Not listed RATIONAL REWRITING OF MOUSE IFN?I LOCUS FOR FUNCTIONAL CHARACTERIZATION - ABSTRACT TYPE I INTERFERON (IFN-I) PLAYS AN ESSENTIAL ROLE IN THE INNATE IMMUNE RESPONSE TO VIRUSES AND OTHER PATHOGENS. UPON DETECTING PATHOGEN-ASSOCIATED MOLECULAR PATTERNS (PAMPS), HOST CELLS WILL EXPRESS AND SECRETE THE IFN-I CYTOKINES, IFN-A AND IFN-B. THESE CYTOKINES FUNCTION IN BOTH AN AUTOCRINE AND PARACRINE MANNER, BINDING TO THE INTERFERON-ALPHA/BETA RECEPTOR, AND INITIATING A SIGNALING PATHWAY THAT TRIGGERS THE EXPRESSION OF HUNDREDS OF INTERFERON-STIMULATED GENES (ISGS). ISGS THEN INHIBIT VIRAL REPLICATION THROUGH A VARIETY OF DIVERSE FUNCTIONS THAT CAN AFFECT BOTH VIRUS AND HOST CELL BIOLOGY. DUE TO THE IMPORTANCE OF THIS SYSTEM IN HOST DEFENSE AGAINST INFECTION, THE PRESENCE OF GENETIC DEFECTS IN THE IFN-I RESPONSE CAN EITHER LEAVE INDIVIDUALS PARTICULARLY SUSCEPTIBLE TO INFECTIONS OR RESULT IN AUTOIMMUNE DISEASES SUCH AS SYSTEMIC LUPUS ERYTHEMATOSUS OR INTERFERONOPATHIES (E.G. AICARDI-GOUTIERES SYNDROME). IN MICE, HUMANS, AND MANY OTHER MAMMALS, ALL IFN-I GENES ARE FOUND WITHIN A SINGLE LOCUS THAT INCLUDES THE GENE FOR IFN-B (IFNB1) AND TWO CLUSTERS OF IFNA GENES SEPARATED BY KLHL9, A GENE INVOLVED IN MITOTIC CHROMOSOME SEGREGATION. THE SIZE OF THIS LOCUS IS ~350KB IN THE GENOMES OF MICE AND ~400KB IN THE HUMANS. THE RELATIVELY LARGE SIZE AND PRESENCE OF AN IMPORTANT CELL GROWTH-RELATED GENE IN THIS MIDDLE OF THIS LOCUS MAKE IT CHALLENGING TO EVALUATE THE ROLES OF INDIVIDUAL IFN-I GENES AND POTENTIAL REGULATORY ELEMENTS BY USING TRADITIONAL GENOME ENGINEERING TECHNIQUES. TO ADDRESS THIS CHALLENGE, WE ARE UTILIZING LARGE-DNA ASSEMBLY AND DELIVERY TECHNIQUES TO REWRITE THE ENTIRE IFN-I LOCUS IN MOUSE EMBRYONIC STEM CELLS (MESCS). WE HAVE BIALLELICALLY DELETED THE WHOLE LOCUS FROM WILD-TYPE MESCS AND USED THESE CELLS TO ESTABLISH THE FIRST COMPLETE IFN-I KNOCKOUT MOUSE MODEL. CREATION OF THIS LINE WAS ACCOMPLISHED BY REPLACING ONE ALLELE OF THE LOCUS WITH A COPY OF THE KLHL9 GENE AND THE OTHER ALLELE WITH A DUAL-USE LANDING PAD SEQUENCE. THIS LANDING PAD IS COMPATIBLE WITH TWO DIFFERENT TECHNIQUES FOR TARGETED LARGE DNA INTEGRATION INTO MAMMALIAN GENOMES, BOTH BIG-IN AND MSWAP-IN (MAMMALIAN SWITCHING OF ANTIBIOTIC-MARKERS PROGRESSIVELY FOR INTEGRATION), WHICH WILL ALLOW FOR EFFICIENT DELIVERY OF SYNTHETIC PAYLOADS OF VARIOUS SIZES. WE HAVE DESIGNED AND WILL ASSEMBLE FOUR SYNTHETIC IFN-I LOCI THAT WILL ENABLE A FULL IFNA KNOCKOUT, TWO IFNA GENE CLUSTER KNOCKOUTS, AND THE DELETION OF A LARGE FRAGMENT SPANNING IFN-I GENES. UPON CONFIRMATION OF CORRECT DELIVERY OF THESE LOCI INTO OUR LANDING PAD, THESE MESCS WILL BE DIFFERENTIATED IN VITRO OR USED TO GENERATE NOVEL MOUSE LINES. THESE DIFFERENTIATED CELLS AND MICE WILL THEN BE EXPOSED TO IMMUNE STIMULI TO INTERROGATE THE ROLES OF DIFFERENT IFNA GENES AND POTENTIAL REGULATORY ROLES OF NON-CODING REGIONS SPANNING IFN-I GENE CLUSTERS. $0 6/3/26 Not listed RATIONAL REWRITING OF MOUSE IFN?I LOCUS FOR FUNCTIONAL CHARACTERIZATION - ABSTRACT TYPE I INTERFERON (IFN-I) PLAYS AN ESSENTIAL ROLE IN THE INNATE IMMUNE RESPONSE TO VIRUSES AND OTHER PATHOGENS. UPON DETECTING PATHOGEN-ASSOCIATED MOLECULAR PATTERNS (PAMPS), HOST CELLS WILL EXPRESS AND SECRETE THE IFN-I CYTOKINES, IFN-A AND IFN-B. THESE CYTOKINES FUNCTION IN BOTH AN AUTOCRINE AND PARACRINE MANNER, BINDING TO THE INTERFERON-ALPHA/BETA RECEPTOR, AND INITIATING A SIGNALING PATHWAY THAT TRIGGERS THE EXPRESSION OF HUNDREDS OF INTERFERON-STIMULATED GENES (ISGS). ISGS THEN INHIBIT VIRAL REPLICATION THROUGH A VARIETY OF DIVERSE FUNCTIONS THAT CAN AFFECT BOTH VIRUS AND HOST CELL BIOLOGY. DUE TO THE IMPORTANCE OF THIS SYSTEM IN HOST DEFENSE AGAINST INFECTION, THE PRESENCE OF GENETIC DEFECTS IN THE IFN-I RESPONSE CAN EITHER LEAVE INDIVIDUALS PARTICULARLY SUSCEPTIBLE TO INFECTIONS OR RESULT IN AUTOIMMUNE DISEASES SUCH AS SYSTEMIC LUPUS ERYTHEMATOSUS OR INTERFERONOPATHIES (E.G. AICARDI-GOUTIERES SYNDROME). IN MICE, HUMANS, AND MANY OTHER MAMMALS, ALL IFN-I GENES ARE FOUND WITHIN A SINGLE LOCUS THAT INCLUDES THE GENE FOR IFN-B (IFNB1) AND TWO CLUSTERS OF IFNA GENES SEPARATED BY KLHL9, A GENE INVOLVED IN MITOTIC CHROMOSOME SEGREGATION. THE SIZE OF THIS LOCUS IS ~350KB IN THE GENOMES OF MICE AND ~400KB IN THE HUMANS. THE RELATIVELY LARGE SIZE AND PRESENCE OF AN IMPORTANT CELL GROWTH-RELATED GENE IN THIS MIDDLE OF THIS LOCUS MAKE IT CHALLENGING TO EVALUATE THE ROLES OF INDIVIDUAL IFN-I GENES AND POTENTIAL REGULATORY ELEMENTS BY USING TRADITIONAL GENOME ENGINEERING TECHNIQUES. TO ADDRESS THIS CHALLENGE, WE ARE UTILIZING LARGE-DNA ASSEMBLY AND DELIVERY TECHNIQUES TO REWRITE THE ENTIRE IFN-I LOCUS IN MOUSE EMBRYONIC STEM CELLS (MESCS). WE HAVE BIALLELICALLY DELETED THE WHOLE LOCUS FROM WILD-TYPE MESCS AND USED THESE CELLS TO ESTABLISH THE FIRST COMPLETE IFN-I KNOCKOUT MOUSE MODEL. CREATION OF THIS LINE WAS ACCOMPLISHED BY REPLACING ONE ALLELE OF THE LOCUS WITH A COPY OF THE KLHL9 GENE AND THE OTHER ALLELE WITH A DUAL-USE LANDING PAD SEQUENCE. THIS LANDING PAD IS COMPATIBLE WITH TWO DIFFERENT TECHNIQUES FOR TARGETED LARGE DNA INTEGRATION INTO MAMMALIAN GENOMES, BOTH BIG-IN AND MSWAP-IN (MAMMALIAN SWITCHING OF ANTIBIOTIC-MARKERS PROGRESSIVELY FOR INTEGRATION), WHICH WILL ALLOW FOR EFFICIENT DELIVERY OF SYNTHETIC PAYLOADS OF VARIOUS SIZES. WE HAVE DESIGNED AND WILL ASSEMBLE FOUR SYNTHETIC IFN-I LOCI THAT WILL ENABLE A FULL IFNA KNOCKOUT, TWO IFNA GENE CLUSTER KNOCKOUTS, AND THE DELETION OF A LARGE FRAGMENT SPANNING IFN-I GENES. UPON CONFIRMATION OF CORRECT DELIVERY OF THESE LOCI INTO OUR LANDING PAD, THESE MESCS WILL BE DIFFERENTIATED IN VITRO OR USED TO GENERATE NOVEL MOUSE LINES. THESE DIFFERENTIATED CELLS AND MICE WILL THEN BE EXPOSED TO IMMUNE STIMULI TO INTERROGATE THE ROLES OF DIFFERENT IFNA GENES AND POTENTIAL REGULATORY ROLES OF NON-CODING REGIONS SPANNING IFN-I GENE CLUSTERS. $76.8k 6/3/26 Not listed RATIONAL REWRITING OF MOUSE IFN?I LOCUS FOR FUNCTIONAL CHARACTERIZATION - ABSTRACT TYPE I INTERFERON (IFN-I) PLAYS AN ESSENTIAL ROLE IN THE INNATE IMMUNE RESPONSE TO VIRUSES AND OTHER PATHOGENS. UPON DETECTING PATHOGEN-ASSOCIATED MOLECULAR PATTERNS (PAMPS), HOST CELLS WILL EXPRESS AND SECRETE THE IFN-I CYTOKINES, IFN-A AND IFN-B. THESE CYTOKINES FUNCTION IN BOTH AN AUTOCRINE AND PARACRINE MANNER, BINDING TO THE INTERFERON-ALPHA/BETA RECEPTOR, AND INITIATING A SIGNALING PATHWAY THAT TRIGGERS THE EXPRESSION OF HUNDREDS OF INTERFERON-STIMULATED GENES (ISGS). ISGS THEN INHIBIT VIRAL REPLICATION THROUGH A VARIETY OF DIVERSE FUNCTIONS THAT CAN AFFECT BOTH VIRUS AND HOST CELL BIOLOGY. DUE TO THE IMPORTANCE OF THIS SYSTEM IN HOST DEFENSE AGAINST INFECTION, THE PRESENCE OF GENETIC DEFECTS IN THE IFN-I RESPONSE CAN EITHER LEAVE INDIVIDUALS PARTICULARLY SUSCEPTIBLE TO INFECTIONS OR RESULT IN AUTOIMMUNE DISEASES SUCH AS SYSTEMIC LUPUS ERYTHEMATOSUS OR INTERFERONOPATHIES (E.G. AICARDI-GOUTIERES SYNDROME). IN MICE, HUMANS, AND MANY OTHER MAMMALS, ALL IFN-I GENES ARE FOUND WITHIN A SINGLE LOCUS THAT INCLUDES THE GENE FOR IFN-B (IFNB1) AND TWO CLUSTERS OF IFNA GENES SEPARATED BY KLHL9, A GENE INVOLVED IN MITOTIC CHROMOSOME SEGREGATION. THE SIZE OF THIS LOCUS IS ~350KB IN THE GENOMES OF MICE AND ~400KB IN THE HUMANS. THE RELATIVELY LARGE SIZE AND PRESENCE OF AN IMPORTANT CELL GROWTH-RELATED GENE IN THIS MIDDLE OF THIS LOCUS MAKE IT CHALLENGING TO EVALUATE THE ROLES OF INDIVIDUAL IFN-I GENES AND POTENTIAL REGULATORY ELEMENTS BY USING TRADITIONAL GENOME ENGINEERING TECHNIQUES. TO ADDRESS THIS CHALLENGE, WE ARE UTILIZING LARGE-DNA ASSEMBLY AND DELIVERY TECHNIQUES TO REWRITE THE ENTIRE IFN-I LOCUS IN MOUSE EMBRYONIC STEM CELLS (MESCS). WE HAVE BIALLELICALLY DELETED THE WHOLE LOCUS FROM WILD-TYPE MESCS AND USED THESE CELLS TO ESTABLISH THE FIRST COMPLETE IFN-I KNOCKOUT MOUSE MODEL. CREATION OF THIS LINE WAS ACCOMPLISHED BY REPLACING ONE ALLELE OF THE LOCUS WITH A COPY OF THE KLHL9 GENE AND THE OTHER ALLELE WITH A DUAL-USE LANDING PAD SEQUENCE. THIS LANDING PAD IS COMPATIBLE WITH TWO DIFFERENT TECHNIQUES FOR TARGETED LARGE DNA INTEGRATION INTO MAMMALIAN GENOMES, BOTH BIG-IN AND MSWAP-IN (MAMMALIAN SWITCHING OF ANTIBIOTIC-MARKERS PROGRESSIVELY FOR INTEGRATION), WHICH WILL ALLOW FOR EFFICIENT DELIVERY OF SYNTHETIC PAYLOADS OF VARIOUS SIZES. WE HAVE DESIGNED AND WILL ASSEMBLE FOUR SYNTHETIC IFN-I LOCI THAT WILL ENABLE A FULL IFNA KNOCKOUT, TWO IFNA GENE CLUSTER KNOCKOUTS, AND THE DELETION OF A LARGE FRAGMENT SPANNING IFN-I GENES. UPON CONFIRMATION OF CORRECT DELIVERY OF THESE LOCI INTO OUR LANDING PAD, THESE MESCS WILL BE DIFFERENTIATED IN VITRO OR USED TO GENERATE NOVEL MOUSE LINES. THESE DIFFERENTIATED CELLS AND MICE WILL THEN BE EXPOSED TO IMMUNE STIMULI TO INTERROGATE THE ROLES OF DIFFERENT IFNA GENES AND POTENTIAL REGULATORY ROLES OF NON-CODING REGIONS SPANNING IFN-I GENE CLUSTERS. $76.8k 6/3/26