Xkr4 polypeptide, XRCC4 polypeptide, and method for identifying gene corresponding to phenotype of interest

Abstract

To provide a method to identify molecules involved in lipid scrambling activity.SOLUTION: The present invention provides an Xkr4 polypeptide, which is (A) a polypeptide comprising a specific amino acid sequence, or (B) a polypeptide, comprising an amino acid sequence with deletion, substitution, or addition or their combination in the amino acid sequence of the (A) polypeptide, and exhibiting at least 80% sequential identity to the amino acid sequence of the (A) polypeptide, and demonstrating lipid scramble activity in a cell membrane without interaction with an XRCC4 polypeptide.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to Xkr4 or XRCC4 polypeptides, polynucleotides comprising nucleotide sequences encoding them, expression vectors comprising said polynucleotides, or host cells comprising said polynucleotides or expression vectors.The present disclosure also relates to a method for screening substances that modulate (e.g., inhibit or induce) the lipid scrambling activity of Xkr4.The present disclosure also relates to a composition for activating Xkr4.The present disclosure also relates to a kit for screening substances that inhibit the lipid scrambling activity of Xkr4.The present disclosure further relates to a method for identifying genes corresponding to a phenotype of interest. [Background technology]

[0002] During brain development, a large amount of cell death is observed, and most of the cell death is apoptotic. Apoptosis is a type of cell death characterized by nuclear fragmentation, cytoplasmic clumping and fragmentation. In apoptosis, cells are rapidly removed from tissues compared to other cell deaths such as necrosis. Defects in apoptosis during brain development may be associated with neurodevelopmental or behavioral disorders, including attention-deficit hyperactivity disorder (ADHD). Excessive apoptosis may be associated with neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS).

[0003] The cell membrane of eukaryotes is composed of a phospholipid bilayer, in which phospholipids are distributed asymmetrically. This asymmetric distribution of phospholipids is thought to be maintained by flippase, an enzyme that transports aminophospholipids such as phosphatidylserine to the inside (cytoplasmic side) of the cell membrane. When a cell receives an apoptotic stimulus, the asymmetric distribution of phospholipids collapses, and phosphatidylserine that was on the inside of the cell membrane is exposed on the cell surface and is phagocytosed by phagocytes. Xkr8, a protein involved in the exposure of phosphatidylserine due to apoptosis, has been identified (Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] WO 2014 / 077279 Summary of the Invention [Problem to be solved by the invention]

[0005] The present disclosure has an object to identify a molecule related to lipid scrambling activity. The present disclosure has an object to provide a polynucleotide comprising a nucleotide sequence encoding a molecule related to lipid scrambling activity, an expression vector comprising said polynucleotide, and a host cell comprising said polynucleotide or expression vector. The present disclosure has an object to provide a method for screening a substance that regulates (e.g., inhibits or induces) lipid scrambling activity. The present disclosure has an object to provide a composition for activating Xkr4. The present disclosure has an object to provide a kit for screening a substance that inhibits lipid scrambling activity. The present disclosure also has an object to provide a method for identifying a gene corresponding to a phenotype of interest. [Means for solving the problem]

[0006] The present disclosure provides the following inventions. [Section 1] 1. An Xkr4 polypeptide comprising: (A) a polypeptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 3 to 5 and 37 to 39; or (B) A polypeptide having an amino acid sequence including deletions, substitutions, or additions or a combination thereof in the amino acid sequence of the polypeptide of (A), which shows at least 80% sequence identity with the amino acid sequence of (A), and which can exhibit lipid scrambling activity in a cell membrane without interacting with the XRCC4 polypeptide. An Xkr4 polypeptide. [Section 2] 1. An XRCC4 polypeptide comprising: (a) a polypeptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 7 to 12 and 42; or (b) a polypeptide having an amino acid sequence including deletions, substitutions, or additions or a combination thereof in the amino acid sequence of the polypeptide of (a), which shows at least 80% sequence identity with the amino acid sequence of (a), and which can induce lipid scrambling activity in a cell membrane by interacting with a C-terminal truncated Xkr4 polypeptide. The XRCC4 polypeptide. [Section 3] Item 3. The XRCC4 polypeptide according to Item 2, comprising an arginine at position 270 according to the numbering of amino acid residues in the amino acid sequence set forth in SEQ ID NO:6. [Section 4] Item 4. The XRCC4 polypeptide according to Item 2 or 3, which does not contain a methionine at position 265 according to the numbering of amino acid residues in the amino acid sequence set forth in SEQ ID NO:6. [Section 5] A polynucleotide comprising a nucleotide sequence encoding the Xkr4 polypeptide according to Item 1 or the XRCC4 polypeptide according to any one of Items 2 to 4. [Section 6] Item 6. An expression vector comprising the polynucleotide according to Item 5. [Section 7] A host cell comprising the polynucleotide of Item 5, or the expression vector of Item 6. [Section 8] A composition for activating Xkr4, comprising at least one selected from the group consisting of an XRCC4 polypeptide described in any one of items 2 to 4; a polynucleotide comprising a nucleotide sequence encoding the XRCC4 polypeptide; and an expression vector comprising the polynucleotide. [Section 9] Item 9. The composition according to item 8, which is for treating or preventing a condition or disease involving the inhibition of apoptosis.

[0007] [Section 10] A method for screening for a substance that modulates the lipid scrambling activity of Xkr4, comprising the steps of: (1) Contacting a cell expressing the Xkr4 polypeptide according to item 1 with a candidate substance that may modulate the lipid scrambling activity of Xkr4; (2) measuring lipid scrambling activity in the cells contacted with the candidate substance; and (3) selecting the candidate substance as a substance that regulates the lipid scrambling activity of Xkr4 when the lipid scrambling activity is lower or higher than the lipid scrambling activity in the cells not contacted with the candidate substance; A screening method comprising: [Section 11] The screening method described in Item 10, wherein, when the lipid scrambling activity of the substance that regulates the lipid scrambling activity of Xkr4 is lower in the comparison than the lipid scrambling activity in the cells that have not been contacted with the candidate substance, the regulating substance is a drug candidate for the treatment or prevention of a condition or disease involving the promotion of apoptosis. [Section 12] A method for screening a substance that induces the lipid scrambling activity of Xkr4, comprising the steps of: (1) contacting a cell expressing a C-terminally truncated Xkr4 polypeptide, the C-terminally truncated Xkr4 polypeptide being (α) a C-terminally truncated Xkr4 polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or 36, or (β) a C-terminally truncated Xkr4 polypeptide having an amino acid sequence including deletions, substitutions, or additions or a combination thereof in the amino acid sequence of the (α) polypeptide, and exhibiting at least 80% sequence identity with the amino acid sequence of the (α) polypeptide, and capable of exhibiting lipid scrambling activity in the cell membrane through interaction with an XRCC4 polypeptide, with a candidate substance that may induce the lipid scrambling activity of Xkr4; (2) measuring lipid scrambling activity in the cells contacted with the candidate substance; and (3) selecting the candidate substance as a substance that induces the lipid scrambling activity of Xkr4 when the lipid scrambling activity is higher than the lipid scrambling activity in the cells not contacted with the candidate substance; A screening method comprising:

[0008] [Section 13] A method for screening for a substance that inhibits the lipid scrambling activity of Xkr4, comprising the steps of: (1) contacting a cell into which the XRCC4 polypeptide according to any one of items 2 to 4 has been introduced and which expresses a C-terminal truncated Xkr4 polypeptide with a candidate substance that may inhibit the lipid scrambling activity of Xkr4; (2) measuring lipid scrambling activity in the cells; and (3) selecting the candidate substance as a substance that inhibits the lipid scrambling activity of Xkr4 when the lipid scrambling activity is lower than the lipid scrambling activity in the cells not contacted with the candidate substance; A screening method comprising: [Section 14] A method for screening for a substance that inhibits the lipid scrambling activity of Xkr4, comprising the steps of: (1) contacting the XRCC4 polypeptide according to any one of items 2 to 4 with a C-terminal truncated Xkr4 polypeptide in the presence of a candidate substance that may inhibit the lipid scrambling activity of Xkr4; (2) measuring binding between the XRCC4 polypeptide and the C-terminal truncated Xkr4 polypeptide; and (3) selecting the candidate substance as a substance that inhibits the lipid scrambling activity of Xkr4 when the binding between the XRCC4 polypeptide and the C-terminal truncated Xkr4 polypeptide is lower than the binding when the XRCC4 polypeptide and the C-terminal truncated Xkr4 polypeptide are contacted in the absence of the candidate substance; A screening method comprising: [Section 15] Item 15. The screening method according to any one of Items 10 to 14, wherein the lipid scrambling activity is an activity of taking up a phospholipid or glycolipid, or an activity of exposing a phospholipid located on the inner side of a cell membrane.

[0009] [Section 16] A kit for screening a substance that inhibits the lipid scrambling activity of Xkr4 according to item 14 or 15, A kit comprising at least one selected from the group consisting of the XRCC4 polypeptide according to any one of Items 2 to 4; a polynucleotide comprising a nucleotide sequence encoding the XRCC4 polypeptide; an expression vector comprising the polynucleotide; and a host cell comprising the polynucleotide or a host cell comprising the expression vector. [Section 17] A method for identifying a gene corresponding to a phenotype of interest, comprising the steps of: (a) providing a guide RNA library, the guide RNA comprising at least one guide sequence corresponding to a nucleotide sequence of a plurality of genes in genomic DNA of a cell; (b) obtaining a cell population into which the guide RNA library has been introduced; (c) selecting cells from said cell population based on a phenotype of interest; (d) recovering genomic DNA from the selected cells; (e) preparing a new guide RNA library from the collected genomic DNA and performing steps (b) to (d) using the new guide RNA library until steps (b) to (d) are performed a predetermined number of times; (f) determining the guide sequence in the recovered genomic DNA when steps (b) to (d) have been performed a predetermined number of times; and (g) identifying a gene corresponding to the determined guide sequence as a gene corresponding to the desired phenotype, A method for identifying, wherein the predetermined number of times is at least two. [Brief description of the drawings]

[0010] [Figure 1] Figure 1(a) is a graph showing the lipid scrambling assay in PLB cells (parental). Figure 1(b) is a graph showing the lipid scrambling assay in PLB cells expressing Xkr4WT. Figure 1(c) is a graph showing the lipid scrambling assay in PLB cells expressing Xkr4ΔC. Figure 1(d) is a graph showing the results of a fluorescent Annexin V staining assay in PLB cells (parental). Figure 1(e) is a graph showing the results of a fluorescent Annexin V staining assay in PLB cells expressing Xkr4WT. Figure 1(f) is a graph showing the results of a fluorescent Annexin V staining assay in PLB cells expressing Xkr4ΔC. Light gray indicates no STS treatment (control), and dark gray indicates STS treatment (apoptotic stimulus). Bars indicate areas positive for PC uptake and PS exposure. [Diagram 2] The top row of Fig. 2 shows micrographs of live PLB cells expressing Xkr4WT-GFP. The bottom row of Fig. 2 shows confocal micrographs of live PLB cells expressing Xkr4ΔC-GFP. The left column of Fig. 2 shows micrographs of GFP-derived fluorescence. The middle column of Fig. 2 shows micrographs of differential interference contrast (DIC). The right column of Fig. 2 shows a superimposition of the fluorescence and DIC photographs. The scale bar indicates 10 μm.

[0011] [Diagram 3] Figure 3(a) is a graph showing the results of a lipid scramble assay in Xkr4WT-expressing cells in the absence of a caspase inhibitor. Figure 3(b) is a graph showing the results of a lipid scramble assay in Xkr4WT-expressing cells in the presence of a caspase inhibitor. Figure 3(c) is a graph showing the results of a lipid scramble assay in Xkr4Δ-expressing cells in the absence of a caspase inhibitor. Figure 3(d) is a graph showing the results of a lipid scramble assay in Xkr4ΔC-expressing cells in the presence of a caspase inhibitor. Light grey indicates no STS treatment (control) and dark grey indicates STS treatment (apoptotic stimulus). Bars indicate areas positive for PC uptake. [Figure 4] Figure 4(a) is a photograph showing Western blotting after BN-PAGE of cell membrane lysates from Xkr4WT or Xkr4ΔC expressing cells. Black arrowheads indicate Xkr4 monomers, and gray arrowheads indicate Xkr4 dimers. Figure 4(b) (top) is a photograph showing Western blotting after SDS-PAGE of cell membrane lysates from Xkr4WT or Xkr4ΔC expressing cells. Figure 4(b) (bottom) is a photograph showing CBB-stained PVDF membranes as a loading control.

[0012] [Diagram 5] Figure 5(a) is a schematic diagram showing a strategy to establish cells that constitutively exhibit lipid scrambling activity using Ba / F3 cells (BDKO) lacking two ubiquitous scramblase enzymes, Xkr8 and TMEM16F, and Figure 5(b) is a series of graphs showing the results of a strategy to select cells that constitutively incorporate PC from a population of BDKO cells expressing Xkr4ΔC. [Figure 6] Figure 6(a) is a graph showing PC uptake in transfectants (parental) expressing Xkr4ΔC. Figure 6(b) is a graph showing PC uptake in PC6, which was sorted six times. Figure 6(c) is a graph showing PC uptake in PC cells expressing sgRNA against Xkr4 (PC6+sgXkr4). The upper part of Figure 6(d) is a photograph showing Western blotting for detecting Xkr4. The lower part of Figure 6(d) is a photograph showing a CBB-stained PVDF membrane as a loading control. [Figure 7] Figure 7(a) is a graph showing the results of a lipid scramble assay in 1LPC0. Figure 7(b) is a graph showing the results of a lipid scramble assay in 1LPC3. Figure 7(c) is a graph showing the results of a lipid scramble assay in 2LPC0. Figure 7(d) is a graph showing the results of a lipid scramble assay in 2LPC1. The bars indicate the areas where PC uptake is positive.

[0013] [Figure 8] Figure 8(a) is a graph showing the results of a PC uptake assay in BDKO cells expressing full-length Xkr4 or various Xkr4 variants, and Figure 8(b) is a graph showing the results of a fluorescent Annexin V staining assay. Bars indicate areas positive for PS exposure. [Figure 9] The top panel of Fig. 9(a) shows Western blotting of cell lysates of transfectants expressing Xkr4 variants after BN-PAGE. The black arrowheads indicate Xkr4 monomers, and the grey arrowheads indicate Xkr4 dimers. The middle panel of Fig. 9(a) shows Western blotting of cell lysates of transfectants expressing selected Xkr4 variants after SDS-PAGE. The bottom panel of Fig. 9(a) shows a PVDF membrane stained with CBB as a loading control. The top panel of Fig. 9(b) shows Western blotting of cell lysates of transfectants expressing selected Xkr4 variants after SDS-PAGE. The bottom panel of Fig. 9(b) shows a PVDF membrane stained with CBB as a loading control.

[0014] [Figure 10] FIG. 10(a) is a graph showing PC uptake under conditions in which neither magnesium ions nor calcium ions are present outside the cells. FIG. 10(b) is a graph showing PC uptake under conditions in which magnesium ions are present outside the cells but not calcium. FIG. 10(c) is a graph showing PC uptake under conditions in which magnesium ions are not present outside the cells but calcium ions are present. FIG. 10(d) is a graph showing PC uptake under conditions in which both magnesium ions and calcium ions are present outside the cells. In FIG. 10(a)-(d), the bars indicate areas where PC uptake is positive. FIG. 10(e) is a graph showing PC uptake depending on the extracellular calcium concentration. [Figure 11]Figure 11(a) is a graph showing the effect of calcium ionophore on PC uptake. Figure 11(b) is a graph showing the effect of extracellular calcium on Fluo4-AM as well as calcium ionophore. Figure 11(c) is a series of graphs showing PC uptake activity in transfectants expressing Xkr4WT or Xkr4ΔC. In Figure 11(c), light grey indicates no STS treatment, dark grey indicates STS treatment, and bars indicate areas positive for PC uptake. [Figure 12] FIG. 12 is a flow diagram of revival screening.

[0015] [Figure 13] Figure 13(a) shows sequence analysis of established CAD KO PLB cells. The underlined sequence in CAD+ / + indicates the sgRNA target sequence. The dashed region in CAD- / - indicates the deleted nucleotides (7 bps) in both alleles. Figure 13(b) (top) shows Western blotting of CAD. Whole cell lysates from parental and CAD KO PLB cells were subjected to SDS-PAGE, followed by Western blotting using anti-CAD antibody. Figure 13(b) (bottom) shows a PVDF membrane stained with CBB as a loading control. Figure 13(c) shows a DNA fragmentation assay. Parental and CAD KO PLB cells were stimulated with STS for 4 hours, solubilized in solubilization buffer, and applied to an agarose gel. [Figure 14]FIG. 14(a) is a graph showing the results of flow cytometry of a cell population (sgPC0: unsorted) containing cells that are Xkr4ΔC-RFP positive and PC uptake negative in revival screening. FIG. 14(b) is a graph showing the results of flow cytometry of a cell population (sgPC2: sorted twice) containing cells that are Xkr4ΔC-RFP positive and PC uptake negative in revival screening. FIG. 14(c) is a graph showing the results of flow cytometry of a cell population (sgPC3: sorted three times) containing cells that are Xkr4ΔC-RFP positive and PC uptake negative in revival screening. The bars indicate areas where PC uptake is negative.

[0016] [Figure 15] Figure 15 is a graph showing NGS analysis of the total sgRNAs integrated into the genomic DNA of the sgPC4 cell population. The X-axis shows the list of genes for which more than three out of six target sgRNAs were recovered. The Y-axis shows the total value of different sgRNAs corresponding to the same gene in the reads. Each term (CYCS, APAF1) is written near the dots corresponding to cytochrome C (CYCS) and APAF1. [Figure 16] Figure 16(a) is a graph showing PC uptake by flow cytometry in sgPC6 cell populations. Figure 16(b) is a graph showing caspase 3 activity by flow cytometry in sgPC6 cell populations. The area surrounded by the solid line indicates the sorted area. [Figure 17] FIG. 17 is a bar graph showing the ratio of total reads of target sgRNAs in sgPC7 cells to total reads of target sgRNAs in sgPC4 cells.

[0017] [Figure 18]The top of Figure 18(a) is a graph showing caspase 3 activity in PLB cells. The bottom of Figure 18(a) is a graph showing PC uptake in PLB cells. The top of Figure 18(b) is a graph showing caspase 3 activity in PLB cells using sgRNA against XRCC4. The bottom of Figure 18(b) is a graph showing PC uptake in PLB cells using sgRNA against XRCC4. The top of Figure 18(c) is a graph showing caspase 3 activity in PLB cells using sgRNA against CYCS. The bottom of Figure 18(c) is a graph showing PC uptake in PLB cells using sgRNA against CYCS. The top of Figure 18(d) is a graph showing caspase 3 activity in PLB cells using sgRNA against APAF1. The bottom of Figure 18(d) is a graph showing PC uptake in PLB cells using sgRNA against APAF1. The bars in the top of Figures 18(a) to (d) indicate areas positive for caspase 3 activity. The bars in the lower rows of Figure 18(a) to (d) indicate areas positive for PC uptake.

[0018] [Figure 19]FIG. 19(a) shows sequence analysis of XRCC4 knockout PLB cells. The underlined nucleotide sequence in XRCC4+ / + indicates the sgRNA target sequence for XRCC4. The dashed line in XRCC4- / - indicates the deleted nucleotide (4bp) sequence, and the underlined nucleotide indicates the added nucleotide (1pb). FIG. 19(b) shows Western blotting of CAD, V5, and XRCC4. Whole cell lysates were prepared from parental cells, CAD KO (CAD- / -) PLB cells, CAD / XRCC4 double KO (CAD- / - XRCC4- / -) PLB cells, and CAD- / - XRCC4- / - PLB cells expressing V5-Xkr4ΔC-FLAG, and the whole cell lysates were subjected to SDS-PAGE, followed by Western blotting using anti-V5, anti-CAD, and anti-XRCC4 antibodies. The lower part of Figure 19(b) is a photograph showing a CBB-stained PVDF membrane as a loading control. The upper part of Figure 19(c) is a photograph showing Western blotting for XRCC4. The middle part of Figure 19(c) is a photograph showing Western blotting for activated caspase 3. PLB cells expressing XRCC4 WT or XRCC4 2DA fused with RFP at the C-terminus were stimulated to induce apoptosis using STS. Total cell lysates were prepared from the cells, and the cell lysates were subjected to SDS-PAGE, followed by Western blotting using a given antibody. The lower part of Figure 19(c) is a photograph showing a CBB-stained PVDF membrane as a loading control.

[0019] [Figure 20]FIG. 20(a) is a graph showing PC uptake in XRCC4- / - cells. FIG. 20(b) is a graph showing PC uptake in XRCC4- / - cells expressing XRCC4 WT. FIG. 20(c) is a graph showing PC uptake in XRCC4- / - cells expressing XRCC4 2DA. Light gray indicates control, and dark gray indicates STS treatment. Bars indicate areas positive for PC uptake. FIG. 20(d) is a graph showing PC uptake in Xkr4ΔC Q332E expressing cells. FIG. 20(e) is a graph showing PC uptake in Xkr4ΔC Q332E expressing cells transfected with sgRNA against XRCC4. FIG. 20(f) is a graph showing PC uptake in Xkr4ΔC Q332E expressing cells transfected with sgRNA against CYCS. Light gray indicates PC uptake in Xkr4ΔCQ332E-expressing cells without sgRNA. [Figure 21] Figure 21 (top) is a photograph showing Western blotting of V5 (Xkrs). Figure 21 (middle) is a photograph showing Western blotting of RFP (XRCC4). Total cell lysates from XRCC4 KO PLB cells expressing Xkrs tagged with V5 at the N-terminus, with or without co-expression of XRCC4 WT-RFP, were applied to SDS-PAGE and Western blotting. Figure 21 (bottom) is a photograph showing a CBB-stained PVDF membrane as a loading control.

[0020] [Figure 22]FIG. 22(a) is a graph showing PC uptake in XRCC4 KO PLB cells expressing Xkr4. FIG. 22(b) is a graph showing PC uptake in XRCC4 KO PLB cells expressing Xkr8. FIG. 22(c) is a graph showing PC uptake in XRCC4 KO PLB cells expressing Xkr9. FIG. 22(d) is a graph showing PC uptake in XRCC4 KO PLB cells co-expressing Xkr4 and XCCR4 WT. FIG. 22(e) is a graph showing PC uptake in XRCC4 KO PLB cells co-expressing Xkr8 and XCCR4 WT. FIG. 22(f) is a graph showing PC uptake in XRCC4 KO PLB cells co-expressing Xkr9 and XCCR4 WT. In Figure 22(a)-(f), light grey indicates no STS treatment (control), dark grey indicates STS treatment (apoptotic stimulus), and bars indicate areas positive for PC uptake. Figure 22(g) is a graph showing caspase 3 activity in XRCC4 KO PLB cells expressing Xkr4. Figure 22(h) is a graph showing caspase 3 activity in XRCC4 KO PLB cells expressing Xkr8. Figure 22(i) is a graph showing caspase 3 activity in XRCC4 KO PLB cells expressing Xkr9. Figure 22(j) is a graph showing caspase 3 activity in XRCC4 KO PLB cells co-expressing Xkr4 and XRCC4. Figure 22(k) is a graph showing caspase 3 activity in XRCC4 KO PLB cells co-expressing Xkr8 and XRCC4. Figure 22(l) is a graph showing caspase-3 activity in XRCC4 KO PLB cells co-expressing Xkr9 and XRCC4. In Figure 22(g)-(l), light grey indicates no STS treatment (control), dark grey indicates STS treatment (apoptotic stimulus), and bars indicate areas positive for caspase-3 activity.

[0021] [Figure 23]FIG. 23(a) is a micrograph of a transfectant expressing XRCC4 WT tagged with RFP at the C-terminus (XRCC4 WT-RFP). FIG. 23(b) is a micrograph of a transfectant expressing XRCC4 WT tagged with RFP at the N-terminus (RFP-XRCC4 WT). FIG. 23(c) is a micrograph of a transfectant expressing XRCC4 2DA tagged with RFP at the C-terminus (XRCC4 2DA-RFP). FIG. 23(d) is a micrograph of a transfectant expressing XRCC4 2DA tagged with RFP at the N-terminus (RFP-XRCC4 2DA). Arrowheads indicate cytoplasmic XRCC4. Scale bars indicate 10 μm.

[0022] [Figure 24] The top panel of Fig. 24(a) is a photograph showing Western blotting of RFP-XRCC4 WT or 2DA. XRCC4 KO PLB cells expressing V5-Xkr4ΔC-FLAG and XRCC4 WT fused with RFP at the N-terminus, and XRCC4 KO PLB cells expressing V5-Xkr4ΔC-FLAG and non-caspase-cleavable XRCC4 2DA fused with RFP at the N-terminus were stimulated with apoptosis by STS. Total cell lysates from the cells were prepared, and the cell lysates were subjected to SDS-PAGE and then Western blotting using anti-RFP antibody. The bottom panel of Fig. 24(a) is a photograph showing a PVDF membrane stained with CBB as a loading control. Fig. 24(b) is a graph showing the PC uptake activity of XRCC4 KO PLB cells expressing V5-Xkr4ΔC-FLAG and XRCC4 WT fused with RFP at the N-terminus. Figure 24(c) is a graph showing the PC uptake activity of XRCC4 KO PLB cells expressing V5-Xkr4ΔC-FLAG and N-terminal RFP-fused XRCC4 2DA. In Figures 24(b) and (c), light grey indicates no STS treatment (control), dark grey indicates STS treatment (apoptotic stimulus), and bars indicate areas positive for PC uptake. [Diagram 25]Figure 25 is a schematic diagram of the XRCC4 domain. XLF indicates the XLF binding domain. Dimer indicates the dimerization domain. DNA Lig IV indicates the DNA LigIV binding domain. Between amino acids 265 and 266 is a caspase recognition site. Amino acids 270-275 indicate the nuclear localization signal (NLS) region.

[0023] [Figure 26]FIG. 26(a) is a graph showing PC uptake in XRCC4 KO PLB cells (XRCC4- / -). FIG. 26(b) is a graph showing PC uptake in XRCC4 KO PLB cells expressing XRCC4 WT (1-336). FIG. 26(c) is a graph showing PC uptake in XRCC4 KO PLB cells expressing XRCC4 (116-336) with a partially deleted N-terminus. FIG. 26(d) is a graph showing PC uptake in XRCC4 KO PLB cells expressing XRCC4 (156-336) with a partially deleted N-terminus. FIG. 26(e) is a graph showing PC uptake in XRCC4 KO PLB cells expressing XRCC4 (204-336) with a partially deleted N-terminus. FIG. 26(f) is a graph showing PC uptake in XRCC4 KO PLB cells expressing N-terminally deleted XRCC4(248-336). FIG. 26(g) is a graph showing PC uptake in XRCC4 KO PLB cells expressing N-terminally deleted XRCC4(256-336). FIG. 26(h) is a graph showing PC uptake in XRCC4 KO PLB cells expressing N-terminally deleted XRCC4(226-336) (also referred to as "ΔN"). FIG. 26(i) is a graph showing PC uptake in XRCC4 KO PLB cells expressing C-terminally deleted XRCC4(1-305). FIG. 26(j) is a graph showing PC uptake in XRCC4 KO PLB cells expressing C-terminally deleted XRCC4(1-285). Figure 26(k) is a graph showing PC uptake in XRCC4 KO PLB cells expressing a partially C-terminally deleted XRCC4(1-265) (also called "ΔC"). Figure 26(l) is a graph showing PC uptake in XRCC4 KO PLB cells expressing an XRCC4 fragment (also called "Mini") that induces PC uptake. PC uptake assays were performed with 10 μM STS. Light grey indicates no STS treatment (control) and dark grey indicates STS treatment (apoptotic stimulus). Bars indicate areas positive for PC uptake.

[0024] [Figure 27]The top panel of Figure 27(a) shows a photograph of Western blotting of XRCC4-RFP with a partially deleted N-terminus. The open arrowhead indicates the predicted protein size of XRCC4-RFP. The black arrowhead indicates the C-terminal fragment of XRCC4 cleaved by caspase. The asterisk indicates the degradation product. Figure 27(b) shows an intrinsically disordered region (IDR) analysis of XRCC4. The amino acid sequence of XRCC4 was analyzed using the software IUPred2A, and the results are shown. The arrow indicates the caspase 3 cleavage site. Figure 27(c) shows a photograph of Western blotting of XRCC4-RFP with a partially deleted C-terminus. Figure 27(d) shows a photograph of Western blotting of XRCC4ΔC-RFP and XRCC4ΔN-RFP. Figure 27(e) shows an alignment of XRCC4 amino acid sequences in a given biological species. The underline indicates the isoleucine after the caspase cleavage site in human XRCC4, which is changed to the similar hydrophobic amino acid valine in some species. The amino acid sequence enclosed in a solid line is conserved in a given species. Figure 27(f) is a photograph showing Western blotting of XRCC4(Mini)-RFP. In Figure 27(a, c, d, and f), XRCC4 KO PLB cells expressing Xkrc4 and a given XRCC4 fused to RFP at the C-terminus were stimulated with apoptosis by STS. Total cell lysates from the cells were prepared, and the cell lysates were subjected to SDS-PAGE and then Western blotting with anti-RFP antibody. The lower panels of Figure 27(a, c, d, and f) are photographs showing CBB-stained PVDF membranes as loading controls. The loading amount of total cell lysate is shown at the bottom (μg).

[0025] [Figure 28]Figure 28(a) shows the amino acid sequences of synthetic peptides C20 and C21. The underline indicates the caspase recognition site. The amino acid sequence surrounded by a solid line indicates the NLS. Figure 28(b) is a graph showing the scrambled activity of synthetic peptides C20 and C21 to expose PS. Figure 28(c) is a bar graph showing the quantification of PS exposure activity (arbitrary units, AU). 1 (AU) corresponds to the fluorescence intensity of Xkr4ΔC expressing cells transfected with C20. [Figure 29] FIG. 29(a) is a micrograph showing the intracellular distribution of XRCC4 WT fused to RFP at the C-terminus. FIG. 29(b) is a micrograph showing the intracellular distribution of XRCC4 mutant (R270A) fused to RFP at the C-terminus. FIG. 29(c) is a micrograph showing the intracellular distribution of XRCC4 mutant (K271A) fused to RFP at the C-terminus. FIG. 29(d) is a micrograph showing the intracellular distribution of XRCC4 mutant (R272A) fused to RFP at the C-terminus. FIG. 29(e) is a micrograph showing the intracellular distribution of XRCC4 mutant (R273A) fused to RFP at the C-terminus. FIG. 29(f) is a micrograph showing the intracellular distribution of XRCC4 mutant (R275A) fused to RFP at the C-terminus. XRCC4 KO PLB cells expressing V5-Xkr4ΔC-FLAG were transfected with the indicated RFP-fused XRCC4 mutants, and the subcellular distribution of the RFP-fused XRCC4 mutants was analyzed by confocal microscopy. DRAQ5 was used for nuclear staining. DIC indicates differential interference contrast. Scale bar indicates 20 μm.

[0026] [Diagram 30]FIG. 30(a) is a graph showing PC uptake in XRCC4 KO PLB cells expressing XRCC4 WT. FIG. 30(b) is a graph showing PC uptake in XRCC4 KO PLB cells expressing XRCC4 mutant (R270A). FIG. 30(c) is a graph showing PC uptake in XRCC4 KO PLB cells expressing XRCC4 mutant (K271A). FIG. 30(d) is a graph showing PC uptake in XRCC4 KO PLB cells expressing XRCC4 mutant (R272A). FIG. 30(e) is a graph showing PC uptake in XRCC4 KO PLB cells expressing XRCC4 mutant (R273A). FIG. 30(f) is a graph showing PC uptake in XRCC4 KO PLB cells expressing XRCC4 mutant (R275A). PC uptake assay was performed using 10 μM STS. Light grey indicates no STS treatment (control), dark grey indicates STS treatment (apoptotic stimulus). Bars indicate areas positive for PC uptake.

[0027] [Diagram 31]The top panel of Fig. 31(a) shows a photograph of Western blotting of XRCC4 mutants. XRCC4 KO PLB cells expressing V5-Xkr4ΔC-FLAG and XRCC4-RFP were stimulated with STS to induce apoptosis. Whole cell lysates were prepared from the cells, and the cell lysates were subjected to SDS-PAGE and Western blotting using anti-RFP antibody. The bottom panel of Fig. 31(a) shows a photograph of a PVDF membrane stained with CBB as a loading control. Fig. 31(b) shows a bar graph showing the scramble activity of synthetic peptides to expose PS. The underlined amino acid sites are replaced with alanine. XRCC4 fragment mutants C20-R270A and C20-K271A were electroporated into BDKO cells or cells expressing V5-Xkr4ΔC-FLAG(ΔC) and a PS exposure assay was performed. Quantification of PS exposure activity is shown as the fluorescence intensity of C20-transfected V5-Xkr4ΔC-FLAG expressing cells defined as 1 (arbitrary unit, AU). Error bars indicate the mean ± SD of triplicate samples. P values ​​were calculated according to Student's t-test. NS indicates not significant. [Diagram 32]The top panel of Fig. 32(a) is a photograph showing Western blotting of XRCC4 KO PLB cells expressing SPOT-Xkr4ΔC-FLAG and XRCC4-WT-2A-RFP or XRCC4-R270A-2A-RFP using an anti-FLAG antibody. The middle panel of Fig. 32(b) is a photograph showing Western blotting of XRCC4 KO PLB cells expressing SPOT-Xkr4ΔC-FLAG and XRCC4-WT-2A-RFP or XRCC4-R270A-2A-RFP using an anti-XRCC4 antibody. The bottom panel of Fig. 32(c) is a photograph showing a PVDF membrane stained with CBB as a loading control. Fig. 32(b) is a graph showing PC uptake in XRCC4 KO PLB cells expressing SPOT-Xkr4ΔC-FLAG and XRCC4-WT-2A-RFP or XRCC4-R270A-2A-RFP. PC uptake assay was performed with 10 μM STS. In Figure 32(b), light grey indicates no STS treatment (control), dark grey indicates STS treatment (apoptotic stimulus), and bars indicate areas positive for PC uptake. Median fluorescence intensity was 139 in WT and 29.2 in R270A. [Diagram 33] Figure 33 is a distribution diagram showing label-free quantification of Xkr4 interactors. Plasma membrane fractions of PLB cells were solubilized and immunoprecipitated with anti-SPOT nanobody-conjugated beads to precipitate Xkr4 interactors. The precipitates were then subjected to mass spectrometry. The X-axis shows the number of peptides of XRCC4 WT with STS divided by the number of peptides of XRCC4 WT without STS. The Y-axis shows the number of peptides of XRCC4 WT with STS divided by the number of peptides of XRCC4 R270A with STS. [Diagram 34]FIG. 34(a) is a schematic diagram showing the regions of two XRCC4 peptides identified by immunoprecipitation with Xkr4. FIG. 34(b) is an extracted ion chromatogram of the transition of XRCC4 peptide 1. Peptide 1 was analyzed by targeted mass spectrometry using the PRM method. FIG. 34(c) is a bar graph showing the relative amount of XRCC4 normalized by the relative amount of Xkr4. FIG. 34(d) is an extracted ion chromatogram of the transition of XRCC4 peptide 2. Peptides were analyzed by targeted mass spectrometry using the PRM method. FIG. 34(e) is a bar graph showing the relative amount of XRCC4 normalized by the relative amount of Xkr4. In FIG. 34(c) and (e), the relative amount of XRCC4 was normalized by the relative amount of Xkr4 so that the relative amount of XRCC4 precipitated with Xkr4 in live cells is 1. [Diagram 35] FIG. 35(a) is a photograph of Western blotting showing the detection of XRCC4 in Xkr4 immunoprecipitates. XRCC4 WT immunoprecipitates recovered together with SPOT-fused Xkr4 were detected. FIG. 35(b) is a micrograph showing the localization of the XRCC4 peptide. C20-TMR was introduced into HCT116 cells expressing Xkr4WT-GFP or Xkr4ΔC-GFP by electroporation. The resulting cells were observed under a confocal microscope. FIG. 35(c) is a graph showing the results of line-scan analysis along the white dotted arrow in the photograph shown in the right column (Marged) of FIG. 35(b). The fluorescence intensity derived from GFP and the fluorescence intensity derived from TMR are shown. In FIG. 35(c), the Y-axis indicates arbitrary units (AU), and the scale bar indicates 20 μm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Xkr4 Polypeptide "Xkr4" is a membrane protein belonging to the Xkr family and having 10 transmembrane domains. Xkr4 is expressed in specific tissues, such as the brain or skin. Xkr4 may be Xkr4 from mammals, including humans, mice, and pigs. Xkr4 may be a homologous protein of Xkr4 in birds, reptiles, amphibians, or fish. The amino acid sequence of Xkr4 is available, for example, from publicly available databases, for example, from GeneBank. Without wishing to be bound by theory, Xkr4 may be partially cleaved by caspases to form a dimer, for example, in cells that have received an apoptotic stimulus. The C-terminally truncated Xkr4 that has formed a dimer may exhibit lipid scrambling activity through interaction with the XRCC4 polypeptide described below.

[0029] Xkr4 is, for example, mouse-derived Xkr4 consisting of or containing the amino acid sequence set forth in SEQ ID NO:1 shown below. TIFF2025011252000001.tif81165

[0030] Xkr4 is, for example, human-derived Xkr4 consisting of or containing the amino acid sequence set forth in SEQ ID NO:35 shown below. TIFF2025011252000002.tif81165

[0031] Xkr4 consists of or comprises an amino acid sequence containing 1 to 100, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 4, 3, 2 or 1 amino acid deletions, substitutions or additions, or a combination thereof, in the amino acid sequence set forth in, for example, SEQ ID NO: 1 or 35. Xkr4 consists of or comprises an amino acid sequence showing at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in, for example, SEQ ID NO: 1 or 35.

[0032] An "Xkr4 polypeptide" is (A) a polypeptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 3 to 5 and 37 to 39 (specific amino acid sequences are shown below); or (B) a polypeptide having an amino acid sequence that includes deletions, substitutions, or additions or a combination thereof in the amino acid sequence of the polypeptide (A), which shows at least 80% sequence identity with the amino acid sequence of (A), and which can exhibit lipid scrambling activity in a cell membrane without interacting with an XRCC4 polypeptide. TIFF2025011252000003.tif187165

[0033] TIFF2025011252000004.tif197165

[0034] In comparison with the amino acid sequence set forth in SEQ ID NO:2, the amino acid sequence set forth in SEQ ID NO:3 has an isoleucine (I) at position 322 replaced by serine (S) (I322S) according to the numbering of amino acid residues in the amino acid sequence set forth in SEQ ID NO:1.

[0035] Compared to the amino acid sequence set forth in SEQ ID NO:2, the amino acid sequence set forth in SEQ ID NO:4 has a substitution of leucine (L) at position 331 with phenylalanine (F) (L331F) according to the numbering of amino acid residues in the amino acid sequence set forth in SEQ ID NO:1.

[0036] Compared to the amino acid sequence set forth in SEQ ID NO:2, the amino acid sequence set forth in SEQ ID NO:5 has a substitution of glutamine (Q) at position 332 with glutamic acid (E) (Q332E) according to the numbering of amino acid residues in the amino acid sequence set forth in SEQ ID NO:1.

[0037] Compared to the amino acid sequence set forth in SEQ ID NO: 36, the amino acid sequence set forth in SEQ ID NO: 37 has an isoleucine (I) at position 325 replaced by serine (S) according to the numbering of the amino acid residues in the amino acid sequence set forth in SEQ ID NO: 35 (I325S).

[0038] Compared to the amino acid sequence set forth in SEQ ID NO: 36, the amino acid sequence set forth in SEQ ID NO: 38 has a substitution of leucine (L) at position 334 with phenylalanine (F) (L334F) according to the numbering of the amino acid residues in the amino acid sequence set forth in SEQ ID NO: 35.

[0039] Compared to the amino acid sequence set forth in SEQ ID NO: 36, the amino acid sequence set forth in SEQ ID NO: 39 has a substitution of glutamine (Q) at position 335 with glutamic acid (E) according to the numbering of the amino acid residues in the amino acid sequence set forth in SEQ ID NO: 35 (Q335E).

[0040] The Xkr4 polypeptide can exhibit lipid scrambling activity in the cell membrane without interacting with the XRCC4 polypeptide described below. The lipid scrambling activity by the Xkr4 polypeptide is, for example, lipid scrambling activity (e.g., PC uptake activity) in human PLB985 cells (also referred to as "PLB cells" in this specification) (Reference 26).

[0041] "Peptide", "polypeptide" and "protein" refer to polymers of amino acids of any length, including amino acids linked together by peptide bonds or modified peptide bonds. "Peptide", "polypeptide" and "protein" are used interchangeably. Amino acids include the 20 genetically encoded amino acids and other modified amino acids. Peptides, polypeptides and proteins may be derived from, for example, natural sources (cells) or may be artificially prepared, such as by recombinant gene technology. Peptides, polypeptides and proteins may be isolated or purified, for example, from natural sources or from reagents after artificial preparation. Methods for isolating or purifying peptides, polypeptides and proteins are well known and may be, for example, using HPLC.

[0042] In this specification, the Xkr4 polypeptide according to (B) above is also referred to as a "mutant Xkr4 polypeptide".

[0043] Xkr4 polypeptide can be prepared, for example, by genetic engineering technology. Xkr4 polypeptide can be prepared, for example, by transcription and translation from a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence described herein. Xkr4 polypeptide can be used, for example, in the screening method for a substance that inhibits or induces the lipid scrambling activity of Xkr4 described later.

[0044] A mutant Xkr4 polypeptide has, for example, in the amino acid sequence set forth in SEQ ID NO: 1, at least one amino acid substitution selected from the group consisting of serine for isoleucine at position 322, phenylalanine for leucine at position 331, and glutamic acid for glutamine at position 332, according to the numbering of the amino acid residues in the amino acid sequence set forth in SEQ ID NO: 1. A mutant Xkr4 polypeptide has, for example, in the amino acid sequence set forth in SEQ ID NO: 35, at least one amino acid substitution selected from the group consisting of serine for isoleucine at position 325, phenylalanine for leucine at position 334, and glutamic acid for glutamine at position 335, according to the numbering of the amino acid residues in the amino acid sequence set forth in SEQ ID NO: 35.

[0045] An amino acid "deletion" means that an amino acid residue at any position in a given amino acid sequence is deleted. For example, a mutant Xkr4 polypeptide may have an amino acid deleted at the N-terminus, C-terminus, and / or between the N-terminus and C-terminus in the amino acid sequence of the Xkr4 polypeptide according to (A) above. In one embodiment, a mutant Xkr4 polypeptide having an amino acid deletion may have an amino acid deleted at the N-terminus or between the N-terminus and C-terminus in the amino acid sequence of the Xkr4 polypeptide according to (A) above.

[0046] A mutant Xkr4 polypeptide having an amino acid deletion in the amino acid sequence set forth in any one of SEQ ID NOs: 3 to 5 may be, for example, 500 to 563 amino acids long, 520 to 563 amino acids long, 540 to 563 amino acids long, 550 to 563 amino acids long, 560 to 563 amino acids long, 561 amino acids long, 562 amino acids long, or 563 amino acids long. A mutant Xkr4 polypeptide having an amino acid deletion in the amino acid sequence set forth in any one of SEQ ID NOs: 37 to 38 may be, for example, 500 to 566 amino acids long, 520 to 566 amino acids long, 540 to 566 amino acids long, 550 to 566 amino acids long, 560 to 566 amino acids long, or 565 or 566 amino acids long.

[0047] The term "addition" of an amino acid means that an amino acid residue has been added or inserted at any position in a given amino acid sequence. A mutant Xkr4 polypeptide may have an amino acid added or inserted at the N-terminus, C-terminus, and / or between the N-terminus and C-terminus of the amino acid sequence of the Xkr4 polypeptide according to (A) above. In one embodiment, a mutant Xkr4 polypeptide may have an amino acid added or inserted at the N-terminus or between the N-terminus and C-terminus of the amino acid sequence of the Xkr4 polypeptide according to (A) above.

[0048] A mutant Xkr4 polypeptide having an amino acid addition in the amino acid sequence set forth in any one of SEQ ID NOs: 3 to 5 may be, for example, 565 to 600 amino acids long, 565 to 590 amino acids long, 565 to 580 amino acids long, 565 to 570 amino acids long, 569 amino acids long, 568 amino acids long, 567 amino acids long, 566 amino acids long, or 565 amino acids long. A mutant Xkr4 polypeptide having an amino acid addition in the amino acid sequence set forth in any one of SEQ ID NOs: 37 to 38 may be, for example, 568 to 600 amino acids long, 568 to 590 amino acids long, 568 to 580 amino acids long, 568 to 570 amino acids long, 569 amino acids long, or 568 amino acids long.

[0049] An amino acid "substitution" means that an amino acid residue at any position in a given amino acid sequence is replaced with another amino acid residue. The amino acid substitution may be, for example, a conservative substitution. An amino acid "conservative substitution" means that an amino acid residue in a polypeptide is replaced with another amino acid residue from a group of amino acids having similar characteristics of the side chain (e.g., charge, side chain size, hydrophobicity / hydrophilicity). For example, the group of amino acids with aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic hydroxyl side chains consists of serine and threonine; the group of amino acids with amide-containing side chains consists of asparagine and glutamine; the group of amino acids with aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains consists of lysine, arginine, and histidine; the group of amino acids with acidic side chains consists of glutamate and aspartic acid; and the group of amino acids with sulfur-containing side chains consists of cysteine ​​and methionine.Exemplary conservative substitutions of amino acids are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

[0050] The Xkr4 polypeptide may be fused with a functional polypeptide (e.g., a tag sequence such as SPOT or FLAG, or a labeling protein such as green fluorescent protein). A mutant of the Xkr4 polypeptide in the fusion protein has an amino acid sequence that is the same as the amino acid sequence of the fusion protein except for the amino acid sequence of the functional polypeptide.

[0051] "Sequence identity" refers to the sequence similarity between two optimally aligned polynucleotide sequences or two amino acid strings. If a position in both of the two compared sequences has the same base or amino acid, the two molecules are identical at that position. The percentage of identity is the number of identical positions in the two sequences divided by the total number of positions compared, multiplied by 100. Sequence identity can be calculated using commercially or publicly available software, such as BLAST+.

[0052] In one embodiment, the sequence identity between the amino acid sequence of a mutant Xkr4 polypeptide and the amino acid sequence of the Xkr4 polypeptide is calculated from the amino acid sequence of the mutant Xkr4 polypeptide and the amino acid sequence set forth in any one of SEQ ID NOs: 3 to 5 and 37 to 39 (e.g., SEQ ID NO: 37).

[0053] "Amino acid residue numbering" is established by assigning numbers in ascending order from the N-terminus (left end) to the C-terminus (right end) in a particular sequence. For example, in the sequence ASG, the leftmost amino acid, alanine (A), is assigned position 1, and the rightmost amino acid, glycine (G), is assigned position 3.

[0054] The "lipid scrambling activity" of Xkr4 includes, for example, the uptake activity of phospholipids or glycolipids, or the exposure activity of phospholipids located inside the cell membrane. "Phospholipid" refers to a phospholipid that constitutes a cell membrane. Phospholipids are, for example, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, or phosphatidylinositol. "Glycolipid" refers to a glycolipid that constitutes a cell membrane. Glycolipids are, for example, glyceroglycolipids or sphingoglycolipids.

[0055] The "uptake activity" of phospholipids or glycolipids is the action of taking up phospholipids or glycolipids present outside the cell into the cell membrane of the cell. The uptake activity of phospholipids or glycolipids can be measured, for example, using fluorescently labeled phospholipids or glycolipids. The uptake activity of phospholipids or glycolipids can be measured, for example, by adding fluorescently labeled phospholipids or glycolipids to the culture medium of cells expressing Xkr4 polypeptide (e.g., PLB cells), allowing the fluorescently labeled phospholipids or glycolipids to be taken up into the cell membrane of the cell, and then detecting the fluorescence derived from the fluorescently labeled phospholipids or glycolipids that have been taken up. As the fluorescent label, for example, fluorescent substances such as nitrobenzoxadiazole (NBD) and TopFluor can be used. Fluorescent labeling of phospholipids or glycolipids can be performed using known methods. Fluorescently labeled phospholipids or glycolipids are commercially available.

[0056] The uptake activity of phospholipids or glycolipids can be measured, for example, by culturing PLB cells that constitutively express Xkr4 polypeptide in a microwell plate, adding fluorescently labeled phospholipids or glycolipids to the culture solution, and allowing them to be incorporated into the cell membrane. After a predetermined time has elapsed, the culture medium containing unreacted fluorescently labeled phospholipids or glycolipids is replaced with a new culture medium, and then the fluorescence from the fluorescently labeled phospholipids or glycolipids incorporated into the cell membrane can be measured, for example, with a plate reader. The measured fluorescence intensity can be compared with the fluorescence intensity from a negative control to determine the presence or absence or degree of uptake of phospholipids or glycolipids. When the fluorescence intensity in PLB cells that constitutively express Xkr4 polypeptide is higher than that in the negative control, for example, at least 2-fold, 3-fold, 5-fold, or 10-fold higher, it can be determined that phospholipids or glycolipids have been uptaken.

[0057] The uptake activity of phospholipids or glycolipids can be measured, for example, by recovering PLB cells constitutively expressing Xkr4 polypeptides incorporating fluorescently labeled phospholipids or glycolipids from a microwell plate in the same manner as described above, and measuring the recovered cell suspension by flow cytometry. The proportion of a cell population exhibiting a predetermined fluorescence intensity can be compared with the proportion of a cell population derived from a negative control to determine the presence or absence or the degree of uptake of phospholipids or glycolipids. The proportion of a cell population exhibiting a predetermined fluorescence intensity can be calculated, for example, by setting a predetermined gate in a distribution diagram of cells exhibiting fluorescence intensity per cell, and dividing the number of cells exceeding the gate by the total number of cells measured. The predetermined gate can be set, for example, at a position where the proportion of a cell population exhibiting a predetermined fluorescence intensity is 3% or less in a distribution diagram of cells exhibiting fluorescence intensity per cell of a negative control. Uptake of phospholipids or glycolipids may be determined to occur when the proportion of cell population in the specified gate in PLB cells that constitutively express Xkr4 polypeptide is higher, for example at least 2-fold, 3-fold, 5-fold, or 10-fold higher, than the proportion of cell population in the specified gate in a negative control.

[0058] In one embodiment, the uptake activity of a phospholipid or glycolipid may be PC or PS uptake activity using NBD-labeled phosphatidylcholine (NBD-PC) or phosphatidylserine (NBD-PS).

[0059] The "exposure activity" of phospholipids is an action of exposing phosphatidylserine (PS) or phosphatidylethanolamine (PE) located on the inside of a cell membrane showing phospholipid asymmetry to the outside of the cell membrane. The exposure activity of phospholipids can be measured, for example, using a detection reagent fluorescently labeled with a molecule that binds to PS or PE. The exposure activity of phospholipids can be measured, for example, by adding a detection reagent to a culture medium of cells expressing Xkr4 polypeptide (for example, PLB cells) and detecting the fluorescence derived from the detection reagent bound to the cell membrane after a predetermined time has elapsed. For example, fluorescent substances such as Cy5 and Alexa657 can be used as the fluorescent label. For example, annexin V can be used as the molecule that binds to PS or PE. Fluorescent labeling of PS or PE can be performed using a known method. Fluorescently labeled annexin V is commercially available.

[0060] For example, the exposure of phospholipids can be performed by culturing PLB cells that constitutively express Xkr4 polypeptide in a microwell plate, adding a detection reagent to the culture medium, and binding it to PS or PE exposed on the outside of the cell membrane. After a predetermined time has passed, the culture medium containing the unreacted detection reagent is replaced with a new culture medium, and then the fluorescence from the detection reagent bound to the cell membrane can be measured, for example, with a plate reader. The measured fluorescence intensity can be compared with the fluorescence intensity from a negative control to determine the presence or absence or the degree of phospholipid exposure activity. The negative control may be, for example, the result of a test performed in the same manner except that wild-type PLB cells (not expressing Xkr4 polypeptide) are used.

[0061] The phospholipid exposure activity can be measured, for example, by recovering PLB cells constitutively expressing Xkr4 polypeptide bound to a detection reagent from a microwell plate, and measuring the recovered cell suspension by flow cytometry. The percentage of a cell population showing a predetermined fluorescence intensity can be compared with the percentage of a cell population derived from a negative control to determine the presence or absence or the degree of phospholipid exposure activity. The percentage of a cell population showing a predetermined fluorescence intensity can be calculated, for example, by setting a predetermined gate in a distribution diagram of cells showing the fluorescence intensity per cell, and dividing the number of cells exceeding the gate by the total number of cells measured. The predetermined gate can be set at a position where the percentage of a cell population showing a predetermined fluorescence intensity is 3% or less in a distribution diagram of cells showing the fluorescence intensity per cell of a negative control.

[0062] In one embodiment, the Xkr4 polypeptide mutant has an amino acid sequence of, for example, 500 to 600 contiguous amino acids, 520 to 580 amino acids, 530 to 570 amino acids, 540 to 570 amino acids, or 550 to 570 amino acids in the amino acid sequence set forth in SEQ ID NO: 1, and has at least one amino acid substitution selected from the group consisting of serine instead of isoleucine at position 322, phenylalanine instead of leucine at position 331, and glutamic acid instead of glutamine at position 332, according to the numbering of amino acid residues in the amino acid sequence set forth in SEQ ID NO: 1, and has at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity between the amino acid sequence of the mutant and the amino acid sequence of the Xkr4 polypeptide; and the mutant exhibits lipid scrambling activity in a cell membrane without interaction with the XRCC4 polypeptide.

[0063] In one embodiment, the Xkr4 polypeptide mutant has an amino acid sequence of, for example, 500 to 600 contiguous amino acids, 520 to 580 amino acids, 530 to 570 amino acids, 540 to 570 amino acids, or 550 to 570 amino acids in the amino acid sequence set forth in SEQ ID NO: 35, and has at least one amino acid substitution selected from the group consisting of serine instead of isoleucine at position 325, phenylalanine instead of leucine at position 334, and glutamic acid instead of glutamine at position 335, according to the numbering of amino acid residues in the amino acid sequence set forth in SEQ ID NO: 35, and has at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity between the amino acid sequence of the mutant and the amino acid sequence of the Xkr4 polypeptide; and the mutant exhibits lipid scrambling activity in a cell membrane without interaction with the XRCC4 polypeptide.

[0064] "Can exhibit lipid scrambling activity in cell membrane without interaction with XRCC4 polypeptide" means that the mutant Xkr4 polypeptide exhibits PC uptake activity in the cell membrane of a cell expressing the mutant Xkr4 polypeptide in the absence of the XRCC4 polypeptide described below. For example, the mutant Xkr4 polypeptide is identified as being capable of exhibiting lipid scrambling activity in cell membrane without interaction with XRCC4 polypeptide when the PC uptake activity of the test subject is higher than that of the negative control, for example, at least 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold, when the PC uptake activity of the test subject is higher than that of the negative control, by comparing the PC uptake activity of the test subject in the absence of XRCC4 polypeptide (for example, a method using flow cytometry). The PC uptake activity can be measured by the method disclosed herein (for example, a method using flow cytometry).

[0065] The term "negative control cells" with respect to the mutant Xkr4 polypeptide refers to cells that are the same as the cells expressing the mutant Xkr4 polypeptide used to measure PC uptake activity, except that the mutant Xkr4 polypeptide is not expressed. The negative control cells may be, for example, wild-type PLB cells (not expressing the Xkr4 polypeptide).

[0066] Variants of Xkr4 polypeptides have, for example, any combination of amino acid length, amino acid substitution, sequence identity, and lipid scrambling activity (eg, PC uptake activity) disclosed herein.

[0067] XRCC4 Polypeptides "XRCC4" is a DNA repair protein, and in humans, it is encoded by the XRCC4 gene. XRCC4 may be localized in the nucleus. XRCC4 may be XRCC4 from mammals, including humans, mice, and pigs. XRCC4 may be a homologous protein of XRCC4 in birds, reptiles, amphibians, or fish. The amino acid sequence of XRCC4 is available, for example, from publicly available databases, for example, from GeneBank. Without wishing to be bound by theory, XRCC4 is partially cleaved by caspases, for example, in cells that have received an apoptotic stimulus, and the cleaved XRCC4 polypeptide diffuses into the cytoplasm. XRCC4 polypeptide may induce the lipid scrambling activity of Xkr4 through interaction with a dimerized C-terminal truncated Xkr4.

[0068] XRCC4 is, for example, human-derived XRCC4 consisting of or containing the amino acid sequence set forth in SEQ ID NO:6 shown below. TIFF2025011252000005.tif49165

[0069] XRCC4 consists of or comprises an amino acid sequence containing 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 4, 3, 2, or 1 amino acid deletion, substitution, or addition, or a combination thereof, in the amino acid sequence set forth in, for example, SEQ ID NO: 6. XRCC4 consists of or comprises an amino acid sequence showing at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in, for example, SEQ ID NO: 6.

[0070] An "XRCC4 polypeptide" is (a) a polypeptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 7 to 12 and 42 (specific amino acid sequences are shown below); or (b) a polypeptide having an amino acid sequence that includes deletions, substitutions, or additions or a combination thereof in the amino acid sequence of the polypeptide (a), which shows at least 80% sequence identity with the amino acid sequence of (a), and which can induce lipid scrambling activity in the cell membrane by interacting with a C-terminally truncated Xkr4 polypeptide. TIFF2025011252000006.tif56165

[0071] The amino acid sequence of SEQ ID NO:7 is an amino acid sequence of 20 consecutive amino acids from positions 266 to 285 according to the numbering of the amino acid sequence of SEQ ID NO:6.

[0072] Compared to the amino acid sequence set forth in SEQ ID NO:7, the amino acid sequence set forth in SEQ ID NO:8 has a substitution of lysine (K) at position 271 with alanine (A) (K271A) according to the numbering of the amino acid sequence set forth in SEQ ID NO:6.

[0073] Compared to the amino acid sequence set forth in SEQ ID NO:7, the amino acid sequence set forth in SEQ ID NO:9 has an arginine (R) at position 272 replaced by alanine (A) according to the numbering of the amino acid sequence set forth in SEQ ID NO:6 (R272A).

[0074] Compared to the amino acid sequence set forth in SEQ ID NO:7, the amino acid sequence set forth in SEQ ID NO:10 has an arginine (R) at position 273 replaced by alanine (A) (K273A) according to the numbering of the amino acid sequence set forth in SEQ ID NO:6.

[0075] Compared to the amino acid sequence set forth in SEQ ID NO:7, the amino acid sequence set forth in SEQ ID NO:11 has an arginine (R) at position 275 replaced by alanine (A) (K275A) according to the numbering of the amino acid sequence set forth in SEQ ID NO:6.

[0076] The amino acid sequence of SEQ ID NO:12 is an amino acid sequence of 30 consecutive amino acids from positions 256 to 285 according to the numbering of the amino acid sequence of SEQ ID NO:6.

[0077] The amino acid sequence of SEQ ID NO:42 is an amino acid sequence of 81 consecutive amino acids from positions 256 to 336 according to the numbering of the amino acid sequence of SEQ ID NO:6.

[0078] XRCC4 polypeptide can exhibit the lipid scrambling activity of Xkr4 in cell membranes by interacting with C-terminal truncated Xkr4 that forms a dimer. The lipid scrambling activity of Xkr4 by XRCC4 polypeptide is, for example, lipid scrambling activity (e.g., PC uptake activity) in PLB cells.

[0079] In this specification, the XRCC4 polypeptide according to (b) above is also referred to as a "mutant XRCC4 polypeptide".

[0080] XRCC4 polypeptide can be prepared, for example, according to known methods (for example, genetic engineering technology or chemical synthesis technology). XRCC4 polypeptide can be prepared, for example, by transcription and translation from a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence described herein. XRCC4 polypeptide can be used, for example, in the screening method for a substance that inhibits the lipid scrambling activity of Xkr4 described below, or as one component of a composition for activating Xkr4.

[0081] The XRCC4 polypeptide variant may be, for example, an amino acid sequence of 18 to 100 amino acids, 18 to 80 amino acids, 18 to 60 amino acids, or 18 to 50 amino acids; 19 to 100 amino acids, 19 to 80 amino acids, 19 to 60 amino acids, or 19 to 50 amino acids; or 20 to 100 amino acids, 20 to 80 amino acids, 20 to 60 amino acids, or 20 to 50 amino acids. The XRCC4 polypeptide variant may be, for example, an amino acid sequence of 50 to 80 consecutive amino acids in the amino acid sequence set forth in SEQ ID NO: 42, and may be, for example, an amino acid sequence of 50 to 100 amino acids, 60 to 100 amino acids, or 70 to 100 amino acids; 50 to 90 amino acids, 60 to 90 amino acids, or 70 to 90 amino acids; or 70 to 100 amino acids, 70 to 90 amino acids, 75 to 90 amino acids, or 75 to 85 amino acids. A mutant of XRCC4 polypeptide comprises, for example, the amino acid sequence set forth in SEQ ID NO: 7 or 12, and consists of an amino acid sequence that is 18 to 100 amino acids in length, 18 to 80 amino acids in length, 18 to 60 amino acids in length, or 18 to 50 amino acids in length; 19 to 100 amino acids in length, 19 to 80 amino acids in length, 19 to 60 amino acids in length, or 19 to 50 amino acids in length; or 20 to 100 amino acids in length, 20 to 80 amino acids in length, 20 to 60 amino acids in length, or 20 to 50 amino acids in length.

[0082] A mutant of XRCC4 polypeptide includes, for example, the amino acid sequence of SEQ ID NO: 7 or 12, and consists of an amino acid sequence of 18 to 100 contiguous amino acids, 18 to 80 amino acids, 18 to 60 amino acids, or 18 to 50 amino acids; 19 to 100 amino acids, 19 to 80 amino acids, 19 to 60 amino acids, or 19 to 50 amino acids; or 20 to 100 amino acids, 20 to 80 amino acids, 20 to 60 amino acids, or 20 to 50 amino acids in length in the amino acid sequence of SEQ ID NO: 6.

[0083] The XRCC4 polypeptide mutant contains 1 to 5, 1 to 4, 1 to 3, 2 or 1 amino acid addition, substitution or deletion, or a combination thereof, in the amino acid sequence of the XRCC4 polypeptide according to (a) above, when the amino acid length of the mutant is, for example, 20 to 50. The XRCC4 polypeptide mutant contains 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 2 or 1 amino acid addition, substitution or deletion, or a combination thereof, in the amino acid sequence of the XRCC4 polypeptide according to (a) above, when the amino acid length of the mutant is, for example, 51 to 100.

[0084] In one embodiment, the variant of the XRCC4 polypeptide comprises an arginine at position 270, according to the numbering of the amino acid residues of the amino acid sequence set forth in SEQ ID NO: 6. In one embodiment, the variant of the XRCC4 polypeptide does not comprise a methionine at position 265, according to the numbering of the amino acid residues of the amino acid sequence set forth in SEQ ID NO: 6. In one embodiment, the variant of the XRCC4 polypeptide comprises an arginine at position 270 and does not comprise a methionine at position 265, according to the numbering of the amino acid residues of the amino acid sequence set forth in SEQ ID NO: 6.

[0085] In one embodiment, the variant of the XRCC4 polypeptide comprises at least one amino acid substitution selected from the group consisting of K271A, R272A, K273A and K275A according to the numbering of the amino acid sequence set forth in SEQ ID NO: 6. In one embodiment, the variant of the XRCC4 polypeptide comprises an arginine at position 270 and either or both of at least one amino acid substitution selected from the group consisting of the following, and does not comprise a methionine at position 265, for example according to the numbering of the amino acid residues of the amino acid sequence set forth in SEQ ID NO: 6: K271A, R272A, K273A and K275A according to the numbering of the amino acid sequence set forth in SEQ ID NO: 6.

[0086] The XRCC4 polypeptide may be fused with a functional polypeptide (e.g., a tag sequence such as SPOT or FLAG, or a labeling protein such as green fluorescent protein). A mutant of the XRCC4 polypeptide in the fusion protein has an amino acid sequence that is the same as the amino acid sequence of the fusion protein except for the amino acid sequence of the functional polypeptide.

[0087] The XRCC4 polypeptide variant has an amino acid sequence comprising deletions, substitutions, or additions, or a combination thereof, in the amino acid sequence set forth in any one of SEQ ID NOs: 7 to 12; the variant is 18 to 100 amino acids long, 18 to 80 amino acids long, 18 to 60 amino acids long, or 18 to 50 amino acids long; 19 to 100 amino acids long, 19 to 80 amino acids long, 19 to 60 amino acids long, or 19 to 50 amino acids long; or 20 to 100 amino acids long, 20 to 80 amino acids long, 20 to 60 amino acids long, or 20 to 50 amino acids long; the amino acid sequence of the variant has at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity with the amino acid sequence of the XRCC4 polypeptide; and the variant is capable of inducing lipid scrambling activity in a cell membrane by interacting with a C-terminally truncated Xkr4 polypeptide.

[0088] In one embodiment, the sequence identity between the amino acid sequence of a mutant XRCC4 polypeptide and the amino acid sequence of the XRCC4 polypeptide is calculated from the amino acid sequence of the mutant XRCC4 polypeptide and the amino acid sequence set forth in any of SEQ ID NOs: 7 to 12 (e.g., SEQ ID NO: 12).

[0089] "Can induce lipid scrambling activity in cell membranes by interaction with C-terminal truncated Xkr4 polypeptide" means that the mutant XRCC4 polypeptide induces PC uptake activity in the cell membrane of a cell into which the mutant XRCC4 polypeptide is introduced and expresses the C-terminal truncated Xkr4 polypeptide. For example, the PC uptake activity of PLB cells (subject) into which the mutant XRCC4 polypeptide is introduced and expresses the C-terminal truncated Xkr4 polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 is compared with the PC uptake activity of PLB cells (negative control) that are identical except that the mutant is not introduced, and if the PC uptake activity of the subject is higher than that of the negative control, for example, at least 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold higher, the mutant XRCC4 polypeptide is identified as being capable of inducing lipid scrambling activity in cell membranes by interaction with C-terminal truncated Xkr4 polypeptide. The PC uptake activity can be measured by the method disclosed herein (e.g., a method using flow cytometry).

[0090] Variants of XRCC4 polypeptides have, for example, any combination of amino acid length, amino acid substitutions, sequence identity, and lipid scrambling activity (eg, PC uptake activity) disclosed herein.

[0091] "Introduction" or "introducing" a peptide or protein into a cell refers to making the peptide or protein present in the cell. Introduction of the peptide or protein into a cell includes bringing the peptide or protein into the cell from outside the cell and expressing the peptide or protein in the cell. The introduction of the peptide or protein into a cell can be carried out according to a known method. The introduction of the peptide or protein into the cell from outside the cell can be carried out, for example, by lipofection or electroporation. The expression of the peptide or protein in the cell can be carried out, for example, by transfecting a cell with a polynucleotide comprising a nucleotide sequence encoding the peptide or protein or an expression vector comprising the polynucleotide, and expressing the peptide or protein in the cell. In one embodiment, the XRCC4 polypeptide or XRCC4 polypeptide can be introduced into a cell by lipofection or electroporation.

[0092] Polynucleotides A "polynucleotide" is a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Polynucleotides include, for example, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers that contain purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. Polynucleotides include single-stranded polynucleotides (such as sense or antisense) and double-stranded polynucleotides. Polynucleotides containing a given nucleotide sequence can be prepared, for example, according to known methods (such as genetic engineering or chemical synthesis techniques) or are commercially available.

[0093] In one embodiment, the polynucleotide comprises a nucleotide sequence encoding the Xkr4 polypeptide disclosed herein. In one embodiment, the polynucleotide comprises a nucleotide sequence encoding the XRCC4 polypeptide disclosed herein. In one embodiment, the polynucleotide comprises a nucleotide sequence encoding the Xkr4 polypeptide and a nucleotide sequence encoding the XRCC4 polypeptide in the same polynucleotide or in separate polynucleotides.

[0094] Expression vector An "expression vector" is a genetic element that allows the introduction, replication or expression of an exogenous polynucleotide in a host cell. An expression vector can, for example, allow another polynucleotide, i.e., an insert, to be operably linked to a promoter, thereby allowing the replication, transcription or expression of said other polynucleotide in a host cell. An expression vector can, for example, be stably integrated into the genome of a host cell or exist as an independent genetic element (e.g., episome, plasmid). An expression vector can, for example, be a plasmid, a phage, a virus or a cosmid. Expression vectors for linking another polynucleotide can, for example, be prepared according to known methods (e.g., genetic engineering techniques) or are commercially available.

[0095] "Operably linked" indicates that the linked polynucleotide is capable of functioning in the intended manner, i.e., being transcribed and optionally translated in the intended manner. The intended manner indicates conditions that permit transcription, optionally translation, of the linked polynucleotide, e.g., when an additive, such as tetracycline, is present, or an amount of expression, such as high or low expression.

[0096] In one embodiment, the expression vector comprises a polynucleotide disclosed herein. In one embodiment, the expression vector comprises a polynucleotide comprising a nucleotide sequence encoding the Xkr4 polypeptide disclosed herein. In one embodiment, the expression vector comprises a polynucleotide comprising a nucleotide sequence encoding the XRCC4 polypeptide disclosed herein. In one embodiment, the expression vector may comprise a polynucleotide comprising a nucleotide sequence encoding the Xkr4 polypeptide and a polynucleotide comprising a nucleotide sequence encoding the XRCC4 polypeptide in the same expression vector or in separate expression vectors.

[0097] host cell A "host cell" is a cell into which a vector molecule can be inserted, i.e., a eukaryotic or prokaryotic cell. A host cell can be, for example, a eukaryotic or prokaryotic cell. Eukaryotic cells include, for example, cells from mammals, birds and fish, or plant cells, including, for example, eukaryotic algal cells. Mammalian host cells include, for example, human PLB985 cells, Chinese hamster ovary (CHO) cells, COS cells, Vero cells, SP2 / 0 cells, NS / 0 myeloma cells, human embryonic kidney (HEK293) cells and baby hamster kidney (BHK) cells, HeLa cells, human B cells, CV-1 / EBNA cells, L cells, 3T3 cells, HEPG2 cells, PerC6 cells and MDCK cells. Prokaryotic host cells include, for example, yeast cells and E. coli cells. Host cells can be, for example, cells from the brain or skin. Host cells can be, for example, cells of the nervous system. The host cell may be, for example, a cell in which either or both of XRCC4 and Xkr4 have been knocked out. The host cell may, for example, be publicly available or commercially available.

[0098] In one embodiment, a host cell comprises a polynucleotide or an expression vector disclosed herein. The host cell comprises, for example, a genome into which part or all of the polynucleotide is integrated. The host cell comprises, for example, a genome into which part or all of the expression vector is integrated. The host cell comprises, for example, the expression vector as a genetic element independent of the genome.

[0099] Compositions for activating Xkr4 The "composition for activating Xkr4" comprises an element capable of inducing lipid scrambling activity by Xkr4. The element is, for example, the XRCC4 polypeptide disclosed herein, a polynucleotide comprising a nucleotide sequence encoding the polypeptide, or an expression vector comprising the polynucleotide. The composition can be prepared, for example, by mixing the element with an additive such as an excipient, a preservative, a buffer, or a pH adjuster.

[0100] In one embodiment, the composition for activating Xkr4 comprises at least one selected from the group consisting of the XRCC4 polypeptide disclosed herein; a polynucleotide comprising a nucleotide sequence encoding the XRCC4 polypeptide; and an expression vector comprising the polynucleotide. The composition for activating Xkr4 can be used to treat or prevent, for example, the conditions or diseases associated with the inhibition of apoptosis described below.

[0101] A "condition or disease involving inhibition of apoptosis" is a condition or disease that can be induced by inhibition of apoptosis. A condition or disease involving inhibition of apoptosis is, for example, a condition or disease that can be induced by inhibition of apoptosis in an organ or cell of the nervous system. A condition or disease involving inhibition of apoptosis is, for example, cancer in an organ or cell of the nervous system, elevated thyroid stimulating hormone, or a neurodevelopmental or behavioral disorder, including attention deficit hyperactivity disorder (ADHD).

[0102] "Treatment" refers to performing a procedure with the goal of maintaining, reducing, or eliminating a given condition or symptom. A procedure with the goal of maintaining, reducing, or eliminating a given condition or symptom includes, for example, administering a given composition. A therapeutic composition can be administered, for example, during or after the onset of a condition or disease.

[0103] "Prevention" means performing a treatment with the intent of inhibiting the onset of a given condition or symptom. Treatment with the intent of inhibiting the onset of a given condition or symptom includes, for example, administering a given composition. A preventative composition can be administered, for example, prior to the onset of a condition or disease.

[0104] Screening method for substances that modulate the lipid scrambling activity of Xkr4 A method for screening for a substance that regulates the lipid scrambling activity of Xkr4 includes: (1) contacting cells expressing the Xkr4 polypeptide disclosed herein with a candidate substance that may regulate the lipid scrambling activity of Xkr4; (2) measuring the lipid scrambling activity in the cells contacted with the candidate substance; and (3) selecting the candidate substance as a substance that regulates the lipid scrambling activity of Xkr4 if the lipid scrambling activity is lower or higher compared to the lipid scrambling activity in the cells not contacted with the candidate substance.

[0105] A "cell expressing an Xkr4 polypeptide" can be prepared by introducing a polynucleotide comprising a nucleotide sequence encoding the Xkr4 polypeptide disclosed herein or an expression vector comprising said polynucleotide into a host cell. In one embodiment, the cell expresses the Xkr4 polypeptide without receiving an apoptotic stimulus (e.g., an apoptotic stimulus using staurosporine (STS)).

[0106] "Potential candidate substance that may regulate the lipid scrambling activity of Xkr4" is, for example, a substance that is expected to directly or indirectly affect C-terminal truncated Xkr4 polypeptide, thereby inducing or increasing, or inhibiting or reducing the lipid scrambling activity of Xkr4. The potential candidate substance that may regulate may be, for example, a small molecule compound, a protein (e.g., an antibody), DNA, RNA, small interfering RNA, or an antisense oligonucleotide.

[0107] The candidate substances that may regulate include candidate substances that may induce the lipid scrambling activity of Xkr4 or candidate substances that may inhibit the lipid scrambling activity of Xkr4. A "candidate substance that may induce the lipid scrambling activity of Xkr4" is a substance that is expected to induce or increase the lipid scrambling activity of Xkr4 in cells or animals. A "candidate substance that may inhibit the lipid scrambling activity of Xkr4" is a substance that is expected to inhibit or reduce the lipid scrambling activity of Xkr4 in cells or animals.

[0108] "Contacting" or "contacting" the cells with the candidate substance means placing the cells under conditions that allow contact between the cells and a candidate substance that may regulate (e.g., induce or inhibit) the lipid scrambling activity of Xkr4. The contacting may be, for example, adding the candidate substance to a culture medium in which the cells are cultured.

[0109] The lipid scrambling activity is, for example, the uptake activity of phospholipids or glycolipids, or the exposure activity of phospholipids located on the inner side of the cell membrane. The lipid scrambling activity can be measured according to the method disclosed herein (for example, a method using flow cytometry).

[0110] In one embodiment, the screening method for the substance that regulates the lipid scrambling activity of Xkr4 is a screening method for the substance that inhibits the lipid scrambling activity of Xkr4.The screening method for the substance that inhibits the lipid scrambling activity of Xkr4 includes, for example, (1) contacting the cell that expresses the Xkr4 polypeptide disclosed herein with a candidate substance that may inhibit the lipid scrambling activity of Xkr4; (2) measuring the lipid scrambling activity in the cell that has been contacted with the candidate substance; and (3) selecting the candidate substance as a substance that inhibits the lipid scrambling activity of Xkr4 when the lipid scrambling activity is 50%, 40%, 30%, 20%, 10%, or 5% or less compared with the lipid scrambling activity in the cell that has not been contacted with the candidate substance.The candidate substance that may inhibit the lipid scrambling activity of Xkr4 can be used as a drug candidate for treating or preventing a condition or disease that involves the promotion of apoptosis.

[0111] "Condition or disease associated with the promotion of apoptosis" refers to a condition or disease that can be induced by the promotion of apoptosis.Condition or disease associated with the promotion of apoptosis refers to a condition or disease that can be induced by the promotion of apoptosis in, for example, nervous system organs.Condition or disease associated with the promotion of apoptosis refers to neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS).

[0112] In one embodiment, the method for screening the substance that regulates the lipid scrambling activity of Xkr4 is a method for screening the substance that induces the lipid scrambling activity of Xkr4.The method for screening the substance that induces the lipid scrambling activity of Xkr4 includes, for example, (1) contacting the cell that expresses the Xkr4 polypeptide disclosed herein with a candidate substance that may induce the lipid scrambling activity of Xkr4; (2) measuring the lipid scrambling activity in the cell that has been contacted with the candidate substance; and (3) selecting the candidate substance as a substance that induces the lipid scrambling activity of Xkr4 when the lipid scrambling activity is at least 2-fold, 3-fold, 4-fold, 5-fold, 7-fold, 9-fold, or 10-fold higher than the lipid scrambling activity in the cell that has not been contacted with the candidate substance.The candidate substance that may induce the lipid scrambling activity of Xkr4 can be used as a drug candidate for treating or preventing a condition or disease that involves the suppression of apoptosis.

[0113] "Condition or disease associated with the suppression of apoptosis" refers to a condition or disease that can be induced by the promotion of apoptosis. A condition or disease associated with the promotion of apoptosis is, for example, a condition or disease that can be induced by the promotion of apoptosis in an organ of the nervous system. A condition or disease associated with the promotion of apoptosis can be, for example, a condition or disease disclosed herein, such as ADHD.

[0114] Screening method for substances that induce lipid scrambling activity of Xkr4 A method for screening for a substance that induces the lipid scrambling activity of Xkr4 includes: (1) contacting a cell expressing a C-terminally truncated Xkr4 polypeptide, wherein (α) the C-terminally truncated Xkr4 polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 2 or 36, or (β) the C-terminally truncated Xkr4 polypeptide has an amino acid sequence that includes deletions, substitutions, or additions or a combination thereof in the amino acid sequence of the (α) polypeptide, and shows at least 80% sequence identity with the amino acid sequence of (α), and can exhibit lipid scrambling activity in the cell membrane through interaction with an XRCC4 polypeptide, with a candidate substance that may induce the lipid scrambling activity of Xkr4; (2) measuring the lipid scrambling activity in the cells contacted with the candidate substance; and (3) selecting the candidate substance as a substance that induces the lipid scrambling activity of Xkr4 if the lipid scrambling activity is higher than the lipid scrambling activity in the cells not contacted with the candidate substance.

[0115] "C-terminal truncated Xkr4 polypeptide" is a fragment of Xkr4 that can form a dimer in the cell membrane of a cell. Without being bound by theory, C-terminal truncated Xkr4 polypeptide can exhibit lipid scrambling activity through interaction with the XRCC4 polypeptide disclosed herein. C-terminal truncated Xkr4 polypeptide is, for example, a polypeptide in which Xkr4 disclosed herein is cleaved by caspase.

[0116] A C-terminally truncated Xkr4 polypeptide is, for example, (α) a C-terminally truncated Xkr4 polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or 36 (specific amino acid sequences are shown below), or (β) a polypeptide having an amino acid sequence containing deletions, substitutions, or additions or a combination thereof in the amino acid sequence of the (α) polypeptide, which shows at least 80% sequence identity with the amino acid sequence of the (α) polypeptide, and which can exhibit lipid scrambling activity in the cell membrane by interacting with the XRCC4 polypeptide. TIFF2025011252000007.tif134165

[0117] C-terminal truncated Xkr4 polypeptides can be prepared, for example, by genetic engineering techniques. C-terminal truncated Xkr4 polypeptides can be prepared, for example, by transcription and translation from a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence described herein.

[0118] The C-terminal truncated Xkr4 polypeptide, for example, in the amino acid sequence set forth in SEQ ID NO: 2, is not serine at position 322, phenylalanine at position 331, and glutamine at position 332 according to the numbering of the amino acid residues in the amino acid sequence set forth in SEQ ID NO: 1. The C-terminal truncated Xkr4 polypeptide, for example, in the amino acid sequence set forth in SEQ ID NO: 36, is not serine at position 325, phenylalanine at position 334, and glutamic acid at position 335 according to the numbering of the amino acid residues in the amino acid sequence set forth in SEQ ID NO: 35.

[0119] The C-terminal truncated Xkr4 polypeptide consists of or comprises an amino acid sequence that contains 1 to 100, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 4, 3, 2 or 1 amino acid deletions, substitutions or additions, or a combination thereof, in the amino acid sequence set forth in, for example, SEQ ID NO: 2 or 36. The C-terminal truncated Xkr4 polypeptide consists of or comprises an amino acid sequence that shows at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in, for example, SEQ ID NO: 2 or 36.

[0120] In this specification, the C-terminally truncated Xkr4 polypeptide according to (β) above is also referred to as a "mutant C-terminally truncated Xkr4 polypeptide".

[0121] C-terminally truncated Xkr4 polypeptides have, for example, any combination of amino acid length, amino acid substitutions, and sequence identity disclosed for the Xkr4 polypeptides herein.

[0122] "Can exhibit lipid scrambling activity in cell membranes through interaction with XRCC4 polypeptide" means that in the presence of the XRCC4 polypeptide disclosed herein, the mutant C-terminal truncated Xkr4 polypeptide exhibits PC uptake activity in the cell membrane of a cell expressing the mutant C-terminal truncated Xkr4 polypeptide. For example, if the PC uptake activity of a PLB cell expressing the mutant C-terminal truncated Xkr4 polypeptide in the presence of XRCC4 polypeptide is higher than the PC uptake activity under the same conditions except for the absence of the XRCC4 polypeptide, for example, at least 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold higher, the mutant C-terminal truncated Xkr4 polypeptide is identified as being capable of exhibiting lipid scrambling activity in cell membranes through interaction with XRCC4 polypeptide. The PC uptake activity can be measured by the method disclosed herein (for example, a method using flow cytometry).

[0123] Candidate substances identified as being capable of exhibiting lipid scrambling activity in cell membranes through interaction with the XRCC4 polypeptide can be used as drug candidates for the treatment or prevention of conditions or diseases involving the inhibition of apoptosis.

[0124] Screening method for substances that inhibit the lipid scrambling activity of Xkr4 In one embodiment, a method for screening a substance that inhibits the lipid scrambling activity of Xkr4 includes: (1) contacting a cell into which the XRCC4 polypeptide disclosed herein has been introduced and which expresses a C-terminally truncated Xkr4 polypeptide with a candidate substance that may inhibit the lipid scrambling activity of Xkr4; (2) measuring the lipid scrambling activity in the cell; and (3) selecting the candidate substance as a substance that inhibits the lipid scrambling activity of Xkr4 if the lipid scrambling activity is lower than the lipid scrambling activity in the cell not contacted with the candidate substance.

[0125] A "cell into which an XRCC4 polypeptide has been introduced and which expresses a C-terminally truncated Xkr4 polypeptide" can be prepared, for example, by introducing an XRCC4 polypeptide or a mutant thereof into a cell expressing a C-terminally truncated Xkr4 polypeptide by lipofection or electroporation. A cell into which an XRCC4 polypeptide or a mutant thereof has been introduced and which expresses a C-terminally truncated Xkr4 polypeptide can be prepared, for example, by introducing into a host cell one or more polynucleotides comprising nucleotide sequences encoding an XRCC4 polypeptide or a mutant thereof and a C-terminally truncated Xkr4 polypeptide, or one or more expression vectors comprising said polynucleotides.

[0126] The contact of the cells with a candidate substance that may inhibit the lipid scrambling activity of Xkr4 can be carried out, for example, by adding the candidate substance to a culture medium in which the cells are cultured. The contact of the cells with a candidate substance that may inhibit the lipid scrambling activity of Xkr4 may include, for example, removing the culture medium after a predetermined time has elapsed since the addition of the candidate substance in order to remove unreacted candidate substance.

[0127] In the above embodiment, the substance selected as the substance that inhibits the lipid scrambling activity of Xkr4 is, for example, a substance that can prevent or reduce direct or indirect interaction between the XRCC4 polypeptide and the XRCC4 polypeptide that has formed a dimer in the cell membrane.

[0128] In one embodiment, a method for screening a substance that inhibits the lipid scrambling activity of Xkr4 includes: (1) contacting an XRCC4 polypeptide disclosed herein with a C-terminally truncated Xkr4 polypeptide in the presence of a candidate substance that may inhibit the lipid scrambling activity of Xkr4; (2) measuring the binding between the XRCC4 polypeptide and the C-terminally truncated Xkr4 polypeptide; and (3) selecting the candidate substance as a substance that inhibits the lipid scrambling activity of Xkr4 if the binding between the XRCC4 polypeptide and the C-terminally truncated Xkr4 polypeptide is lower than the binding when the XRCC4 polypeptide and the C-terminally truncated Xkr4 polypeptide are contacted in the absence of the candidate substance, for example, by 50%, 40%, 30%, 20%, 10%, or 5% or less.

[0129] Step (1) according to the embodiment may be, for example, adding a candidate substance that may inhibit the lipid scrambling activity of Xkr4 and an XRCC4 polypeptide to a C-terminal truncated Xkr4 polypeptide immobilized on a support. In one example, step (1) according to the embodiment may be, for example, immobilizing a C-terminal truncated Xkr4 polypeptide on the plate surface (support) of a microwell plate by adsorption or the like. To the microwell plate on which the C-terminal truncated Xkr4 polypeptide is immobilized, a solution containing a candidate substance that may inhibit the lipid scrambling activity of Xkr4 and an XRCC4 polypeptide labeled with a fluorescent substance may be added. In this example, step (2) may be measuring the fluorescence from the fluorescent substance-labeled XRCC4 polypeptide remaining in the microwell plate after removing the unbound fluorescent substance-labeled XRCC4 polypeptide.

[0130] Screening kits A kit is provided for screening the substance that inhibits the lipid scrambling activity of Xkr4.The kit comprises at least one, two or three selected from the group consisting of the XRCC4 polypeptide disclosed herein; the polynucleotide comprising the nucleotide sequence encoding the XRCC4 polypeptide; the expression vector comprising the polynucleotide; and the host cell comprising the polynucleotide or the host cell comprising the expression vector.

[0131] The components of the kit may be, for example, in separate containers or may be mixed in a single container. The kit may include instructions for use in screening for substances that inhibit the lipid scrambling activity of Xkr4, for example.

[0132] Methods for identifying genes responsible for a desired phenotype One embodiment of the present invention provides a method for identifying genes corresponding to a phenotype of interest.

[0133] "Phenotype" refers to the trait of an organism that results from the action of genes in a given environment. A phenotype of interest can be, for example, a response such as lipid scrambling activity during apoptosis, a protein that is induced in the presence of a particular substance, or a substance that is secreted after a particular stimulus.

[0134] "Gene" refers to the basic unit that controls traits in genetic phenomena. A gene is a unit that carries genetic information that codes for, for example, a protein. Genes are broadly classified into structural genes that code for proteins or enzymes that constitute living organisms, and regulatory genes that code for protein factors that control the expression of structural genes. A trait is controlled by multiple structural genes and regulatory genes.

[0135] A "gene corresponding to" a desired phenotype is a gene that affects the desired phenotype. The corresponding gene may, for example, act directly or indirectly on the desired phenotype. When the desired phenotype is a protein that is expressed under a specific environment, the structural gene encoding the protein may be a gene that acts directly on the desired phenotype. In the above example, a regulatory gene encoding a protein factor that controls the expression of the protein may be a gene that acts indirectly on the desired phenotype.

[0136] <Process a> The method of identifying includes (a) providing a guide RNA library comprising guide RNAs including at least one guide sequence corresponding to the nucleotide sequences of multiple genes in the genomic DNA of a cell.

[0137] The "genomic DNA" of a cell means all the genes that the cell has. Information including sequence information of each gene on the genomic DNA, information on the animal species, and information on the phenotype (e.g., protein) corresponding to the gene is available, for example, from a publicly provided database. The genomic DNA of a cell is, for example, the genomic DNA of a cell derived from a specific biological species. The genomic DNA of a cell may be, for example, homogeneous genomic DNA derived from a specific individual of a specific biological species, or heterogeneous genomic DNA derived from multiple separate individuals of a specific biological species. In one embodiment, the genomic DNA of a cell is homogeneous genomic DNA derived from a specific individual of a specific biological species. In one embodiment, the genomic DNA of a cell may be heterogeneous genomic DNA derived from multiple separate individuals of a specific biological species.

[0138] "Guide RNA" refers to a sgRNA (single-guide RNA) in which a crRNA (CRISPR-RNA) linked to a guide sequence and a tracrRNA (trans-activating crRNA) are linked. The guide RNA includes, for example, a nucleotide sequence encoding Cas9. When referring to a guide RNA incorporated into genomic DNA, the guide RNA encompasses a DNA that includes a nucleotide sequence that is transcribed into the guide RNA.

[0139] "Cas9" is C RISPR as Cas9 is a nuclease called associated protein 9, and has a molecular weight of about 160 kilodaltons. The amino acid sequence or nucleotide sequence of Cas9 is known, and the sequence can be obtained from a publicly available database. Polynucleotides containing a nucleotide sequence encoding Cas9 are commercially available.

[0140] A "guide sequence" includes a sequence that can hybridize to a target sequence in the nucleotide sequence of a given gene in the genomic DNA of a particular organism, and is complementary to the target sequence. The guide sequence has, for example, a length of 10 to 30 nucleotides, a length of 15 to 25 nucleotides, a length of 18 to 22 nucleotides, or a length of 20 to 21 nucleotides. The sequence identity between the guide sequence and its corresponding target sequence is, for example, 80% to 100%, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 99% or more, or 100%. The optimal alignment of the guide sequence and the target sequence can be determined using any algorithm for aligning sequences (e.g., NCBIBLAST).

[0141] A "guide RNA library" includes guide RNAs for at least one target sequence in the nucleotide sequence of a plurality of genes in the genomic DNA of a cell. Each guide RNA in the guide RNA library may be stored inside a lentivirus or retrovirus, for example. In this case, the guide RNA library is also referred to as a virus-type guide RNA library (for example, a lentivirus-type or retrovirus-type guide RNA library). For example, when a cell of a particular species contains 22,000 genes in its genomic DNA and the guide RNA library contains guide RNAs for each gene in the genomic DNA, and the guide RNA contains guide sequences for six different target sequences for each gene, the guide RNA library contains 132,000 types of sgRNAs. The guide RNA library can be prepared according to a known method, and the guide RNA library can be prepared according to the method described in, for example, Wang, et al. (2014). Genetic screens in human cells using the CRISPR-Cas9 system. Science 343, 80. The guide RNA library is, for example, commercially available. The lentiviral guide RNA library may be, for example, the Gecko sgRNA library (Addgene#1000000049).

[0142] The "preparation" or "preparing" of a guide RNA library includes, for example, placing the guide RNA library in a state in which it can be introduced into a cell. For example, if the guide RNA library is frozen in a liquid, it includes thawing the frozen material containing the guide RNA library. The preparation of a guide RNA library may include, for example, preparing the guide RNA library.

[0143] <Process b> The identification method includes (b) obtaining a cell population into which the guide RNA library has been introduced.

[0144] The "introduction" or "introducing" of a guide RNA library includes, for example, contacting a cell population with a guide RNA library (for example, a lentivirus-type guide RNA library). In a cell population into which a guide RNA library has been introduced, the introduced guide RNA is incorporated into the genome of the cells. In a cell population into which a guide RNA library has been introduced, the guide RNA library is introduced at a ratio such that one type of guide RNA is introduced per cell. The ratio of one type of guide RNA being introduced per cell can be achieved, for example, by introducing a guide RNA library into a cell population such that the number of cells constituting the cell population is, for example, 5 times, 10 times, or 15 times the number of guide RNAs constituting the guide RNA library.

[0145] When using a virus-type guide RNA library, the guide RNA library can be introduced into a cell population by infecting the cell population with the virus-type guide RNA library. When using a lentivirus-type guide RNA library, the guide RNA can be efficiently incorporated into the genome of cells. When using a lentivirus-type guide RNA library, the guide RNA can be efficiently incorporated into the genome of cells even if the cell population is a population of cells with low or almost no proliferation rate (e.g., nerve cells, cells undergoing apoptosis, cells in tissue). In one embodiment, the cell population into which the guide RNA library is introduced is a population of cells with low or almost no proliferation rate.

[0146] The cell population into which the guide RNA library is introduced expresses Cas9. The Cas9 may be expressed, for example, by incorporating a guide RNA containing a nucleotide sequence encoding Cas9 into the genomic DNA of the cells of the cell population. The Cas9 may be expressed, for example, by incorporating a nucleotide sequence encoding Cas9 into the genomic DNA of the cell population into which the guide RNA library is introduced.

[0147] The "cell population" into which the guide RNA library is introduced may be, for example, a cultured cell or a cell population in a tissue. The cell population in a tissue may be, for example, a cell population in a tissue taken out of the body; a cell population in a tissue in a non-human mammal, bird, reptile, or fish; or a cell population in a tissue constructed by tissue culture. When the cell population is a cultured cell, the cultured cell may incorporate a nucleotide sequence encoding Cas9. When the cell population is a cell population in a tissue, the guide RNA in the guide RNA library introduced into the cell population may include a nucleotide sequence encoding Cas9.

[0148] For example, a predetermined stimulus may be applied to the cell population into which the guide RNA library is introduced to induce a desired phenotype. For example, if the desired phenotype is an effect observed in the apoptosis process (e.g., lipid scrambling activity), the cell population into which the guide RNA library is introduced may be subjected to an apoptosis stimulus (e.g., an apoptosis stimulus by adding staurosporine (STS)).

[0149] <Process c> The method includes (c) selecting cells from the cell population based on a desired phenotype. The variety of guide RNAs introduced into the cells selected in step (c) is expected to be enriched for guide RNAs containing sequences complementary to the sequences of genes corresponding to the desired phenotype, compared to the variety of guide RNAs introduced into the cell population in step (b).

[0150] Step (c) includes, for example, distinguishing between cells exhibiting a desired phenotype and cells not exhibiting the phenotype, and recovering cells not exhibiting the desired phenotype. Step (c) includes, for example, distinguishing between cells exhibiting a desired phenotype and cells not exhibiting the phenotype, and recovering cells exhibiting the desired phenotype. When the cell population is a population of individually separated cells, step (c) can be performed, for example, using fluorescence activated cell sorting (FACS) or magnetic activated cell sorting (MACS). For example, when a fluorescent reagent that binds to cells exhibiting a desired phenotype is used, cells exhibiting a fluorescence derived from the fluorescent reagent that is equal to or greater than a predetermined value and cells exhibiting a value less than the predetermined value can be distinguished, and cells not exhibiting the desired phenotype can be recovered.

[0151] When the cell population is a population of cells in tissue, step (c) can be carried out, for example, by using laser microdissection. For example, when a fluorescent reagent that binds to cells that do not show the desired phenotype is used, the cells or regions that emit fluorescence from the fluorescent reagent can be distinguished from the cells or regions that do not emit fluorescence under a microscope, and the cells that do not show the desired phenotype can be collected by cutting them out with a laser.

[0152] <Process d> The method for identifying the target cell further comprises (d) recovering genomic DNA from the selected cell, wherein the genomic DNA recovered in step (d) is enriched for guide RNAs that include a sequence complementary to a sequence of a gene corresponding to a target phenotype, as compared to the variety of guide RNAs introduced into the cell population in step (b).

[0153] Genomic DNA can be prepared according to known methods, for example, by ethanol precipitation or using a commercially available genomic DNA extraction kit.

[0154] <Process e> The method includes preparing a new guide RNA library from the collected genomic DNA and performing steps (b) to (d) using the new guide RNA library until steps (b) to (d) are performed a predetermined number of times. By performing steps (b) to (d) a predetermined number of times, it is expected that guide RNAs containing sequences complementary to the sequences of genes corresponding to the target phenotype will be further enriched.

[0155] The "predetermined number of times" may be at least two times, for example, two, three, four or five times or more. The more the predetermined number of times, the more the guide RNA containing the sequence complementary to the sequence of the gene corresponding to the target phenotype is expected to be enriched, but on the other hand, more labor is required for implementation. In one embodiment, the predetermined number of times is three times.

[0156] "New guide RNA library" refers to a guide RNA library prepared by recovering the guide RNA incorporated in the genomic DNA recovered in step (d) and using the recovered guide RNA. The guide RNA incorporated in the genomic DNA can be recovered, for example, by amplifying a region containing the guide RNA by PCR and recovering the amplified PCR product. In this example, the guide RNA further includes a sequence for amplifying the guide RNA by PCR. When preparing a lentivirus-type guide RNA library, the recovered guide RNA is inserted into a lentivirus vector to prepare a plasmid. A lentivirus-type guide RNA library can be prepared by transfecting host cells with the prepared plasmid and culturing the transfected host cells to generate lentivirus.

[0157] <Process f> The method includes (f) determining the guide sequence in the collected genomic DNA when steps (b) to (d) have been performed a predetermined number of times. In step (f), for example, the region containing the guide RNA incorporated in the genomic DNA collected in step (d) is amplified by PCR, and the amplified PCR product is subjected to a sequencer to determine the guide sequence contained in the guide RNA.

[0158] <Process g> The identification method includes a step (g) of identifying a gene corresponding to the determined guide sequence as a gene corresponding to the desired phenotype. Step (g) can be carried out by, for example, searching a database containing information on the genomic DNA of the cell (cell of a specific species) in step (a) to find a gene containing a sequence showing a predetermined sequence identity (e.g., 90%, 95%, 97%, 98%, 99% or 100%) with the guide sequence of the guide RNA determined in step (f), and identifying the found gene as a gene corresponding to the desired phenotype.

[0159] By carrying out the method for identifying a gene corresponding to a desired phenotype disclosed herein, a cell population enriched in cells in which a gene corresponding to a desired phenotype has been identified can be obtained. One embodiment of the present invention provides a method for producing a cell population enriched in cells in which a gene corresponding to a desired phenotype has been identified. Also, by carrying out the method for identifying a gene corresponding to a desired phenotype disclosed herein, a cell population enriched in cells in which a gene corresponding to a desired phenotype has been identified and which exhibit or do not exhibit the phenotype can be obtained. One embodiment of the present invention provides a method for producing a cell population enriched in cells in which a gene corresponding to a desired phenotype has been identified and which exhibit or do not exhibit the phenotype.

[0160] Any definitions or specific examples of terms set forth herein apply, as appropriate, to the corresponding terms in any aspect or embodiment disclosed herein.

[0161] The following examples are intended to illustrate the invention and are not intended to limit it in any way. The compositions and methods described in the examples form a part of the invention.

[0162] [Example] material and method In the examples described below, the following materials and methods were used. (cell culture) Mouse interleukin 3 (IL-3)-dependent Ba / F3 cells (Reference 24) were cultured in RPMI1640 (WAKO) containing 10% FCS (Gibco), antibiotics (Nacalai), 55 μM β2-mercaptoethanol, and 45 units / ml of IL-3 (Reference 25). Human PLB985 cells were cultured in RPMI1640 containing 10% FCS, antibiotics, and 55 μM β2-mercaptoethanol. HEK293T and HCT116 (RIKEN BioResource Center) cells were maintained in DMEM (WAKO) containing 10% FCS and antibiotics.

[0163] (plasmid) DNA encoding mouse Xkr4 tagged with monomeric EGFP at the N-terminus and C-terminus, and its caspase-cleaved form Xkr4ΔC, was introduced into the lentiCas9-blast vector (also referred to as "plenti" herein) from which Cas9 and Blast had been removed (Reference 15, Addgene #52962). DNA encoding mouse Xkr4 tagged with V5 at the N-terminus and FLAG at the C-terminus (V5-Xkr4-FLAG), Xkr4ΔC, and Xkr4 mutants with point mutations (glutamine at position 332 is replaced by glutamic acid (Q332E), leucine at position 331 is replaced by phenylalanine (L331F), and isoleucine at position 322 is replaced by serine (I322S)) were introduced into the pNEF vector (Reference 10) and pMXs-puro vector (Reference 27), respectively. Human Xkr4, Xkr8, and Xkr9 cDNAs were tagged with V5 at their N-terminus and introduced into plenti carrying an endogenous ribosome entry site (IRES)-driven puromycin resistance gene. V5-Xkr4-FLAG and V5-Xkr4ΔC-FLAG were also introduced into lentiviral vectors. Mouse Xkr4ΔC tagged with SPOT at the N-terminus and FLAG at the C-terminus (SPOTXkr4ΔC-FLAG) and mouse Xkr4ΔC tagged with V5 at the N-terminus and tagRFP (Evrogen) at the C-terminus (Xkr4ΔC-RFP) were inserted into plenti vectors.

[0164] Human XRCC4 cDNA was amplified by PCR using first strand cDNA generated from PLB cells as a template. The following XRCC4 mutants were fused to tagRFP at the N- or C-terminus in a plenti vector: wild type (WT), caspase-uncleaved (2DA), caspase-cleaved N-terminal fragment (ΔC, 1-265) and C-terminal fragment (ΔN, 266-336), deletion mutants (116-336, 156-336, 204-336, 246-336, 256-336, 1-305, 1-285 ... 285), and mutants in the nuclear localization signal (arginine at position 270 is replaced with alanine (R270A), lysine at position 271 is replaced with alanine (K271A), arginine at position 272 is replaced with alanine (R272A), arginine at position 273 is replaced with alanine (R273A), and arginine at position 275 is replaced with alanine (R275A)). A self-cleaving 2A peptide (T2A; EGRGSLLTCGDVEENPGP (SEQ ID NO: 15)) sequence was introduced between the XRCC4 cDNA and TagRFP to create XRCC4-WT-2A-RFP or XRCC4-R270A-2A-RFP. When the short guide RNA (sgRNA) for XRCC4 was stably expressed in cells, a silencing mutation was introduced into the XRCC4-sgRNA (XRCC4 sgRNA target sequence 5'-tggagactgatctttataa in XRCC4 cDNA). gcgg -3' (SEQ ID NO: 16) to 5'-ttagaaacagatctatacaaa cgt -3' (SEQ ID NO: 17). The underline indicates the PAM sequence).

[0165] The FLAG sequence in lentiCas9-Blast was replaced with HA to express Cas9-HA. Lentiviral expression vectors for sgRNAs against mouse Xkr4 (5'-gcggcgctgtgcctgcgcct-3': SEQ ID NO: 18), human XRCC4 (5'-tggagactgatctttataag-3': SEQ ID NO: 19), human APAF1 (5'-agcattgtagaatgatacgt-3': SEQ ID NO: 20), and human cytochrome C (5'-acagccgccaataagaacaa-3': SEQ ID NO: 21) were constructed using the plentiGuide-Puro vector (Reference 15, Addgene #52963). To establish XRCC4 KO cells, sgRNA against XRCC4 was inserted into the plentiGuide (puro-) vector, which was constructed as follows: the EF1α promoter and puromycin resistance gene in plentiGuide-puro were removed by excision at the SmaI and MluI sites, and blunt ends were generated using T4 polymerase, followed by ligation with T4 ligase. The pX330 vector (Reference 28) was used to express sgRNA against human CAD (5'-gaacatcgcggccgagaccc-3': SEQ ID NO: 22) and mouse Xkr8 (5'-cttagacgtggtcgtaggcc-3': SEQ ID NO: 23).

[0166] (Establishment of cell lines) Ba / F3 cells lacking TMEM16F were prepared (Reference 29). A pX330 vector encoding an sgRNA against Xkr8 was electroporated into TMEM16F KO Ba / F3 cells, and limiting dilution was performed 3 days later. Genomic DNA was collected from each clone, and the Xkr8 locating region in the genomic DNA was amplified for sequencing to confirm that Xkr8 was knocked out (referred to as "BDKO" in this specification). A pX330 vector encoding an sgRNA against CAD was electroporated into PLB cells transfected with CRISPR / Cas9, and limiting dilution was performed to obtain a single clone. CAD deletion was confirmed by genome sequencing, Western blotting, and DNA fragmentation assay after apoptotic stimulation. XRCC4 knockout cells were established by infection with a lentivirus encoding an sgRNA against XRCC4. Single clones were obtained by limiting dilution, and knockout of XRCC4 was confirmed by genomic sequencing and Western blotting with anti-XRCC4 antibody.

[0167] Retroviruses were prepared by transfecting HEK293T cells with the retroviral vectors pMXs-puro, pGag-pol IRES-bsr (gift from Dr. Toshio Kitamura) and pCMV-VSV-G (RIKEN). 48 hours after transfection, the culture supernatant containing the retrovirus was collected, filtered, concentrated by centrifugation at 6,000 × g for 16 hours, and used to infect Ba / F3 cells. Stable transfectants were obtained by selection with 2.0 μg / ml puromycin.

[0168] Lentiviruses for the expression of sgRNA or cDNA of Cas9-HA, Xkrs, XRCC4 and their mutants were produced by transfecting HEK293T cells with lentiviral vectors pCMV-VSVG-RSV-Rev (RIKEN) and pCAG-HIVgp (RIKEN). Culture supernatants containing lentiviruses were collected, filtered, concentrated by centrifugation at 6,000 × g for 16 hours, and used to infect BDKO, PLB and HCT116 cells. In some experiments, stable transfectants were selected by culturing the cells with 2.0 μg / ml puromycin or 10 μg / ml blasticidin.

[0169] (Apoptosis stimulation by staurosporine (STS)) PLB cells, 5 x 10 5 The next day, cells were counted and plated at 1 × 10 6 The cells were resuspended in fresh medium pre-warmed to 37°C at 100 cells / ml, and the cell suspension medium was incubated in a culture incubator for 2 hours. STS (LC Laboratories) was added to the cell suspension medium at a final concentration of 10 μM, and the cells were incubated for the specified time. In some experiments, the cells were pretreated with 20 μM Q-VD-OPh (Cayman), a pan-caspase inhibitor, 1 hour before apoptotic stimulation.

[0170] (Lipid scrambling assay) 1×10 6Cells were washed with HBSS (Gibco) containing 1 mM CaCl2 and MgCl2 (HBSS / Ca / Mg), resuspended in 500 μl HBSS / Ca / Mg, and incubated on ice for 7 min. An equal volume of HBSS / Ca / Mg containing 1 μM nitrobenzoxadiazole-conjugated phosphatidylcholine (also referred to herein as "NBD-PC", Avanti) was added to the cell suspension and incubated on ice for 10 min. Then, HBSS / Ca / Mg containing 5 mg / ml fatty acid-free BSA (Sigma) was mixed with 1 μM DAPI (Dojindo) for 5 min to remove unincorporated NBD-PC. Residual fluorescent signal with incorporated NBD-PC was measured in non-necrotic cell population (DAPI negative) by flow cytometry. Depending on the cell number, other parameters were changed to give the same ratio. NBD-PC incorporation was performed at either 4°C or 15°C incubation temperature.

[0171] To observe the extracellular divalent cations required for lipid scrambling, cells were resuspended in 500 μl of HEPES buffer (10 mM Hepes-NaOH (pH 7.4) and 140 mM NaCl) with or without the indicated concentrations of CaCl2 and / or MgCl2 and incubated on ice for 10 min before lipid scrambling assay. An equal volume of HEPES buffer containing 1 μM NBDPC was added and incubated on ice for the indicated time. After incubation with NBD-PC, samples were mixed with HEPES buffer containing 5 mg / ml fatty acid-free BSA and 1 μM DAPI and analyzed by flow cytometry. In some experiments, 2 μM A23187 (Sigma) was added to the cell suspension in buffer containing NBD-PC and incubated on ice for 4 min.

[0172] (Measurement of intracellular calcium influx) 2.5 million BDKO cells expressing Xkr4ΔC Q332E were pretreated with 1 μM Fluo4-AM (Invitrogen) in RPMI1640 containing 10% FBS and incubated at 37°C for 30 min. After incubation, 5 × 10 5 Cells were washed once with HEPES buffer and resuspended in HEPES buffer containing 0.125 mM calcium or 2 mM calcium. After incubation on ice for 10 min, cells were mixed with HEPES buffer containing 10 μg / ml PI (Dojindo) with or without 2 μM A23187 for 1 min. Fluo4 signals were observed using a flow cytometer.

[0173] (Annexin V staining) PS exposure was measured by staining with Annexin V-Cy5 (BioVision). 5 Cells were resuspended in 200 μl ice-cold annexin buffer (10 mM Hepes-NaOH (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2) and mixed with an equal volume of annexin buffer containing annexin V-Cy5. After 15 min incubation on ice, DAPI was added to a final concentration of 1 μM. Annexin V-Cy5 signal in the non-necrotic cell population (DAPI negative) was analyzed by flow cytometry.

[0174] (Construction of cDNA library and identification of Xkr4ΔC mutant) To prepare a cDNA library, total RNA was diluted with 10 7PC6 cells were prepared and mRNA purification was performed using Oligotex-dT30<super>mRNA purification kit (Takara). A cDNA library was constructed from the purified mRNA using the In-Fusion SMARTer Directional cDNA Library Construction Kit (Clontech) with some modifications to the manufacturer's instructions. Briefly, the mRNA was applied to first strand cDNA generation by reverse transcriptase using the In-Fusion SMARTer CDS primer and SMARTer oligos provided in the kit, followed by second strand cDNA generation by Primestar GXL DNA Polymerase (Takara) using primers 5'-AAGCAGTGGTATCAACGCAGAGT-3' (SEQ ID NO: 24) and 5'-CGGGGTACGATGAGACACCA-3' (SEQ ID NO: 25). The amplified cDNA was loaded onto a 1% agarose gel, stained with ethidium bromide, and separated under LED light into two parts: low molecular weight (LMW) (1.0-2.5 kbps) and high molecular weight (HMW) (2.5-6.0 kbps). The cDNA was purified from the excised gel and inserted into the NcoI-cut pMXs-HiFi vector using NEBuilder HiFi DNA assembly (New England Biolabs) (ref. 30), electroporated into ElectroMAX DH10B competent cells (Thermo Fisher Scentific), and plated onto ten 15 cm Lysogeny broth (LB) agar plates for each of the HMW and LMW cDNA libraries. The next day, colonies were collected in LB medium and applied to Maxiprep (Qiagen). The number of clones for each library was 21 million for HMW and 6.1 million for LMW. The pMXs-HiFi vector was constructed by inserting the following sequences into the BamHI and SalI sites of the pMXs vector (Reference 27) and annealing them. 5'-ggatcccagaagcagtggtatcaaccatggtctcatcgtaccccgccagcacagtggtcgac-3' (SEQ ID NO: 26)

[0175] (cDNA library screening) The HMW cDNA library was transfected into ten 10 cm plates of HEK293T cells (2 × 10 6 Two days after transfection, the supernatant was collected, filtered through a 0.22 μm filter, and centrifuged (6,000 × g, 16 h, 4 °C). The next day, the virus pellet was resuspended in medium at a concentration 20 times that of the supernatant and incubated at 1 × 10 6 The cells were mixed with BDKO cells at a concentration of 1000 cells / ml. Six hours after infection, the medium was replaced with fresh medium, and the cells were cultured for three days and then applied to the lipid scramble assay. Then, the cells were sorted by flow cytometry FACS Aria II (Beckton Dickinson). After several rounds (3 to 4 times) of sorting, 10 7 Genomic DNA was prepared from the cells by phenol / chloroform extraction as described below, and the integrated cDNA was amplified by PCR using Primestar GXL DNA polymerase and primers 5'-cccatatggccatatgagatctta-3' (SEQ ID NO: 27) and 5'-caaaatggcgttacttaagctagc-3' (SEQ ID NO: 28). The amplified product was then inserted into pCX4BSR (Reference 31) cut with BglII and NheI using NEBuilder. After electroporation into E. coli DH10B, the second cDNA library was purified and applied to the next round of screening. After screening, genomic DNA was prepared and applied to PCR amplification of the integrated cDNA, excised from the agarose gel and inserted into the pCX4BSR vector. The sequence inserted into the vector was subjected to sequencing, and the obtained sequences were analyzed by Blast search.

[0176] (Genomic DNA purification) After cDNA library screening, genomic DNA was prepared as follows. Briefly, 10 million cells were washed twice with PBS, resuspended in 1 ml of Genome Lysis buffer (100 mM Tris-HCl (pH 8.5), 200 mM NaCl, 5 mM EDTA, 0.2% SDS) containing 100 μg / ml proteinase K, and incubated at 55°C for 3 hours. An equal volume of isopropanol was then added and gently inverted. The resulting spooled DNA was collected, washed with 70% ethanol, resuspended in 1 ml of TE containing 50 μg / ml RNase A, incubated at 37°C for 1 hour, and rotated overnight at room temperature. The next day, TE-saturated phenol was added to the DNA solution, mixed well, centrifuged to collect the supernatant, and then subjected to TE-saturated phenol extraction again. The supernatant containing DNA was then mixed with an equal volume of phenol / chloroform / isoamyl alcohol and centrifuged. The supernatant (approximately 500 μl) was mixed with 1 / 10 volume of 3M sodium acetate and 2 volumes of 100% ethanol and gently inverted. The DNA slope was collected, washed with 70% ethanol, resuspended in 100 μl of TE, incubated at 65°C for 10 min, and stored at 4°C.

[0177] (Establishment of cells with strong lipid scrambling activity) To perform lipid scrambling assay, BDKO cells were transfected with V5-Xkr4ΔCFLAG and Cas9-HA. 40 million cells were subjected to lipid scrambling assay with NBD-PC at a final concentration of 1 μM. After incubation with NBD-PC for 7 min at 15 °C, lipids in the outer layer on the cell membrane were removed by fatty acid-free BSA for 5 min, and cells that had taken up NBD-PC were sorted using FACS AriaII and dead cells stained with DAPI were gated out. The collected cells were expanded and applied for the next sorting. Sorting was repeated six times to obtain cells with strong lipid scrambling activity. The obtained high lipid-scrambling cells are called PC6.

[0178] (Revival screening using CRISPR sgRNA libraries) CAD KO PLB cells expressing Cas9-HA and Xkr4ΔC-RFP were infected with Gecko sgRNA library A&B (reference 15, Addgene #1000000049) at an infection efficiency of approximately 40%. After culturing the cells with 2.0 μg / ml puromycin for 2 days and without puromycin for 3 days, the cells were transferred to 150 ml of fresh medium at 5 × 10 5 The following day, 20 million PLB cells in 20 ml medium were cultured in a culture incubator for 3 hours, stimulated with 20 μl of 10 mM STS (final concentration 10 μM) and incubated for 3 hours. The cells were then subjected to an NBD-PC uptake assay, and cells lacking NBD-PC uptake were collected by flow cytometry. Approximately 50,000 cells were collected per experiment, which was performed five times per day: a total of 1 × 10 8Cells were subjected to primary screening. A total of 270,000 cells were collected by flow cytometry and applied to the Geno Plus Genomic DNA Extraction Kit (Viogene), and the resulting genomic DNA (total 3 μg) was subjected to PCR to amplify the integrated sgRNA encoding using Primestar GXL polymerase and primers 5'-gttttaaaatggactatcatatgc-3' (SEQ ID NO: 29) and 5'-tatccatctttgcacccgggc-3' (SEQ ID NO: 30) in a 25 μl scale for 32 tubes. PCR conditions were as follows: denaturation at 95°C for 3 minutes, followed by 28 cycles of 98°C for 10 seconds, 55°C for 15 seconds, 68°C for 30 seconds, and a final extension at 68°C for 30 seconds. The PCR reaction mixture was pooled into one 1.5 ml tube, from which 160 μl was loaded onto an agarose gel to excise the 425 bps band. The PCR products were purified using a Gel / PCR extraction kit (Fastgene), mixed with plentiGuide-puro vector (250 ng) cut with NdeI and SmaI, incubated with NEBuilder for 1 h at 50°C, and subjected to ethanol precipitation. The precipitate was then resuspended in 157.5 μl of 10% glycerol, from which 103 μl of the solution was mixed with 63 μl of DH10B. Three 70 μl mixtures were transferred to electroporation cuvettes, pulsed at 2700 V using a NEPA Porator (Nepa Gene), resuspended in 5 ml SOC, incubated at 37°C for 2 h with gentle shaking, and plated on 15 cm LB plates (1 ml per plate). The next day, colonies were counted (2.2 million clones), harvested in PBS, and subjected to Midiprep kit (Roche). The resulting plasmids were used to generate lentiviruses and subjected to subsequent rounds of sorting until a clear NBD-PC-negative cell population emerged.

[0179] Cells negative for NBD-PC incorporation and active caspase-3 were collected by flow cytometry as follows: sgRNA library was infected at low infectivity (10%) into CAD KO PLB cells expressing Xkr4ΔC-RFP, and each cell was transduced with a single sgRNA lentivirus. After 1 week of puromycin selection, RFP-positive and PC-incorporation-negative cells were collected by flow cytometry, fixed with 1% paraformaldehyde in PBS for 2 min, permeabilized with methanol, stained with anti-activated caspase-3 antibody, and sorted for cells positive for caspase-3 activity.

[0180] (Localization study) The localization of EGFP fusion of Xkr4 or tagRFP fusion of XRCC4 was observed by Nikon A1 confocal microscope using CFI Plan Apo 100× oil immersion objective, 1.40NA or CFI Plan Apo 40× dry objective, 0.95NA. To visualize the localization of tagRFP fusion of XRCC4, PLB cells expressing XRCC4 and its mutants were treated with or without 10 μM STS for 2.5 h and resuspended in ice-cold HBSS containing 5 mg / ml fatty acid-free BSA and 20 μM DRAQ5 (BioStatus). Before observation, cells were seeded on glass-bottom dishes (IWAKI) and fluorescent images were acquired. Differential interference contrast (DIC) images were also acquired to visualize the morphology of the cells. Images were processed by imageJ1.52p.

[0181] (DNA fragmentation assay) 5×10 5PLB and CAD KO cells were stimulated with 10 μM STS for 4 h. Cells were collected, washed once with PBS, lysed in 500 μL of Genome Lysis buffer containing 100 μg / ml Proteinase K, and incubated overnight at 55 °C. To precipitate genomic DNA, 500 μl of isopropanol was added, mixed, and centrifuged at 20,000 × g for 5 min. The precipitate was rinsed with 70% ethanol and resuspended in 20 μl of TE buffer. Samples were loaded onto a 1% agarose gel, and DNA fragmentation was visualized by staining with ethidium bromide under UV light.

[0182] (Electroporation of synthetic peptides) Synthetic peptides corresponding to amino acids 266-285 of XRCC4 (C20: IAPSRKRRQRMQRNLGTEPK (SEQ ID NO: 7)), C20 with methionine at the N-terminus (C21: MIAPSRKRRQRMQRNLGTEPK (SEQ ID NO: 13)), C20 with arginine at position 270 substituted with alanine (C20-R270A: IAPSAKRRQRMQRNLGTEPK (SEQ ID NO: 14)), and C20 with lysine at position 271 substituted with alanine (C20-K271A: IAPSRARRQRMQRNLGTEPK (SEQ ID NO: 8)) were obtained from SCRUM Co., Ltd. (Tokyo, Japan). These peptides were diluted with 1 × 10 5 ribozymes in 100 μl of Opti-MEM (Gibco) at a final concentration of 10 μM. 6 Electroporation was performed using ELEPO21 (Nepa Gene). After electroporation, the cells were immediately mixed with RPMI1640 containing 10% FBS pre-warmed to 37°C. Half of the mixture (5 × 10 5 Cells) were stained with Annexin V-Cy5 as described above and analyzed by flow cytometry.

[0183] C20 localization was observed in HCT116 cells expressing Xkr4WT-GFP or Xkr4ΔCGFP. 4 Cells were cultured in 5 μg / cm pepsin-solubilized rat collagen type I (Nippi). 2The cells were seeded on coated glass-bottom 24-well plates (IWAKI) and cultured for 2 days. Tetramethylrhodamine-labeled C20 (C20-TMR) was diluted in 300 μl of Opti-MEM to a final concentration of 2 μM and electroporated in an ELEPO21 equipped with a CUY900-13-3-5 adherent cell electrode. After electroporation, the cells were fixed with 4% PFA in PBS for 10 min at RT and observed under a confocal microscope.

[0184] (Blue Native PAGE (BN-PAGE)) BN-PAGE was performed using the NativePAGE Novex Bis-Tris Gel System (Life Technologies). 8The cells were resuspended in 6 ml of cold buffer A (10 mM Tris-HCl (pH 7.4), 1 mM CaCl2, 1 mM p-APMSF, 1 mM NaF) and disrupted using Dounce homogenization (60 strokes) on ice. An equal volume of cold buffer B (10 mM Tris-HCl (pH 7.4), 0.1 M NaCl, 0.5 M sucrose, 1 mM CaCl2, 10 mM MgCl2, 1 mM p-APMSF, 1 mM NaF) was then added and centrifuged at 800 x g for 10 min at 4 °C. The supernatant was collected and further centrifuged at 8,000 x g for 10 min at 4 °C. After removing the pellet, the supernatant was subjected to ultracentrifugation at 100,000 x g for 50 min at 4 °C. The pelleted membrane fraction was solubilized by incubation with solubilization buffer (20 mM Tris-HCl (pH 7.4), 100 mM 6-aminocaproic acid, 50 mM NaCl, 10% (vol / vol) glycerol, 1 mM CaCl2, 1 mM p-APMSF, protease inhibitor cocktail (Nacalai), 1 mM NaF, and 1% n-dodecyl-β-D-maltoside (DDM) (Dojindo) / 0.01% cholesteryl hemisuccinate (CHS) (Sigma)) for 3 h at 4 °C. Insoluble material was removed by centrifugation at 20,000 × g for 30 min at 4 °C. The amount of solubilized membrane fraction was measured by BCA Protein Assay kit (Thermo Fisher Scientific) and adjusted to 0.6 μg / μl with solubilization buffer. Samples were then loaded onto a NativePAGE Novex 4-16% (wt / vol) Bis-Tris gel and separated by electrophoresis at 150 V for 35 min at 4° C. The CBB G-250 concentration in the cathode buffer was changed from 0.02 to 0.002% and samples were further electrophoresed for 120 min at 150 V. The gel was incubated with SDS running buffer (25 mM Tris, 190 mM glycine, 0.1% SDS, (pH 8.3)) for 20 min at RT and then transferred to a PVDF membrane for Western blotting at 0.1 A for 1 h.

[0185] (SDS-PAGE)Whole cell lysates were prepared by incubation at 4°C for 1 h in solubilization buffer (20 mM Tris-HCl (pH 7.4), 100 mM 6-aminocaproic acid, 50 mM NaCl, 10% (vol / vol) glycerol, 1 mM CaCl2, 1 mM p-APMSF, 1 mM NaF, and 1% DDM / 0.01% CHS) or RIPA buffer (50 mM Hepes-NaOH buffer (pH 8.0), 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate) containing a protease inhibitor cocktail (Nacalai). After incubation, samples were centrifuged at 20,000 × g for 20 min at 4°C. The supernatants were collected, and protein concentrations were measured by BCA Protein Assay kit. The samples were then adjusted to 0.1-1.0 μg / μl using solubilization buffer and 5×SDS sample buffer (200 mM Tris-HCl (pH 6.8), 10% SDS, 25% glycerol, 5% mercaptoethanol, 0.05% bromophenol blue) and loaded onto a 10% polyacrylamide gel (BIOCRAFT). The samples were electrophoresed at 35 mA for 40 minutes and transferred to a PVDF membrane at 0.1 A for 60 minutes. The membrane was probed with 1:5,000 dilution of anti-V5-HRP (Invitrogen), 1:5,000 dilution of anti-FLAG-HRP (Sigma), 1:4,000 dilution of anti-tRFP rabbit pAb (Evrogen, AB233), 1:2,000 dilution of anti-XRCC4 mouse mAb (SantaCruz, C-4), 1:500 dilution of anti-CAD mouse mAb (SantaCruz, F-11), 1:4,000 dilution of anti-cleaved caspase 3 rabbit mAb (Cell Signaling Technology #9661), or 1:5,000 dilution of antiserum against Xkr4 raised in rabbits immunized with GST-Xkr4 N-terminus (amino acids 1-110), followed by 1:10,000 dilution of HRP-conjugated goat anti-mouse or rabbit IgG (Dako). Chemiluminescent signals were detected using Immobilon Western chemiluminescent HRP substrate (Millipore) with a FUSION chemiluminescence imaging system (Vilber).Total protein on the PVDF membrane after chemiluminescence was visualized by CBB staining (WAKO). The membrane was incubated with CBB staining buffer (0.25% CBB R250, 50% methanol, 10% acetic acid) at room temperature for 2-30 minutes, and then washed with destaining buffer (10% methanol and 10% acetic acid).

[0186] (Immunoprecipitation) A SPOT tag (ref. 32) was fused to the N-terminus of Xkr4. Ten million XRCC4 KO PLB cells expressing SPOT-Xkr4ΔC-FLAG and XRCC4-RFP were harvested by centrifugation at 400g for 5 min and washed with annexin buffer after apoptotic stimulation with 10 μM STS for 2.5 h at 37°C. Membrane fractions were then prepared and solubilized as described above. Membrane lysates were incubated with anti-SPOT nanobody-conjugated magnetic agarose beads (SPOT-Trap, ChromoTek) and rotated at 4°C for 2 h. The beads were washed three times with chilled wash buffer (20 mM Tris-HCl (pH 7.5), 100 mM 6-aminocaproic acid, 50 mM NaCl, 1 mM CaCl2, and 0.03% DDM / 0.003% CHS) and subjected to mass spectrometry or eluted by boiling for 5 min at 95°C with sample buffer (40 mM Tris-HCl (pH 6.8), 2% SDS, 5% glycerol, 0.01% bromophenol blue). Eluted samples were analyzed by SDS-PAGE and Western blotting.

[0187] (mass spectrometry) After immunoprecipitation, the magnetic agarose beads SPOT-Trap were washed twice more with 50 mM ammonium bicarbonate. The proteins on the beads were digested by adding 200 ng of trypsin / Lys-C mix (Promega) for 16 hours at 37°C. The digested products were reduced, alkylated, acidified, and desalted using GL-Tip SDB (GL Sciences). The eluate was evaporated in a SpeedVac concentrator and dissolved in 0.1% trifluoroacetic acid and 3% acetonitrile (ACN). LCMS / MS analysis of the resulting peptides was performed on an EASY-nLC 1200 UHPLC connected to an Orbitrap Fusion mass spectrometer via a nanoelectrospray ion source (Thermo Fisher Scientific). Peptides were separated on a 75 μm i.d. × 150 mm 18 reversed-phase column (Nikkyo Technos) using a direct ACN gradient from 4 to 32% in 0–100 min, followed by increasing ACN to 80% in 10 min. The mass spectrometer was operated in data-dependent acquisition mode with a maximum duty cycle of 3 s. MS1 spectra were captured at a resolution of 120,000, 4 × 10 5 HCD MS / MS spectra were recorded with an automatic gain control (AGC) target of 1 × 10 and a mass range of 375–1,500 m / z. 4The data were acquired in a linear ion trap with an isolation window of 1.6 m / z, a maximum injection time of 100 ms, and a normalized collision energy of 30. Dynamic exclusion was set to 20 s. Raw data were analyzed directly against the SwissProt database, supplemented with mouse Xkr4 protein and restricted to human (H. sapiens), using Proteome Discoverer version 2.3 (Thermo Fisher Scientific) and the SequestHT search engine. Search parameters were: (a) trypsin as enzyme with a maximum of two missed cleavages; (b) precursor mass tolerance of 10 ppm; (c) fragment mass tolerance of 0.6 Da; (d) carbamidomethylation of cysteine ​​as fixed modification; and (e) acetylation of the protein N-terminus and oxidation of methionine as variable modifications. Peptides were filtered using the percolator node at a false discovery rate of 1%. Label-free precursor ion quantification was performed using the Precursor Ion Quantification node and normalized to ensure that the sum of the abundance values ​​of each sample across all peptides was equal.

[0188] Selected peptides of human XRCC4 and mouse Xkr4 were measured by PRM, an MS / MS-based targeted quantification method using high-resolution MS. LC-MS / MS analysis was performed on an EASY-nLC 1200 UHPLC coupled to a Q Exactive Plus mass spectrometer via a nanoelectrospray ion source (Thermo Fisher Scientific). Targeted MS / MS scans were performed with a resolution of 70,000 and an AGC target of 2 × 10 5 The data were acquired with a time-scheduled inclusion list with an isolation window of 4.0 m / z, a maximum injection time of 2 s, and a normalized collision energy of 27. Time alignment and relative quantification of transitions were performed using PinPoint version 1.4 (Thermo Fisher Scientific).

[0189] (Mapping of NGS data) Raw data were received as a fastq file (containing sgRNA sequences and barcode sequences) and two reference csv files (containing reference sgRNA sequences). In the reference files, each reference sgRNA sequence is listed according to the original gene from which it was obtained and assigned a unique identification tag (UID). In both cases, six unique reference sgRNA sequences (with different UIDs) were derived from the same gene.

[0190] The fastq and reference csv files were processed using three in-house computer codes. The first code (ref. 33) removes barcode sequences from genes in the fastq files and then converts all sequences (both genes in the fastq files and sgRNA sequences in the reference files) to integer sequences (cytosine=1, adenine=2, etc.). This code outputs two lists of integer sequences, one for the genes contained in the fastq files and one for the sgRNAs contained in the two reference files. These outputs are then read by a second code (written in C++), which counts the number of times each sgRNA in the reference list occurs among the genes in the fastq files. These counts are then converted to a third code (written in R), which creates a spreadsheet showing the number of times each sgRNA from the reference list occurs among the genes in the fastq files. To create this spreadsheet, we first group the reference sgRNAs according to the gene from which they were originally obtained. The groups are then ranked according to the number of reference sgRNAs that appeared at least once among the genes in the fastq file.

[0191] The above mapping procedure takes advantage of R's ability to read and process string data (raw gene sequences) and the speed of C++ when looping through large amounts of integer data.

[0192] [Example 1] Xkr4 mutants that exhibit constitutive lipid scrambling activity Xkr4 is cleaved at its C-terminus by caspases during the apoptosis process. C-terminally cleaved Xkr4 forms a dimer and generates scramblase activity. This causes phosphatidylserine (PS) located on the inner side of the cell membrane to be exposed on the outer side of the cell membrane. However, cleavage of the C-terminus of Xkr4 alone is not sufficient to expose PS on the outer side of the cell membrane (Reference 12).

[0193] A plasmid carrying a gene encoding wild-type Xkr4 (Xkr4WT) and a plasmid carrying a gene encoding Xkr4 whose C-terminus has been cleaved by caspase (also referred to as ``Xkr4ΔC'' herein) (Reference 15) were transfected into PLB cells to prepare transfectants that constitutively express Xkr4WT and Xkr4ΔC, respectively.

[0194] The PLB cells and the transfectants were each stimulated with apoptosis by staurosporine (STS), and the uptake of nitrobenzoxadiazole-conjugated phosphatidylcholine (NBD-PC) by each cell was measured using flow cytometry (Figures 1(a) to (c)) (see (Apoptosis stimulation by STS) and (Lipid scramble assay) in "Materials and Methods" above).

[0195] In PLB cells (parental), no phosphatidylcholine (PC) uptake was observed in the cells, whether they were given the apoptotic stimulus with STS (Control) or not (STS) (Fig. 1(a)). In transfectants expressing Xkr4WT (also referred to as "Xkr4WT-expressing cells" herein), PC uptake was observed in the cells when they were given the apoptotic stimulus with STS (Fig. 1(b)STS). These results indicate that Xkr4WT generates lipid scramblase activity in response to the apoptotic stimulus.

[0196] In transfectants expressing Xkr4ΔC (also referred to as "Xkr4ΔC-expressing cells" herein), no PC uptake was observed in the cells when they were not stimulated with apoptosis by STS (Fig. 1(c) Control). This result indicates that the C-terminal truncation of Xkr4 alone is not sufficient to generate scramblase activity for the uptake of PC added to the culture medium. When Xkr4ΔC-expressing cells were stimulated with apoptosis by STS, PC uptake was observed in the cells (Fig. 1(c) STS). This result indicates that Xkr4ΔC generates lipid scrambling activity in response to apoptosis stimulation, similar to Xkr4WT, which generates scrambling activity in response to apoptosis stimulation.

[0197] Annexin V staining was performed on PLB cells (parental) and transfectants expressing Xkr4WT and Xkr4ΔC (Fig. 1(d)-(f)) (see Annexin V staining in Materials and Methods above). Fig. 1(e) and (f) show that PS is exposed in transfectants Xlr4WT and Xkr4ΔC in which PC uptake had occurred (Fig. 1(b) and (c)). These results indicate that Xkr4ΔC causes PS exposure not only in response to PC uptake but also in response to apoptotic stimuli.

[0198] PLB cells expressing Xkr4WT and Xkr4ΔC, which are C-terminally tagged with EGFP, were prepared. After apoptotic stimulation with STS, Xkr4ΔC was localized on the plasma membrane, similar to Xkr4WT (Figure 2). This result indicates that Xkr4ΔC, like Xkr4WT, produces lipid scrambling activity on the plasma membrane upon apoptotic stimulation.

[0199] PLB cells expressing Xkr4WT or Wkr4ΔC were cultured with the total caspase inhibitor Q-VD-OPh and then stimulated with STS for 3 hours (Figure 3). When Xkr4WT-expressing cells were pretreated with the caspase inhibitor, lipid scrambling activity was not induced by apoptotic stimulation with STS (Figures 3(a) and (b)). The results in Wkr4ΔC-expressing cells were similar to those in Xkr4WT-expressing cells (Figures 3(c) and (d)). The results shown in Figures 1 to 3 indicate that Xkr4ΔC, like Xkr4WT, produces lipid scrambling activity on the cell membrane by caspases induced by apoptotic stimulation.

[0200] We prepared plasma membrane lysates from PLB cells expressing Xkr4WT or Wkr4ΔC and performed biochemical analysis by blue native PAGE (BN-PAGE) or SDS-PAGE (Figure 4). Biochemical analysis by BN-PAGE and Western blotting with anti-Xkr4 antibody show that before apoptotic stimulation, Xkr4WT exists as a monomer and Xkr4ΔC exists as a dimer (Figure 4(a)). Biochemical analysis by SDS-PAGE shows that Xkr4 and Xkr4ΔC have corresponding molecular weights (Figure 4(b)). These results indicate that Xkr4ΔC, like Xkr4WT, forms dimers on the plasma membrane, but this is not sufficient to generate lipid scrambling activity.

[0201] (Establishment of cells with strong lipid scrambling activity) To understand how Xkr4 is activated, we obtain cells that exhibit constitutive lipid scrambling activity. We prepared transfectants stably expressing Xkr4ΔC in Ba / F3 cells (BDKO) lacking two ubiquitous scramblase enzymes, Xkr8 (reference 10) and TMEM16F (reference 9). We performed lipid scrambling assays on the transfectants grown in culture, and collected transfectants with high NBD-PC-derived fluorescence intensity, i.e., cells that take up PC, by flow cytometry. This process was repeated six times (Figure 5(a)). By repeating the process, we obtained transfectants with high NBD-PC-derived fluorescence intensity (Figure 5(b)). Each graph in Figure 5(b) shows the results of flow cytometry according to the number of times the process was performed (PC0 to PC5), and the square area surrounded by a solid line in each graph indicates cells that take up PC (PC-uptake-positive cells) in the non-necrotic cell population.

[0202] The number of transfectants in this region increased with the number of repetitions of the process (PC0 to PC5). Starting from a transfectant expressing Xkr4ΔC (Figure 6(a)), the process was repeated six times to obtain cells (PC6) that constitutively showed lipid scrambling activity without apoptotic stimulation (Figure 6(b)). PC6 cells were infected with sgRNA lentivirus against Xkr4. No PC uptake was observed in the resulting infected PC6 cells (PC6+sgXrk4) (Figure 6(c)). Total cell lysates of PC6 or PC6+sgXkr4 were subjected to SDS-PAGE and Western blotting (WB) using anti-V5 antibody (Figure 6(d)). The WB detects V5-Xkr4ΔC-FLAG. The WB results show that PC6+sgXrk4 does not express Xkr4. These results indicate that constitutive lipid scrambling activity in PC6 cells is dependent on Xkr4.

[0203] (Construction of cDNA library and identification of Xkr4ΔC mutant) The constitutive lipid scrambling activity dependent on Xkr4 in PC6 may be due to the introduction of a mutation in a gene encoding an endogenous protein acting on Xkr4ΔC. Total RNA was extracted from PC6 cells, and the first strand of cDNA was generated using reverse transcriptase. The resulting first strand of cDNA was then generated by PCR using primers 5'-AAGCAGTGGTATCAACGCAGAGT-3' (SEQ ID NO: 31) and 5'-CGGGGTACGATGAGACACCA-3' (SEQ ID NO: 32). The resulting second strand of cDNA was separated into low molecular weight (LMW) (1.0-2.5 kbps) cDNA and high molecular weight (HMW) (2.5-6.0 kbps) cDNA by agarose gel electrophoresis. Plasmids carrying LMW and HMW, respectively, were used to transduce ElectroMAX DH10B. This resulted in a LMW cDNA library containing 6.1 million transductant clones for LMW and a LMW cDNA library containing 21 million transductant clones for HMW.

[0204] (cDNA library screening) The first HMW cDNA library was transfected into HEK293T cells using the retroviral vectors pGag-pol IRES-bsr and pCMV-VSV-G. Retroviruses were collected from the transfectants. The collected retroviruses were then used to infect BDKO cells to obtain a 1LPC0 cell population. The 1LPC0 cell population was subjected to a lipid scrambling assay, and cells with lipid scrambling activity (0.6%) were obtained by flow cytometry (Figure 7(a)). The obtained cell population was cultured and subjected to a lipid scrambling assay again to obtain a cell population with lipid scrambling activity. This process was repeated three times, and cells with lipid scrambling activity (91.9%) were obtained in the 1LPC3 cell population (Figure 7(b)). Genomic DNA of the cells obtained from the 1LPC3 cell population was prepared (see (Genomic DNA production) described in "Materials and Methods" above), and the cDNA from the first HMW cDNA library integrated in the genomic DNA was amplified by PCR using primers 5'-cccatatggccatatgagatctta-3' (SEQ ID NO: 33) and 5'-caaaatggcgttacttaagctagc-3' (SEQ ID NO: 34). Several bands were observed when the obtained PCR fragments were subjected to agarose gel electrophoresis. Therefore, a second cDNA library was prepared using the PCR fragments.

[0205] In the same manner as for the first cDNA library, retrovirus was prepared from the second cDNA library, and the prepared virus was used to infect BDKO cells to obtain the 2LPC0 cell population. The 2LPC0 cell population was subjected to a lipid scrambling assay, and cells (11.7%) with lipid scrambling activity were obtained by flow cytometry (Figure 7(c)). The obtained cell population (2LPC1) was cultured and subjected to a lipid scrambling assay, and 92.5% of the cells had lipid scrambling activity (Figure 7(d)). The cDNA from the second cDNA library integrated into the genomic DNA of the cells obtained from the 2LPC1 cell population was amplified by PCR. The obtained PCR fragments were subjected to agarose gel electrophoresis, and a nearly single band was observed. The obtained PCR fragments were sequenced. As a result, all of the identified genes encoded Xkr4ΔC mutants with single point mutations (I322S, L331F, or Q332E) in Xker4. These results indicate that the Xkr4-dependent constitutive lipid scrambling activity in PC6 is caused by alterations in Xkr4ΔC itself due to the identified point mutations.

[0206] Xkr4 has the first to tenth transmembrane domains, with I322S being a mutation in the fourth transmembrane domain, and L331F and Q332E being mutations in the fifth transmembrane domain.

[0207] [Example 2] Characterization of constitutively active Xkr4 mutants Transfectants expressing full-length (FL) Xkr4, Xkr4ΔC (also referred to as "ΔC"), Xkr4ΔC with the above-mentioned point mutations (referred to as "ΔC I322S," "ΔC L331F," and "ΔC Q332E," respectively), and FL Q332E, which has the point mutation Q332E in full-length Xkr4, were prepared in BDKO cells (parental).

[0208] The prepared transfectants were subjected to lipid scrambling assay (Figure 8(a)). In this assay, apoptotic stimulation by STS was not given. After starting the lipid scrambling assay, it was observed that PC uptake increased over time in BDKO cells expressing ΔC I322S, ΔC L331F and ΔC Q332E (Figure 8(a)). To test whether PS located on the inner side of the cell membrane was exposed on the outer side of the cell membrane, the prepared transfectants were subjected to Annexin V staining (Figure 8(b)) (see (Annexin V staining) in "Materials and Methods" above). Figure 8(b) shows that PS was exposed in transfectants with increased PC uptake (ΔC I322S, ΔC L331F and ΔC Q332E). In full-length Xkr4 with Q332E (FL Q332E), neither PC uptake nor PS exposure was observed (Figures 8(a) and (b)). These results indicate that C-terminal truncation of Xkr4 in combination with the identified point mutations (ΔC I322S, ΔC L331F, or ΔC Q332E) results in lipid scrambling activity and PS exposure by Xkr4 even in living cells.

[0209] Whole cell lysates were prepared from the prepared transfectants, and the cell lysates were subjected to SDS-PAGE and Western blotting using anti-V5 antibody to detect V5-Xkr4-FLAG to detect Xkr4. The results showed that each transfectant expressed Xkr4 with the corresponding molecular weight (Figure 9(a) upper panel). Whole cell lysates were prepared from transfectants expressing Xkr4FL, Xkr4FL with Q332E (Xkr4FL-Q332E), Xkr4ΔC, and Xkr4ΔC with Q332E (Xkr4ΔC-Q332E), and the cell lysates were subjected to BN-PAGE and Western blotting using anti-V5 antibody to detect V5-Xkr4-FLAG to detect Xkr4. As a result, Xkr4ΔC-Q322E formed a dimer, similar to Xkr4ΔC (Figure 9(b) upper panel). This indicates that dimerization by Xkr4ΔC is not affected by the identified point mutation Q322E. The results shown in Figures 8 and 9 indicate that Xkr4 that formed a dimer by truncation of the C-terminus can be activated by changing its three-dimensional structure due to the identified point mutation. These results suggest that Xkr4 that formed a dimer by truncation of the C-terminus may generate lipid scrambling activity due to a structural change regulated by some activator.

[0210] Using the identified Xkr4 mutants with point mutations, we identify the components essential for lipid scrambling activity. Xkr4ΔC Q332E expressing cells in a buffer containing neither calcium ions nor magnesium ions did not take up PC even when NBD-PC was added to the buffer (Figure 10(a)). By adding calcium ions to the buffer, Xkr4ΔC Q332E expressing cells in the buffer took up PC (Figures 10(c) and (d)). Increasing the concentration of calcium ions in the buffer increased the amount of PC taken up by Xkr4ΔC Q332E expressing cells (Figure 10(e)).

[0211] Transfectants expressing Xkr4ΔC Q332E were mixed with NBD-PC under conditions containing 0 mM, 0.125 mM, or 2 mM extracellular calcium ion concentrations, and PC uptake by the transfectants was examined. In the PC uptake assay, there was no change in PC uptake with (A23) or without (-) 2 μM calcium ionophore A23187 (Figure 11(a)). Transfectants expressing Xkr4ΔC Q332E were incubated with 1 μM Fluo4-AM for 30 min, stimulated with 2 μM A23187 in the presence of 0.125 mM or 2 mM extracellular calcium, and analyzed by flow cytometry. There was no change in Fluo4-AM uptake with or without stimulation with A23187 (Figure 11(b)). These results show that calcium ionophore A23187 does not enhance scrambling activity by Xkr4, suggesting that calcium in the extracellular domain of Xkr4 is important. Transfectants expressing Xkr4WT or Xkr4ΔC were cultured with STS in medium containing 0.4 mM calcium for 3 h, and then PC uptake assays were performed with or without 2.5 mM extracellular calcium. Both Xkr4FL and Xkr4ΔC activated by apoptotic stimulation with STS required calcium for lipid scrambling activity (Figure 11(c)). Thus, the importance of calcium in the extracellular domain of Xkr4 for lipid scrambling activity is not specific to the Xkr4ΔC mutant in living cells.

[0212] [Example 3] Identification of Xkr4 activators by revival screening In the presence of extracellular calcium, Xkr4 is activated in two steps. One step is dimerization induced by C-terminal cleavage. The other step is a conformational change regulated by some activator, as suggested in Example 2. In order to identify the activator in dying cells, CRISPR sgRNA library is used. However, it is generally difficult to enrich target sgRNA using dying cells because the cells do not proliferate. To overcome this difficulty, a new screening system called "revival screening" was developed (see (revival screening using CRISPR sgRNA library) in "Materials and Methods" above).

[0213] In revival screening, the target sgRNA that was integrated into the genomic DNA of the cell by the lentivirus infection is revived and enriched in the sgRNA library for the next round of screening. In this method, the sgRNA of the lentivirus is infected into the cell, and the sgRNA is integrated into the genomic DNA of the infected cell. By selecting cells into which the sgRNA has been integrated based on a specific indicator, the sgRNA corresponding to the indicator is enriched from the cells. Genomic DNA is prepared from the selected infected cells, and the sgRNA integrated into the genomic DNA is recovered to generate a new lentivirus sgRNA library. As a result, in the revival screening, the sgRNA corresponding to the specific indicator is enriched in the sgRNA library prepared for the next round of screening (Figure 12).

[0214] (Revival screening using CRISPR sgRNA libraries) To facilitate revival screening, PLB cells lacking the apoptotic DNase CAD (Reference 14) were established. CAD KO PLB cells were established by electroporating a pX330 vector encoding sgRNA against CAD into PLB cells transfected with CRISPR / Cas9 and performing limiting dilution to obtain single clones (Figure 13). A plasmid was prepared in which a DNA sequence encoding Xkr4ΔC fused to tagRFP at the C-terminus (Xkr4ΔC-RFP) was inserted. CAD KO PLB cells were transfected with the plasmid. The resulting transfectants were infected with a lentiviral sgRNA library. The lentiviral sgRNA library contains six different target sgRNAs for each gene of all human genes (Reference 15).

[0215] Cells infected with the lentiviral sgRNA library were stimulated with STS for 3 hours to provide an apoptotic stimulus, and then a PC uptake assay was performed. A cell population that was RFP positive and negative for PC uptake was collected by flow cytometry (Figure 14(a)). Genomic DNA was prepared from the collected cells, and the sgRNA region incorporated into the genomic DNA was collected to prepare a lentiviral sgRNA library again. The prepared lentiviral sgRNA library was infected into the transfectant, and the infected cells were given an apoptotic stimulus with STS, and then a PC uptake assay was performed. This cycle was repeated three times to obtain a cell population (sgPC3) containing 24.5% PC uptake-negative cells (Figure 13(c)). This cycle was repeated once more to obtain a sgPC4 cell population.

[0216] Genomic DNA of the sgPC4 cell population was collected, and the region encoding the sgRNA integrated in the collected genomic DNA was amplified, and the amplified fragment was subjected to next-generation sequencing (NGS) analysis to perform gene mapping (see (Mapping of NGS data) in "Materials and Methods" above). After excluding genes corresponding to one or two sgRNAs, the obtained genes were ranked based on the score of the total number of reads (Figure 13). The top 14 ranked genes are summarized in the table below. [Table 1]

[0217] As shown in Table 1, cytochrome C (CYCS) and APAF1 were found in the top 14 gene list. CYCS is released from mitochondria upon apoptotic stimuli. CYCS then binds to APAF1 and sequentially activates caspase 9 (Casp9) and caspase 3 (Casp3). Thus, both CYCS and APAF1 are necessary factors for the activation of caspase 9. This result indicates that the revival screen is functioning to explore some activators that regulate the lipid scrambling activity by dimeric Xkr4 in the apoptotic process.

[0218] Further screening was performed to narrow down the candidate genes for the activating factor. Among the cells into which the sgRNA library was introduced, a cell population (sgPC5) containing cells positive for RFP and negative for PC uptake was collected by flow cytometry. The collected sgPC6 cells were fixed and permeabilized. The treated cells were then stained with an anti-activated caspase 3 antibody to classify caspase 3-activated cells. A lentiviral sgRNA library was prepared from the sgPC6 cells, and further screening was performed using the sgPC6 cell population obtained using the lentiviral sgRNA (Figure 16).

[0219] The region containing the sgRNA integrated into the genomic DNA from the sgPC7 cells obtained by the screening was subjected to NGS analysis in the same manner as described above. The genes obtained by NGS analysis were ranked based on the score of the total number of reads, and the top 8 genes are summarized in the following table. [Table 2]

[0220] As shown in Table 2, the gene designated XRCC4 was higher in total reads than CYCS and APAF1. The total reads of XRCC4 shown in the NGS analysis of sgPC7 (Table 2) were enriched more than 40-fold compared to the total reads of XRCC4 shown in the NGS analysis of sgPC4 (Table 1) (FIG. 17). This result indicates that XRCC4 is the most likely candidate as an activator of Xkr4.

[0221] [Example 4] Regulation of Xkr4 by caspase-cleaved XRCC4 In Example 3, the PLB cells into which sgRNAs against the top three candidate genes shown to be activators of Xkr4 were introduced were stimulated with apoptosis by STS, and the effects on caspase 3 activity and PC uptake in the cells were examined (Figure 18). Even when sgRNAs against XRCC4 were used, no effect on caspase 3 activity in the cells was observed (Figure 18(a) upper row and Figure 18(b) upper row). When sgRNAs against CYCS or APAF1 were used, caspase 3 activity in the cells was reduced (Figure 18(c) upper row and Figure 18(d) upper row). These results indicate that in the apoptosis process, CYCS and APAF1 are factors that affect caspase 3 upstream, and XRCC4 is a factor that affects caspase 3 downstream. When sgRNAs against the three genes were used, PC uptake in the cells was reduced (Figures 18(a) to (d) lower row). These results indicate that XRCC4 is a factor that exerts its effect downstream of caspase 3 in the apoptotic process, thereby affecting lipid scrambling activity.

[0222] To investigate the effect of XRCC4 on PC uptake in cells stimulated with apoptosis, XRCC4 knockout PLB cells (XRCC4 - / - The prepared XRCC4 cells were used (Figure 19(a)). - / - The absence of XRCC4 expression in the cells was confirmed by Western blotting using an anti-CAD antibody (Figure 19(b)). XRCC4 has a caspase-3 cleavage site at amino acid 265 (References 16, 17), and is cleaved by an apoptotic stimulus. Mutation of the two aspartic acids at this cleavage site to alanine (2DA) renders the cells resistant to cleavage by caspases (Figure 19(c) top, XRCC4 2DA(STS+)). - / -Cells were transfected with a plasmid carrying the gene encoding wild-type XRCC4 (WRCC4 WT) or caspase-uncleaved XRCC4 (XRCC4 2DA). The resulting transfectants were stimulated with STS to induce apoptosis, and the effect on PC uptake in the transfectants was examined by lipid scrambling assay using NBD-PC (Figures 20(a)-(c)). XRCC4 - / - In cells expressing XRCC4 WT, no PC uptake was observed after apoptosis stimulation with STS (Figure 20(a)). - / - In cells expressing XRCC4 2DA, PC uptake was observed after apoptosis stimulation with STS (Figure 20(b)). These results indicate that XRCC4 is involved in lipid scrambling activity induced by apoptosis stimulation. - / - In cells, no PC uptake was observed after apoptotic stimulation with STS (FIG. 20(c)). This result indicates that cleavage of XRCC4 by caspases regulates the lipid scrambling activity of Xkr4 upon apoptotic stimulation.

[0223] sgRNAs against XRCC4 and CYCS were introduced into PLB cells expressing a C-terminal truncated Xkr4 mutant (Xkr4ΔC Q322E) with a point mutation (Q322E) that confers constitutive lipid scrambling activity. The cells were stimulated with apoptosis by STS, and the effect on PC uptake in the cells was examined (Figure 20(e)-(f)). PC uptake in the cells was not affected by either sgRNA against XRCC4 or sgRNA against CYCS. These results support the above suggestion that cleavage of XRCC4 by caspases regulates the lipid scrambling activity of Xkr4 by apoptotic stimuli. These results also show that the point mutation that confers constitutive lipid scrambling activity in Xkr4ΔC can avoid the involvement of XRCC4 in lipid scrambling activity by apoptotic stimuli.

[0224] XRCC4 KO PLB cells were transfected with plasmids encoding Xkr4, Xkr8, and Xkr8 tagged with V5 at the N-terminus to generate transfectants expressing each Xkr. Furthermore, transfectants were transfected to express XRCC4 WT-RFP to generate transfectants co-expressing each Xkr and XRCC4 WT. XRCC4 KO PLB cells (XRCC4 - / - ), transfectants expressing each Xkr, and transfectants co-expressing each Xkr and XRCC4 WT were prepared, and the cell lysates were subjected to SDS-PAGE and Western blotting. Anti-V5 antibody (Figure 21, top) and anti-RFP antibody (Figure 21, middle) were used for Western blotting. Figure 21 shows that the desired transfectants were prepared.

[0225] A lipid scrambling assay was performed on the transfectants thus prepared (FIG. 22). Upon apoptotic stimulation with STS, caspase 3 activity was observed in all the transfectants tested (FIG. 22(g)-(l)). When Xkr4 was expressed in XRCC4 knockout PLB cells and apoptotic stimulation with STS was administered, no PC uptake was observed (FIG. 22(a)). Furthermore, when XRCC4 WT was expressed, PC uptake was observed (FIG. 22(d)). These results support the above suggestion that cleavage of XRCC4 by caspase regulates the lipid scrambling activity of Xkr4 upon apoptotic stimulation.

[0226] In XRCC4 KO PLB cells expressing Xkr8 or Xkr9 instead of Xkr4, PC uptake was observed upon apoptotic stimulation with STS, even in the absence of co-expression of XRCC4 WT (Figures 22(b) and (c)). These results indicate that caspase-mediated cleavage of XRCC4 is an Xkr4-specific activator in regulating lipid scrambling activity upon apoptotic stimulation.

[0227] XRCC4 is a component of the nuclear DNA repair complex (References 18, 19). In transfectants expressing XRCC4 WT tagged with RFP at the C-terminus (XRCC4 WT-RFP), XRCC4 was observed only in the nucleus (Figure 23(a) top). When the transfectants expressing XRCC4 WT-RFP were stimulated with apoptosis by STS, XRCC4 was also observed in the cytoplasm (Figure 23(a) bottom). In transfectants expressing XRCC4 WT tagged with RFP at the N-terminus (RFP-WRCC4 WT), XRCC4 was observed only in the nucleus even when stimulated with apoptosis by STS (Figure 23(b)). These results indicate that XRCC4 was cleaved by the apoptosis stimulus, and the C-terminal fragment diffused into the cytoplasm.

[0228] In transfectants expressing caspase-uncleaved XRCC4 tagged with RFP at the C-terminus or N-terminus (XRCC4 2DA-RFP or RGP-XRCC4 2DA), XRCC4 was observed only in the cells, regardless of whether apoptosis was given (STS+) or not (STS-) (Figures 23(c) and (d)). Caspase-uncleaved XRCC4 2DA was not cleaved by apoptosis stimulation with STS (fourth lane from the left in Figure 24(a)) and did not incorporate PC (Figure 24(c)). XRCC4 WT was cleaved by apoptosis stimulation with STS (second lane from the left in Figure 24(a)) and incorporated PC (Figure 24(b)). These results indicate that the diffusion of the caspase-generated C-terminal fragment of XRCC4 from the nucleus to the cytoplasm is important for regulating the lipid scrambling activity of Xkr4 by apoptosis stimulation.

[0229] [Example 5] Regulation of Xkr4 by cytoplasmic XRCC4 fragments XRCC4 / N functions as a dimerization domain and a binding domain for DNA repair proteins (References 18, 20, 21) (Figure 25). To determine the minimal region of XRCC4 that regulates Xkr4, we generated XRCC4 deletion mutants with partial deletions of the N-terminus from the full-length wild-type XCCR4 (1-336) (116-336, 156-336, 204-336, 248-336, 256-336, and 226-336 (ΔN)), XRCC4 deletion mutants with partial deletions of the C-terminus (1-305, 1-285, and 1-265 (ΔC)), and XRCC4 KO expressing a short-length XRCC4 fragment (Mini). PLB cells were prepared. A lipid scramble assay was performed on the cells (Figure 26). XRCC4 / 256-336, which contains 10 amino acids on the N-terminal side of the caspase cleavage site (between amino acids 265 and 266), showed PC uptake (Figure 26(g) and Figure 27(a)). Compared with XRCC4 / N, which is on the N-terminal side of the caspase cleavage site, XRCC4 / C, which is on the C-terminal side of the caspase cleavage site, did not have any known binding partners with intrinsically disordered regions (Figure 27(b)). XRCC4 / 1-285, which contains only 20 amino acids on the C-terminal side of the caspase cleavage site, showed PC uptake (Figure 26(j) and Figure 27(c)). Caspase cleavage The ΔN form did not show complete PC uptake (Fig. 26(h)). The caspase-cleaved ΔC form did not show PC uptake (Fig. 26(k)). These results indicate that the isoleucine at amino acid 266 must be exposed in the cleaved C-terminal fragment of XRCC4. Indeed, XRCC4 / 256-285(Mini) (containing 20 amino acids C-terminal and 10 amino acids N-terminal of the caspase cleavage site) showed PC uptake (Fig. 26(l) and Fig. 27(f)). These results indicate that the C-terminal fragment of XRCC4 containing the sequence at amino acids 256-285 is sufficient to induce the lipid scrambling activity of Xkr4 by apoptotic stimuli.

[0230] To investigate the amino acid sequence of XRCC4 / 256-285(Mini) that induces the lipid scrambling activity of Xkr4 by apoptotic stimulation, shorter peptide fragments were prepared. A 20 amino acid peptide (C20) consisting of amino acid positions 266 to 285 of XRCC4 and a peptide (C21) containing an additional methionine at the N-terminus of C20 were artificially synthesized (Figure 28(a)). Synthetic peptides C20 or C21 were introduced by electroporation into BDKO cells expressing Xkr4 WT or Xkr4ΔC, and a PS exposure assay was performed (Figures 28(b) and (c)). PS exposure was observed in the combination of synthetic peptide C20 and Xkr4ΔC. This result indicates that a peptide containing the sequence of amino acid positions 266 to 285 of XRCC4 is sufficient to induce the lipid scrambling activity of Xkr4 by apoptotic stimulation.

[0231] Furthermore, to determine the important amino acid residues for regulating Xkr4 in the synthetic peptide C20, which contains a nuclear localization signal (NLS), we prepared peptides C20-R270A, C20-K271A, C20-R272A, C20-R273A, and C20-R275A in which the positively charged amino acid residues in XRCC4 (arginine at position 270, lysine at position 271, arginine at position 272, arginine at position 273, and arginine at position 275) were replaced with alanine. The XRCC4 fragment mutants C20-R270A, C20-K271A, C20-R272A, and C20-R273A were distributed in the cytoplasm (Figure 29(a-e)), and the XRCC4 fragment mutant C20-R275A was distributed in the nucleus (Figure 29(f)). XRCC4 KO PLB cells expressing V5-Xkr4ΔC-FLAG were transfected with a plasmid encoding the XRCC4 fragment mutant and stimulated with STS to apoptose. PC uptake activity was observed in the XRCC4 fragment mutants C20-K271A, C20-R272A, C20-R273A, and C20-R275A (Figure 30(c-f), Figure 31(a)), whereas PC uptake activity was not observed in the XRCC4 fragment mutant C20-R270A (Figure 30(b), Figure 31(a)). These results indicate that the nuclear distribution of the XRCC4 fragment is not necessary for lipid scrambling activity. The XRCC4 fragment mutant C20-R270A did not fully activate Xkr4 (Figure 31(b)). This result indicates that the arginine residue at position 270 is important for regulating Xkr4.

[0232] [Example 6] Direct binding of Xkr4 to the C-terminal fragment of XRCC4 To investigate how Xkr4 is activated by the cytoplasmic XRCC4 fragment, we expressed Xkr4ΔC fused with SPOT and FLAG tags (SPOT-Xkr4ΔC-FLAG) in XRCC4 WT or XRCC4 R270A-expressing XRCC4 KO PLB cells (Figure 32(a)). As expected, when subjected to apoptotic stimulation with STS, SPOT-Xkr4ΔC-FLAG efficiently induced lipid scrambling activity in combination with XRCC4 WT (Figure 32(b) top), whereas it did not sufficiently induce lipid scrambling activity in combination with XRCC4 R270A (Figure 32(b) bottom).

[0233] The cell membrane lysate was prepared, and the cell membrane lysate was subjected to immunoprecipitation with anti-SPOT nanobody and subjected to mass spectrometry. This mass spectrometry is expected to identify peptides associated with SPOT-Xkr4ΔC-FLAG recovered by immunoprecipitation with anti-SPOT nanobody. Label-free quantification revealed that XRCC4 WT is one of the most reliable apoptosis-dependent Xkr4 interactors (Figure 33). Two adjacent tryptic digest peptides of XRCC4 / C (amino acid positions 286-296: MAPQENQLQEK (SEQ ID NO: 40)) and peptide 2 (amino acid positions 297-310: ENSRPDSSLPETSK (SEQ ID NO: 41)) were identified by mass spectrometry (Figure 34(a)). Targeted quantification using parallel reaction monitoring (PRM) showed that the two peptides were increased after apoptotic stimulation with STS in cells expressing XRCC4 WT (Figure 34(b-e)).

[0234] The interaction between XRCC4 WT-derived peptide fragments and Xkr4 was confirmed by Western blotting to detect XRCC4 in the precipitate obtained by Xkr4 immunoprecipitation of the whole cell library from cells stimulated with apoptosis by STS (Figure 35(a)). To confirm the interaction in intact cells, fluorescent TMR-conjugated C20 peptide (C20-TMR) was introduced by electroporation into HCT116 cells expressing Xkr4 WT-GFP or Xkr4ΔC-GFP, and the localization of the introduced C20-TMR with Xkr4 WT-GFP or Xkr4ΔC-GFP was observed (Figures 35(b) and (c)). The fluorescence intensity derived from GFP reflects the intracellular distribution of Xkr4. The peaks in the fluorescence intensity derived from GFP in the middle and lower rows of Figure 35(c) reflect the cell membrane. The lower panel of Figure 35(d) shows that the peaks of fluorescence intensity derived from C20-TEM overlap with the peaks of fluorescence intensity derived from Xkr4ΔC-GFP. This result indicates that the peptide fragments of each protein, XRCC4, directly associate with the plasma membrane protein Xkr4 in cells undergoing cell death, thereby inducing lipid scrambling activity.

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Claims

1. 1. An Xkr4 polypeptide, (A) a polypeptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 3 to 5 and 37 to 39; or (B) A polypeptide having an amino acid sequence including deletions, substitutions, or additions or a combination thereof in the amino acid sequence of the polypeptide of (A), and exhibiting at least 80% sequence identity with the amino acid sequence of (A), and capable of exhibiting lipid scrambling activity in a cell membrane without interacting with the XRCC4 polypeptide. The Xkr4 polypeptide.

2. 1. An XRCC4 polypeptide, comprising: (a) a polypeptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 7 to 12 and 42; or (b) a polypeptide having an amino acid sequence including deletions, substitutions, or additions or a combination thereof in the amino acid sequence of the polypeptide of (a), which shows at least 80% sequence identity with the amino acid sequence of (a), and which can induce lipid scrambling activity in a cell membrane by interacting with a C-terminal truncated Xkr4 polypeptide. The XRCC4 polypeptide,

3. 3. The XRCC4 polypeptide of claim 2, comprising an arginine at position 270 according to the numbering of the amino acid residues in the amino acid sequence set forth in SEQ ID NO:

6.

4. 4. The XRCC4 polypeptide of claim 2 or 3, which does not contain a methionine at position 265 according to the numbering of the amino acid residues in the amino acid sequence set forth in SEQ ID NO:

6.

5. A polynucleotide comprising a nucleotide sequence encoding the XRCC4 polypeptide of claim 1 or the XRCC4 polypeptide of any one of claims 2 to 4.

6. An expression vector comprising the polynucleotide of claim 5.

7. A host cell comprising the polynucleotide of claim 5 or the expression vector of claim 6.

8. A composition for activating XRCC4, comprising at least one selected from the group consisting of an XRCC4 polypeptide according to any one of claims 2 to 4; a polynucleotide comprising a nucleotide sequence encoding said XRCC4 polypeptide; and an expression vector comprising said polynucleotide.

9. The composition according to claim 8, for treating or preventing a condition or disease involving the inhibition of apoptosis.

10. A method for screening a substance that regulates the lipid scrambling activity of Xkr4, comprising the steps of: (1) contacting a cell expressing the Xkr4 polypeptide of claim 1 with a candidate substance that may regulate the lipid scrambling activity of Xkr4; (2) measuring lipid scrambling activity in the cells contacted with the candidate substance; and (3) selecting the candidate substance as a substance that regulates the lipid scrambling activity of Xkr4 when the lipid scrambling activity is lower or higher than the lipid scrambling activity in the cells not contacted with the candidate substance; A screening method comprising:

11. The screening method of claim 10, wherein the substance that regulates the lipid scrambling activity of Xkr4 is a drug candidate for the treatment or prevention of a condition or disease involving the promotion of apoptosis, if, in the comparison, the lipid scrambling activity is lower than the lipid scrambling activity in the cells not contacted with the candidate substance.

12. A method for screening a substance that induces the lipid scrambling activity of Xkr4, comprising the steps of: (1) contacting a cell expressing a C-terminal truncated Xkr4 polypeptide, the C-terminal truncated Xkr4 polypeptide being (α) a C-terminal truncated Xkr4 polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or 36, or (β) a C-terminal truncated Xkr4 polypeptide having an amino acid sequence including deletions, substitutions, or additions or a combination thereof in the amino acid sequence of the (α) polypeptide, and exhibiting at least 80% sequence identity with the amino acid sequence of the (α) polypeptide, and capable of exhibiting lipid scrambling activity in the cell membrane by interacting with the XRCC4 polypeptide, with a candidate substance that may induce the lipid scrambling activity of Xkr4; (2) measuring lipid scrambling activity in the cells contacted with the candidate substance; and (3) selecting the candidate substance as a substance that induces the lipid scrambling activity of Xkr4 when the lipid scrambling activity is higher than the lipid scrambling activity in the cells not contacted with the candidate substance; A screening method comprising:

13. A method for screening a substance that inhibits the lipid scrambling activity of Xkr4, comprising the steps of: (1) contacting a cell in which the XRCC4 polypeptide according to any one of claims 2 to 4 has been introduced and which expresses a C-terminal truncated Xkr4 polypeptide with a candidate substance that may inhibit the lipid scrambling activity of Xkr4; (2) measuring lipid scrambling activity in the cells; and (3) selecting the candidate substance as a substance that inhibits the lipid scrambling activity of Xkr4 when the lipid scrambling activity is lower than the lipid scrambling activity in the cells not contacted with the candidate substance; A screening method comprising:

14. A method for screening a substance that inhibits the lipid scrambling activity of Xkr4, comprising the steps of: (1) contacting the XRCC4 polypeptide according to any one of claims 2 to 4 with a C-terminal truncated Xkr4 polypeptide in the presence of a candidate substance that may inhibit the lipid scrambling activity of Xkr4; (2) measuring the binding between the XRCC4 polypeptide and the C-terminal truncated Xkr4 polypeptide; and (3) selecting the candidate substance as a substance that inhibits the lipid scrambling activity of Xkr4 when the binding between the XRCC4 polypeptide and the C-terminal truncated Xkr4 polypeptide is lower than the binding when the XRCC4 polypeptide and the C-terminal truncated Xkr4 polypeptide are contacted in the absence of the candidate substance; A screening method comprising:

15. The method of any one of claims 10 to 14, wherein the lipid scrambling activity is an activity of incorporating a phospholipid or glycolipid, or an activity of exposing a phospholipid located on the inner side of a cell membrane.

16. A kit for screening a substance that inhibits the lipid scrambling activity of Xkr4 according to claim 14 or 15, comprising: A kit comprising at least one selected from the group consisting of an XRCC4 polypeptide according to any one of claims 2 to 4; a polynucleotide comprising a nucleotide sequence encoding the XRCC4 polypeptide; an expression vector comprising the polynucleotide; and a host cell comprising the polynucleotide or a host cell comprising the expression vector.

17. A method for identifying a gene corresponding to a phenotype of interest, comprising the steps of: (a) providing a guide RNA library, the guide RNA comprising at least one guide sequence corresponding to a nucleotide sequence of a plurality of genes in the genomic DNA of a cell; (b) obtaining a cell population into which the guide RNA library has been introduced; (c) selecting cells from said cell population based on a phenotype of interest; (d) recovering genomic DNA from the selected cells; (e) preparing a new guide RNA library from the collected genomic DNA and performing steps (b) to (d) using the new guide RNA library until steps (b) to (d) have been performed a predetermined number of times; (f) determining the guide sequence in the recovered genomic DNA when steps (b) to (d) have been performed a predetermined number of times; and (g) identifying a gene corresponding to the determined guide sequence as a gene corresponding to the desired phenotype, A method for identifying, wherein the predetermined number of times is at least two.

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