drawn to scale). (B) Schematic diagram of the domain arrangement of TALhesin[hsCentro](Rec8) targeting human centromere repeats (not drawn to scale). (C) mRNA of components A and B of TALhesin[mmMajSat](Rec8) analyzed by 1% agarose gel electrophoresis. (D) mRNA of components A and B of TALhesin[hsCentro](Rec8) (including spy-catcher 001) analyzed by 1% agarose gel electrophoresis, used for microinjection of human oocytes. For (C) to (D), the domain composition and expected size of each transcript are shown next to the image annotated with nucleotides (nt). (E) Mobility of TALhesin[mmMajSat](Rec8) chromosome focus, as measured by FRAP, compared to TALhesin[mmMajSat] and TAL[mmMajSat] effector alone. Bars represent the standard deviation from the mean of 30 replicates for each condition. [Figure 8B]This involves the incorporation of the Rec8 motif into an artificial complex structure. (A) A schematic diagram of the domain arrangement of TALhesin[mmMajSat](Rec8) targeting mouse centromere repeats (not drawn to scale). (B) A schematic diagram of the domain arrangement of TALhesin[hsCentro](Rec8) targeting human centromere repeats (not drawn to scale). (C) mRNA of components A and B of TALhesin[mmMajSat](Rec8) analyzed by 1% agarose gel electrophoresis. (D) mRNA of components A and B of TALhesin[hsCentro](Rec8) (including spy-catcher 001) analyzed by 1% agarose gel electrophoresis, used for microinjection of human oocytes. For (C) to (D), the domain composition and expected size of each transcript are shown next to the image annotated with nucleotides (nt). (E) Mobility of TALhesin[mmMajSat](Rec8) chromosome focus, as measured by FRAP, compared to TALhesin[mmMajSat] and TAL[mmMajSat] effector alone. Bars represent the standard deviation from the mean of 30 replicates for each condition. [Figure 8C]This involves the incorporation of the Rec8 motif into an artificial complex structure. (A) A schematic diagram of the domain arrangement of TALhesin[mmMajSat](Rec8) targeting mouse centromere repeats (not drawn to scale). (B) A schematic diagram of the domain arrangement of TALhesin[hsCentro](Rec8) targeting human centromere repeats (not drawn to scale). (C) mRNA of components A and B of TALhesin[mmMajSat](Rec8) analyzed by 1% agarose gel electrophoresis. (D) mRNA of components A and B of TALhesin[hsCentro](Rec8) (including spy-catcher 001) analyzed by 1% agarose gel electrophoresis, used for microinjection of human oocytes. For (C) to (D), the domain composition and expected size of each transcript are shown next to the image annotated with nucleotides (nt). (E) Mobility of TALhesin[mmMajSat](Rec8) chromosome focus, as measured by FRAP, compared to TALhesin[mmMajSat] and TAL[mmMajSat] effector alone. Bars represent the standard deviation from the mean of 30 replicates for each condition. [Figure 8D]This involves the incorporation of the Rec8 motif into an artificial complex structure. (A) A schematic diagram of the domain arrangement of TALhesin[mmMajSat](Rec8) targeting mouse centromere repeats (not drawn to scale). (B) A schematic diagram of the domain arrangement of TALhesin[hsCentro](Rec8) targeting human centromere repeats (not drawn to scale). (C) mRNA of components A and B of TALhesin[mmMajSat](Rec8) analyzed by 1% agarose gel electrophoresis. (D) mRNA of components A and B of TALhesin[hsCentro](Rec8) (including spy-catcher 001) analyzed by 1% agarose gel electrophoresis, used for microinjection of human oocytes. For (C) to (D), the domain composition and expected size of each transcript are shown next to the image annotated with nucleotides (nt). (E) Mobility of TALhesin[mmMajSat](Rec8) chromosome focus, as measured by FRAP, compared to TALhesin[mmMajSat] and TAL[mmMajSat] effector alone. Bars represent the standard deviation from the mean of 30 replicates for each condition. [Figure 8E]This involves the incorporation of the Rec8 motif into an artificial complex structure. (A) A schematic diagram of the domain arrangement of TALhesin[mmMajSat](Rec8) targeting mouse centromere repeats (not drawn to scale). (B) A schematic diagram of the domain arrangement of TALhesin[hsCentro](Rec8) targeting human centromere repeats (not drawn to scale). (C) mRNA of components A and B of TALhesin[mmMajSat](Rec8) analyzed by 1% agarose gel electrophoresis. (D) mRNA of components A and B of TALhesin[hsCentro](Rec8) (including spy-catcher 001) analyzed by 1% agarose gel electrophoresis, used for microinjection of human oocytes. For (C) to (D), the domain composition and expected size of each transcript are shown next to the image annotated with nucleotides (nt). (E) Mobility of TALhesin[mmMajSat](Rec8) chromosome focus, as measured by FRAP, compared to TALhesin[mmMajSat] and TAL[mmMajSat] effector alone. Bars represent the standard deviation from the mean of 30 replicates for each condition.

[0022] [Figure 9A] Artificial complexes containing the incorporated Rec8 motif reduce PSSCs.

[0023] (A) The reduction in PSSC in oocytes from 20-month-old mice treated with 0.02 or 0.05 amol of TALhesin[mmMajSat](Rec8) mRNA, compared to oocytes from untreated young mice and 20-month-old mice. (B) The percentage of oocytes from 20-month-old mice treated with TALhesin[mmMajSat](Rec8) with a PSSC error of 0 (white bar), 1 (light gray bar), 2 (dark gray bar), or more than 2 (filled bar), compared to oocytes from untreated young mice and 20-month-old mice. Oocytes expressing only 0.05 amol of TALhesin[mmMajSat](Rec8) component B mRNA are shown on the far right. The number of oocytes analyzed is shown below each condition. (C) Increase in error-free oocytes from 20-month-old mice treated with 0.02 or 0.05 amol of TALhesin[mmMajSat](Rec8) mRNA, compared to untreated oocytes from young mice and 20-month-old mice. (D) Inter-kinetochore spacing (iKt) between sister chromatids in oocytes from young mice (white bars), mice treated with TALhesin[mmMajSat](Rec8) for 20 months (light gray bars), or untreated mice (dark gray bars, left). The iKt of oocytes expressing only 0.05 amol of TALhesin[mmMajSat](Rec8) component B mRNA is shown on the right as a dark gray bar. Statistical values ​​were measured by a two-sample t-test (equal variances not assumed). The median is shown as a line. The mean is shown as a black square. Bars represent SD, and mos. represents months. [Figure 9B]Artificial complexes containing the incorporated Rec8 motif reduce PSSC. (A) Reduction in PSSC in oocytes from 20-month-old mice treated with 0.02 or 0.05 amol of TALhesin[mmMajSat](Rec8) mRNA, compared to oocytes from untreated young and 20-month-old mice. (B) Percentage of oocytes from 20-month-old mice treated with TALhesin[mmMajSat](Rec8) with a PSSC error of 0 (open bar), 1 (light gray bar), 2 (dark gray bar), or more than 2 (filled bar), compared to oocytes from untreated young and 20-month-old mice. Oocytes expressing only 0.05 amol of TALhesin[mmMajSat](Rec8) component B mRNA are shown on the far right. The number of oocytes analyzed is shown below each condition. (C) Increase in error-free oocytes from 20-month-old mice treated with 0.02 or 0.05 amol of TALhesin[mmMajSat](Rec8) mRNA, compared to untreated oocytes from young mice and 20-month-old mice. (D) Inter-kinetochore spacing (iKt) between sister chromatids in oocytes from young mice (white bars), mice treated with TALhesin[mmMajSat](Rec8) for 20 months (light gray bars), or untreated mice (dark gray bars, left). The iKt of oocytes expressing only 0.05 amol of TALhesin[mmMajSat](Rec8) component B mRNA is shown on the right as a dark gray bar. Statistical values ​​were measured by a two-sample t-test (equal variances not assumed). The median is shown as a line. The mean is shown as a black square. Bars represent SD, and mos. represents months. [Figure 9C]Artificial complexes containing the incorporated Rec8 motif reduce PSSC. (A) Reduction in PSSC in oocytes from 20-month-old mice treated with 0.02 or 0.05 amol of TALhesin[mmMajSat](Rec8) mRNA, compared to oocytes from untreated young and 20-month-old mice. (B) Percentage of oocytes from 20-month-old mice treated with TALhesin[mmMajSat](Rec8) with a PSSC error of 0 (open bar), 1 (light gray bar), 2 (dark gray bar), or more than 2 (filled bar), compared to oocytes from untreated young and 20-month-old mice. Oocytes expressing only 0.05 amol of TALhesin[mmMajSat](Rec8) component B mRNA are shown on the far right. The number of oocytes analyzed is shown below each condition. (C) Increase in error-free oocytes from 20-month-old mice treated with 0.02 or 0.05 amol of TALhesin[mmMajSat](Rec8) mRNA, compared to untreated oocytes from young mice and 20-month-old mice. (D) Inter-kinetochore spacing (iKt) between sister chromatids in oocytes from young mice (white bars), mice treated with TALhesin[mmMajSat](Rec8) for 20 months (light gray bars), or untreated mice (dark gray bars, left). The iKt of oocytes expressing only 0.05 amol of TALhesin[mmMajSat](Rec8) component B mRNA is shown on the right as a dark gray bar. Statistical values ​​were measured by a two-sample t-test (equal variances not assumed). The median is shown as a line. The mean is shown as a black square. Bars represent SD, and mos. represents months. [Figure 9D]Artificial complexes containing the incorporated Rec8 motif reduce PSSC. (A) Reduction in PSSC in oocytes from 20-month-old mice treated with 0.02 or 0.05 amol of TALhesin[mmMajSat](Rec8) mRNA, compared to oocytes from untreated young and 20-month-old mice. (B) Percentage of oocytes from 20-month-old mice treated with TALhesin[mmMajSat](Rec8) with a PSSC error of 0 (open bar), 1 (light gray bar), 2 (dark gray bar), or more than 2 (filled bar), compared to oocytes from untreated young and 20-month-old mice. Oocytes expressing only 0.05 amol of TALhesin[mmMajSat](Rec8) component B mRNA are shown on the far right. The number of oocytes analyzed is shown below each condition. (C) Increase in error-free oocytes from 20-month-old mice treated with 0.02 or 0.05 amol of TALhesin[mmMajSat](Rec8) mRNA, compared to untreated oocytes from young mice and 20-month-old mice. (D) Inter-kinetochore spacing (iKt) between sister chromatids in oocytes from young mice (white bars), mice treated with TALhesin[mmMajSat](Rec8) for 20 months (light gray bars), or untreated mice (dark gray bars, left). The iKt of oocytes expressing only 0.05 amol of TALhesin[mmMajSat](Rec8) component B mRNA is shown on the right as a dark gray bar. Statistical values ​​were measured by a two-sample t-test (equal variances not assumed). The median is shown as a line. The mean is shown as a black square. Bars represent SD, and mos. represents months.

[0024] [Figure 10] The chromatin-binding component (TAL protein) fused with N-securin (aa1-101) is degraded in mouse oocytes during anaphase I.

[0025] The relative fluorescence intensity of the N-securin (aa1-101)-mClover-TAL [mmMajSat] focus (black circle) around the centromere decreases before and after the onset of late I (dotted line). The relative fluorescence intensity of the chromosome reporter H2B-miRFP is unrelated to late I (white square). The time to the left of the dotted line is before the onset of late I. The time to the right of the dotted line is after the onset of late I.

[0026] [Figure 11] TAL proteins fused with N-cyclin B1 (aa1-79) are degraded in mouse oocytes during anaphase I.

[0027] The relative fluorescence intensity of the N-cyclin B 1(aa1-79)-mClover-TAL[mmMajSat] focus (black circle) around the centromere decreases before and after the onset of late I (dotted line). The relative fluorescence intensity of the chromosome reporter H2B-miRFP is unrelated to late I (white square). The time to the left of the dotted line is before the onset of late I. The time to the right of the dotted line is after the onset of late I.

[0028] [Figure 12] NS3 / 4A protease degrades artificial complexes on mouse oocyte chromosomes.

[0029] The fluorescent chromosome focus signal of TALhesin[mmMajSat]-A / B containing a 2×NS3 / 4A proteolytic motif decreases several hours after washout with BILN-2061 compared to chromosome dye (SiR-DNA) (black diamond). Uncleavable TALhesin does not decrease after washout with BILN-2061 (white circle). The number of cells analyzed, n, was 10. Bars indicate standard deviation (SD). [Modes for carrying out the invention]

[0030] Disclosed is a complex suitable for condensation of chromatids or chromosomes, comprising (I) one or more first proteins and (II) one or more second proteins, as further defined in the claims. In the complex, (I) the first and (II) the second proteins each comprise at least three portions. Specifically, (I) the first and (II) the second proteins each comprise, as further defined in the claims, (i) a chromatin-binding component, (ii) a protein-binding region (PBN region) at the N-terminus of the chromatin-binding component, and (iii) a protein-binding region (PBC region) at the C-terminus of the chromatin-binding component. According to this disclosure, the chromatin-binding components of (i) the first and second proteins can bind to target sequences on chromatids or chromosomes. Furthermore, (I)(ii) the PBN region of the first protein can bind to (II)(ii) the PBN region of the second protein, (I)(iii) the PBC region of the first protein can bind to (II)(iii) the PBC region of the second protein, or (I)(ii) the PBN region of the first protein can bind to (II)(iii) the PBC region of the second protein, and (I)(iii) the PBC region of the first protein can bind to (II)(ii) the PBN region of the second protein. As further defined in the claims, binding to chromatids or chromosomes, and the binding of the PBN and PBC regions of the first and second proteins, reversibly tethers the chromatids or chromosomes. Figure 1b shows the overall configuration of the complex. The complex is an artificial protein complex. In other words, the complex does not exist in nature. The complex is obtained ex vivo, or the nucleic acid encoding the complex is obtained ex vivo. The complex or the nucleic acid encoding the complex can be obtained, for example, by an in vitro method. Those skilled in the art will recognize the existence of commercial suppliers, such as IDT or ProMab, who can be provided with sequences to obtain purified proteins and / or desired nucleic acid sequences.

[0031] The technical advantage of this arrangement is that the complex can tether sister chromatids or chromosomes to reduce the risk of premature separation, such as premature separation of sister chromatids. As a result, the risk of aneuploidy, for example, is reduced.

[0032] As used herein, “component” is part of a larger whole. In the present invention, a component is a part of the first and second proteins. A “chromatin-binding component” is a part of the first and second proteins that can bind to chromatin. As is known in the art, chromatin refers to the mixture of DNA from chromosomes and proteins found in eukaryotic cells. Thus, a chromatin-binding component can bind to DNA or DNA-related proteins. Those skilled in the art can determine whether a component is bound to DNA or DNA-related proteins by standard methods in the art. A chromatin-binding component may comprise, and ideally may consist of, one or more domains. Preferably, a chromatin-binding component is a chromatin-binding domain. Generally, a domain is a region of a polypeptide chain that is self-stable and folds independently of the rest. Generally, each domain forms a compact, folded three-dimensional structure.

[0033] As used herein, “region” refers to a part of a larger whole. In the present invention, the first and second proteins each contain at least two regions. Thus, a “protein-binding region” is an amino acid sequence that can bind to another protein. Ideally, a protein-binding region can bind to another protein-binding region. According to the present invention, the protein-binding region is the N-terminus and C-terminus of a chromatin-binding component. Each of the first and second proteins contains one protein-binding region N-terminus of a chromatin-binding component and one protein-binding region C-terminus of a chromatin-binding region. Ideally, the PBN region is located at the N-terminus of the first and second proteins. In particular, the PBC region may be located at the C-terminus of the first and second proteins.

[0034] Combination of PBN and PBC regions In the first embodiment, (I)(ii) the PBN region of the first protein can bind to (II)(ii) the PBN region of the second protein, and (I)(iii) the PBC region of the first protein can bind to (II)(iii) the PBC region of the second protein. This arrangement is also shown in Figure 1b. In this embodiment, the protein-binding regions bind to each other without induction. This is advantageous because no further trigger for induction is required. Ideally, the (I)(ii) PBN region of the first protein and the (I)(iii) PBC region of the first protein have at least 80% sequence identity, preferably at least 83%, more preferably at least 86%, more preferably at least 90%, more preferably at least 93%, even more preferably at least 96%, and most preferably 100% sequence identity. Furthermore, the PBN region of the second protein (II)(ii) and the PBC region of the second protein (II)(iii) may have at least 80% sequence identity, preferably at least 83%, more preferably at least 86%, more preferably at least 90%, more preferably at least 93%, even more preferably at least 96%, and most preferably 100% sequence identity. Those skilled in the art can evaluate the sequence identity of amino acid sequences using online tools such as ExPASy (SIM, a protein sequence alignment tool).

[0035] In embodiments, the PBN and PBC regions of a first protein can form covalent bonds with the PBN and PBC regions of a second protein. As known in the art, a covalent bond is a chemical bond that involves the sharing of electrons to form an electron pair between atoms. For example, amine bonds are preferred covalent bonds. Ideally, the PBN and PBC regions of a first protein can be reconstituted with the PBN and PBC regions of a second protein. As used herein, “reconstituted” means that the two protein-binding regions interact to form a covalent amine bond. The PBN and PBC regions of a first protein can be conjugated with the PBN and PBC regions of a second protein. As used herein, “conjugated” means that the two protein-binding regions irreversibly interact with each other to form an intermolecular isopeptide bond between them. As known in the art, an isopeptide bond is an amide bond that can be formed between the carboxyl group of one amino acid and the amino group of another amino acid, where at least one of these linking groups is part of one side chain of these amino acids. For example, spy tags and spy catchers conjugate to form isopeptide bonds. The PBN and PBC regions of the first and second proteins can split protein fragments. As is known in the art, split protein fragments can be reconstituted to rebuild an intact protein from two fragments, i.e., split protein fragments. In general, splitting bioactive proteins into reconstituted fragments is a powerful strategy for constructing tools for controlling biological systems. Those skilled in the art are familiar with the corresponding split protein fragments, and further examples are provided below. For example, the PBN and PBC regions of the first protein may be spy tags. The PBN and PBC regions of the second protein may be spy catchers to conjugate to the second protein.

[0036] In particular, (I)(ii) the PBN region of the first protein may be selected from a spy tag, snoop-tag, dog-tag, and split-intane N-terminal fragment, preferably (I)(ii) the PBN region of the first protein is a spy tag. Preferably (I)(iii) the PBC region of the first protein may be selected from a spy tag, snoop-tag, dog-tag, and split-intane N-terminal fragment, preferably (I)(iii) the PBC region of the first protein is a spy tag. Furthermore, (I)(ii) the PBN region of the first protein and (I)(iii) the PBC region of the first protein may be selected from a spy tag, snoop-tag, dog-tag, and split-intane N-terminal fragment, preferably (I)(ii) the PBN region of the first protein and (I)(iii) the PBC region of the first protein are spy tags.

[0037] The spy tag is particularly preferably selected from spy tag 001, spy tag 002, and spy tag 003, and more preferably the spy tag is spy tag 001. Example 1 shows that spy tag 001 is particularly suitable for the complex. In embodiments in which (I)(ii) the PBN region of the first protein and / or (I)(iii) the PBC region of the first protein is a split intein N-terminal fragment, the split intein N-terminal fragment may be a split DnaE intein N-terminal fragment, and preferably the split DnaE intein N-terminal fragment is derived from Nostoc punctiforme (Npu).

[0038] (II)(ii) With respect to the PBN region of the second protein, the protein-binding region may be selected from among the spycatcher, snoopcatcher, dogcatcher, and split-intane C-terminal fragment. In embodiments, (II)(iii) the PBC region of the second protein is selected from among the spycatcher, snoopcatcher, dogcatcher, and split-intane C-terminal fragment. Ideally, (II)(ii) the PBN region of the second protein and (II)(iii) the PBC region of the second protein are selected from among the spycatcher, snoopcatcher, dogcatcher, and split-intane C-terminal fragment.

[0039] In embodiments where (II)(ii) the PBN region of the second protein and / or (II)(iii) the PBC region of the second protein is a spycatcher, the spycatcher is ideally selected from spycatcher 001, spycatcher 002, and spycatcher 003. Most preferably, the spycatcher is spycatcher 001. In embodiments where (II)(ii) the PBN region of the second protein and / or (II)(iii) the PBC region of the second protein is a split intein C-terminal fragment, the split DnaE intein C-terminal fragment is preferably derived from Nostoc punctiforme (Npu).

[0040] According to this first aspect of the present invention, (I)(ii) the PBN region of the first protein can bind to the PBN region of the (II)(ii) second protein, and (I)(iii) the PBC region of the first protein can bind to the PBC region of the (II)(iii) second protein. Thus, the corresponding protein binding regions are suitable for binding to each other. Regarding split inteins, the corresponding split protein fragments, i.e., the split intein N-terminal and C-terminal fragments that can be reconstituted, are known in the art. It is known in the art that an intein is a protein segment that can bind adjacent residues via a peptide bond. In this process known as protein splicing, seamless peptide ligation is achieved. For example, Pinto et al. (2020) 52 provides 15 examples of split inteins that can be reconstituted. For example, the split intein N-terminal and C-terminal fragments can be SspDnaB Δ275 M86, Npu N / Ssp C DnaE, gp41-1, gp41-8, NrdJ-1, IMPDH-1, SspDnaX Δ297 SspGyrB Δ279 TerThyX Δ134 TvoVMA, PhoRadA, CIV RIR1, CthATCC27405 TerA, MP-Be DnaB, MP-Catera gp206, PfuRIR1-1, and MjaKlbA.

[0041] In a preferred embodiment, the (I)(ii)PBN region and (I)(iii)PBC region of the first protein are spy tags. Specifically, the (I)(ii)PBN region and (I)(iii)PBC region of the first protein may have sequence identity of at least 70%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 94%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, and most preferably 100% with respect to SEQ ID NO: 4 or 8. Furthermore, the (II)(ii)PBN region and (II)(iii)PBC region of the second protein may be spy catchers. In particular, the (II)(ii)PBN region and (II)(iii)PBC region of the second protein may have sequence identity of at least 70%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 94%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, and most preferably 100% with respect to SEQ ID NO: 2 or 6. As shown in Example 1, the spy tag binds to the spycatcher to form a stable covalent isopeptide bond between the first protein and the second protein. 24 .

[0042] In a second aspect of the present invention, (I)(ii) the PBN region of the first protein can bind to (II)(iii) the PBC region of the second protein, and (I)(iii) the PBC region of the first protein can bind to (II)(ii) the PBN region of the second protein. Ideally, this binding between the PBN region and the PBC region is inducible. An advantage of this aspect is that the first and second proteins may have the same or very similar sequences. Therefore, only one type of artificial protein or the nucleic acid encoding the artificial protein needs to be introduced into the cells to form a complex. This has the particular advantage of requiring only a single introduction, e.g., a single injection. This is particularly advantageous when dealing with oocytes or egg cells in order to minimize damage to the oocytes or egg cells.

[0043] Exemplary inducible protein binding is dependent on the presence of the compound or is photoinducible. Having an inducible system is particularly advantageous because binding can be introduced at a desired time. Ideally, (I)(ii) the PBN region of the first protein can be reconstituted with (II)(iii) the PBC region of the second protein, and (I)(iii) the PBC region of the first protein can be reconstituted with (II)(ii) the PBN region of the second protein.

[0044] In preferred embodiments, the PBN region of (I)(ii) the first protein and the PBN region of (II)(ii) the second protein have at least 80% sequence identity, preferably at least 83%, more preferably at least 86%, more preferably at least 90%, more preferably at least 93%, even more preferably at least 96%, and most preferably 100% sequence identity. Preferably, the PBC region of (I)(iii) the first protein and the PBC region of (II)(iii) the second protein have at least 80% sequence identity, preferably at least 83%, more preferably at least 86%, more preferably at least 90%, more preferably at least 93%, even more preferably at least 96%, and most preferably 100% sequence identity.

[0045] Furthermore, the (I)(ii)PBN region and (I)(iii)PBC region of the first protein can form covalent bonds with the (II)(ii)PBN region and (II)(iii)PBC region of the second protein. In this embodiment, the (I)(ii)PBN region of the first protein can form covalent bonds with the (II)(ii)PBN region of the second protein, and the (I)(iii)PBC region of the first protein can form covalent bonds with the (II)(iii)PBC region of the second protein.

[0046] Ideally, the PBN and PBC regions of the first protein can be reconstituted with the PBN and PBC regions of the second protein.

[0047] Regarding inductivity, the binding of the PBN region and the PBC region is inductive by light and / or chemically induced proximity. This has the advantage that an inducer, such as light, induces the binding of the protein-binding regions. Inductive binding has the advantage that the first and second proteins may exist without forming a complex until the inducer is present. Therefore, complex formation can be induced by an inducer at a desired time, for example, at a specific point in time during meiosis I or II.

[0048] When binding is induced by light, the binding may be induced by blue, green, or red light, preferably by blue light. There are photo-inducible binding systems known in the art. In certain embodiments, the (I)(ii)PBN region and (I)(iii)PBC region of a first protein can form a covalent bond with the (II)(ii)PBN region and (II)(iii)PBC region of a second protein, and the bond is photo-inducible. There are photo-inducible binding systems known in the art. For example, the (I)(ii)PBN region of the first protein and / or the (II)(ii)PBN region of the second protein may be a photo-inducible spy tag, preferably a blue light-inducible spy tag (BLISS), and the (I)(iii)PBC region of the first protein and / or the (II)(iii)PBC region of the second protein may be a spycatcher. Exemplary spycatcher protein and nucleic acid sequences can be found herein, such as SEQ ID NOs: 1, 2, 5, and 6. Further details regarding BLISS can be found in Hartzell et al. (2021). 53 .

[0049] In certain embodiments, (I)(ii) the PBN region of the first protein has sequence identity of at least 70%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 94%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, and most preferably 100% with respect to SEQ ID NO: 40, and (I)(iii) the PBC region of the first protein has sequence identity of at least 70%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 94%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, and most preferably 100% with respect to SEQ ID NO: 40. An exemplary nucleic acid sequence encoding the sequence of SEQ ID NO: 40 can be found in SEQ ID NO: 39.

[0050] In certain embodiments, (II)(ii) the PBN region of the second protein has sequence identity of at least 70%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 94%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, and most preferably 100% with respect to SEQ ID NO: 40, and (II)(iii) the PBC region of the second protein has sequence identity of at least 70%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 94%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, and most preferably 100% with respect to SEQ ID NO: 40. An exemplary nucleic acid sequence encoding the sequence of SEQ ID NO: 40 can be found in SEQ ID NO: 39.

[0051] In further embodiments, the binding between the PBN and PBC regions is inducible by chemically induced proximity (CIP). As is known in the art, physical distance or proximity between molecules often leads to biological events. Also, as is known in the art, CIP generally relies on membrane-permeable compounds that reversibly regulate molecular proximity. Numerous examples of CIP are presented by Stanton et al. (2018). 54This is outlined in [reference]. The technical advantage of using CIP in situations where chromatids and chromosomes are artificially tethered is that binding and release can be chemically induced by the presence and absence of the compound (see also Figure 1b). In practice, the compound can be added, removed, or, for example, washed away at the desired time. Since the compound is generally membrane-permeable, oocytes or oocytes are disturbed only minimally. Note that when cells are present ex vivo, the compound can be added to the cells and removed from the cells, for example, by washing. Therefore, any such method is an in vitro method. In addition, the compound may be contained in the culture medium. For removal, the cells can be washed with a medium that no longer contains the compound.

[0052] Ideally, the chemically induced proximity is induced by a compound. In particular, the compound may be membrane-permeable. Furthermore, the compound is a small molecule compound. An example of a small molecule compound for CIP is Stanton et al. (2018). 54 They can also be found in the following contexts. In this context, the small molecule compound may be selected from one or two, preferably one, of rapamycin, FK506, FK506-cyclosporine, FK1012, coumamycin, gibberellin, S-(+)-abscisic acid, BisMTX, TMP-Halo (TMP-Halo) (trimethoprim fused to a halo ligand), caffeine, and cannabidiol. For example, based on Stanton et al. (2018), those skilled in the art know which protein pairs exhibit inductive binding when one of the above small molecule compounds is added.

[0053] Furthermore, the following examples are provided: [Table 1]

[0054] Specifically, (I)(ii) the PBN region of the first protein and (II)(iii) and the PBC region of the second protein can be selected from any of the following: FRB and FKBP; FKBP and calcineurin A; FKBP and cyclophyllin; FKBP and FKBP; GyrB and GyrB; GID1 and GAI; PYL1 and ABI1; DHFR and DHFR; AcVHH and AcVHH; or nanobodies that can be heterodimerized with cannabidiol.

[0055] In particular, (I)(iii) the PBC region of the first protein and (II)(ii) and the PBN region of the second protein can be selected from FRB and FKBP; FKBP and calcineurin A; FKBP and cyclophyllin; FKBP and FKBP; GyrB and GyrB; GID1 and GAI; PYL1 and ABI1; DHFR and DHFR; AcVHH and AcVHH; or nanobodies capable of heterodimerization with cannabidiol.

[0056] Further information regarding nanobodies that can undergo heterodimerization with cannabidiol can be found in Kang et al. (2019). 61 It is available to [username].

[0057] The PBN and PBC regions are more preferably inducible by chemically induced dimerization. Generally, “dimerization” refers to the process by which two identical parts, such as monomers, react to form a dimer, i.e., a molecule containing two identical or similar parts. As shown in the table above, there are examples of chemically induced dimerization, i.e., chemically induced binding of two identical or similar proteins, such as DHFR, AcVHH, FKBP, and GyrB.

[0058] For example, AcVHH dimerizes during chemically induced dimerization by caffeine. Further information on AcVHH dimerization upon caffeine binding can be found in Lesne et al. (2019). 55 It can be found there.

[0059] In certain embodiments, chemically induced proximitys are photoswitchable. Those skilled in the art are generally aware of photoswitchable chemically induced proximitys. In photoswitchable chemically induced proximitys, compounds are typically photoswitchable. Such proximitys are induced by "switching on" the compound, i.e., by obtaining a compound that can induce proximity. Those skilled in the art are aware that photoswitchable compounds are generally commercially available. For example, compounds include TMP-halo(trimethoprim fused to a halo ligand; chemical name: 4,5-dimethoxy-2-nitrobenzyl(4-amino-5-(4-((21-chloro-5,8-dioxo-12,15-dioxa-4,9-diazahenicosyl)oxy)-3,5-dimethoxybenzyl)pyrimidine-2-yl)carbamate, photocaged rapamycin, and coumarin-caged -TMP-halo(CTH; chemical name: One or two, preferably one, of CTH(7-(diethylamino)-2-oxo-2H-chromen-4-yl)methyl(4-amino-5-(4-((21-chloro-5,8-dioxo-12,15-dioxa-4,9-diazahenicosyl)oxy)-3,5-dimethoxybenzyl)pyrimidine-2-yl)carbamate) is selected. For example, photocaged rapamycin is also described by Karginov et al. (2011). 56 Further details are provided there.

[0060] Alternatively, the compound may be the TMP-halo(NTH) of the NVOC cage. The chemical name of the TMP-halo(NTH) of the NVOC cage is 4,5-dimethoxy-2-nitrobenzyl(4-amino-5-(4-((21-chloro-5,8-dioxo-12,15-dioxa-4,9-diazahenicosyl)oxy)-3,5-dimethoxybenzyl)pyrimidine-2-yl)carbamate. In particular, light removes the NVOC cage from the TMP-halo. Then, the TMP binds to the DHFR protein domain, and the halo portion binds to the halo-tag. Further information on TMP-halo inductive binding of the NVOC cage can be found in Ballister et al. (2014). 67 It can be found there.

[0061] Specific examples of optically switchable, chemically induced proximity in the context of this disclosure are provided as follows: [Table 2]

[0062] In certain embodiments, (I)(ii) the PBN region of the first protein and (II)(iii) the PBC region of the second protein are selected from FRB and FKBP; and DHFR and halotag, respectively. In further specific embodiments, (I)(iii) the PBC region of the first protein and (II)(ii) the PBN region of the second protein are selected from FRB and FKBP; and DHFR and halotag, respectively. Furthermore, photoswitchable chemically induced dimerization is also described by Chen et al. (2018). 60 Further details are provided there.

[0063] According to a second aspect of the present disclosure, the first and second proteins may have at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and even more preferably at least 100% sequence identity.

[0064] Reversible tethering According to the first and second aspects of the present invention, the binding of the PBN region and the PBC region of the first and second proteins reversibly tethers the chromatid or chromosome. Ideally, such tethering is (a) cleavage of a complex, wherein the first and second proteins of the complex further comprise protease cleavage sites; and / or (b) Degradation of the complex, wherein the first and / or second protein of the complex further comprises a ubiquitination site; and / or (c) Disruption of the binding between the PBN region and the PBC region of the first and second proteins, the disruption being reversible and mediated by chemically induced proximity.

[0065] In certain embodiments, tethering is reversible by one of options (a) to (c), for example, by cleavage of the complex via a protease cleavage site. In other embodiments, tethering is reversible by two of options (a) to (c). In some embodiments, all three options (a) to (c) are present. Experimental data for using a protease cleavage site are provided in Example 1 of this specification. Experimental data for using a ubiquitination site are provided in Example 2.

[0066] As used herein, the term “reversible” means that something can be reversed so that a previous state is restored. With respect to this disclosure, this means that “reversible” tethering means that tethering can be reversed. In other words, tethering can be released. After release, chromosomes or chromatids are no longer tethered.

[0067] Protease cleavage site Chromatid or chromosome tethering is preferably reversible by cleavage of the complex. Such cleavage preferably occurs at protease cleavage sites. Ideally, (a) the protease cleavage sites are located between (ii) the PBN region and (i) the chromatin-binding component of the first or second protein, or between (i) the chromatin-binding component and (iii) the PBC region. In particular, (a) the protease cleavage sites are located between (ii) the PBN region and (i) the chromatin-binding component of the first or second protein, or between (i) the chromatin-binding component and (iii) the PBC region.

[0068] Ideally, the complex is cleavable in its natural environment by a naturally occurring protease. In certain embodiments, the protease cleavage site is cleavable by a naturally occurring protease in the fertilized zygote. It is more preferable that the protease cleavage site is a separase-cleavable site, and even more preferable that the separase-cleavable site is a Rec8 site. Those skilled in the art can identify the Rec8 site by using online tools such as Uniprot. Furthermore, Kudo et al. (2009) 57 This provides further information regarding Rec8 cleavage sites. For example, Kudo et al. (2009) describe various separase-cleavable sites, including the mouse Rec8 site.

[0069] Preferably, the Rec8 site is a mammalian Rec8 site. The Rec8 site may be derived from a veterinary animal. In particular, mammalian Rec8 sites may be derived from humans, cats, dogs, horses, cattle, sheep, goats, or pigs. The Rec8 site may be derived from endangered animals such as pandas, orangutans, saola, tigers, rhinos, leopards, gorillas, elephants, bonobos, or dolphins. Even more preferably, the Rec8 site is derived from humans, cats, dogs, or horses. Ideally, the Rec8 site is a human Rec8 site.

[0070] It is also preferable that the first and second proteins each contain at least two Rec8 cleavage sites. Ideally, the first and second proteins each contain two Rec8 cleavage sites. Experimental data supporting the presence of two Rec8 cleavage sites are provided in Example 1 of this specification. In particular, the first and second proteins each contain a protein cleavable site further comprising a Rec8 leucine-proline-glutamate motif (LPE motif). Those skilled in the art will see Rosen et al. (2019). 37 Further information regarding the motif is provided therein. In some embodiments, at least one protease cleavage site has at least 75% identity with SEQ ID NOs. 30 or 32. The corresponding nucleic acid sequences can be found in SEQ ID NOs. 29 and 31.

[0071] The first and second proteins may contain two or more protease cleavage sites. Preferably, the two or more protease cleavage sites are two Rec8 cleavage sites. As shown in Example 1, preferably, the two Rec8 cleavage sites further contain a Rec8 LPE motif.

[0072] The first and second proteins each preferably contain two protease cleavage sites having at least 70% sequence identity with respect to SEQ ID NO: 28, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 93%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, and most preferably at least 100% sequence identity. An example nucleic acid sequence is provided in SEQ ID NO: 27.

[0073] In certain embodiments, the protease is a non-natural enzyme in the fertilized zygote. In other words, such protease cleavage sites are not spontaneously cleaved in the fertilized zygote. Such protease cleavage sites may be cleavable by cysteine ​​or serine proteases. Exemplary proteases are TEV or HCV NS3 / 4 proteases.

[0074] In some embodiments, proteases can be inhibited by small molecule inhibitors. An exemplary inhibitor is BILN-2061. Such inhibitors are commercially available. Example 2 provides experimental evidence that the NS3 / 4 protease cleavage site and the inhibitor BILN-2061 function in the context of artificial cohesin.

[0075] Ubiquitination sites Chromodide or chromosome tethering is preferably reversible by the presence of (b) ubiquitination sites in the first or second protein. Chromodide or chromosome tethering may be reversible by the presence of (b) ubiquitination sites in both the first and second proteins. In such cases, the first and / or second proteins of the complex further contain ubiquitination sites. This arrangement is also shown in Figure 1b. Experimental evidence is provided in Example 2.

[0076] Ubiquitination can be applied to various amino acids in the first and / or second protein. For example, the ubiquitination site may contain one or more lysines. Preferably, the ubiquitination site contains one lysine. Alternatively, the ubiquitination site may contain two lysines.

[0077] During meiosis, securin inhibits separase, which cleaves the cohesin ring that holds chromosomes together. To ensure that chromosome segregation occurs in anaphase, securin must be depleted before anaphase. In cells, securin is naturally depleted by proteolysis; that is, securin is ubiquitinated and thereby degraded. Naturally, securin is ubiquitinated by APC / C. Those skilled in the art will recognize that APC / C is an abbreviation for Anaphase-Promoting Complex / Cyclosome. APC / C is an E3 ubiquitin ligase that marks target cell cycle proteins for degradation. The ubiquitination sites ubiquitinated by APC / C are degraded by the 26S proteasome in the cell. One of the primary targets of degradation by APC / C is securin and cyclin (including cyclin B). Securin releases separase, a protease. Next, the separase induces the cleavage of cohesin, a protein complex that binds sister chromatids together.

[0078] The inventors have adapted this idea to an artificial cohesin and, importantly, provided proof-of-concept data in Example 2 of this specification. Specifically, the first and / or second protein of the complex may contain a ubiquitination site. According to the present invention, the ubiquitination site can be ubiquitinated and thereby degraded. Specifically, the ubiquitination site may be ubiquitinated by APC / C. According to this disclosure, the ubiquitination site is, ideally, an amino acid sequence of securin that can be ubiquitinated by APC / C. According to this disclosure, the ubiquitination site is, ideally, an amino acid sequence of cyclin, preferably cyclin B, more preferably cyclin B2, that can be ubiquitinated by APC / C. Those skilled in the art can trace securin and cyclin sequences via online tools such as Uniprot. The sequence of human securin can be found, for example, via Uniprot identifier O95997. The sequence of human cyclin B1 can be found, for example, via the Uniprot identifier P14635. Similarly, corresponding sequences for mouse, cat, dog, and other animals can be found. If the artificial complex contains a ubiquitination site, the ubiquitination site must be adapted to the animal in which the artificial cohesin will be used. A particular advantage of release via a ubiquitination site that is naturally ubiquitinated by APC / C, for example, is that the artificial cohesin is released at the start of the later stage by the intrinsic enzyme in the cell. Thus, the complex is degraded at the same time as or at the same point in time as the intrinsic aggregation system degrades. Therefore, no further intervention is required, and thus these embodiments omit the step of inducing release.

[0079] Example 2 provides experimental evidence that when an artificial cohesin contains a ubiquitination site that can be ubiquitinated by APC / C, it is degraded at the start of anaphase. Once the artificial cohesin is degraded, it no longer tethers chromosomes or chromatids.

[0080] The amino acid sequence of securin preferably has at least 25 amino acids, preferably at least 40 amino acids, more preferably at least 60 amino acids, more preferably at least 80 amino acids, and even more preferably 80 to 150 amino acids. Ideally, the amino acid sequence of securin is located at the N-terminus of securin. Ideally, the securin sequence is derived from humans, cats, dogs, horses, cattle, sheep, goats, or pigs. The securin sequence may be derived from veterinary animals. The securin sequence may be derived from endangered animals such as pandas, orangutans, saola, tigers, rhinos, leopards, gorillas, elephants, bonobos, or dolphins. Even more preferably, the securin sequence is derived from humans, cats, dogs, or horses. Ideally, the securin sequence is a human securin sequence.

[0081] In particular, the sequence may be derived from humans, cats, dogs, or horses. In certain embodiments, the sequence is derived from humans. The amino acid sequence of securin is preferably ubiquitinated when it enters the later stages.

[0082] The amino acid sequence of cyclin preferably has at least 25 amino acids, preferably at least 40 amino acids, more preferably at least 60 amino acids, and even more preferably 60 to 300 amino acids. Ideally, the amino acid sequence of cyclin is located at the N-terminus of cyclin. Cyclin B is particularly preferred, and cyclin B1 is even more preferred. Ideally, the cyclin sequence is derived from humans, cats, dogs, horses, cattle, sheep, goats, or pigs. The cyclin sequence may be derived from veterinary animals. The cyclin sequence may be derived from endangered animals such as pandas, orangutans, saola, tigers, rhinos, leopards, gorillas, elephants, bonobos, or dolphins. Even more preferably, the cyclin sequence is derived from humans, cats, dogs, or horses. Ideally, the cyclin sequence is a human cyclin sequence. The amino acid sequence of cyclin is preferably ubiquitinated when it enters the later stages.

[0083] As described above, chromatid or chromosome tethering is preferably reversible by the presence of (b) ubiquitination sites in the first and / or second protein. Such tethering may be reversible by targeted proteolysis. Targeted proteolysis is an overall process that targets a protein for destruction. Thus, the first and / or second protein of the complex may contain ubiquitination sites that are ubiquitinated via targeted proteolysis. Ideally, the first and / or second protein further contain a PROTAC binding site. A proteolysis target chimera (PROTAC) is a heterobifunctional molecule consisting of two active domains and a linker that can bind to a PROTAC binding site and an E3 ubiquitin ligase. In the presence of a PROTAC, the first and / or second protein is ubiquitinated by the E3 ligase, thereby causing the first and / or second protein to proteolysis. In these embodiments, the target of the targeted proteolysis is the first and / or second protein. In embodiments of this disclosure, the complex further comprises a PROTAC binding site. In other words, the PROTAC binds to the PROTAC binding site, thereby recruiting an E3 ubiquitin ligase. In preferred embodiments, the presence of the PROTAC results in ubiquitination of the complex. Target protein degradation has the particular advantage that the presence of the PROTAC allows degradation to be induced at a desired time, for example, at the start of late-stage degradation. The PROTAC may be membrane-permeable so that it can be added to the culture medium at a desired time to release tethering. Thus, release can be induced at any desired time.

[0084] For example, the PROTAC binding site may be selected from the group consisting of androgen receptor domains, estrogen receptor domains, BRD9 domains, B-cell giant domains (BCL-xL), IRAK4 domains, STAT3 domains, BTK domains, EGFR domains, and tropomyosin receptor domains. Further information on PROTACs can be found in Bekes et al. (2022). 58 It is outlined in [the document].

[0085] Chemically induced proximity Chromodid or chromosome tethering is preferably reversible by disruption of the binding between the PBN and PBC regions of the first and second proteins, and this binding is mediated by chemically induced proximity. As described above, the principle of chemically induced proximity (CID) is known in the art. However, CID has not been used in the context of artificial cohesin until now.

[0086] In particular, the binding of the PBN and PBC regions of the first protein to the PBN and PBC regions of the second protein can be mediated by chemically induced proximity, and the binding of the PBN and PBC regions of the first and second proteins can be disrupted by the absence of a small molecule compound or by functionally inactivating the small molecule compound. Those skilled in the art will recognize examples of chemically induced proximity. The examples provided in the table above can also be used for release. In this case, release is achieved, for example, by washing out the compound mediating the chemically induced proximity. Thus, the protein-binding regions of the first and second proteins release their binding, and therefore tethering is reversed. Ideally, the compound mediating the chemically induced proximity is a small molecule compound.

[0087] The small molecule compound is preferably a photoswitchable small molecule compound. A photoswitchable compound can be inactivated by light. Therefore, the inactivated compound can no longer undergo CID mediated by light. In other words, CID is destroyed by inactivating the compound. This releases chromosome or chromatid tethering. Examples of such compounds are known in the art. Preferably, the compound is selected from one of MeNV-HaXS and TMP-NVOC linker-halo.

[0088] In the context of this disclosure, an example of a photo-switchable, chemically induced proximity for releasing tethering is provided as follows: [Table 3]

[0089] Generally, these optically switchable compounds are commercially available. Further information on MeNV-HaXS can be found, for example, in Zimmermann et al. (2014). 59 It can be found there.

[0090] The chemical name of TMP-NVOC linker-halo(TNH) is 4-((18-chloro-3,6,9,12-tetraoxaoctadecyl)oxy)-5-methoxy-2-nitrobenzyl(3-(4-((2,4-diaminopyrimidine-5-yl)methyl)-2,6-dimethoxyphenoxy)propyl)carbamate.

[0091] Chromatin binding components As defined above, the "chromatin-binding component" is a part of the first and second proteins that can bind to chromatin. As is known in the art, chromatin refers to the mixture of DNA from chromosomes and proteins found in eukaryotic cells. Therefore, the chromatin-binding component can bind to DNA or DNA-related proteins. Ideally, the chromatin-binding component is a DNA-binding domain. The chromatin-binding component can bind to DNA-related proteins.

[0092] When the chromatin-binding component is a DNA-binding domain, the DNA-binding domain can be selected from the group consisting of a TAL protein domain, a zinc finger protein, a leucine zipper, and dCas9 complexed with gRNA. Preferably, the DNA-binding component is selected from the group consisting of a TAL protein domain and a zinc finger protein. As shown in Example 1, the DNA-binding component can be a TAL protein domain. As shown in Figure 1e, the first protein contained TAL protein domains flanking the spy tag on both sides (N-terminus and C-terminus), and the second protein contained TAL protein domains flanking the spy catcher on both sides (N-terminus and C-terminus).

[0093] In some embodiments, the chromatin-binding component is a DNA-binding domain suitable for helically traveling at least half a turn around a chromatid or chromosome. For example, a TAL protein domain helically travels around DNA. Ideally, the DNA-binding domain helically travels at least 3 / 4 of a turn, preferably a full turn, more preferably at least 1.5 turns, and more preferably at least 2 turns around a chromatid or chromosome. In particular, the DNA-binding domain can helically travel up to 20 turns, preferably up to 10 turns, around a chromatid or chromosome.

[0094] Alternatively, the chromatin-binding component can bind to DNA-related proteins. Those skilled in the art are familiar with DNA-related proteins. Exemplary DNA-related proteins include CENP-C, CENP-T, CENP-H, TRF1, TRF2, Dsn1, and histones. The chromatin-binding component may bind to any of these exemplary proteins.

[0095] As further defined in the claims, the chromatin-binding component can bind to a target sequence on a chromatid of a chromosome. Ideally, such a target sequence is located at a specific position on the chromosome or chromatid. For example, such a specific position may be a centromere or telomere. Ideally, the target sequence on each chromatid or chromosome is a repeating DNA sequence. Those skilled in the art recognize repeating sequences along a chromatid or chromosome. For example, such a repeating sequence may be a satellite repeat around the centromere, a satellite repeat around the centromere, or a telomere repeat. It is preferable that the repeating sequence is a satellite repeat around the centromere. As experimentally demonstrated in Example 1, the artificial cohesin successfully tethers the chromatid when using a satellite repeat around the centromere.

[0096] Premature separation of sister chromatids (PSSCs) is caused by the loss of cohesin in the centromere region, making centromere satellite repeats particularly preferable. 12,15,20,21 Therefore, in Example 1, the inventors targeted a 15 bp sequence in the major pericentromere satellite DNA of repeating centromeres with an artificial aggregation system. The complex according to this disclosure efficiently binds to the pericentromere region of mouse chromosomes and is well tolerated by meiotic oocytes. 22,23 .

[0097] Ideally, the repetitive DNA sequence is derived from humans, cats, dogs, horses, cattle, sheep, goats, or pigs. Ideally, the repetitive DNA sequence is derived from humans, cats, dogs, horses, cattle, sheep, goats, or pigs. The repetitive DNA sequence may be derived from veterinary animals. The repetitive DNA sequence may be derived from endangered animals such as pandas, orangutans, saola, tigers, rhinos, leopards, gorillas, elephants, bonobos, or dolphins. More preferably, the repetitive DNA sequence is derived from humans, cats, dogs, or horses. Ideally, the repetitive DNA sequence is a human repetitive DNA sequence.

[0098] In certain embodiments, the chromatin-binding components of the first and second proteins bind to the same target sequence, for example, the repeats around the centromere as shown in Example 1.

[0099] The chromatin-binding components of the first and second proteins may have at least 70% sequence identity with respect to SEQ ID NO: 25. Ideally, this sequence identity is at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 94%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, and most preferably at least 100%.

[0100] The chromatin-binding components of the first and second proteins may have at least 90%, preferably at least 92%, more preferably at least 94%, and even more preferably at least 96% amino acid sequence identity, and most preferably, the first and second proteins may have 100% amino acid sequence identity.

[0101] As described above, the complex described herein is an artificial complex. In other words, the complex does not exist in nature.

[0102] With respect to the number of first and second proteins in a single complex, the number is at least one of the first proteins and at least one of the second proteins. Ideally, the complex contains at least two first proteins and at least two second proteins, preferably at least three first proteins and at least three second proteins, more preferably at least four first proteins and at least four second proteins, and more preferably at least five first proteins and at least five second proteins. In practice, the complex or the nucleic acid encoding the complex can be delivered to cells via in vitro methods. Thus, many first and second proteins are present in cells. Furthermore, two, three, four, five, six, seven, eight, nine, ten or more first and second proteins can form a single complex tethering chromosomes or chromatids together. In such cases, several first and second proteins can be obtained from an artificial aggregation ring of four to possibly 400,000 proteins. In other words, the first and second proteins can bind in a multimerous manner. This means that several first and second proteins can tether chromosomes or chromatids. A single complex for tethering chromosomes or chromatids may contain up to 1,000,000 first proteins and up to 1,000,000 second proteins.

[0103] As shown in Examples 1 and 2, the complex may further contain a fluorescent marker. The marker may be located between the PBN region and the chromatin-binding component, or between the PBC region and the chromatin-binding component. For example, the fluorescent marker may be detectable by fluorescence microscopy.

[0104] nucleic acid molecule As described above, this disclosure also includes nucleic acid molecules encoding the complex disclosed herein. Alternatively, two nucleic acid molecules may encode the complex disclosed herein. For example, a first nucleic acid may encode the first protein of the complex, and a second nucleic acid may encode the second protein of the complex. Alternatively, a single nucleic acid molecule may encode both the first and second proteins. This can be achieved, for example, by encoding the first and second proteins successively. Alternatively, the amino acid sequences of the first and second proteins may be identical. Thus, the nucleic acid sequences may also be identical such that only one nucleic acid molecule encodes both the first and second proteins.

[0105] Preferably, the nucleic acid molecule is an RNA or DNA molecule. RNA molecules are more preferred because RNA molecules can be directly translated into proteins in oocytes or eggs. Ideally, the RNA molecule is an mRNA molecule. In other words, the RNA molecule is translatable to form complexes according to this disclosure.

[0106] RNA molecules may contain stabilization modifications. Those skilled in the art are aware of stabilization modifications. Generally, such modifications inhibit endonucleases and / or exonucleases. In other words, such stabilization modifications inhibit enzymatic degradation within cells. Therefore, stabilized RNA molecules have a longer half-life within cells. Consequently, such RNA molecules can be translated over a longer period on average, resulting in more first and second protein expression over a longer period. Exemplary stabilization modifications include pseudouridine, 2-thiouridine, 5-methyluridine, 5-methylcytidine, N6-methyladenosine, N-methylguanosine, and / or 1-methyl-pseudridine. One or more of these modifications may be present within an RNA molecule. Preferably, the RNA molecule contains a 5'-end stabilization modification. For example, such a 5'-end stabilization modification may be 7-methylguanosine (m 7 It could be G).

[0107] Ideally, the total size of the nucleic acid molecules encoding the first and / or second protein of the complex is at most 10 kb, preferably at most 9 kb, more preferably at most 8 kb, more preferably at most 7 kb, and most preferably at most 6 kb. This maximum size has the advantage that they are easier to produce by in vitro methods and easier to introduce into cells. Thus, the overall handling of the nucleic acids becomes less cumbersome.

[0108] In particular, the complexes or nucleic acid molecules described herein may be contained within oocytes or egg cells.

[0109] Further embodiments of the complex and / or nucleic acid molecules encoding the complex In certain embodiments, the complex tethers chromatids or chromosomes during meiosis. For example, this tethering may occur pre-fertilization. The tethering may also occur during meiosis I.

[0110] The complex or the nucleic acid molecule encoding the complex may be introduced into an oocyte or egg by microinjection, transfection, transduction, or electroporation. The complex is preferably introduced by microinjection. Those skilled in the art will recognize, for example, the methodological steps of microinjection into oocytes and / or egg cells using a microinjection needle. Such a microinjection needle can introduce 1 to 25 picoliters into an oocyte or egg cell. Other delivery methods may also be used to introduce proteins and / or nucleic acid molecules into oocytes and / or egg cells. Generally, electroporation is a method of introducing proteins or nucleic acid molecules into cells by electric current. According to this disclosure, such cells may be oocytes or egg cells.

[0111] Alternatively, the complex or the nucleic acid molecule encoding the complex may be introduced by transfection using liposomes. Generally, transfection is the process of intentionally introducing a nucleic acid molecule or protein into a eukaryotic cell. Furthermore, those skilled in the art know that liposomes fuse with cell membranes and release their cargo into the cell. According to this disclosure, liposomes may fuse with oocytes or egg cell membranes and release the complex or the nucleic acid molecule encoding the complex. In other words, the complex or nucleic acid molecule may be the cargo. Liposomes can fuse with oocytes or egg cells. Liposomes may further encapsulate the complex or the nucleic acid molecule encoding the complex. An exemplary liposome contains a Sendai virus coat protein. For example, the liposome may be a Hemagglutinating virus of Japan (HVJ) envelope vector. Ideally, a Sendai virus (HVJ) envelope cell fusion kit may be used for the introduction of the complex.

[0112] In particular, the complex may be suitable for use during meiosis. Meiosis can be subdivided into meiosis I and meiosis II. Ideally, the complex is suitable for use before fertilization. Fertilization is when sperm binds to an egg cell. More specifically, the complex may be suitable for use during meiosis I. In meiosis I, the chromosomes of a diploid cell reseparate, producing haploid daughter cells. In a haploid cell, only a single set of chromosomes exists. In other words, homologous pairs separate during the first round of cell division, i.e., meiosis I. Using the complex during meiosis I has the technical advantage that the chromosomes are tethered and released during the process, ensuring that all haploid daughter cells contain one complete set of chromosomes. If an error occurs during meiosis, the daughter cells may contain too many or too few chromosomes. These resulting daughter cells are prone to aneuploidy. To reduce the risk of aneuploidy, the complex disclosed herein tethers the chromosomes and releases the tethering during anaphase I. During meiosis II, sister chromatids separate.

[0113] Complexes or nucleic acid molecules for use in reproductive medicine are also disclosed herein. Complexes or nucleic acid molecules suitable for use in reproductive medicine are intended herein. Ideally, such complexes or nucleic acid molecules may be for use in assisted reproductive technology (ART). In particular, such complexes or nucleic acid molecules may be suitable for use in assisted reproductive technology (ART). ART includes fertilization procedures that handle either oocytes or zygotes. ART does not include somatic cell nuclear transfer, etc. According to this disclosure, ART may be performed in vitro, for example, when introducing a complex or a nucleic acid molecule encoding such a complex into an oocyte or egg cell. Alternatively, ART may be performed, for example, when the complex or a nucleic acid molecule encoding such a complex may be for use in reducing the risk of aneuploidy in zygotes. Furthermore, the complexes or nucleic acid molecules disclosed herein may be for use in chromatid or chromosome aggregation in zygotes. Specifically, the complexes or nucleic acid molecules disclosed herein may be for use in reducing the risk of aneuploidy in zygotes. Aneuploidy is the occurrence of one or more extra chromosomes or chromosomal deletions that result in an unbalanced chromosomal complement, or any number of chromosomes that is not an exact multiple of the haploid number. The haploid number in humans is 23. In other words, aneuploidy is the presence of an abnormal number of chromosomes in a cell. Aneuploidy is a major cause of infertility and hereditary congenital defects. Therefore, reducing the risk of aneuploidy has the medical benefit of having more fertilized oocytes or egg cells and a lower risk of congenital defects.

[0114] Oocytes or egg cells can be obtained, for example, from a woman. In other words, such oocytes or egg cells may be ex vivo. Such oocytes or egg cells may be in vitro. Starting from such oocytes or egg cells, the complexes or nucleic acids disclosed herein can be introduced into the oocytes or egg cells. An in vitro method for introducing the complexes or nucleic acid molecules disclosed herein into oocytes or egg cells is disclosed herein. Such method may include the step of introducing the complexes or nucleic acid molecules into the oocytes or egg cells. The term “introduce” refers to any process for transferring the complexes or nucleic acid molecules into the oocytes or egg cells, such as microinjection or electroporation.

[0115] Nucleic acid molecules can be translated to form the complexes disclosed herein. Ideally, the complexes or translated nucleic acid molecules disclosed herein stabilize the aggregation of chromatids or chromosomes in oocytes or egg cells. In certain embodiments, a suitable amount of the complex or nucleic acid molecule is introduced into human oocytes or egg cells. As shown in Example 1, the amount of the complex or nucleic acid molecule varies the result. When too much of the complex or nucleic acid molecule is introduced, for example, chromatin clustering may result. Specifically, a suitable amount can be evaluated by titration experiments. For example, if chromosome clustering, broken kinetochores, decreased spacing between chromatids or chromosome kinetochores, decreased recovery of chromosome localization fluorescence signals after photobleaching, resistance to sister chromosome segregation after acute depletion of cohesin subunits, decreased chromosomal errors in metaphase II oocytes, and / or other chromatid or chromosome aggregation defects are detected using fluorescence microscopy or polarizing microscopy, the amount is too high. For example, if the early separation of sister chromatids is not reduced compared to cells that have not been introduced with the complex or the nucleic acid molecule encoding the complex, then the amount is too small.

[0116] Exemplary preferred amounts of nucleic acid molecules are in the range of 0.0002 to 0.02 atmoles, preferably 0.0004 to 0.01 atmoles, more preferably 0.0008 to 0.005 atmoles, more preferably 0.0009 to 0.004 atmoles, more preferably 0.001 to 0.003 atmoles, and most preferably about 0.002 atmoles. Example 1 provides an exemplary titration of the atomolecules of nucleic acid for introduction into oocytes or egg cells.

[0117] Exemplary preferred amounts of the complex are in the range of 0.001 to 25 pg per oocyte, preferably 0.005 to 10 pg, more preferably 0.01 to 8 pg, more preferably 0.02 to 5 pg, more preferably 0.04 to 3 pg, and even more preferably 0.06 to 2 pg.

[0118] Also disclosed herein is an in vitro method for stabilizing the aggregation of chromatids or chromosomes in oocytes or egg cells, the method comprising the step of introducing the complex or nucleic acid molecule according to the present disclosure into the oocyte or egg cell. Ideally, the complex or nucleic acid molecule is introduced into human oocytes or human egg cells. In particular, the in vitro method does not alter human germline identity.

[0119] This method can reduce premature separation of sister chromatids (PSSCs) in oocytes compared to oocytes that have not been introduced with the complexes or nucleic acid molecules disclosed herein. Specifically, PSSCs can be reduced by at least 10%, preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, and even more preferably at least 100%. Example 1 shows that PSSCs were reduced by at least 100% in mouse oocytes. Similar levels are expected for human oocytes, as the process is largely conserved. In particular, PSSCs can be reduced by up to 1000%. As shown in Example 1, for example in Figures 7D and 7E, the duration of late II can be at least 15 minutes. The duration of late II can be up to 40 minutes. Preferably, the duration of late II is less than 20 minutes.

[0120] Ideally, the complexes or nucleic acid molecules disclosed herein are delivered before metaphase II arrest. In particular, the complexes or nucleic acid molecules disclosed herein may be delivered during meiosis I. The term “deliver” refers to any process for transferring the complexes or nucleic acid molecules into oocytes or egg cells, e.g., microinjection or electroporation. Ideally, the complexes or nucleic acid molecules disclosed herein are delivered before nuclear membrane collapse of vesicle-stage oocytes, and the resumption of meiosis is determined by nuclear membrane collapse. It is even more preferable that the complexes or nucleic acid molecules disclosed herein be delivered after nuclear membrane collapse but before anaphase I. In particular, the complexes or nucleic acid molecules disclosed herein may be delivered in metaphase I. Delivery after nuclear membrane collapse but before anaphase I has the particular advantage that the complexes can function during chromosome segregation in meiosis I, thereby reducing the risk of aneuploidy in meiosis I.

[0121] An in vitro method for stabilizing the aggregation of chromatids or chromosomes in oocytes or egg cells is also disclosed. The method comprises the step of delivering a complex or nucleic acid molecule disclosed herein to a human oocyte or egg cell, the nucleic acid molecule being translated into a complex disclosed herein, the complex thereby stabilizing the aggregation of chromatids or chromosomes in the human oocyte or egg cell. Preferably, the method comprises an additional step carried out as a first step, in which the oocyte or egg cell is provided in vitro.

[0122] In particular, this method does not alter the germline identity of humans or animals. Ideally, the oocytes or eggs are derived from humans, cats, dogs, horses, cattle, sheep, goats, or pigs. In particular, the oocytes or eggs may be derived from humans, cats, dogs, or horses. Preferably, the oocytes or eggs are derived from humans.

[0123] This in vitro method can reduce premature separation of sister chromatids (PSSCs) in oocytes compared to oocytes that have not been introduced with the complex or nucleic acid molecules disclosed herein. Specifically, PSSCs can be reduced by at least 10%, preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, and even more preferably at least 100%. Example 1 shows that PSSCs were reduced by at least 100% in mouse oocytes. Similar levels are expected for human oocytes, as the process is largely conserved. In particular, PSSCs can be reduced by up to 1000%.

[0124] Ideally, the complexes or nucleic acid molecules disclosed herein are delivered before metaphase II arrest. In particular, the complexes or nucleic acid molecules disclosed herein may be delivered during meiosis I. Ideally, the complexes or nucleic acid molecules disclosed herein are delivered before nuclear membrane breakdown of the vesicle-stage oocyte, and the restart of meiosis is determined by nuclear membrane breakdown. It is even more preferable that the complexes or nucleic acid molecules disclosed herein be delivered after nuclear membrane breakdown and before anaphase I. In particular, the complexes or nucleic acid molecules disclosed herein may be delivered during metaphase I.

[0125] The following methods are also disclosed, and the embodiments described herein with respect to complexes, nucleic acid molecules, and methods and medical uses also apply to the following methods.

[0126] Methods in reproductive medicine are also disclosed, which include the step of introducing a complex or nucleic acid molecule described herein into an oocyte, egg cell, or zygote. Specifically, such methods may be assisted reproductive technologies.

[0127] Also disclosed is a method for increasing chromatid or chromosome aggregation in oocytes, oocytes, or zygotes, comprising the step of introducing a complex or nucleic acid molecule described herein into the oocytes, oocytes, or zygotes. Specifically, chromatid or chromosome aggregation is increased compared to oocytes, oocytes, or zygotes that have not been introduced the complex or nucleic acid molecule described herein.

[0128] A method for reducing the risk of aneuploidy in oocytes, oocytes, or zygotes, comprising the step of introducing the complex or nucleic acid molecule described herein into oocytes, oocytes, or zygotes, wherein the risk of aneuploidy is reduced compared to oocytes or zygotes that have not been introduced the complex or nucleic acid molecule described herein.

[0129] A method for reducing the probability of aneuploidy in oocytes, oocytes, or zygotes, comprising the step of introducing the complex or nucleic acid molecule described herein into oocytes, oocytes, or zygotes, wherein the risk of aneuploidy is reduced compared to oocytes or zygotes that have not been introduced with the complex or nucleic acid molecule described herein.

[0130] Finally, this disclosure also describes a kit comprising the complex or nucleic acid molecule described herein. As used herein, “kit” generally refers to a set of parts. In other words, a kit may comprise several parts. The complex or nucleic acid molecule may be suitable for introduction into oocytes. The complex or nucleic acid molecule may be suitable for introduction into egg cells. The kit may comprise a device for introducing the complex or the nucleic acid molecule encoding the complex into egg cells or oocytes. For example, the device may be suitable for microinjection. The device may be a microinjection needle. Ideally, the microinjection needle comprises a suitable pump suitable for transferring the complex or the nucleic acid molecule encoding the complex into oocytes or egg cells. Preferably, the suitable pump is suitable for transferring 1 to 25 picoliters. In certain embodiments, the kit comprises a liposome suitable for transfection of the complex or nucleic acid molecule. The liposome may encapsulate the complex or the nucleic acid molecule encoding the complex. Specifically, the liposomes may contain Sendai virus coat proteins. The liposomes may be Sendai virus (HVJ) envelope vectors.

[0131] Furthermore, the kit may include a container. Ideally, the complex or nucleic acid molecule is stored in the container. This has the advantage of allowing for easy transport of the complex or nucleic acid molecule. Preferably, the complex or nucleic acid molecule is stored at a temperature of 4°C or below (at most 4 °C). In particular, the complex or nucleic acid molecule may be stored at a temperature of -20°C or below (at most -20 °C). Preferably, the complex or nucleic acid molecule may be stored at a temperature of at least -196°C.

[0132] The present invention is further described by the following embodiments. Embodiment 1. A complex suitable for chromatid or chromosome aggregation, comprising (I) one or more first proteins and (II) one or more second proteins, (I) The first protein and (II) the second protein each include (i) a chromatin-binding component, (ii) a protein-binding region (PBN region) at the N-terminus of the chromatin-binding component, and (iii) a protein-binding region (PBC region) at the C-terminus of the chromatin-binding component; The (i) chromatin-binding components of the first and second proteins can bind to target sequences on chromatids or chromosomes; (I)(ii) the PBN region of the first protein can bind to (II)(ii) the PBN region of the second protein, (I)(iii) the PBC region of the first protein can bind to (II)(iii) the PBC region of the second protein, or (I)(ii) the PBN region of the first protein can bind to (II)(iii) the PBC region of the second protein, and (I)(iii) the PBC region of the first protein can bind to (II)(ii) the PBN region of the second protein; A complex in which binding to chromatids or chromosomes, and the binding of the PBN and PBC regions of the first and second proteins, reversibly tethers the chromatids or chromosomes.

[0133] Embodiment 2. The complex according to Embodiment 1, wherein (I)(ii) the PBN region of the first protein can bind to (II)(ii) the PBN region of the second protein, and (I)(iii) the PBC region of the first protein can bind to (II)(iii) the PBC region of the second protein.

[0134] Embodiment 3. The complex according to Embodiment 2, wherein the PBN region of the first protein (I)(ii) and the PBC region of the first protein (I)(iii) have at least 90% sequence identity, preferably 100% sequence identity.

[0135] Embodiment 4. The complex according to Embodiment 2 or 3, wherein the PBN region of the second protein (II)(ii) and the PBC region of the second protein (II)(iii) have at least 90% sequence identity, preferably 100% sequence identity.

[0136] Embodiment 5. The complex according to any one of Embodiments 2 to 4, wherein the PBN region and PBC region of the first protein can form a covalent bond with the PBN region and PBC region of the second protein.

[0137] Embodiment 6. The complex according to any one of Embodiments 2 to 5, wherein the PBN region and PBC region of the first protein can be reconstituted with the PBN region and PBC region of the second protein.

[0138] Embodiment 7. The complex according to any one of Embodiments 2 to 6, wherein the PBN region of the first protein (I)(ii) and / or the PBC region of the first protein (I)(iii) are selected from spytag, snooptag, dogtag, and split-intine N-terminal fragment, preferably the PBN region of the first protein (I)(ii) and / or the PBC region of the first protein (I)(iii) are spytag.

[0139] Embodiment 8. The composite according to Embodiment 7, wherein the spy tag is selected from spy tag 001, spy tag 002, and spy tag 003, and more preferably the spy tag is spy tag 001.

[0140] Embodiment 9. The complex according to Embodiment 7, wherein the split intein N-terminal fragment is a split DnaE intein N-terminal fragment, and preferably the split DnaE intein N-terminal fragment is derived from Nostoc punctiforme (Npu).

[0141] Embodiment 10. The complex according to any of Embodiments 2 to 9, wherein (II)(ii) the PBN region of the second protein and / or (II)(iii) the PBC region of the second protein is selected from spycatcher, snoopcatcher, dogcatcher, and split-intine C-terminal fragments.

[0142] Embodiment 11. The composite according to Embodiment 10, wherein the spy catcher is selected from spy catcher 001, spy catcher 002, and spy catcher 003, and preferably the spy catcher is spy catcher 001.

[0143] Embodiment 12. The complex according to Embodiment 10, wherein the split intein C-terminal fragment is a split DnaE intein C-terminal fragment, and preferably the split DnaE intein C-terminal fragment is derived from Nostoc punctiforme (Npu).

[0144] Embodiment 13. The N-terminal and C-terminal fragments of the split intein are SspDnaB Δ275 M86, NPU N / Ssp C DnaE, ​​gp41-1, gp41-8, NrdJ-1, IMPDH-1, SspDnaX Δ297 SspGyrB Δ279 TerThyX Δ134 The composite according to Embodiments 9 and / or 12, selected from any of TvoVMA, PhoRadA, CIV RIR1, CthATCC27405 TerA, MP-Be DnaB, MP-Catera gp206, PfuRIR1-1, and MjaKlbA.

[0145] Embodiment 14. The (I)(ii)PBN region and (I)(iii)PBC region of the first protein have sequence identity of at least 70%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 94%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, and most preferably 100% with respect to SEQ ID NO: 4 or 8, and / or The complex according to any of Embodiments 2 to 13, wherein the (II)(ii)PBN region and (II)(iii)PBC region of the second protein have sequence identity of at least 70%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 94%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, and most preferably 100% with respect to SEQ ID NO: 2 or 6.

[0146] Embodiment 15.(I)(ii) The PBN region of the first protein can bind to (II)(iii) the PBC region of the second protein, and (I)(iii) The PBC region of the first protein can bind to (II)(ii) the PBN region of the second protein, The complex according to Embodiment 1, wherein the binding between the PBN region and the PBC region is inducible.

[0147] Embodiment 16. (I)(ii) The PBN region of the first protein can be reconstituted with (II)(iii) the PBC region of the second protein. (I)(iii) The complex according to Embodiment 15, wherein the PBC region of the first protein can be reconstituted with (II)(ii) the PBN region of the second protein.

[0148] Embodiment 17.(I)(ii) The PBN region of the first protein and (II)(ii) the PBN region of the second protein have at least 90% sequence identity, preferably 100% sequence identity; and / or The complex according to Embodiment 15 or 16, wherein the PBC region of the first protein (I)(iii) and the PBC region of the second protein (II)(iii) have at least 90% sequence identity, preferably 100% sequence identity.

[0149] Embodiment 18. The complex according to any one of Embodiments 15 to 17, wherein the (I)(ii)PBN region and the (I)(iii)PBC region of the first protein can form covalent bonds with the (II)(ii)PBN region and the (II)(iii)PBC region of the second protein.

[0150] Embodiment 19. The complex according to any one of Embodiments 15 to 18, wherein the PBN region and PBC region of the first protein can be reconstituted with the PBN region and PBC region of the second protein.

[0151] Embodiment 20. The composite according to any one of Embodiments 15 to 19, wherein the binding between the PBN region and the PBC region is inducible by photo and / or chemically induced proximity.

[0152] Embodiment 21. The composite according to Embodiment 20, wherein the bonding is inducible by light, preferably blue, green, or red light, more preferably blue light.

[0153] Embodiment 22. The complex according to Embodiment 20 or 21, wherein the (I)(ii)PBN region and (I)(iii)PBC region of the first protein can form covalent bonds with the (II)(ii)PBN region and (II)(iii)PBC region of the second protein, and such bonds are light-inducible.

[0154] Embodiment 23.(I)(ii) The PBN region of the first protein and / or (II)(ii) The PBN region of the second protein is a light-inducible spy tag, preferably a blue light-inducible spy tag (BLISS). The complex according to any of Embodiments 20 to 22, wherein (I)(iii) the PBC region of the first protein and / or (II)(iii) the PBC region of the second protein is a spycatcher.

[0155] Embodiment 24.(I)(iii) The PBC region of the first protein and / or (II)(iii) The PBC region of the second protein is a light-inducible spy tag, preferably a blue light-inducible spy tag (BLISS). (I)(ii) The PBN region of the first protein and / or (II)(ii) The PBN region of the second protein is a spycatcher, as described in any of Embodiments 20 to 22.

[0156] Embodiment 25.(I)(ii) The PBN region of the first protein and / or (II)(ii) The PBN region of the second protein have sequence identity of at least 70%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 94%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, and most preferably 100% with respect to SEQ ID NO: 40, and / or The complex according to any of Embodiments 22 to 24, wherein (I)(iii) the PBC region of the first protein and / or (II)(iii) the PBC region of the second protein have sequence identity of at least 70%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 94%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, and most preferably 100% with respect to SEQ ID NO: 40.

[0157] Embodiment 26. The complex according to any one of Embodiments 20 to 25, wherein the binding between the PBN region and the PBC region is induceable by chemically induced proximity, preferably by chemically induced dimerization.

[0158] Embodiment 27. The complex according to Embodiment 26, wherein the chemically induced proximity is induced by a compound, preferably the compound is a small molecule compound, and more preferably the small molecule compound is selected from one or two, preferably one, of rapamycin, FK506, FK506-cyclosporine, FK1012, coumamycin, gibberellin, S-(+)-abscisic acid, BisMTX, TMP-halo, caffeine, and cannabidiol.

[0159] Embodiment 28.(I)(ii) The complex according to Embodiment 26 or 27, wherein the PBN region of the first protein and the (II)(iii) and PBC regions of the second protein are respectively selected from FRB and FKBP; FKBP and calcineurin A; FKBP and cyclophyllin; FKBP and FKBP; GyrB and GyrB; GID1 and GAI; PYL1 and ABI1; DHFR and DHFR; AcVHH and AcVHH; or nanobodies capable of heterodimerization with cannabidiol.

[0160] Embodiment 29.(I)(iii) A complex according to any one of Embodiments 26 to 28, wherein the PBC region of the first protein and the (II)(ii) and PBN regions of the second protein are respectively selected from FRB and FKBP; FKBP and calcineurin A; FKBP and cyclophyllin; FKBP and FKBP; GyrB and GyrB; GID1 and GAI; PYL1 and ABI1; DHFR and DHFR; AcVHH and AcVHH; or nanobodies capable of heterodimerization with cannabidiol.

[0161] Embodiment 30. The composite according to Embodiment 26, wherein the chemically induced proximity is a photoswitchable chemically induced dimerization.

[0162] Embodiment 31. The complex according to Embodiment 30, wherein the photoswitchable, chemically induced dimerization is inducible by a small molecule compound, more preferably the compound is selected from one or two, preferably one, of TMP-halo (trimethoprim fused to a halo ligand), photocaged rapamycin, and coumarin-caged TMP-halo (CTH).

[0163] Embodiment 32.(I)(ii) The complex according to Embodiment 30 or 31, wherein the PBN region of the first protein and the (II)(iii) and PBC regions of the second protein are selected from FRB and FKBP; and DHFR and halotag; respectively.

[0164] Embodiment 33.(I)(iii) The complex according to Embodiment 30 or 31, wherein the PBC region of the first protein and the (II)(ii) and PBN regions of the second protein are selected from FRB and FKBP; and DHFR and halotag; respectively.

[0165] Embodiment 34. The complex according to any one of Embodiments 15 to 33, wherein the first and second proteins have sequence identity of at least 70%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and even more preferably 100%.

[0166] Embodiment 35. Tethering is (a) cleavage of a complex, wherein the first and second proteins of the complex further include protease cleavage sites; (b) Degradation of the complex, wherein the first and / or second protein of the complex further comprises a ubiquitination site; and / or (c) The complex according to any one of Embodiments 1 to 34, wherein the binding between the PBN region and the PBC region of the first and second proteins is reversible by disruption, which is mediated by chemically induced proximity.

[0167] Embodiment 36.(a) The complex according to Embodiment 35, wherein the protease cleavage site is located between (ii) the PBN region and (i) the chromatin-binding component, or between (i) the chromatin-binding component and (iii) the PBC region of the first and / or second protein.

[0168] Embodiment 37. The complex according to Embodiment 36, wherein the protease cleavage site is cleavable by a naturally occurring enzyme in the fertilized zygote, preferably the protease cleavage site is a separase cleavage site, and more preferably the separase cleavage site is a Rec8 site.

[0169] Embodiment 38. The composite according to Embodiment 37, wherein the Rec8 site is a mammalian Rec8 site, preferably derived from a human, cat, dog, horse, cattle, sheep, goat, or pig, and more preferably derived from a human, cat, dog, or horse.

[0170] Embodiment 39. The complex according to Embodiment 37 or 38, wherein the Rec8 site is a human Rec8 site.

[0171] Embodiment 40. The complex according to any one of Embodiments 37 to 39, wherein the first and second proteins each contain at least two Rec8 cleavage sites, preferably the first and second proteins each contain two Rec8 cleavage sites.

[0172] Embodiment 41. The complex according to any one of Embodiments 37 to 40, wherein the first and second proteins each further comprise a Rec8 leucine-proline-glutamic acid motif (LPE motif).

[0173] Embodiment 42. The complex according to any one of Embodiments 37 to 41, wherein at least one protease cleavage site has at least 75% identity with SEQ ID NO: 30 or 32.

[0174] Embodiment 43. The complex according to any one of Embodiments 37 to 42, wherein the first and second proteins comprise two or more protease cleavage sites, preferably the two or more protease cleavage sites comprise two Rec8 cleavage sites, and more preferably the two Rec8 cleavage sites further comprise a Rec8 LPE motif.

[0175] Embodiment 44. The complex according to any one of Embodiments 37 to 43, wherein the first and second proteins each contain two protease cleavage sites having at least 70%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 94%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, and most preferably 100% sequence identity with respect to SEQ ID NO: 28.

[0176] Embodiment 45.(a) The complex according to Embodiment 36, wherein the protease cleavage site is an enzyme that does not naturally exist in the fertilized zygote.

[0177] Embodiment 46. The complex according to Embodiment 45, wherein the protease cleavage site is cleavable by a cysteine ​​or serine protease, and preferably the protease is a TEV or HCV NS3 / 4 protease.

[0178] Embodiment 47. The complex of Embodiment 45 or 46, wherein the protease can be inhibited by a small molecule inhibitor, preferably BILN206.

[0179] Embodiment 48. The complex according to Embodiment 35, wherein the first and second proteins of the complex further comprise (b) a ubiquitination site.

[0180] Embodiment 49. The complex according to Embodiment 48, wherein the ubiquitination site is an amino acid sequence of securin that can be ubiquitinated by APC / C, preferably having at least 25 amino acids of securin, preferably at least 40 amino acids, more preferably at least 60 amino acids, more preferably at least 80 amino acids, even more preferably 80 to 150 amino acids, and most preferably the amino acid sequence of securin is located at the N-terminus of securin.

[0181] Embodiment 50. The complex according to Embodiment 49, wherein the sequence of securin is derived from human, cat, dog, horse, cattle, sheep, goat, or pig, more preferably from human, cat, dog, or horse, and even more preferably from human.

[0182] Embodiment 51. The complex of Embodiment 49 or 50, wherein the amino acid sequence of securin is ubiquitinated when it enters the late stage.

[0183] Embodiment 52. The complex according to Embodiment 48, wherein tethering is reversible by degradation of the target protein.

[0184] Embodiment 53. The complex according to Embodiment 52, further comprising a PROTAC binding site.

[0185] Embodiment 54. The complex according to Embodiment 53, wherein the presence of PROTAC results in ubiquitination of the complex.

[0186] Embodiment 55. The complex according to either Embodiment 53 or 54, wherein the PROTAC binding site is selected from the group consisting of an androgen receptor domain, an estrogen receptor domain, a BRD9 domain, a B cell giant domain (BCL-xL), an IRAK4 domain, a STAT3 domain, a BTK domain, an EGFR domain, and a tropomyosin receptor domain.

[0187] Embodiment 56. The complex according to Embodiment 35, wherein the binding of the PBN and PBC regions of the first protein to the PBN and PBC regions of the second protein is mediated by chemically induced proximity, and the binding of the PBN and PBC regions of the first and second proteins is disrupted by the absence of a small molecule compound or by functionally inactivating the small molecule compound.

[0188] Embodiment 57. The complex according to Embodiment 56, wherein the small molecule compound is a photoswitchable small molecule compound, and preferably the compound is selected from one of MeNV-HaXS, TMP-halo(NTH) of an NVOC cage, and TMP-NVOC linker-halo.

[0189] Embodiment 58. The complex according to any one of Embodiments 1 to 57, wherein the chromatin-binding component is a DNA-binding domain and / or each chromatin-binding component can bind to a DNA-related protein.

[0190] Embodiment 59. The complex according to Embodiment 58, wherein the chromatin-binding component is a DNA-binding domain, and preferably each DNA-binding domain is individually selected from the group consisting of a TAL protein domain, a zinc finger protein, a leucine zipper, and dCas9 complexed with gRNA, and preferably each DNA-binding component is individually selected from the group consisting of a TAL protein domain and a zinc finger protein.

[0191] Embodiment 60. The complex according to Embodiment 58, wherein each chromatin-binding component can bind to a DNA-related protein, and preferably the DNA-related protein is selected from CENP-C, CENP-T, CENP-H, TRF1, TRF2, Dsn1, and histones.

[0192] Embodiment 61. The complex according to any one of Embodiments 1 to 60, wherein each target sequence on a chromatid or chromosome is a repetitive DNA sequence, preferably a satellite repeat around a centromere, a centromere-satellite repeat, or a telomere repeat.

[0193] Embodiment 62. The complex according to any one of Embodiments 1 to 61, wherein each target sequence on a chromatid or chromosome is a satellite repeat around the centromere.

[0194] Embodiment 63. The complex according to Embodiment 61 or 62, wherein the repetitive DNA sequence is derived from human, cat, dog, horse, cattle, sheep, goat, or pig, more preferably from human, cat, dog, or horse, and even more preferably from human.

[0195] Embodiment 64. The complex according to any one of Embodiments 1 to 63, wherein the chromatin-binding components of the first and second proteins bind to the same target sequence.

[0196] Embodiment 65. The complex according to any one of Embodiments 1 to 64, wherein the chromatin-binding components of the first and second proteins may have sequence identity of at least 70%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 94%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, and most preferably 100% with respect to SEQ ID NO: 25.

[0197] Embodiment 66. The complex according to any one of Embodiments 1 to 65, wherein the chromatin-binding components of the first and second proteins have at least 90%, preferably at least 92%, more preferably at least 94%, and even more preferably at least 96% amino acid sequence identity, and most preferably the first and second proteins have 100% amino acid sequence identity.

[0198] Embodiment 67. A non-natural composite according to any one of Embodiments 1 to 66.

[0199] Embodiment 68. The complex according to any one of Embodiments 1 to 67, wherein the complex comprises at least one first protein and at least one second protein, preferably the complex comprises at least two first proteins and at least two second proteins, more preferably the complex comprises at least three first proteins and at least three second proteins, more preferably the complex comprises at least four first proteins and at least four second proteins, more preferably the complex comprises at least five first proteins and at least five second proteins, and the complex comprises up to 1,000,000 first proteins and up to 1,000,000 second proteins.

[0200] Embodiment 69. The complex according to any one of Embodiments 1 to 68, wherein the complex further comprises a fluorescent marker, preferably located between the PBN region and the chromatin-binding component, or between the PBC region and the chromatin-binding component, and more preferably the fluorescent marker is detectable by fluorescence microscopy.

[0201] Embodiment 70. A nucleic acid molecule or two nucleic acid molecules encoding the complex described in any of Embodiments 1 to 69.

[0202] Embodiment 71. The nucleic acid molecule or two nucleic acid molecules according to Embodiment 70, wherein the nucleic acid molecule is an RNA or DNA molecule, preferably an RNA molecule, and more preferably an mRNA molecule.

[0203] Embodiment 72. A nucleic acid molecule or two nucleic acid molecules according to Embodiment 70 or 47, wherein the nucleic acid molecule is RNA, and the RNA molecule includes a stabilizing modification.

[0204] Embodiment 73. The nucleic acid molecule or two nucleic acid molecules according to embodiment 72, wherein the RNA molecule contains one or more pseudouridines, 2-thiouridine, 5-methyluridine, 5-methylcytidine, N6-methyladenosine, N-methylguanosine and / or 1-methyl-pseudouridine.

[0205] Embodiment 74. The nucleic acid molecule or two nucleic acid molecules according to embodiment 72 or 73, wherein the RNA molecule contains a 5'-end stabilization modification, preferably, the 5'-end stabilization modification is 7-methylguanosine (m 7 G).

[0206] Embodiment 75. The nucleic acid molecule or two nucleic acid molecules according to any one of embodiments 70 to 74, with a total size of at most 10 kb, preferably at most 9 kb, more preferably at most 8 kb, more preferably at most 7 kb, and most preferably at most 6 kb.

[0207] Embodiment 76. The nucleic acid molecule or two nucleic acid molecules according to any one of embodiments 70 to 75, wherein the nucleic acid molecule is RNA and the RNA molecule is translatable to form the complex according to any one of embodiments 1 to 69.

[0208] Embodiment 77. The complex or nucleic acid molecule according to any one of embodiments 1 to 69 or the nucleic acid molecule according to any one of embodiments 70 to 76, wherein the complex or nucleic acid molecule is contained within an oocyte or egg.

[0209] Embodiment 78. The complex or nucleic acid molecule according to any one of embodiments 1 to 69 or the nucleic acid molecule according to any one of embodiments 70 to 76, wherein the complex tethers chromatids or chromosomes during meiosis, preferably before fertilization, more preferably during meiosis I.

[0210] Embodiment 79. The complex or the nucleic acid molecule encoding the complex according to any one of Embodiments 1 to 69 or any one of Embodiments 70 to 76, wherein the complex or the nucleic acid molecule encoding the complex is introduced into an oocyte or egg by microinjection, liposome-based transfection, transduction, or electroporation, preferably by microinjection or liposome-based transfection, more preferably by microinjection.

[0211] Embodiment 80. The complex according to any of Embodiments 1 to 69 or the nucleic acid molecule according to any of Embodiments 70 to 76, wherein the complex is suitable for use during meiosis, particularly before fertilization, and more specifically during meiosis I.

[0212] Embodiment 81. A complex according to any one of Embodiments 1 to 69 or a nucleic acid molecule according to any one of Embodiments 70 to 76, suitable for use in reproductive medicine, preferably in assisted reproductive technology.

[0213] Embodiment 82. A complex according to any one of Embodiments 1 to 69 or a nucleic acid molecule according to any one of Embodiments 70 to 76 for use in the aggregation of chromatids or chromosomes in a zygote.

[0214] Embodiment 83. A complex according to any of Embodiments 1 to 69 or a nucleic acid molecule according to any of Embodiments 70 to 76, for use in reducing the risk of aneuploidy in a conjugate.

[0215] Embodiment 84. A complex according to any of Embodiments 1 to 69 or a nucleic acid molecule according to any of Embodiments 70 to 76, for use in reducing the probability of aneuploidy in a conjugate.

[0216] Embodiment 85. An in vitro method for introducing a complex described in any of Embodiments 1 to 69 or a nucleic acid molecule described in any of Embodiments 70 to 76 into an oocyte or egg cell, the method comprising the step of introducing the complex or nucleic acid molecule into an oocyte or egg cell.

[0217] Embodiment 86. The method according to Embodiment 85, wherein the complex or translated nucleic acid molecule stabilizes the aggregation of chromatids or chromosomes in oocytes or egg cells.

[0218] Embodiment 87. The method according to Embodiment 85 or 86, wherein a suitable amount of the complex or nucleic acid molecule is introduced into a human oocyte or human egg cell.

[0219] Embodiment 88. The method according to any one of Embodiments 85 to 87, wherein the preferred amount of nucleic acid molecules is in the range of 0.0002 to 0.02 atmoles, preferably 0.0004 to 0.01 atmoles, more preferably 0.0008 to 0.005 atmoles, more preferably 0.0009 to 0.004 atmoles, more preferably 0.001 to 0.003 atmoles, and most preferably about 0.002 atmoles.

[0220] Embodiment 89. The method according to any one of Embodiments 85 to 88, wherein the preferred amount of the complex is in the range of 0.001 to 25 pg per oocyte, preferably 0.005 to 10 pg, more preferably 0.01 to 8 pg, more preferably 0.02 to 5 pg, more preferably 0.04 to 3 pg, and even more preferably 0.06 to 2 pg.

[0221] Embodiment 90. The method according to any one of Embodiments 85 to 89, wherein a suitable amount is too much when, as evaluated by titration, preferably using fluorescence microscopy or polarizing microscopy, chromosome clustering, fractured kinetochores, a decrease in the spacing between chromosome kinetochores or chromatids, a decrease in the recovery of chromosome localization fluorescence signals after photobleaching, resistance to sister chromosome segregation after acute depletion of cohesin subunits, and a decrease in chromosome errors in metaphase II oocytes, and / or other chromatid or chromosome aggregation defects are detected.

[0222] Embodiment 91. An in vitro method for stabilizing chromatid or chromosome aggregation in oocytes or egg cells, comprising the step of introducing a complex according to any of Embodiments 1 to 69 or a nucleic acid molecule according to any of Embodiments 70 to 76 into oocytes or egg cells.

[0223] Embodiment 92. The method according to Embodiment 91, wherein the complex or nucleic acid molecule is introduced into a human oocyte or human egg cell.

[0224] Embodiment 93. The method according to Embodiment 91 or 92, which does not alter human germline identity.

[0225] Embodiment 94. The method according to any one of Embodiments 91 to 93, which reduces premature separation of sister chromatids (PSSCs) in oocytes or egg cells compared to oocytes or egg cells that have not been introduced with the complex described in any one of Embodiments 1 to 69 or the nucleic acid molecule described in any one of Embodiments 70 to 76.

[0226] Embodiment 95. The method according to Embodiment 94, wherein the PSSC is reduced by at least 10%, preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, even more preferably at least 100%, and / or up to 1000%.

[0227] Embodiment 96. The method according to any one of Embodiments 91 to 96, wherein the complex described in any one of Embodiments 1 to 69 or the nucleic acid molecule described in any one of Embodiments 70 to 76 is delivered before metaphase II arrest, preferably during meiosis I.

[0228] Embodiment 97. The method according to Embodiment 96, wherein the complex according to any one of Embodiments 1 to 69 or the nucleic acid molecule according to any one of Embodiments 70 to 76 is delivered before the nuclear envelope breakdown of the germinal vesicle stage oocyte, and the resumption of meiosis is determined by the nuclear envelope breakdown, and more preferably, it is determined after the nuclear envelope breakdown and before prometaphase I, and most preferably, it is determined at metaphase I.

[0229] Embodiment 98. An in vitro method for stabilizing the condensation of chromatids or chromosomes of an oocyte or egg cell, comprising the step of delivering the complex according to any one of Embodiments 1 to 69 or the nucleic acid molecule according to any one of Embodiments 70 to 76 to a human oocyte or egg cell, and the nucleic acid molecule is translated into the complex according to any one of Embodiments 1 to 69, Thereby, the complex stabilizes the condensation of chromatids or chromosomes in the human oocyte or egg cell.

[0230] Embodiment 99. The method according to Embodiment 98, which does not modify the germline identity of a human or animal.

[0231] Embodiment 100. The method according to Embodiment 98 or 99, wherein the oocyte or egg cell is derived from a human, cat, dog, horse, cow, sheep, goat or pig, more preferably a human, cat, dog or horse, and even more preferably a human.

[0232] Embodiment 101. The method according to any one of Embodiments 98 to 100, which reduces premature sister chromatid separation (PSSC) in oocytes as compared to oocytes into which the complex according to any one of Embodiments 1 to 69 or the nucleic acid molecule according to any one of Embodiments 70 to 76 has not been introduced.

[0233] Embodiment 102. The method according to Embodiment 101, wherein the PSSC is reduced by at least 10%, preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, even more preferably at least 100%, and / or up to 1000%.

[0234] Embodiment 103. The method according to any one of Embodiments 98 to 102, wherein the complex described in any one of Embodiments 1 to 69 or the nucleic acid molecule described in any one of Embodiments 70 to 76 is delivered before metaphase II arrest, preferably during meiosis I.

[0235] Embodiment 104. The method according to Embodiment 103, wherein the complex described in any of Embodiments 1 to 69 or the nucleic acid molecule described in any of Embodiments 70 to 76 is delivered to an embryonic vesicle-stage oocyte before nuclear membrane collapse, and the restart of meiosis is determined by nuclear membrane collapse, more preferably after nuclear membrane collapse, before anaphase I, and most preferably in metaphase I.

[0236] Embodiment 105. A method in reproductive medicine comprising the step of introducing a complex according to any of Embodiments 1 to 69 or a nucleic acid molecule according to any of Embodiments 70 to 76 into an oocyte, egg cell, or zygote.

[0237] Embodiment 106. A method for increasing chromatid or chromosome aggregation in oocytes, oocytes, or zygotes, comprising the step of introducing a complex according to any of Embodiments 1 to 69 or a nucleic acid molecule according to any of Embodiments 70 to 76 into oocytes, oocytes, or zygotes, wherein chromatid or chromosome aggregation is increased compared to oocytes, oocytes, or zygotes that have not been introduced the complex according to any of Embodiments 1 to 69 or the nucleic acid molecule according to any of Embodiments 70 to 76.

[0238] Embodiment 107. A method for reducing the risk of aneuploidy in oocytes, oocytes or zygotes, comprising the step of introducing a complex described in any of Embodiments 1 to 69 or a nucleic acid molecule described in any of Embodiments 70 to 76 into oocytes, oocytes or zygotes, wherein the risk of aneuploidy is reduced compared to oocytes or zygotes that have not been introduced the complex described in any of Embodiments 1 to 69 or the nucleic acid molecule described in any of Embodiments 70 to 76.

[0239] Embodiment 108. A method for reducing the probability of aneuploidy in oocytes, oocytes or zygotes, comprising the step of introducing a complex described in any of Embodiments 1 to 69 or a nucleic acid molecule described in any of Embodiments 70 to 76 into oocytes, oocytes or zygotes, wherein the risk of aneuploidy is reduced compared to oocytes or zygotes that have not been introduced the complex described in any of Embodiments 1 to 69 or the nucleic acid molecule described in any of Embodiments 70 to 76.

[0240] Embodiment 109. A kit comprising a complex according to any of Embodiments 1 to 69 or a nucleic acid molecule according to any of Embodiments 70 to 76.

[0241] Embodiment 110. The kit according to Embodiment 109, wherein the complex or nucleic acid molecule is suitable for introduction into oocytes.

[0242] Embodiment 111. The kit according to Embodiment 109, wherein the complex or nucleic acid molecule is suitable for introduction into egg cells.

[0243] Embodiment 112. The kit according to any one of Embodiments 109 to 111, wherein the kit comprises a device for introducing a complex or a nucleic acid molecule encoding the complex, preferably the device is suitable for microinjection, more preferably the device is a microinjection needle, and even more preferably the microinjection needle is equipped with a compatible pump suitable for transferring the complex or a nucleic acid molecule encoding the complex into an oocyte or egg cell, and most preferably the compatible pump is suitable for transferring 1 to 25 picoliters of the complex or a nucleic acid molecule encoding the complex.

[0244] Embodiment 113. A kit according to any one of Embodiments 109 to 111, wherein the kit comprises liposomes suitable for transfection of a complex or nucleic acid molecule, preferably the liposomes encapsulate the complex or a nucleic acid molecule encoding the complex, more preferably the liposomes comprise a Sendai virus coat protein, and more preferably the liposomes comprise a Sendai virus (HVJ) envelope vector.

[0245] Embodiment 114. The kit according to any one of Embodiments 109 to 113, wherein the kit includes a container.

[0246] Embodiment 115. The kit according to Embodiment 114, wherein the complex or nucleic acid molecule is stored in a container.

[0247] Embodiment 116. The kit according to Embodiment 115, wherein the complex or nucleic acid molecules are stored at a maximum temperature of 4°C, preferably a maximum of -20°C and / or a minimum of -196°C. [Examples]

[0248] The following examples are intended to further illustrate the present invention, but are not limited thereto. The examples describe technical features, and the present invention also relates to combinations of the technical features presented in this section.

[0249] Example 1 Aneuploidy increases the risk of infertility and birth defects in older women; therefore, it is necessary to prevent premature separation of sister chromatids and chromosomes, or reverse their loss, by preserving cohesin. To the best of our knowledge, there are currently no available systems that can improve chromosome or chromatid aggregation. As a result, there are no available treatments for age-related infertility, and many women over 35 are unable to conceive.

[0250] To address this need, the inventors developed an artificial aggregation system to reduce aging-related PSSCs in oocytes (lower panel, Figure 1a). Because human oocytes are difficult to obtain, the inventors primarily used mouse oocytes as a model for PSSCs (Figure 1c, Figure 1d). PSSCs are caused by the loss of cohesin in the pericentromere region. 12,15,20,21 In this example, an artificial aggregation system is targeted to these regions by utilizing a transcription activator-like (TAL) effector protein that targets a 15 bp sequence in the major satellite DNA around the repeating centromere (Figure 1e). This TAL protein efficiently binds to the centromere region of mouse chromosomes and is well tolerated by meiotic oocytes. 22,23 The inventors modified the TAL protein to create the TALhesin (TALcohesin) system. This system consists of TALhesin component A, which has spy-tag domains adjacent on both sides, and TALhesin component B, which has spy-catcher domains adjacent on both sides (Figure 1e; Figures 2a, 2b). The spy-tag binds to the spy-catcher, forming a stable covalent isopeptide bond between TALhesin component A and spy-B component. 24 The inventors named TALhesin according to its designated DNA target for the purposes of this embodiment. Thus, TALhesin[mmMajSat]A / B is a pair of TALhesin components -A and -B that target the major satellite sequence of the mouse (mus musculus). 22 .

[0251] To characterize the TALhesin system, the inventors isolated and arrested immature mouse oocytes before the restart of meiosis, injected mRNA encoding TALhesin[mmMajSat]A / B, released the cells from arrest, and allowed them to proceed through meiosis I (see Methods). TALhesin[mmMajSat]A / B localized to the pericentromere region within 1.5–2 hours after nuclear membrane breakdown (NEBD) and remained on the sister chromatids throughout meiosis I until metaphase II arrest (Figure 1f, f; Figure 2c). TALhesin[mmMajSat]A / B did not hinder the progress of meiosis I (Figure 2d) and did not interfere with the unpairing of sister kinetochores that occurs when oocytes progress from meiosis I to meiosis II (Figure 1g). 25,26 Fluorescence recovery after photobleaching (FRAP) revealed a lower turnover of TALhesin[mmMajSat]A / B on meiotic II chromosomes compared to TAL-mClover fusion proteins completely lacking the Spy interaction domain (Figure 1h). Therefore, TALhesin[mmMajSat]A / B stably associates with its sister chromatid, similar to cohesin. 6,8,9,27 .

[0252] To determine whether TALhesin[mmMajSat]A / B reduces sister chromatid separation, the inventors first tested the spacing between sister kinetochores (kinetochore spacing) in mouse meiotic II chromosomes. Low levels of condensation in the pericentromere region, as observed in aged oocytes, resulted in large kinetochore spacing, while high levels of condensation, as observed in young oocytes, correlated with small kinetochore spacing. 12,15,20,28The inventors injected different amounts of TALhesin[mmMajSat]A / B mRNA into young oocytes and found that at intermediate levels of TALhesin[mmMajSat]A / B mRNA (0.5-0.2 amol), the average interkinetochore spacing was reduced by more than half compared to young oocytes that were not injected (Figure 1i). At low mRNA concentrations (0.02-0.05 amol), the interkinetochore spacing was similar to that of uninjected cells, but at TALhesin[mmMajSat]A / B mRNA levels more than 10 times higher (≧0.2 amol), the chromosomes formed densely clustered groups, and kinetochores were sometimes detached from the chromosomes during late I (Figures 2e, 2f). Occasionally, TALhesin[mmMajSat]A / B-labeled chromosomes in the first polar body remained linked to the chromosomes in the oocyte (Figure 2g). Overall, it can be concluded that TALhesin[mmMajSat]A / B can form stable links between sister chromatids, and that intermediate levels of TALhesin[mmMajSat]A / B have the ability to bring sister kinetochores closer together.

[0253] Next, we examined whether TALhesin[mmMajSat]A / B could compensate for the rapid depletion of cohesin. 29 Rec8 is an essential component of the cohesin complex, and by depleting Rec8 using Trim-Away, sister chromatids are separated. 29To determine whether the TALhesin system can mediate aggregation on its own, the inventors depleted Rec8 from young oocytes injected with a large amount (2 amol) of TALhesin[mmMajSat]A / B mRNA or TALhesin[mmMajSat]-B alone, and tested for sister chromatid separation (Figure 3a). Indeed, in cells expressing TALhesin[mmMajSat]A / B, most sister chromatids remained together after Rec8 depletion, but none remained in cells expressing only TALhesin-B[mmMajSat] (Figure 3b; Figure 4a). To test whether aggregation mediated by TALhesin[mmMajSat]A / B can be mitigated, TALhesin[mmMajSat]A / B was depleted using anti-GFP Trim-Away. Depletion of TALhesin[mmMajSat]A / B alone would not affect chromosome condensation when endogenous cohesin remains intact (Figure 3c, left panel), but in the absence of Rec8, it would eliminate condensation and induce sister chromatid separation (Figure 3c, right panel). Consistent with this hypothesis, we found that depletion of Rec8, followed by depletion of TALhesin[mmMajSat]A / B, resulted in sister chromatid separation over a period of 6 hours (bottom panel, Figure 3d), while in cells depleted with TALhesin[mmMajSat]A / B alone, sister chromatids remained intact for the same period (top panel, Figure 3d). Overall, these data demonstrate that TALhesin[mmMajSat]A / B maintains sister chromatid condensation after depletion of Rec8-cohesin, confirming that TALhesin can functionally replace endogenous cohesin.

[0254] Considering that the TALhesin system can maintain sister chromatid aggregation, we hypothesized that TALhesin[mmMajSat]A / B could reduce PSSC errors in oocytes from aged mice. To this end, we compared the number of chromosomes with PSSCs in untreated or TALhesin[mmMajSat]A / B mRNA-treated oocytes from aged mice. As a control, untreated oocytes from young mice (2-3 months old) were tested (Figure 3e). Aged oocytes were treated with two low doses of TALhesin[mmMajSat]A / B mRNA (0.02 and 0.05 amol), which maintained similar interkinetochore spacing to that of young oocytes without inducing chromosome clustering, fragmented kinetochores, or other effects observed at higher mRNA levels (≥0.2 amol) (Figure 1h, Figures 2d-2g).

[0255] PSSC errors were defined as kinetocoreal sister chromatids separated at intervals of ≥3 μm or appearing as single arms with a single kinetocore (Figure 3f). Surprisingly, the frequency of PSSCs was significantly reduced in TALhesin[mmMajSat]A / B-treated oocytes isolated from 13, 17, and 20-month-old mice, approaching the low PSSC error rates observed in younger oocytes (Figures 3g, 3h; 4b-4i). Furthermore, the percentage of aged oocytes completely free of PSSC errors increased from 46.7% to 74.1% after treatment with TALhesin[mmMajSat]A / B (Figure 3i), similar to the percentage of PSSC-free oocytes from younger mice. Treatment with 0.05 amol of TALhesin[mmMajSat]A / B mRNA resulted in a 97.6% recovery of error-free oocytes from 17-month-old mice compared to oocytes from younger mice (Figure 3j). A similar effect was observed in TALhesin[mmMajSat]A / B-treated oocytes from 13-month-old mice (Figures 4b-4e). This effect was less pronounced but still significant for oocytes from the 20-month-old group (Figures 4f-4i). Consistent with results from younger oocytes, injection of small amounts (0.02-0.05 amol) of TALhesin[mmMajSat]A / B mRNA did not have a significant effect on the spacing between kinetochores (Figures 4j-4l). Overall, TALhesin[mmMajSat]A / B treatment reduced the frequency of PSSCs in oocytes from older-age mice and resulted in more PSSC-free oocytes.

[0256] Next, the inventors attempted to apply the TALhesin system to human oocytes. For this purpose, the inventors used a strategy similar to that used for mouse oocytes, but with the addition of a TAL effector domain targeting a 19bp sequence within human centromere satellite repeats. 30The inventors first tested the results of TALhesin in mitotic HeLa cells. Since the duration of mitosis in somatic cells is only about 30 minutes, compared to about 12 hours to complete the first meiosis in mouse oocytes, the inventors used the spy-catcher 003 domain in TALhesin-B to improve the interaction dynamics with spy-tags. 31 These modifications generated TALhesin[hsCentro]A / B (TALhesin directed towards human centromere repeats, Figure 5a). We transfected HeLa cells and observed enrichment of TALhesin[hsCentro]A / B on several chromosomes (arrows, Figure 5a). Some TALhesins had lower chromosomal occupancy, which likely reflects the chromosome-specific density and copy number of the targeted satellite element (Figure 5a). 32 The inventors noticed that newly divided HeLa cells expressing TALhesin[hsCentro]A / B formed a clear intercellular bridge consisting of TALhesin-labeled DNA (Figure 6b). It is well known that HeLa cells spontaneously form chromosome bridges during mitosis. 33 The inventors observed a significant increase in these bridges, from approximately 20% in untransfected cells and 32% in cells expressing only TALhesin[hsCentro]-B, to 94.7% in cells expressing both TALhesin[hsCentro]A / B components (Figure 5c). It can be concluded that TALhesin[hsCentro]A / B can stably link chromosomes in human mitotic cells.

[0257] To determine whether the TALhesin system can tether sister chromatids in human oocytes, the dynamics of isopeptide bond formation were modified by incorporating the spy-catcher 001 domain into TALhesin[hsCentro]-B (Figures 6b and 6c). Donated human oocytes were injected with 0.0006 amol of TALhesin[hsCentro]A / B mRNA and matured in vitro (Figure 5d). TALhesin[hsCentro]A / B localized to the pericentromere region on most human meiotic I and II chromosomes and did not hinder progression through the first meiotic division (Figures 5e and 5f). In addition to oocyte-specific variability in expression and maturation timing, variability between donor ages (Figure 6d) resulted in heterogeneous levels of TALhesin[hsCentro] protein, which constrained the analysis. It was hypothesized that sister chromatid pairs would exhibit higher levels of TALhesin[hsCentro]A / B than chromosomes that had undergone PSSC, which is consistent with TALhesin[hsCentro]A / B helping to prevent PSSC in human oocytes. The mean fluorescence intensity of TALhesin[hsCentro]A / B was measured in intact sister chromatid pairs and compared to dissociated chromatids. Indeed, intact chromosomes had higher mean TALhesin[hsCentro]A / B fluorescence intensity compared to those that had undergone PSSC (Figure 5g). Furthermore, chromosomes with smaller interkinetochore spacing had higher mean TALhesin[hsCentro]A / B fluorescence intensity compared to chromosomes with larger interkinetochore spacing or chromosomes that had undergone PSSC (Figures 5h, 6e, and 6f). These data suggest that TALhesin[hsCentro]A / B may reduce the spacing between kinetochores and protect human oocytes from PSSCs.

[0258] Sister chromatids separate when anatropy II is activated by fertilization (Figure 7a). To evaluate sister chromatid separation, the inventors treated mouse and human oocytes expressing the appropriate TALhesin system with compounds that activate anatropy II, thereby simulating sperm fertilization. 34,35The inventors found that even low levels of non-cleavable TALhesin resulted in inefficient sister chromatid separation in late II of activated mice (0.05 amol TALhesin[mmMajSat]A / B) (Figure 7b, center panel), and also inefficient sister chromatid separation in late II of human oocytes (0.0006 amol TALhesin[hsCentro]A / B) (Figure 7c, center panel), accompanied by delayed completion of late II and an increased frequency of chromosomal non-segregation events (Figures 7d-7g).

[0259] The inventors hypothesized that inactivating the TALhesin system is necessary for efficient chromosome segregation in anaphase II. To release TALhesin from sister chromatid tethering during anaphase, the inventors utilized the cell cycle-dependent nature of separatorse activity (Figure 7a). Protease separatorase is activated in anaphase and cleaves the cohesin subunit Rec8 at a specified proteolytic site. 36~38The inventors hypothesized that incorporating a Rec8 proteolytic motif into TALhesin could enhance separatorase cleavage in late II. Using mouse and human Rec8 fragments, respectively, the inventors generated TALhesin[mmMajSat](Rec8)A / B and TALhesin[hsCentro](Rec8)A / B (Figures 8a-8d). In contrast to the non-cleavable TALhesin protein, supplementation with separatorase-cleavable TALhesin[mmMajSat](Rec8)A / B or TALhesin[hsCentro](Rec8)A / B resulted in synchronous chromatid separation in activated young mouse and human oocytes, respectively, as well as in oocytes expressing only TALhesin(Rec8)-B (upper panel, Figures 7b and 7c) (lower panel, Figures 7b and 7c). Furthermore, the time to completion of late II and the frequency of chromosomal non-segregation events were reduced in activated oocytes expressing TALhesin(Rec8) compared to non-cleavable TALhesin in both mouse and human oocytes (Figures 7d-7g). TALhesin[mmMajSat](Rec8)-A / B bound stably to the pericentromere region of chromosomes, similar to non-cleavable TALhesin[mmMajSat] (Figure 1h) (Figure 8e). Importantly, oocytes from 20-month-old mice treated with TALhesin[mmMajSat](Rec8)A / B showed a reduced frequency of PSSC errors (Figure 7h; Figure 9a) and an increased percentage of cells with no PSSC errors (Figures 9b-9d). Therefore, the incorporation of separase-cleavable Rec8 fragments into oocytes allows for inactivation in late II while still reducing PSSC errors in mouse oocytes.

[0260] Example 2 TALhesin functions to tether chromosomes together. However, temporary deactivation of TALhesin is necessary to allow chromosomes to separate freely during late gestation. The inventors have tested several approaches to deactivate TALhesin. In the first approach, the inventors tested whether the introduction of a cell cycle-dependent degradation sequence would cause the TAL[mmMajSat] protein to disappear from the chromosome. As an example, the inventors tested the mouse securin protein. 63 The first 101 amino acids derived from the original protein were introduced at the N-terminus of mClover-TAL[mmMajSat] (N-securin-mClover-TAL[mmMajSat]). Furthermore, the inventors introduced the mouse cyclin B1 protein. 64 An approach was engineered and tested in which the first 79 amino acids derived from mClover-TAL[mmMajSat] were fused to the N-terminus (N-cyclin B1-mClover-TAL[mmMajSat]). Both the N-terminal domains of securin and cyclin B1 contain well-characterized ubiquitin pathway-dependent disruption motifs that are targeted for degradation before and during anaphase I and II. The inventors evaluated the fluorescence signals in mouse oocytes expressing either of these constructs as they progressed through anaphase I (Figures 11, 12). Adding either of the degradation signals to mClover-TAL[mmMajSat] reduced the fluorescence signal before and during anaphase I. Conversely, the chromosome-labeling fluorescent protein reporter H2B-miRFP did not decrease during anaphase I in the same cells. These data indicate that the N-terminal domains of both securin and cyclin B1 target TAL[mmMajSat] for cell cycle-dependent disruption via the ubiquitin pathway.

[0261] Another approach using recombinant NS3 / 4A protease was also tested. This approach relies on expressing the protease from synthetic mRNA together with TALhesin, which has an internal NS3 / 4A cleavage motif. Importantly, the compound BILN-2061 (Ciluprevir) reversibly inhibits NS3 / 4A. 65 BILN-2061's washout allows for timed control of protease activity. 66 This resulted in the inactivation of TALhesin. For both components A and B, cleavage motifs were introduced to both the N and C-terminal flanks of TAL[mmMajSat]. The addition of two cleavage motifs to both TALhesin components produced TALhesin[mmMajSat](2xCS)-A and -B. Overall, each component contains two NS3 / 4A cleavage motifs, thereby maximizing the potential for inactivating TALhesin. Oocytes injected with mRNA encoding TALhesin[mmMajSat](2xCS-A and -B) in addition to mRNA encoding NS3 / 4A were matured in vitro in the presence of BILN-2061, a specific inhibitor of HCV NS3 / 4A protease. After washing out with BILN-2061, the fluorescence signal emanating from TALhesin[mmMajSat](2xCS)-A / B foci on the chromosomes steadily decreased over a 16-hour period (Figure 12). These data demonstrate that TALhesin[mmMajSat] can be inactivated in mouse oocytes using an NS3 / 4A protease and BILN-2061 inhibitor system.

[0262] Materials and methods Isolation and culture of mouse oocytes The Animal Facility of the Max Planck Institute for Multidisciplinary Sciences housed all the mouse strains used in this example. The mice were raised in a pathogen-free environment in accordance with the guidelines and recommendations of the Federation for Laboratory Animal Science Association and the German Welfare Act (Tierschutzgesetz TSchG).

[0263] The mouse strains CD1, 129s6, and B6 / CBA-F1 were used in this study. Figure 1i was prepared using B6 / CBA-F1 mated females. For the aging experiments in Figures 3e-3j and 3b-3l, 2-3 month old 129s6 strain mice were used together with 13, 17, or 20 month old 129s6 strain mice, as indicated. Young CD1 mice, 2-3 months old, were used in all other mouse experiments. Oocytes in the GV stage were cultured in in-house M2 medium supplemented with dibutyryl cyclic AMP (dbcAMP, Sigma-Aldrich: D0627) under NidOil paraffin oil (Nidacon: NO-100). Oocytes from the CD1 and 129s6 strains were cultured with 250 μM dbcAMP, and B6 / CBA-F1 oocytes were cultured with 750 μM dbcAMP. Only mature oocytes with a diameter of approximately 70 μm and a central nucleus and a thick zona pelucida (approximately 5 μm) were selected for the experiment.

[0264] Cloning and mRNA synthesis All constructs were combined into a pGEM-HE vector skeleton. 44To generate TALhesin-A, a custom-synthesized "chassis" containing terminal spy-tag sequences and internal restriction sites (Eurofins) was inserted into pGEM-HE. Subsequently, the sequences of the Gateway cassette (Thermo-Fisher) and mCherry fluorescent protein were inserted frame by frame along with the spy-tag sequences. Plasmids containing the Gateway cassette were grown in ccdB Survival 2 T1® Competent Cells (Thermo-Fisher: A10460) according to the manufacturer's instructions. The TALhesin-B chassis was generated by substituting the terminal spy-tag sequences of TALhesin-A with 5' and 3' terminal spy-catcher sequences, while the mClover3 fluorescent protein was substituted for mCherry. For somatic cell expression, the TALhesin-A and -B chassis containing the internal Gateway cassette and fluorescent proteins were inserted into pCMV vectors by restriction digestion and ligation. Spycatcher 001 (Addgene plasmid #72324) and Spycatcher 003 (Addgene plasmid #133447) were generously donated by Dr. Mark Howarth. 24,31 .

[0265] The transcription activator-like (TAL) sequence targeting the major satellite repeat of mouse, TAL[mmMajSat], pTALYM3B15, was generously donated by Dr. Maria-Elena Torres-Padilla (TALYM3B15; Addgene plasmid #47878). 22 The TAL sequence (TAL[hsCentro]) targeting human centromere repeats was generously donated by Dr. Guang-Hui Liu. 30The TAL sequence lacking the N-terminal NLS was PCR amplified using primers [fwd: 5'-CACCA TGCAG GTGGA TCTAC GCACG CTCGG CTA-3' and rev: 5'-GGAGG AGGCG GTGCC GGA-3'] and cloned into pENTR / D-Topo (Thermo-Fisher) according to the manufacturer's instructions. To generate complete constructs, the TAL sequence was inserted into pGEMHE-TALhesin-A and pGEMHE-TALhesin-B using Gateway cloning with LR clonase II plus (Thermo-Fisher: 12538120). The same reaction was used to generate expression constructs based on the complete pCMV.

[0266] TALhesin[mmMajSat](Rec8) was generated by inserting mouse Rec8(aa417~525) into TALhesin-A and -B. This region contains two xPLE pseudobinding sites in addition to the cleavage motifs C2 and C3. 37 Rec8(aa417-525) was PCR amplified from a pET45b(+)His-tagged FL Rec8 plasmid and inserted into pGEMHE-TALhesin-A and -B in the linker-2 (L2) region with Gibson Assembly Master Mix (New England BioLabs, E2611). TALhesin[hsCentro](Rec8) was similarly generated by inserting human Rec8(aa368-434) into TALhesin[hsCentro]. This region contains cleavage motifs C2 and C3 in addition to a 1×LPE pseudobinding site. 37 The pET45b(+)His-tagged FL Rec8 (Addgene plasmid #45243) and H2B-mScarlet-hRec8(297-506)-mNeonGreen (Addgene plasmid #174717) were generously donated by Dr. Michael Lampson. 12,45The inventors noticed that introducing mCherry between the Gateway cassette and the Rec8 fragment enhanced the expression of TALhesin[MajSat](Rec8)-B (data not shown). Therefore, TALhesin[MajSat](Rec8)-B contains two fluorescent proteins adjacent to the Rec8 fragment.

[0267] The constructs were transcribed in vitro using the HiSscribe T7 ARCA kit (New England BioLabs, E2065S). Briefly, the template plasmid within the pGEM-HE backbone was linearized overnight at 37°C using PacI restriction endonuclease (New England Biolabs, R0547), extracted with phenol / chloroform, precipitated with ethanol in the presence of sodium acetate, washed, and resuspended in endonuclease-free water. pTALYM3B15 was linearized similarly, but using AgeI-HF restriction endonuclease (New England Biolabs: R3552). In vitro mRNA transcription was performed according to the manufacturer's instructions, followed by DNase treatment, phenol / chloroform extraction, precipitation with ethanol in the presence of ammonium acetate, washed, and resuspended in endonuclease-free water. mRNA concentration was measured using high-sensitivity RNA Qubit 4 fluorescence spectroscopy (Thermo-Fisher) and used to calculate the molars of mRNA injected into oocytes. All RNA was analyzed by gel electrophoresis before injection. Briefly, RNA was incubated in formamide loading buffer (Thermo-Fisher: R0641) at 70°C for 5 minutes and run parallel to RibOcruler HR RNA ladder (Thermo-Fisher: SM1821). The gel was visualized with SYBR-safe staining (Thermo-Fisher: S33102).

[0268] mRNA microinjection Using injection settings based on mercury needles, as already described 46Exactly 7 picoliters of mRNA were injected into mouse oocytes. All mRNA amounts reported in this study were calibrated against this injection volume. Briefly, dbcAMP-arrested GV-phase mouse oocytes were placed in a custom injection rack containing fresh, warm M2 medium. Oocytes were injected in small batches to avoid cooling and evaporation of the medium. After injection, the oocytes were washed in oil (NidOil) with 3 drops of fresh M2 medium containing dbcAMP and incubated at 37.5°C for 3 hours to express the protein. The cells were then washed in fresh M2 medium lacking dbcAMP and matured in a 37.5°C incubator for 15 hours, or imaged on a warmed confocal microscope (Zeiss) in M2 medium containing 200 nM SiR-DNA (SpyroChrome: SC007).

[0269] Rec8 antibody generation, Trim-Away and parthenogenesis activation The pET28a(+)-N-Rec8-6xHis plasmid contains antigen proteins corresponding to the mouse Rec8 (aa25-286). 47The proteins were expressed from the following source: Rec8 (aa 25-286) was PCR amplified from pET45b+His-tagged FL Rec8 plasmid (Addgene plasmid #45243) and inserted into a pET28a(+)-6xHis backbone (Merck-Millipore: #69864) by Gibson assembly. The bacteria were cultured in Magic Media (Thermo-Fisher: K6803) at 37°C for 16 hours. The cells were shaken at 4°C for 18 hours in a buffer containing 50 mM Tris, pH 8.0, 250 mM NaCl, and 6 M guanidine HCl, followed by centrifugation at 39,000 × g at 4°C for 2 hours. The supernatant was then passed through a 0.22 μm syringe filter to extract the proteins from the inclusion bodies. The supernatant was diluted 1:10 with dilution buffer (50 mM Tris, pH 8.0; 250 mM NaCl; 15 mM imidazole; 5 mM 2-mercaptoethanol; 6 M urea) and then loaded onto a pre-equilibrated Ni-NTA bead column. The column was washed with dilution buffer and then eluted with elution buffer (50 mM Tris, pH 8.0; 250 mM NaCl; 500 mM imidazole; 5 mM 2-mercaptoethanol; 6 M urea). The protein was then dialyzed for 1 hour in a buffer containing 25 mM Tris, pH 8.0, 250 mM NaCl, 5% glycerol, 5 mM DTT, and 5 M urea, followed by buffer exchange at a rate of 3 mL / min using 3 L of refolding buffer (25 mM Tris, pH 8.0, 250 mM NaCl, 5% glycerol, 5 mM DTT). Next, the protein was concentrated to 1.91 mg / mL using Amicon Ultra-4 10kDa mwco (Merck-Millipore: UFC801024). Antibody generation and affinity column purification of the antibodies were performed in rabbits at Cambridge Research Biochemicals (Cambridge, UK).

[0270] For Trim-away, a mixture containing 2.9 amol of TRIM-21, TALhesin-A, and LA-B mRNA synthesized in vitro as described above was microinjected into GV-phase oocytes (see Methods: mRNA injection). Mature meiotic II oocytes were first incubated in M2 medium (SpyroChrome: SC007) containing 200 nM SiR-DNA. Rabbit anti-Rec8 antibody (for anti-Rec8 Trim-Away) or rabbit anti-GFP antibody (AbCam: 6556) (for anti-GFP Trim-Away targeting TALhesin depletion) was administered as previously described. 29 For trim-away, the cells were buffered in phosphate-buffered saline (PBS). For anti-Rec8 trim-away, MII oocytes were then injected with 7 μg of 1.01 mg / mL anti-Rec8 antibody in PBS containing 0.03% NP-40, along with 43.8 femtograms of dextran-Texas Red, 70 kDa (Thermo-Fisher: D1830) as a tracer for antibody injection (Figure 4a). For TALhesin trim-away, 7 μg of 0.5 mg / mL rabbit anti-GFP antibody in PBS containing 0.03% NP-40 was injected. The cells were then directly imaged in M2 medium containing SiR-DNA.

[0271] For parthenogenesis activation of mouse oocytes, matured oocytes were incubated in M2 medium containing 200 nM SiR-DNA for 1 hour to label the chromosomes. They were then washed in oil with nine 20 μL droplets of calcium-free M2 medium containing SiR-DNA. The oocytes were then washed in oil with nine 20 μL droplets of calcium-free M2 medium supplemented with 10 mM SrCl2 (Sigma-Aldrich: 255521) containing SiR-DNA, and then directly imaged in the same medium. 34 .

[0272] For the activation of parthenogenesis in human oocytes, 0.0002 amol of mRNA was injected into GV or MI-stage oocytes and matured in vitro to MII oocytes. The cells were incubated with 100 nM SiR-DNA in GMOPS-PLUS medium (VitroLife: 10130) for at least 3 hours. They were then washed in nine 20 μL droplets under SiR-DNA / GMOPS-PLUS medium oil containing 10 μM ionomycin (calcium ionophore, MP Biomedicals) and incubated in the final droplet for 10 minutes. 35 The cells were then washed with nine 20 μL droplets of SiR-DNA / GMOPS-PLUS medium under oil and immediately prepared for imaging. Human activation was visualized using a Viventis LS1 live-light sheet microscope (Viventis, Lausanne, Switzerland) in SiR-DNA / GMOPS-PLUS medium under oil at 37.5°C.

[0273] HeLa cell culture HeLa(CCL2) cells were cultured at 37°C in air with 5% carbon dioxide in high-glucose Dulbecco's modified Eagle medium (Thermo-Fisher: 11965084) supplemented with 10% fetal bovine serum (Thermo-Fisher: 26140079). The cells were plated at 120,000 cells / ml onto 4 welldishes (Ibidi: 80427). After culturing the cells for 24 hours, they were transfected with Lipofectamine-LTX (Thermo-Fisher: 15338100) according to the manufacturer's instructions. In short, each well was transfected with either 0.125 μg of TALhesin[hsCentro]-B or 0.125 μg of both TALhesin[hsCentro]-A and TALhesin[hsCentro]-B, diluted in Opti-MEM (Thermo Fisher Scientific: 31985062) supplemented with PLUS reagent (equal to plasmid DNA) and 1 μl of lipofectamine LTX. Eight hours after transfection, the cells were synchronized by treating them with 9 μM RO-3306 (Sigma-Aldrich: SML0569) for 16 hours. After synchronization, the cells were prepared for live-cell microscopy or immunostaining. For live-cell microscopy, the cells were released into a medium supplemented with 10 μM verapamil and 1:10,000 SiR-DNA (Spirochrome: SC007). For immunohistochemical staining, cells were fixed either immediately 16 hours after RO-3306 treatment or after being released into culture medium for 24 hours.

[0274] Immunofluorescence imaging of HeLa cells HeLa cells were fixed in 4% methanol-free formaldehyde in PBS (Polysciences: 040181) for 30 minutes at room temperature. The fixed cells were permeabilized in 0.1% Triton X-100 (Sigma-Aldrich: 93443) in PBS (0.1-PBT) for 1 hour at room temperature. The cells were blocked in 3% BSA / 0.1% PBT for 30 minutes at room temperature. Primary and secondary antibody solutions were prepared in blocking buffer. The cells were stained with primary antibody, anti-GFP (Abcam: Ab6556, 1:100), anti-centromere (Antibodies Incorporated: 15234, 1:200), and anti-tubulin (Bio-Rad: MCA78G, 1:100) for 1 hour at room temperature. The CREST antibody was centrifuged at 16,000 × g for 30 minutes at 4°C to remove antibody aggregates. Cells were washed in PBS and incubated with secondary antibodies, Alexa-Fluor 488 conjugate donkey anti-rabbit (Thermo-Fisher: A21206, 1:400), Alexa-Fluor 568 conjugate goat anti-rat (Thermo-Fisher: A11077, 1:400), and Alexa-Fluor 647 conjugate goat anti-human (Thermo-Fisher: A21445, 1:400), at room temperature for 1 hour. Cells were incubated with Hoechst solution (Thermo-Fisher: 62249) diluted 1:333 in PBS at room temperature for 15 minutes. Cells were then washed in PBS and mounted in 50% glycerol.

[0275] Immunofluorescence imaging of oocytes and eggs Mouse and human oocytes and eggs were fixed in 100 mM HEPES, pH 7.0, 50 mM EGTA, pH 7.0, 10 mM MgSO4, 2% methanol-free formaldehyde, and 0.2% Triton X-100 at 37°C for 30 minutes. The fixed cells were then extracted overnight at 4°C in PBS containing 0.5% Triton X-100 (0.5-PBT) and blocked for at least 6 hours in 0.5% BSA / 0.5-PBT (BSA-0.5PBT) filtered through a 0.22 μm syringe. All primary antibody incubations were performed in BSA-0.5PBT at 4°C for 18 hours. The primary antibodies used were human anti-centromere (Antibodies Incorporated: 15234, 1:10 for mouse cells; 1:20 for human cells), rabbit anti-GFP (Abcam: Ab6556, 1:100 for mouse cells; 1:50 for human cells), and rat anti-tubulin (Bio-Rad: MCA78G, 1:200). Secondary antibodies were incubated at a concentration of 1:200 at room temperature for 1 hour. The secondary antibodies used were Alexa-Fluor 488 conjugate goat anti-human (Thermo-Fisher: A11013), Alexa-Fluor conjugate donkey anti-rat (Thermo-Fisher: A10042), and Alexa-Fluor 647 conjugate goat anti-rat (Thermo-Fisher: A2124). After incubation of the primary and secondary antibodies, each was washed five times in 15 μL of BSA-0.5PBT at room temperature for 2 hours. The cells were stained with 400 μM Hoechst 33342 (Thermo-Fisher: 62249) for 1 hour, washed in PBS, and then directly imaged in PBS.

[0276] Microscopy Live confocal microscopy was performed at 37.0°C using an LSM-800, LSM-880, LSM-900, or LSM-980 equipped with a 40×C-Apochromat 1.2 NA water immersion objective lens. Images were acquired with a 2.0 μm optical slice thickness on confocal sections, with a z-stack spacing of 1.0 μm covering approximately 20 μm. mClover, mCherry, and SiR-DNA were simultaneously imaged using lasers at wavelengths of 488, 561, and 647 nm, respectively, with a frame imaging time of 2.53 seconds and an average setting of 2. Super-resolution immobilized immunofluorescence-labeled cells were imaged using the Airyscan module and processed using ZEN software. Typically, 100–120 z-sections were acquired to reliably detect all chromosomes within the meiotic spindle.

[0277] Fluorescence recovery after photobleaching (FRAP) MII mouse oocytes expressing 0.1 amol of mRNA encoding TAL[mmMajSat] or TALhesin protein were used to determine protein mobility. 10 μm 2 A square region of interest (ROI) corresponding to the specified area was photobleached using 488 nm and 561 nm excitation lasers at 100% power after five preliminary bleeding time points. The time interval between images was 1.26 seconds, and the average was set to 2. The analysis was performed as already described. 48 .

[0278] In situ chromosome analysis Airy-processed super-resolution images were analyzed using Imaris software version 9.1.2 (Bitplane). Automated spot detection was used to identify kinetochore centers based on their maximum size (spot diameter of 360 nm). For some cells, chromosomes were segmented using the automated surface function, followed by masking of channels corresponding to kinetochores and mClover foci. The interval between sister kinetochores (iKt) was obtained by manual pairwise measurements between detected spots. Aneuploidy was recorded when intervals of approximately 20 and 23 iKt could be measured for mouse and human, respectively. PSSC was recorded when an iKt interval ≥ 3.0 μm was measured and / or when a single chromatid with a single kinetochore was observed.

[0279] Semi-automated chromosome segmentation Chromosome segmentation was performed using Imaris 9.3 with a script written in Matlab R2018b. Segmentation combined Otsu thresholding with the watershed algorithm and a "greedy" method to address oversegmentation. First, the segmentation algorithm was initiated by manually pairing sister kinetcores as filaments. The images were linearly interpolated to achieve approximately equal spacing between voxels in each dimension, addressing anisotropy. Artifacts were removed by periodically applying a mask using a maximum estimated volume of 800 μm. Next, image deconvolution to improve segmentation was applied using the Matlab function deconvblind with a 1-micrometer estimated point spread function. This image was then used to create a 5-micrometer Gaussian smoothed image for background subtraction, in...

Claims

1. A complex suitable for chromatid or chromosome aggregation, comprising (I) one or more first proteins and (II) one or more second proteins, The (I) first and (II) second proteins each comprise (i) a chromatin-binding component, (ii) a protein-binding region (PBN region) which is the N-terminus of the chromatin-binding component, and (iii) a protein-binding region (PBC region) which is the C-terminus of the chromatin-binding component; (i) The chromatin-binding components of the first and second proteins can bind to target sequences on chromatids or chromosomes; (I)(ii) the PBN region of the first protein can bind to (II)(ii) the PBN region of the second protein, (I)(iii) the PBC region of the first protein can bind to (II)(iii) the PBC region of the second protein, or (I)(ii) the PBN region of the first protein can bind to (II)(iii) the PBC region of the second protein, and (I)(iii) the PBC region of the first protein can bind to (II)(ii) the PBN region of the second protein; A complex in which the binding to the chromatid or chromosome, and the binding of the PBN region and the PBC region of the first and second proteins, reversibly tether the chromatid or chromosome.

2. (I)(ii) the PBN region of the first protein can bind to (II)(ii) the PBN region of the second protein, (I)(iii) the PBC region of the first protein can bind to (II)(iii) the PBC region of the second protein, and / or The complex according to claim 1, wherein (I)(ii) the PBN region of the first protein and (I)(iii) the PBC region of the first protein have at least 90% sequence identity, preferably 100% sequence identity.

3. The complex according to claim 1 or 2, wherein (I)(ii) the PBN region of the first protein and / or (I)(iii) the PBC region of the first protein is selected from a spy tag, snoop tag, dog tag, and split-intane N-terminal fragment, preferably (I)(ii) the PBN region of the first protein and / or (I)(iii) the PBC region of the first protein is a spy tag.

4. (I)(ii) the PBN region of the first protein can bind to (II)(iii) the PBC region of the second protein, (I)(iii) the PBC region of the first protein can bind to (II)(ii) the PBN region of the second protein, The composite according to claim 1, wherein the bond between the PBN region and the PBC region is inducible, preferably, the bond between the PBN region and the PBC region is inducible by light and / or chemically induced proximity.

5. The complex according to claim 4, wherein the chemically induced proximity is inducible by a compound, preferably the compound is a small molecule compound, and more preferably the small molecule compound is selected from one or two, preferably one, of rapamycin, FK506, FK506-cyclosporine, FK1012, coumamycin, gibberellin, S-(+)-abscisic acid, BisMTX, TMP-halo (trimethoprim fused to a halo ligand), caffeine, and cannabidiol.

6. The aforementioned tethering, (a) cleavage of the complex, wherein the first and second proteins of the complex further include protease cleavage sites; and / or (b) Degradation of the complex, wherein the first and / or second protein of the complex further comprises a ubiquitination site; and / or (c) The complex according to any one of claims 1 to 5, wherein the disruption of the binding between the PBN region and the PBC region of the first and second proteins is reversible by disruption mediated by chemically induced proximity.

7. The complex according to claim 6, wherein the (a) protease cleavage site is located between the (ii) PBN region and the (i) chromatin-binding component, or between the (i) chromatin-binding component and the (iii) PBC region of the first and / or second protein, preferably the protease cleavage site is cleavable by an enzyme naturally present in the fertilized zygote, more preferably the protease cleavage site is a separase-cleavable site, and even more preferably the separase-cleavable site is a Rec8 site.

8. The complex according to claim 6, wherein the first and second proteins of the complex further comprise (b) a ubiquitination site, preferably the ubiquitination site being an amino acid sequence of APC / C cleavable securin or cyclin, preferably the amino acid sequence of securin or cyclin having at least 25 amino acids of securin or cyclin, preferably at least 40 amino acids, more preferably at least 60 amino acids, more preferably at least 80 amino acids, and even more preferably 80 to 150 amino acids, and most preferably the amino acid sequence of securin or cyclin being located at the N-terminus of securin or cyclin.

9. The tethering is reversible by target protein degradation, and preferably the complex further comprises a PROTAC binding site, according to claim 6.

10. The complex according to claim 6, wherein the binding of the PBN region and PBC region of the first protein to the PBN region and PBC region of the second protein is mediated by chemically induced proximity, and the binding of the PBN region and PBC region of the first protein and the second protein is disrupted by the absence of the small molecule compound or by functionally inactivating the small molecule compound.

11. The complex according to any one of claims 1 to 10, wherein the chromatin-binding component is a DNA-binding domain and / or the chromatin-binding component can bind to a DNA-related protein, preferably the chromatin-binding domain is a DNA-binding domain, preferably the DNA-binding domain is selected from the group consisting of a TAL protein domain, a zinc finger protein, a leucine zipper, and dCas9 complexed with gRNA, and preferably the DNA-binding component is individually selected from the group consisting of a TAL protein domain and a zinc finger protein.

12. A nucleic acid molecule or two nucleic acid molecules encoding the complex according to any one of claims 1 to 11, wherein the nucleic acid molecule is an RNA molecule, more preferably an mRNA molecule.

13. A complex according to any one of claims 1 to 11 or a nucleic acid molecule according to claim 12, suitable for use in reproductive medicine, preferably in assisted reproductive technology, and more preferably for use in reducing the risk of aneuploidy in the zygote.

14. An in vitro method for stabilizing the aggregation of chromatids or chromosomes of an oocyte or egg cell, comprising the step of delivering a complex according to any one of claims 1 to 11 or a nucleic acid molecule according to claim 12 to the oocyte or egg cell, wherein the RNA molecule is translated into a complex according to any one of claims 1 to 11. A method wherein the complex stabilizes the aggregation of chromatids or chromosomes in the oocyte or egg cell.

15. The method according to claim 14, wherein the complex according to any one of claims 1 to 11 or the nucleic acid molecule according to claim 12 is delivered before intermediate phase II arrest, preferably before late phase I, and most preferably during intermediate phase I.

Images