Formulations

Abstract

Abstract The invention provides lipid nanoparticle-based compositions with improved properties for delivery of biologically active agents, engineered cells, and methods for delivery of the agents, in particular for the delivery of CRISPR / Cas gene editing components.

Description

Cast) DNA coding sequence 2 Sequence SEQ ID No. ATGGACAAGAAGTACAGCATCGGACTGGACATCGGAACAAAC AGCGTCGGATGGGCAGTCATCACAGACGAATACAAGGTCCCG AGCAAGAAGTTCAAGGTCCTGGGAAACACAGACAGACACAGC ATCAAGAAGAACCTGATCGGAGCACTGCTGTTCGACAGCGGA GAAACAGCAGAAGCAACAAGACTG AAG AGAACAGCAAGAA G AAGATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGA AATCTTCAGCAACGAAATGGCAAAGGTCGACGACAGCTTCTTC CACAGACTGGAAGAAAGCTTCCTGGTCGAAGAAGACAAGAAG CACGAAAGACACCCGATCTTCGGAAACATCGTCGACGAAGTC GCATACCACGAAAAGTACCCGACAATCTACCACCTGAGAAAG AAGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATC TACCTGGC A CTGGC AC AC ATGATC AAGTTC AGAGGACACTTCC TGATCGAAGGAGACCTGAACCCGGACAACAGCGACGTCGACA AGCTGTTCATCCAGCTGGTCCAGACATACAACCAGCTGTTCGA AGAA AACCCGATCA ACGCAAGCGGA GTCGACGCAAAGGCAAT CCTGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCT GATCGCACAGCTGCCGGGAGAAAAGAAGAACGGACTGTTCGG AAACCTGATCGCACTGAGCCTGGGACTGACACCGAACTTCAA GAGCAACTTCGACCTGGCAGAAGACGCAAAGCTGCAGCTGAG CAAGGACACATACGACGACGACCTGGACAACCTGCTGGCACA GATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAA CCTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAAC A C AG AA ATC A C A A A GG C A CCC CTG AG C G C A AG C ATG ATC AA G AGATACGACGAACACCACCAGGACCTGACACTGCTGAAGGCA C TGG TC A G AC A G C A GCTGC CGG A AA AGT AC AAGG AA ATCTTC TTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGA GGAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATC CTGGAAAAGATGGACGGAACAGAAGAACTGCTGGTCAAGCTG AACAGAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAAC GGAAGCATCCCGCACCAGATCCACCTGGGAGAACTGCACGCA ATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGAC AACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCG TACTACGTCGGACCGCTGGCAAGAGGAAACAGCAGATTCGCA TGG ATGACA AG A A AGAGCG AA GA A AC AATC ACA CCGTGGA AC TTCGAAGAAGTCGTCGACAAGGGAGCAAGCGCACAGAGCTTC ATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAA AAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAG TCTACAACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAA TGAGAAAGCCGGCATTCCTGAGCGGAGAACAGAAGAAGGCAA TCGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTCA AGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTCG ACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAA GCCTGGGAACATACCACGACCTGCTGAAGATCATCAAGGACA AGGACTTCCTGGACAACGAAGAAAACGAAGACATCCTGGAAG 2024224004   30 Sep 2024 ACATCGTCCTGACACTGACACTGTTCGAAGACAGAGAAATGAT CGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGACAA GGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGG AAGACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCA GAGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGACGGATT CGCAAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCT GACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCGGACA GGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAG CCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGT CGACGAACTGGTCAAGGTCATGGGAAGACACAAGCCGGAAAA CATCGTCATCGAAATGGCAAGAGAAAACCAGACAACACAGAA GGGACAGAAGAACAGCAGAGAAAGAATGAAGAGAATCGAAG AAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACC CGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGT ACTACCTGCAGAACGGAAGAGACATGTACGTCGACCAGGAAC TGGACATCAACAGACTGAGCGACTACGACGTCGACCACATCG TCCCGCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGG TCCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAAC GTCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGG AGACAGCTGCTGAACGCAAAGCTGATCACACAGAGAAAGTTC GACAACCTGACAAAGGCAGAGAGAGGAGGACTGAGCGAACT GGACAAGGCAGGATTCATCAAGAGACAGCTGGTCGAAACAAG ACAGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAAT GAACACAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGT CAAGGTCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAG AAAGGACTTCCAGTTCTACAAGGTCAGAGAAATCAACAACTA CCA CC A CGC AC A C GA CGCATACCTGAACGCAGTCGTCGGAAC AGCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGT CTACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGC AAAGAGCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTT CTTCTACAGCAACATCATGAACTTCTTCAAGACAGAAATCACA CTGGCAAACGGAGAAATCAGAAAGAGACCGCTGATCGAAACA AACGGAGAA ACAGG AGAAATCGTCTGGG ACAAGGGAAGAG A CTTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAA CATCGTCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAA GGAAAGCATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGC AAGAAAGAAGGACTGGGACCCGAAGAAGTACGGAGGATTCG ACAGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGT CGAAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAAC TGCTGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGA ACCCGATCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCA AGAAGGACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCG A A CT G G A A A A C GG AA G A AAG AG AA TGC TG GCA A GCGC A GG A GAACTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTAC GTCAACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGG GAAGCCCGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAAC AGCACAAGCACTACCTGGACGAAATCATCGAACAGATCAGCG AATTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACA AGGTCCTGAGCGCATACAACAAGCACAGAGAC AAGC CG ATC A GAGAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAA ACCTGGGAGCACCGGCAGCATTCAAGTACTTCGACACAACAA TCGACAGAAAGAGATACACAAGCACAAAGGAAGTCCTGGACG CAACACTGATCCACCAGAGCATCACAGGACTGTACG.AAACAA GAATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCC CGAAGAAGAAGAGAAAGGTCTAG Cas9 DNA ATGGATAAGAAGTACTCAATCGGGCTGGATATCGGAACTAATT z 2024224004   30 Sep 2024 coding sequence 1 CCGTGGGTTGGGCAGTGATCACGGATGAATACAAAGTGCCGT CCAAGAAGTTCAAGGTCCTGGGGAACACCGATAGACACAGCA TCAAGAAAAATCTCATCGGAGCCCTGCTGTTTGACTCCGGCGA AACCGCAGAAGCGACCCGGCTCAAACGTACCGCGAGGCGACG CTACACCCGGCGGAAGAATCGCATCTGCTATCTGCAAGAGATC TTTTCGAACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACC GCCTGGAAGAATCTTTCCTGGTGGAGGAGGACAAGAAGCATG AACGGCATCCTATCTTTGGAAACATCGTCGACGAAGTGGCGTA CCACGAAAAGTACCCGACCATCTACCATCTGCGGAAGAAGTT GGTTGACTCAACTGACAAGGCCGACCTCAGATTGATCTACTTG GCCCTCGCCCATATGATCAAATTCCGCGGACACTTCCTGATCG AAGGCGATCTGAACCCTGATAACTCCGACGTGGATAAGCTTTT CATTCAACTGGTGCAGACCTACAACCAACTGTTCGAAGAAAAC CCAATCAATGCTAGCGGCGTCGATGCCAAGGCCATCCTGTCCG CCCGGCTGTCGAAGTCGCGGCGCCTCGAAAACCTGATCGCACA GCTGCCGGGAGAGAAAAAGAACGGACTTTTCGGCAACTTGAT CGCTCTCTCACTGGGACTCACTCCCAATTTCAAGTCCAATTTTG ACCTGGCCGAGGACGCGAAGCTGCAACTCTCAAAGGACACCT ACGACGACGACTTGGACAATTTGCTGGCACAAAITGGCGATCA GTACGCGGATCTGTTCCTTGCCGCTAAGAACCTI ICGGACGCA ATCTTGCTGTCCGATATCCTGCGCGTGAACACCGAAATAACCA AAGCGCCGCITAGCGCCTCGATGATTAAGCGGTACGACGAGC ATCACCAGGATCTCACGCTGCTCAAAGCGCTCGTGAGACAGCA ACTGCCTGAAAAGTACAAGGAGATCTTCTTCGACCAGTCCAAG AATGGGTACGCAGGGTACATCGATGGAGGCGCTAGCCAGGAA GAGTTCTATAAGTTCATCAAGCCAATCCTGGAAAAGATGGACG G A AC CG AA G AA C T GC TGG TC AAGCTG AAC AG GG AG G ATC TGC TCCGGAAACAGAGAACCTTTGACAACGGATCCATTCCCCACCA GATCCATCTGGGTGAGCTGCACGCCATCTTGCGGCGCCAGGAG GACTTTTACCCATTCCTCAAGGACAACCGGGAAAAGATCGAG AAAATTCTGACGTTCCGCATCCCGTATTACGTGGGCCCACTGG CGCGCGGCAATTCGCGCTTCGCGTGGATGACTAGAAAATCAG AGGAAACCATCACTCCTTGGAATTTCGAGGAAGTTGTGGATAA GGGAGC1TCGGCACAAAGCTTCATCGAACGAATGACCAACTTC GACAAGAATCTCCCAAACGAGAAGGTGCTTCCTAAGCACAGC CTCCTTTACGAATACTTCACTGTCTACAACGAACTGACTAAAG TGAAATACGTTACTGAAGGAATGAGGAAGCCGGCCTTTCTGTC CGGAGAACAGAAGAAAGCAATTGTCGATCTGCTGTTCAAGAC CAACCGCAAGGTGACCGTCAAGCAGCTTAAAGAGGACTACTT CAAGAAGATCGAGTGTTTCGACTCAGTGGAAATCAGCGGGGT GGAGGACAGATTCAACGCTTCGCTGGGAACCTATCATGATCTC CTGAAGATCATCAAGGACAAGGACTTCCTTGACAACGAGGAG AACGAGGACATCCTGGAAGATATCGTCCTGACCTTGACCCTTT TCGAGGATCGCGAGATGATCGAGGAGAGGCTTAAGACCTACG CTCATCTC1TCGACGATAAGGTCATGAAACAACTCAAGCGCCG CCGGTACACTGGTTGGGGCCGCCTCTCCCGCAAGCTGATCAAC GGTATTCGCGATAAACAGAGCGGTAAAACTATCCTGGATTTCC TCAAATCGGATGGCTTCGCTAATCGTAACTTCATGCAATTGAT CCACGACGACAGCCTGACCTTTAAGGAGGACATCCAAAAAGC ACAAGTGTCCGGACAGGGAGACTCACTCCATGAACACATCGC GAATCTGGCCGGTTCGCCGGCGATTAAGAAGGGAATTCTGCA AACTGTGAAGGTGGTCGACGAGCTGGTGAAGGTCATGGGACG GCACAAACCGGAGAATATCGTGATTGAAATGGCCCGAGAAAA CCAGACTACCCAGAAGGGCCAGAAAAACTCCCGCGAAAGGAT G A AGCGGA TCGA AG AAGG AATCA AGGAGCTGGGCAGCCAGAT CCTGAAAGAGCACCCGGTGGAAAACACGCAGCTGCAGAACGA GAAGCTCTACCTGTACTATTTGCAAAATGGACGGGACATGTAC 2024224004   30 Sep 2024 GTGGACC'VvGAGCTGGACATCAATCGGTTGTCTGATTACGACG TGGACCACATCGTTCCACAGTCCTTTCTGAAGGATGACTCGAT CGATAACAAGGTGTTGACTCGCAGCGACAAGAACAGAGGGAA GTCAGATAATGTGCCATCGGAGGAGGTCGTGAAGAAGATGAA GAATTACTGGCGGCAGCTCCTGAATGCGAAGCTGATTACCCAG AGAAAGTTTGACAATCTCACTAAAGCCGAGCGCGGCGGACTC TCAGAGCTGGATAAGGCTGGATTCATCAAACGGCAGCTGGTC GAGACTCGGCAGATTACCAAGCACGTGGCGCAGATCTTGGAC TCCCGCATGAACACTAAATACGACGAGAACGATAAGCTCATC CGGGAAGTGAAGGTGATTACCCTGAAAAGCAAACTTGTGTCG GACTTTCGGAAGGACTTTCAGTTTTACAAAGTGAGAGAAATCA ACAACTACCATCACGCGCATGACGCATACCTCAACGCTGTGGT CGGTACCGCCCTGATCAAAAAGTACCCTAAACTTGAATCGGAG T1TGTGTACGGAGACTACAAGGTCTACGACGTGAGGAAGATG ATAGCCAAGTCCGAACAGGAAATCGGGAAAGCAACTGCGAAA TACTTCTTTTACTCAAACATCATGAACTTTTTCAAGACTGAAAT TACGCTGGCCAATGGAGAAATCAGGAAGAGGCCACTGATCGA AACTAACGGAGAAACGGGCGAAATCGTGTGGGACAAGGGCAG GGACTTCGCAACTGTTCGCAAAGTGCTCTCTATGCCGCAAGTC AATATTGTGAAGAAAACCGAAGTGCAAACCGGCGGATTTTCA AAGGAATCGATCCTCCCAAAGAGAAATAGCGACAAGCTCATT GCACGCAAGAAAGACTGGGACCCGAAGAAGTACGGAGGATTC GATTCGCCGACTGTCGCATACTCCGTCCTCGTGGTGGCCAAGG TGGAGAAGGGAAAGAGCAAAAAGCTCAAATCCGTCAAAGAGC TGCTGGGGATTACCATCATGGAACGATCCTCGTTCGAGAAGAA CCCGATTGATTTCCTCGAGGCGAAGGGTTACAAGGAGGTGAA GAAGGATCTGATCATCAAACTCCCCAAGTACTCACTGTTCGAA CTGGAAAATGGTCGGAAGCGCATGCTGGCTTCGGCCGGAGAA CTCCAAAAAGGAAATGAGCTGGCCTTGCCTAGCAAGTACGTC AACTTCCTCTATCTTGCTTCGCACTACGAAAAACTCAAAGGGT CACCGGAAGATAACGAACAGAAGCAGCTTTTCGTGGAGCAGC ACAAGCATTATCTGGATGAAATCATCGAACAAATCTCCGAGTT TTCAAAGCGCGTGATCCTCGCCGACGCCAACCTCGACAAAGTC CTGTCGGCCTACAATAAGCATAGAGATAAGCCGATCAGAGAA CAGGCCGAGAACATTATCCAC'FTGTTCACCCTGACTAACCTGG GAGCCCCAGCCGCCTTCAAGTACTTCGATACTACTATCGATCG CAAAAGATACACGTCCACCAAGGAAGTTCTGGACGCGACCCT GATCCACCAAAGCATCACTGGACTCTACGAAACTAGGATCGAT CTGTCGCAGCTGGGTGGCGATGGCGGTGGATCTCCGAAAAAG AAGAGAAAGGTGTAATGA Cas9 amiao acid sequence MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIK KNLlGALLFDSGETAEATRLKJkTARRRYTRRKNRFCYLQElFSNE MAKVDDSFFH RLEESF LVEEDKKH ERH Pl FGNIVDEVAY E-IEKYPT lYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLlEGDLNPDNS DVDKLF1QLVQTYNQLFEENPINASGVDAKAJLSARLSKSRRLENL JAQLPGEKKNGLFGNHALSLGLTPNFKSNFDLAEDAKLQLSK.DT YDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA GYIDGGASQEEFYKF1KP1LEKMDGTEELLVKLNREDLLRKQRTF DNGSIPHQJHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYV GPLARGNSRFAWMTRKSEET1TPWNFEEVVDKGASAQSF1ERMT NFDKNLPNEKVLPKHSLL\'EYFTV7YNELTKVKYVTEGMRKPAFL SGEQKKAlVDLLFKTNRKVTVKQLKEDVFKKIECFDSVEiSGVED RFNASLGTYHDLLKIJKDKDFLDNEENEDILEDIVLTLTLFEDREMt 3 2024224004   30 Sep 2024 EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSG K'nLDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH EH JANLAGSPAJKKGlLQTVKVVDELVKVMGRHKPENrVI EMARE NQTTQKGQKNSRERMKRIEEGIKELGSQ1LKEHPVENTQLQNEKL YLYYLQNGRDMYVDQELDTNRLSDYDVDHIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFD NLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKY DENDKL1REVKVJTLKSKLVSDFRKDFQFYKVREINNYHHAHDA YLNAVVGTALlKKYPKLESEFVYGDYKVYDVRKMIAKSEQEtGK ATAKYFFYSN1MNFFKTE1TLANGEIRKRPLIETNGETGEIVWDKG RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDICLrAR KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLG 1T1MERSSFEKNP1DFLEAKGYKEVKKDL1IKLPKYSLFELENGRKR MLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ LFVEQHKHYLDEIIEQISEFSKRV1LADANLDKVLSAYNKHRDKPI REQAEN1IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI HQS1TGLYETR1DLSQLGGDGGGSPKKKRKV Cas9 mRNA open reading frame (ORF) 2 A U GG A C AA G AA G UA CA GCAUC G GAC U GG A CAUC GGAA CAAA CAGCGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCC CGAGCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACAC AGCAUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAG CGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAA GAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUG CAGGAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAG CUUCUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAG ACAAGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUC GACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCA CCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACC UGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUC AGAGGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAA CAGCGACGUCGACAAGCUGUVCAUCCAGCUGGUCCAGACAU ACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGA GUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAG CAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAA AGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUG CK1 AC UGACACCGAACUU CAAGAGCAACU U CGACC UGGC AGA AGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACG ACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCA GACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCU GCUGAGCGACAUCCUGAGAGUCAACACAG AAAU CACA AAG G CACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACAC CACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCA GCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCA AGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAG GAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAU GGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAG ACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUC CCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGA AGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGA AAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACG UCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUG ACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGA AGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCG aaagaaugacaaacuucgacaagaaccugccgaacgaaaag 2024224004   30 Sep 2024 GUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGU CUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAA UGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCA AUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGU CAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCU UCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAAC GCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAA GGACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCC UGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGA GAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUU CGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACA CAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUC AGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAA GAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCC ACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCA CAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGC AAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGC AGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGA A G AC A CA A G CC GG AA A AC AUC GUC AUC G AA AU G GCAA GAGA AAACCAGACAACACAGAAGGGACAG AAG AACAGCAGAGAAA G A AUG AA G A G AA UC G A A G AAGG AAUC AAGG AACUG GGA A GC CAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCA GAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAG ACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGC GACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAG GACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAA GAAC'AGAGGAAAGAGC'GACAACGUCCCGAGCGAAGAAGUCG UCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCA AAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGC AGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCA UCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCAC GUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGA CGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACAC UGAAGAGCAAGC UGGUCAGCGACUUCAGAAAGGACUUCCAG UUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACA CGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCA AGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGAC UACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGA AC ACK) AAA UCGG AA AGGC AACA GCA AAGU AC UUC WCUAC A gcaacaucaugaacuucuucaagacagaaaucacacuggca AACGGAGAAAUCAGAAAGAGACCGCUGAUC GAA ACAAAC GG AGAAACAGGAGAAAUC'GUCUGGGACAAGGG AAG AG ACUUCG CAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUC GUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGA AAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAA GAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGAC AGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGU CGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAAC UGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAG AACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGU CAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGU UCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCA GGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAA GUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGC UGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGC UGUUC GUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACA GAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAA 2024224004   30 Sep 2024 ACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGAC AAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUU CACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACU UCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAG GAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGG ACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAG ACGGAGGAGGAAGCCCGAAGAAGAAGAGAAAGGUCUAG Cas9 mRjNA ORF 1 AUGGAUAAGAAGUACUCAAUCGGGCUGGAUAUCGGAACUAA UUCCGUGGGUUGGGCAGUGAUCACGGAUGAAUACAAAGUGC CGUCCAAGAAGUUCAAGGUCCUGGGGAACACCGAUAGACAC AGCAUCAAGAAAAAUCUCAUCGGAGCCCUGCUGUUUGACUC CGGCGAAACCGCAGAAGCGACCCGGCUCAAACGUACCGCGAG GCGACGCUACACCCGGCGGAAGAAUCGCAUCUGCUAUCUGC AA GAG AUC UUUUCG AAC G AA AU G GC AAAG G U CGA CGAC AG C UUCUUCCACCGCCUGGAAGAAUCUUUCCUGGUGGAGGAGGA CAAGAAGCAUGAACGGCAUCCUAUCUUUGGAAACAUCGUCG ACGAAGUGGCGUACCACGAAAAGUACCCGACCAUCUACCAU CUGCGGAAGAAGUUGGUUGACUCAACUGACAAGGCCGACCU CAGAUUGAUCUACUUGGCCCUCGCCCAUAUGAUCAAAUUCC GCGGACACUUCCUGAUCGAAGGCGAUCUGAACCCUGAUAAC UCCGACGUGGAUAAGCUUUUCAUUCAACUGGUGCAGACCUA CAACCAACUGUUCGAAGAAAACCCAAUCAAUGCUAGCGGCG UCGAUGCCAAGGCCAUCCUGUCCGCCCGGCUGUCGAAGUCGC GGCGCCUCGAAAACCUGAUCGCACAGCUGCCGGGAGAGAAA AAGAACGGACUUUUCGGCAACUUGAUCGCUCUCUCACUGGG ACUCACUCCCAAUUUCAAGUCCAAUUirUGACCUGGCCGAGG ACGCGAAGCUGCAACUCUCAAAGGACACCUACGACGACGAC UUGGACAAUUUGCUGGCACAAAUUGGCGAUCAGUACGCGGA UCUGUUCCUUGCCGCUAAGAACCUUUCGGACGCAAUCUUGC UGUCCGAUAUCCUGCGCGUGAACACCGAAAUAACCAAAGCG CCGCUUAGCGCCUCGAUGAUUAAGCGGUACGACGAGCAUCA CCAGGAUCUCACGCUGCUCAAAGCGCUCGUGAGACAGCAAC UGCCUGAAAAGUACAAGGAGAUCUUCUUCGACCAGUCCAAG AAUGGGUACGCAGGGUACAUCGAUGGAGGCGCUAGCCAGGA AGAGUUCUAUAAGUUCAUCAAGCCAAUCCUGGAAAAGAUGG ACGGAACCGAAGAACUGCUGGUCAAGCUGAACAGGGAGGAU CUGCUCCGGAAACAGAGAACCUUUGACAACGGAUCCAUUCC CCACCAGAUCCAUCUGGGUGAGCUGCACGCCAUCUUGCGGCG CCAGGAGGACUUUUACCCAUUCCUCAAGGACAACCGGGAAA AGAUCGAGAAAAUUCUGACGUUCCGCAUCCCGUAUUACGUG GGCCCACUGGCGCGCGGCAAUUCGCGCUUCGCGUGGAUGAC UAGAAAAUCAGAGGAAACCAUCACUCCUUGGAAUUUCGAGG AAGUUGUGGAUAAGGGAGCUUCGGCACAAAGCUUCAUCGAA CGAAUGACCAACUUCGACAAGAAUCUCCCAAACGAGAAGGU GCUUCCUAAGCACAGCCUCCUUUACGAAUACUUCACUGUCU ACAACGAACUGACUAAAGUGAAAUACGUUACUGAAGGAAUG AGGAAGCCGGCCUUUCUGUCCGGAGAACAGAAGAAAGCAAU UGUCGAUCUGCUGUUCAAGACCAACCGCAAGGUGACCGUCA AGCAGCUUAAAGAGGACUACUUCAAGAAGAUCGAGUGUUUC GACUCAGUGGAAAUCAGCGGGGUGGAGGACAGAUUCAACGC UUCGCUGGGAACCUAUCAUGAUCUCCUGAAGAUCAUCAAGG ACAAGGACUUCCUUGACAACGAGGAGAACGAGGACAUCCUG GAAGAUAUCGUCCUGACCUUGACCCUUUUCGAGGAUCGCGA GAUGAUCGAGGAGAGGCUUAAGACCUACGCUCAUCUCUUCG 5 2024224004   30 Sep 2024 ACGAUAAGGUCAUGAAACAACUCAAGCGCCGCCGGUACACU GGUUGGGGCCGCCUCUCCCGCAAGCUGAUCAACGGUAUUCG cg au aaacagagcgguaaaacuauccuggauuuccucaa.au CGGAUGGCUUCGCUAAUCGUAACUUCAUGCAAUUGAUCCAC GACGACAGCCUGACCUUUAAGGAGGACAUCCAAAAAGCACA AGUGUCCGGACAGGGAGACUCACUCCAUGAACACAUCGCGA AUCUGGCCGGUUCGCCGGCGAUUAAGAAGGGAAUUCUGCAA ACUGUGAAGGUGGUCGACGAGCUGGUGAAGGUCAUGGGACG GCACAAACCGGAGAAUAUCGUGAUUGAAAUGGCCCGAGAAA ACCAGACUACCCAGAAGGGCCAGAAAAACUCCCGCGAAAGG AUGAAGCGGAUCGAAGAAGGAAUCAAGGAGCUGGGCAGCCA GAUCCUGAAAGAGCACCCGGUGGAAAACACGCAGCUGCAGA ACGAGAAGCUCUACCUGUACUAUUUGCAAAAUGGACGGGAC AUGUACGUGGACCAAGAGCUGGACAUCAAUCGGUUGUCUGA UUACGACGUGGACCACAUCGUUCCACAGUCCUUUCUGAAGG AUGACUCGAUCGAUAACAAGGUGUUGACUCGCAGCGACAAG AACAGAGGGAAGUCAGAUAAUGUGCCAUCGGAGGAGGUCGU GAAGAAGAUGAAGAAUUACUGGCGGCAGCUCCUGAAUGCGA AGCUGAUUACCCAGAGAAAGUUUGACAAUCUCACUAAAGCC GAGCGCGGCGGACUCUCAGAGCUGGAUAAGGCUGGAUUCAU CAAACGGCAGCUGGUCGAGACUCGGCAGAUUACCAAGCACG UGGCGCAGAUCUUGGACUCCCGCAUGAACACUAAAUACGAC GAGAACGAUAAGCUCAUCCGGGAAGUGAAGGUGAUUACCCU GAAAAGCAAACUUGUGUCGGACUUUCGGAAGGACUUUCAGU UUUACAAAGUGAGAGAAAUCAACAACUACCAUCACGCGCAU GACGCAUACCUCAACGCUGUGGUCGGUACCGCCCUGAUCAA AAAGUACCCUAAACUUGAAUCGGAGUUUGUGUACGGAGACU ACAAGGUCUACGACGUGAGGAAGAUGAUAGCCAAGUCCGAA CAGGAAAUCGGGAAAGCAACUGCGAAAUACUUCUUUUACUC AAACAUCAUGAACUUUUUCAAGACUGAAAUUACGCUGGCCA AUGGAGAAAUCAGGAAGAGGCCACUGAUCGAAACUAACGGA GAAACGGGCGAAAUCGUGUGGGACAAGGGCAGGGACUUCGC AACUGUUCGCAAAGUGCUCUCUAUGCCGCAAGUCAAUAUUG UGAAGAAAACCGAAGUGCAAACCGGCGGAUUUUCAAAGGAA UCGAUCCUCCCAAAGAGAAAUAGCGACAAGCUCAUUGCACG CAAGAAAGACUGGGACCCGAAGAAGUACGGAGGAUUCGAUU CGCCGACUGUCGCAUACUCCGUCCUCGUGGUGGCCAAGGUG GAGAAGGGAAAGAGCAAAAAGCUCAAAUCCGUCAAAGAGCU GCUGGGGAUUACCAUCAUGGAACGAUCCUCGUUCGAGAAGA ACCCGAUUGAUUUCCUCGAGGCGAAGGGUUACAAGGAGGUG AAGAAGGAUCUGAUCAUCAAACUCCCCAAGUACUCACUGUU CGAACUGGAAAAUGGUCGGAAGCGCAUGCUGGCUUCGGCCG GAGAACUCCAAAAAGGAAAUGAGCUGGCCUUGCCUAGCAAG UACGUCAACUUCCUCUAUCUUGCUUCGCACUACGAAAAACU CAAAGGGUCACCGGAAGAUAACGAACAGAAGCAGCUUUUCG UGGAGCAGCACAAGCAUUAUCUGGAUGAAAUCAUCGAACAA AUCUCCGAGUUUUCAAAGCGCGUGAUCCUCGCCGACGCCAAC CUCGACAAAGUCCUGUCGGCCUACAAUAAGCAUAGAGAUAA GCCGAUCAGAGAACAGGCCGAGAACAUUAUCCACUUGUUCA CCCUGACUAACCUGGGAGCCCCAGCCGCCUUCAAGUACUUCG AUACUACUAUCGAUCGCAAAAGAUACACGUCCACCAAGGAA GUUCUGGACGCGACCCUGAUCCACCAAAGCAUCACUGGACUC UACGAAACUAGGAUCGAUCUGUCGCAGCUGGGUGGCGAUGG CGGUGGAUCUCCGAAAAAGAAGAGAAAGGUGUAAUGA 2024224004   30 Sep 2024 Cas9 nickase (DIO A) amino acid sequence MDKKYSIGLAlGTNSVGWAV!TDE\T<VPSKKn<VLGNTDR.HSIK KNLTGALLFDSGETAEATRLKRTARRRYTRRKNRJCYLQEIFSNE MAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPT IYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS DVDKLF1QLVQTYNQLFEENPINASGVDAKA1LSARLSKSRRLENL IAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDT YDDDLDNLLAQ1GDQYADLFLAAKNLSDA1LLSD1LRVNTE1TKA PLSASM1KRYDE1-IHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA GYIDGGASQEEFYKF1KPILEKMDGTEELLVKLNREDLLRKQRTF DNGSiPHQiHLGELHAJLRRQEDn / PFLKDNREKIEKlLTFRIPYYV GPLARGNSRFAWMTRKSEETITPWNFEEWDKGASAQSFIERMT NFDKNLPNEKVLPKHSLLYEYFT\O7^ELTKVKYVTEGMRK.PAFL SGEQKKAWDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVED RFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMt EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLTNGIRDKQSG KTJLDFLKSDGFANRNFMQLII-IDDSLTFKEDIQKAQVSGQGDSLH EH ianlagspajkkgilqtvkwdelvkvmgrhkpentvi emare NQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKL YLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKL1TQRKFD NLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKY DENDKL1REVKV1TLKSKLVSDFRKDFQFYKVREINNYHHAHDA YLNAVVGTALiKKYPKLESEFVYGDYKVYDVRKMIAKSEQEiGK ATAKYFFYSNIMNFFKTE1TLANGEIRKRPLIETNGETGEIVWDKG RDFATVRKVLSMPQVNrVKKTEVQTGGFSKESILPKRNSDKLlAR KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLG ITIMERSSFEKNPJDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKR MLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ LFVEQHKHYLDEIIEQISEFSKRVlLADANLDKVLSAYNKI-ntDKPI REQAENlIHLFTLTNLGAPAAFKYFD'niDRKRYTSTKEVLDATLI HQS1TGLYETR1DLSQLGGDGGGSPKKKRKV Cas9 nickase (D10A) mRNA ORF A U GG A C A A G A A G U A C A GC AUC G G AC U GGC A A UC G G AA C A AA CAGCGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCC CGAGCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACAC AGCAUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAG CGGAGAAACAGCAGAAGCAACAAGACUGAAGAG AACAGCAA GAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUG CAGGAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAG CUUCUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAG ACAAGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUC GACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCA CCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACC U GAG AC UGA UCUACCUGGCACUGGCA CACAUGAUCAAGUUC AGAGGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAA CAGCGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAU ACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGA GUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAG CAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAA AGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUG GGACUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGA AGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACG ACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCA GACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCU GCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGG 2024224004   30 Sep 2024 CACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACAC CACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCA GCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCA AGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAG GAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAU GGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAG ACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUC CCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGA AGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGA AAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACG UCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUG ACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGA AGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCG AAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAG GUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGU CUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAA UGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCA AUCGUCGACCUGCUGUVCAAGACAAACAGAAAGGUCACAGU C A AGC AGC U GA A G GA A G A CUA CU UCAAGAAGAUCGAAUGCU UCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAAC GCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAA GGACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCC UGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGA GAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUU CGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACA CAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUC AGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAA GAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCC ACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCA CAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGC AAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGC AGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGA AGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGA AAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAA GAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGC CAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCA GAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAG ACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGC GACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAG gacgacagcaucgacaacaagguccugacaagaagcgacaa GAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCG UCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCA AAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGC AGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCA UCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCAC GUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGA CGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACAC UGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAG UUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACA C G AC GC AUA CC UG AA CGCAGUCGUC GG AA CAGCACUGAUCA AGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGAC UACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGA ACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACA GCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCA AACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGG AGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCG CAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUC 2024224004   30 Sep 2024 GUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGA AAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAA GAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGAC AGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGU CGAAAAGGGAAAGAGCAAGAAGCUG AAG AG CGUC AAGG A A C UGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAG AACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGU CAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGU UCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCA GGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAA GUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGC UGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGGUGUUC GUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACA GAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAA ACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGAC AAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUU CACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACU UCGACACAACAAUCGACAGAAAGAGAUACACAAGGACAAAG GAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGG ACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAG A CGG A GG A GGA A GCC C G AAG AAG AA GAG AA AG GUCUAG dCas9 (D10A H840A) amino acid sequence MDKKYSIGLAIGTNSVGWAVJTDEYXVPSKKFKVLGNTDRHSIK KNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNE MAKVDDSFFHRLEESFLVEEDKKHERHPTFGNIVDEVAYHEKYPT IYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS DVDKLF1QLVQTYNQLFEENPINASGVDAKAJLSARLSKSRRLENL 1AQLPGEKK.NGLFGNL1ALSLGLTPMFKSNFDLAEDAKLQLSKDT YDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA PLSASM1KRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA GYIDGGASQEEFYKF1KP1LEKMDGTEELLVKLNREDLLRKQRTF DNGSIPHQ1HLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYV GPLARGNSRFAWMTRKSEETTTPWNFEEWDKGASAQSFIERMT NFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL SGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKiECFDSVEISGVED RFNASLGTYHDLLKIJKDKDFLDNEENEDILEDIVLTLTLFEDREMt EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLiNGIRDKQSG KTJLDFLKSDGFANRNFMQLiHDDSLTFKEDiQKAQVSGQGDSLH EHIANLA GSPAJ KKGILQT VK WDEL VK VMGRHKPENIV (EMARE NQTTQKGQKNSRERMKRIEEGIKELGSQJLKEHPVENTQLQNEKL YLYYLQNGRDMYVDQELDtNRLSDYDVDAiVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKXMKNYWRQLLNAKLJTQRKFD N'LTKAERGGLSELDKAGFrKRQLVETRQlTKHVAQILDSRMNTKY DENDKL1REVKVJTLKSKLVSDFRKDFQFYKVREINNYHHAHDA YLNAVVGTALJKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK ATA KY FF YSN1MNFFKTEH LANG EiRKRPL ETNG ETG EI VWDKG RDFATVRKVLSMPQVNrVKKTEVQTGGFSKESILPKRNSDKLIAR KKDWDPKKYGGFDSPTVAYSVLWAKVEKGKSKJKLKSVKELLG 1T1MERSSFEKNPIDFLEAKGYKEVKKDL1IKLPKYSLFELENGRKR MLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ LFVEQHKHYLDEIIEQISEFSKRV1LADANLDKVLSAYNKHRDKPI REQAEN1IHLFTLTN LG APAAFKYFDTTIDRKRYTSTKEVLDATLI HQS1TGLYETR1DLSQLGGDGGGSPKKKRKV dCas? (DWA AUGGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAA CAGCGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCC 2024224004   30 Sep 2024 H840A) mRNA ORF CGAGCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACAC AGCAUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAG CGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAA GAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUG C A GG A A AUC U UC A G CA A C GAA AUGGC AAAG GUCGA CGA CA G CUUCUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAG acaagaagcacgaaagacacccgaucuucggaaacaucguc GACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCA ccugagaaagaagcuggucgacagcacagacaaggcagacc UGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUC AGAGGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAA CAGCGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAU ACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGA GUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAG CAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAA AGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUG GGAC UGACACCGAACUUCAAGAGCAAC UUCGACCUGGCAGA AGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACG ACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCA GACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCU GCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGG CACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACAC CACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCA GCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCA AGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAG GAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAU GGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAG ACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUC CCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGA AGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGA AAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACG UCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUG ACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGA AGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCG AAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAG GUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGU CUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAA UGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCA AUCGUCGACCUGCUGUVCAAGACAAACAGAAAGGUCACAGU C A AGC AGC U GA A G GA A G A CUA CU UCAAGAAGAUCGAAUGCU UCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAAC GCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAA GG AC AA GG A C UUC C UGG AC AA CG AA G A AAA CGA AGAC AU CC UGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGA GAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUU CGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACA CAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUC AGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAA GAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCC ACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCA CAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGC AAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGC AGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGA AGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGA AAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAA GAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGC CAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCA 2024224004   30 Sep 2024 gaacgaaaagcuguaccuguacuaccugcagaacggaagag ACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGC GACUACGACGUCGACGCAAUCGUCCCGCAGAGCUUCCUGAA GGACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACA A G AA CA GA GG AA A G AGC G A CA AC GUC CC GAGCGAAGA AGUC GUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGC AAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGG CAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUC AUCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCA CGUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACG ACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACA CUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCA GUUC UACAAGG UCAGAGA AAUCAACA AC UACCACCACGCAC ACGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUC AAGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGA CUACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCG AACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUAC AGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGC AAACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACG G A GA A AC A GG AG A A AUC G U CUG GG AC AAGG GAAGAGAC UUC GCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAU CGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGG A A AGC A UCC U GCC GA A G AG AA AC AG C GAC A AGC UGAU CG CA AGAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGA CAGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGG UCGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAA C UGC UGGG A AUC A CA A UC AUGG A AA GAAGCAGCUUCGAAAA GAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAG UCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUG UUCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGC AGGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCA AGUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAG CUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUU CGUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAAC AGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCA AACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGA CAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGU UCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUAC UUCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAA GGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAG GACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGA GACGGAGGAGGAAGCCCGAAGAAG AAG AGA AAGG UCUAG Cas9 bare coding sequence GACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAAACAG CGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGA GCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACACAGC AUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAGCGG A G AAAC AGC A GAAG CAA CAAGAC UGAA GAGAACAGC AAGAA GAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAG GAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUU CUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACA AGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUCGAC GAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCACCU GAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGA GACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGA GGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAG CGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAUACA 10 2024224004   30 Sep 2024 ACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGAGUC GACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAG AAGACUGGAAA.ACCUGAUCGCACAGCUGCCGGGAGAAA.4GA AGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGA CUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGA CGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACC UGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGAC CUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCU GAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCAC CGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCAC CAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCU GCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGA ACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAA GAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGA CGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACC UGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCG CACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAG ACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAA AGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUC GGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGAC AA G A AAG A GC GA A G AA A C AAUC AC A C CG UGGAAC UUCGAAG AAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAA AGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGU CCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCU ACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUG AGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAU CGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCA AGCAGCUGAAGGAAGACUACWCAAGAAGAUCGAAUGCUUC GACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGC AAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGG ACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUG GAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGA AAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCG ACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACA GGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAG AGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGA GCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCAC GACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACA GGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAA ACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAG ACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAG ACACAAGCCGGAAAACAUCGUCAUC GAAAUG GC AAGA GAAA A CC AG AC .AA C A CAGAAGGGACAGAAG AA C AGC AG AG AA AG A AUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCA GAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGA ACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGAC AUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGA CUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGA CGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGA ACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGUC AAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAA GCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAG AGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUC AAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGU CGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGACG AAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACUG AAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUU 2024224004   30 Sep 2024 CUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACG ACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAG AAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACUA CAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAAC AGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGC AACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAA CGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAG AAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGCA ACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCGU CAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAAA GCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGA AAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAG CCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGG UCG AAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUG CUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAA CCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCA AGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUC GAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGGGCAGG AGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAGU ACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCUG AAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGU CGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGA UCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAAC CUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAA GCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCA CACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUC G A C A C AA C A AUCG AC A G AA AG AG AUA CAC AAGC ACAA AG GA AGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGAC UGUACGAAA CAA GA AUCGACCUGAGCCAGCUGGGAGGAGAC GGAGGAGGAAGCCCGAAGAAGAAGAGAAAGGUC Cus9 nickase bare coding sequence GACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAG CGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGA GCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACACAGC AUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAGCGG AGAAA CAGCAGAA GCA ACAAGACUGAAGAGAACAGCAAGAA GAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAG GAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUU CUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACA AGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUCGAC GAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCACCU GAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGA GACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGA GGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAG CGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAUACA ACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGAGUC GACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAG AAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAA AAGA AGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGA CUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGA CGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACC UGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGAC CUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCU GAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCAC CGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCAC 11 2024224004   30 Sep 2024 CAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCU GCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGA ACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAA GAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGA CGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACC UGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCG CACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAG ACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAA AGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUC GGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGAC AAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAG AAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAA AGAAUGACAAACWCGACAAGAACCUGCCGAACGAAAAGGU CCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCU ACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUG AGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAU CGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCA AGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUC GACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGC AAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGG ACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUG GAAGACAUCGUCCUGACACUGACACUGUBCGAAGACAGAGA AAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCG ACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACA GGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAG AGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGA GC GA CGG A U UC GC A AA C A GAA AC GU CAUGCAGCUGAUCCAC GACGACAGCCUGACAUVCAAGGAAGACAUCCAGAAGGCACA GGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAA ACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAG ACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAG ACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAA ACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGA AUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCA GAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGA ACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGAC AUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGA CUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGA CGACAGCAUCGACAACAAGGUCCUGACAAGAAGGGACAAGA ACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGUC AAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAA GCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAG AGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUC AAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGU CGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGACG AAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACUG AAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUU CUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACG ACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAG AAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACUA CAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAAC AGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGC AACAUCAUGAACUUCWCAAGACAGAAAUCACACUGGCAAA CGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAG AAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGCA ACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCGU CAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAAA 2024224004   30 Sep 2024 GCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGA AAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAG CCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCG AAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUG C U GGG AA UC A CA A U CA U GG AA AG AA GC AG CUUCGAAAAGAA CCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCA AGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUC GAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGG AGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAGU ACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCUG AAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGU CGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGA UCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAAC CUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAA GCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCA CACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUC GACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGA AGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGAC UGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGAC GGAGGAGGAAGCCCGAAGAAGAAGAG AAAG GUC dCas9 bare coding sequence GACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAG CGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGA GCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACACAGC AUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAGCGG A G AA A C AGC A GA A G CA A CA AG AC UGAAGAGAACAGCAAGAA GAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAG GAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUU CUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACA AGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUCGAC GAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCACCU GAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGA GACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGA GGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAG CGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAUACA ACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGAGUC GACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAG AAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGA AGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGA CUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGA CG CA AAGCUGCAG CUGA GCAAGGACACAUACGACGACGACC UGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGAC CUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCU GAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCAC CGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCAC CAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCU GCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGA ACGGAUACGCAGGAUACAUCGACGGAGGA GCAAGCCAGGAA GAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGA CGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACC UGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCG CACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAG ACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAA AGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUC GGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGAC AAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAG AAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAA 12 2024224004   30 Sep 2024 AGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGU CCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCU ACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUG AGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAU CGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCA AGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUC GACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGC AAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGG ACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUG GAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGA AAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCG ACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACA GGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAG AGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGA GCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCAC GACGACAGCCUGACA UUCAAGGAAGACAUCCAGAAGGCACA GGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAA ACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAG ACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAG A C AC A A GCC GG A A A A C A UC G U C AUC GAAAUG GC AAGA GAAA A C CA G A C AA C A C A G A A G GG A C A G A AG AACAGCAGAGAAAGA AUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCA GAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGA ACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGAC AUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGA CUACGACGUCGACGCAAUCGUCCCGCAGAGCUUCCUGAAGG ACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAG AACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGU CAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAA AGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCA GAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAU CAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACG UCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGAC GAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACU GAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGU UCUACA AGGUC AG AGA AAUC AACA ACUACC ACC ACGC ACAC GACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAA GAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACU ACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAA CAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAG CAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAA A CGG A GA A A UC A G A AA G A GAC C GC U GAUC GAAACAAACGGA GAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGC AACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCG UCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAA AGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAG AAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACA GCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUC GAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACU GC UGG GA A UC AC A AUC A UGG AAAGAAGCAGCUUCGAAAAGA ACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUC AAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUU CGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAG GAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAG UACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCU GAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCG UCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAG 2024224004   30 Sep 2024 AUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAA CCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACA AGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUC ACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUU CG AC A C A A C A A UC G A C A G A A A G AG A L! AC AC AAGCACA AAG G AAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGA CUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGA CGGAGGAGGAAGCCCGAAGAAGAAGAGAAAGGUC Amino acid sequence of Cas9 (without NLS) MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIK KNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNE MAKVDDSFFHRLEESFLVEEDKKHERHP1FGNIVDEVAYHEKYPT JYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS DVDKLFIQLVQTYNQLFEENPJNASGVDAKA1LSARLSKSRRLENL IAQLPGEKKNGLFGNLL4LSLGLTPNFKSNFDLAEDAKLQLSK DT YDDDLDNLLAQiGDQYADLFLAAKNLSDAILLSDILRVNTEn'KA PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA GYIDGGASQEEFYKFJKP1LEKMDGTEELLVKLNREDLLRKQRTF DNGSIPHQJH LGELH AILRRQEDFYPFLKDNR EKI EKILTFRIPY YV GPLARGNSRFAWMTRKSEET1TPWNFEEVVDKGASAQSF1ERMT NFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL SGEQKKAJVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVED RFNASLGTYHDLLKI i KDKDFLDNEENEDILEDFVLTLTLFEDREM i EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSG KT1LDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH EH IANLAGSPAJKKGILQTVKWDELVKVMGRHKPENTVI emare NQTTQKGQKNSRERMKRrEEGIKELGSQlLKEHPVENTQLQNEKL YLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFD NLTKAERGGLSELDKAGFIKRQLVETRQ1TKHVAQILDSRMNTKY DENDKL1REVKV1TLKSKLVSDFRKDFQFYKVREINNYHHAHDA YLNAWGTALJKKYPKLESEFVYGDYKVYDVRKMTAKSEQEIGK ATAKYFFYSNIMN FFKTE1TLANG EI RKRPLIETNGETGEIVWDKG RDFATVRKVLSMPQVNFVKKTEVQTGGFSKES1LPKRNSDKLIAR KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLG 1TIMERSSFEKNPJDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKR MLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ LFVEQH KHYLDEIIEQISEFSKRVJLADANLDKVLSAYNKHRDK.pl REQAEN1IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI HQSITGLYETR1DLSQLGGD 13 Cas9 mRNA ORF encoding SEQ ID NO: 13 using minimal uridine codons as listed in Table 3, with start and stop codons AUGGACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAAA CAGCGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCC CGAGCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACAC AGCAUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAG CGGAGAAACAGCAGAAGCAACAAGACUGAAGAG AACAGCAA GAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUG CAGGAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAG CUUCUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAG ACAAGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUC GACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCA CCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACC UGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUC AGAGGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAA CAGCGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAU ACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGA 14 2024224004   30 Sep 2024 GUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAG CAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAA AGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUG GGACUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGA AGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACG ACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCA GACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCU GCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGG CACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACAC CACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCA GCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCA AGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAG GAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAU GGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAG ACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUC CCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGA AGACAGGAAGACWCUACCCGUUCCUGAAGGACAACAGAGA AAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACG UCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUG ACA AGAAAGAGCGAAGAAAC'AAUCACACCGUGGAACUUCGA AGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCG AAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAG GUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGU CUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAA UGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCA AUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGU C A AGC AGC UGA A GGAAGACUACUUCAAGAAGAUCGAAUGCU UCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAAC GCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAA GGACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCC UGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGA GAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUU CGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACA CAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUC AGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAA GAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCC ACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCA CAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGC AAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGC AGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGA AGACACAAGCCGGAAAACAUCGUCAUCG AAAUG GCAA GAGA A A ACC AG A C A AC A CA G A AGGG AC AG AAG AACAGCAGAGAAA G A AUG AAGAG AAUCGA AGAAGG AAUC AAGG AACUGGGAAGC CAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCA GAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAG ACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGC GACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAG GACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAA GAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCG UCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCA AAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGC AGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCA UCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCAC GUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGA CGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACAC UGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAG UUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACA 2024224004   30 Sep 2024 ! 15 CGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCA AGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGAC UACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGA ACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACA GCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCA A A CGG AG A A AUC A G AA A G AG A C CGC U G AU C GA A ACA A ACGG AGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCG CAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUC GUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGA AAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAA GAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGAC AGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGU CGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAAC UGCUCGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAG AACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGU CAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGU UCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGGA GGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAA GUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGC UGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUC GUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACA GAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAA ACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGAC AAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUU CACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACU UCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAG GAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGG ACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAG ACUAG Cas9 coding sequence encoding SEQ ID NO: 13 using minimal uridine codons as listed in Table 3 (no start or stop codons; suitable for inclusion in fusion protein coding sequence) GACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAAACAG CGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGA GCAAGAAGUVCAAGGUCCUGGGAAACACAGACAGACACAGC AUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAGCGG AGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAAGAA G A AGAUAC AC AAG AAG A A AG A ACAG AAUC UGCUACCUGCAG GAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUU CUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACA AGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUCGAC GAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCACCU GAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGA GACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGA GGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAG CGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAUACA ACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGAGUC GACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAG AAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGA AGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGA CUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGA CGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACC UGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGAC CUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCU GAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCAC CGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCAC CAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCU 15 2024224004   30 Sep 2024 GCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGA ACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAA GAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGA CGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACC UGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCG CACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAG ACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAA AGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUC GGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGAC AAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAG AAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAA AGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGU CCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCU ACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUG AGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAU CGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCA AGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUC GACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGC AAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGG ACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUG GAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGA AAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCG A C GA C AA GG UC A U GA A GCAGC UG AA GAG A AGAA GAUAC A CA GGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAG AGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGA GCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCAC G A CG A CA GC C UG A CAUUCAAGGAAGACAUCCAGAAGGCACA GGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAA ACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAG ACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAG ACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAA ACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGA AUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCA GAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGA ACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGAC AUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGA CUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGA CGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGA ACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGUC AAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAA GCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAG AGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUC AAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGU CGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGACG AAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACUG AAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUU CUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACG ACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAG AAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACUA CAAGGUCUACGACGUCAGAAA GAUGAUCGCA AAGAGCGAAC AGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGC AACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAA CGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAG AAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGCA ACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCGU CAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAAA GCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGA 2024224004   30 Sep 2024 AAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAG CCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCG AAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUG CUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAA CCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCA AGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUC GAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGG AGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAGU ACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCUG AAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGU CGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGA UCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAAC CUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAA GCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCA CACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUC GACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGA AGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGAC UGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGAC Amino acid sequence of Cas9 nickase (without NLS) MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIK KNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNE MAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPT IYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS DVDKLF1QLVQTYNQLFEENPINASGVDAKA1LSARLSKSRRLENL 1AQLPGEKKNGLFGNLJALSLGLTPNFKSNFDLAEDAKLQLSK.DT YDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA PLSASMlKRYDEfflQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA GYIDGGASQEEFYKF1KPILEKMDGTEELLVKLNREDLLRKQRTF DNGSiPHQlHLGELHAlLRRQEDFYPFLKDNREKIEKiLTFRIPYYV GPLARGNSRFAWMTRKSEET1TPWNFEEVVDKGASAQSF1ERMT NFDKNLPNEKVLPKI-1SLLYEYFTVYNELTKVKYVTEGMRKPAFL SGEQKKAJVDLLFKTNRKVTVK.QLKEDYFKK1ECFDSVEISGVED RFNASLGTYHDLLKI1KOKDFLDNEENEDILEDIVLTLTLFEDREM1 EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLrNGIRDKQSG KT1LDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH EH 1ANLA GSPAJ KKGTLQT VK WDEL VK VMGRHKPENIV (EMARE NQT1QKGQKNSRERMKRJEEG1KELGSQ1LKEHPVENTQLQNEKL YLYYLQNGRDMYVDQELDtNRLSDYDVDHrvPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFD NLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKY DENDKL1REVKV1TLKSKLVSDFRKDFQFYKVREINNYHHAHDA YLNAWGTALJKKYPKLESEFVYGDYKVYDVRKNDAKSEQE1GK ATAKYFFYSNlMNFFKTElTLANGEIRKRPLrETNGETGEIVWDKG RDFATVRKVLSMPQVNrVKKTEVQTGGFSKESILPKRNSDICIJAR KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLG 1T1MERSSFEKNP1DFLEAKGYKEVKKDL11KLPKYSLFELENGRKR MLASAGELQKGNELALPSKYVNFLYLASHYEKLK.GSPEDNEQKQ LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPI REQAENJIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI HQS1TGLYETRJDLSQLGGD 16 Cas9 nickase mRNA ORF encoding AUGGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAA CAGCG UCGGAUGGGCAGUCAUCACA GACG AAUACAAGGUCC CGAGCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACAC 17 2024224004   30 Sep 2024 SEQ ID NO: 16 using minimal uridine codons as listed in Table 3, with start and stop codons agcaucaagaagaaccugaucggagcacugcuguucgacag CGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAA GAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUG CAGGAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAG CUUCUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAG ACAAGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUC GACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCA CCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACC UGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUC AGAGGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAA CAGCGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAU ACAACCAGCUGLTUCGAAGAAAACCCGAUCAACGCAAGCGGA GUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAG CAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAA AGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUG GGACUGACACCGAACWCAAGAGCAACUUCGACCUGGCAGA AGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACG ACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCA GACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCU GC UG A GC G A C AUC C UG A GAG U CA AC A CAG AA AU CACA AAG G CACCGCUGAGCGC AA GC A UG AUC AAG AG AU AC G ACG AA CA C CACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCA GCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCA AGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAG GAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAU GGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAG ACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUC CCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGA AGACAGGAA GACUUCUA CCCGUUCCUG AA GGACAACA GAGA AAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACG UCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUG ACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGA AGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCG AAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAG GUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGU CUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAA UGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCA AUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGU CAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCU UCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAAC GCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAA GG AC A A GG A C UUCCUGGACAACGAAGAAAACGAAGACAUCC UGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGA GAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUU CGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACA CAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUC AGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAA GAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCC ACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCA CAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGC AAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGC AGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGA AGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGA AAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAA GAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGC CAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCA GAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAG 2024224004   30 Sep 2024 ACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGC GACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAG GACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAA GAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCG UCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCA AAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGC AGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCA UCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCAC GUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGA CGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACAC UGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAG UVCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACA CGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCA AGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGAC UACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGA AC AGG AA A UCGG AA AGGC AACA GCA AAGUAC UUC WCUAC A GCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCA AACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGG A G AA A C AGG A GA A A UC G U CUGG GAC AA GGG AAGAG ACUUCG CAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUC GUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGA AAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAA GAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGAC AGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGU CGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAAC UGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAG A A CCC GA UC GA C U UCC UGG A AGC AAAGGGAUACAAGGAAGU CAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGU UCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCA GGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAA GUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGC UGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUC GUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACA GAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAA ACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGAC AAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUU CACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACU UCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAG GAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGG ACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAG ACUAG Cas9 nickase coding sequence encoding SEQ ID NO: 16 using minimal uridine codons as listed in Table 3 (no start or stop codons; suitable for inclusion in GACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAG CGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGA GCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACACAGC AUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAGCGG A G AA A C AGC A GA A G CA A CA AG AC UGAAGAGAACAGCAAGAA G AAG AUAC AC AAG AAG AAAG A ACAG AAUC UG CUA CCUG CA G GAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUU CUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACA AGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUCGAC GAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCACCU GAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGA GACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGA GGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAG CGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAUACA ACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGAGUC 18 2024224004   30 Sep 2024 fusion protein coding sequence) GACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAG AAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGA AGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGA CUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGA CGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACC UGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGAC CUGUUCCUGGCAGCA AA GAACCUGA GCGACGC AAUCCUGCU GAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCAC CGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCAC CAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCU GCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGA ACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGGCAGGAA GAAUUCUACAAGUVCAUCAAGCCGAUCCUGGAAAAGAUGGA CGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACC UGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCG CACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAG ACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAA AGAUCGAAAAGAUCCUGACAUUCAGAA UCCCGUAC UACGUC GGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGAC A A GA A AG A GC GA A G AA A C AAUC AC AC CG UG GAA C UUCGAAG AAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAA AGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGU CCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCU ACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUG AGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAU CGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCA AGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUC GACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGC AAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGG ACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUG GAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGA AAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCG ACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACA GGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAG AGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGA GCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCAC GACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACA GGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAA ACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAG ACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAG ACACAAGCCGGAAAACAUCGUCAUC GAAAUG GC AAGA GA AA A C CA G AC A A CA C A GA A G GG A CAG A AG AA CAGC AGAGAAAGA A U GA AGAGAA UCGA AG AA GGAA UC A AGGAACUGGGAA GC C A GAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGA ACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGAC AUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGA CUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGA CGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGA ACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGUC AAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAA GCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAG AGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUC AAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGU CGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGACG AAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACUG AAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUU CUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACG 2024224004   30 Sep 2024 ACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAG AAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACUA CAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAAC AGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGC AACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAA CGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAG AAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGCA ACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCGU CAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAAA GCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGA AAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAG CCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCG AAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUG CUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAA CCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCA AGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGGCUGUUC GAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGGGCAGG AGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAGU ACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCUG AAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGU CGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGA UCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAAC CUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAA GCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCA CACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUC GACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGA A G UCC UGG A C GC A A CA C UG AU CC ACC AG AG CA UCAC AGGA C UGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGAC Amino acid sequence of dCas9 (without NLS) MDKKYSIGLA1GTNSVGWAV1TDEYKVPSKKFKVLGNTDRHSIK KNL1GALLFDSGETAEATRLKRTARRRYTRRKNR1CYLQE1FSNE MAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPT JYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS DVDKLF1QLVQTYNQLFEENPINASGVDAKAJLSARLSKSRRLENL 1AQLPGEKKNGLFGNLLALSLGLTPNFKSNFDLAEDAKLQLSKDT YDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA PLSASM1KRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA GYIDGGASQEEFYKFJKPJLEKMDGTEELLVKLNREDLLRK.QRTF DNGSIPHQJHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYV GPLARGNSRFAWMTRKSEET1TPWNFEEWDKGASAQSFIERMT N'FDKNLPN'EKVLPKJISLLYEYFTVYNELTKVKYVTEGMRKPAFL SGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKtECFDSVEISGVED RFNASLGTYl-IDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMt EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSG KT1LDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH EH! ANLAGS PA] KKG1LQT VKV VDEL VK VMGRHK PEN iV [EMARE NQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKL YLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNTYWRQLLNAKL1TQRKFD NLTKAERGGLSELDKAGFIKRQLVETRQ1TKHVAQ1LDSRMNTKY DENDKLJREVKV1TLKSKLVSDFRKDFQFYKVREINNYI-I1-IA1-1DA YLNAVVGTALlKKYPKLESEFVYGDYKVYDVRKMIAKSEQEtGK ATAKYFFYSN1MNFFKTE1TLANGEIRKRPL0ETNGETGEIVWDKG RDFATVRKVLSMPQVNrVKKTEVQTGGFSKESILPKRNSDKLIAR KKJDWDPKKYGGFDSPTVAYSVLWAKVEKGKSKKLKSVKELLG 1T1MERSSFEKNP1DFLEAKGYKEVKKDL1IKLPK.YSLFELENGRKR MLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ 19 2024224004   30 Sep 2024 LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPI REQAEN1IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI HQS1TGLYETRJDLSQLGGD mRNA ORF encoding SEQ ID NO: 19 using minimal uridine codons as listed in Table 3, with start assd stop codons auggacaagaaguacagcaucggacuggcaaucggaacaaa CAGCGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCC cgagcaagaaguucaagguccugggaaacacagacagacac AGCAUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAG CGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAA GAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUG CAGGAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAG CUUCUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAG ACAAGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUC GACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCA CC UG AGAAAGAAGC UGG U CG A CAGC AC AGACAAGGCAGACC UGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUC AGAGGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAA CAGCGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAU ACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGA GUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAG C AG A A GA C U GG A A AA C C U GA U CGC A CAGC UG CC GGGAGAAA AGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUG GGACUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGA AGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACG ACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCA GACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCU GCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGG CACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACAC CACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCA GCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCA AGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAG GAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAU GGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAG ACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUC CCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGA AGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGA AA AGAUCGAAAAGAUCCUGACAUUCAGAAU CCCGUACUACG UCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUG ACAAGAAAGAGCGAAGAAAC'AAUCACACCGUGGAACUUCGA AGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCG A A AG A AUG A C A A A C U UC G AC A A G A A CC UG CCG A ACG A A AA» G GUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGU CUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAA UGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCA AUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGU CAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCU UCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAAC GCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAA G G AC .AA GG A C UU C C UG G AC AA CG AA GA AAACG A AGACAUCC UGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGA GAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUU CGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACA CAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUC AGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAA GAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCC 20 2024224004   30 Sep 2024 ACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCA CAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGC AAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGC AGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGA A G AC A CA A G C CGG AA A AC AUC GUC A UC G AAAUG GCAAGAGA AAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAA GAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGC CAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCA GAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAG ACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGC GACUACGACGUCGACGCAAUCGUCCCGCAGAGCUUCCUGAA GGACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACA AGAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUC GUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGC AAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGG CAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUC AUCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCA CGUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACG ACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACA CUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCA GUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCAC ACGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUC A A G A A GU A CC CG A A GC U GG A A AGC G AAUUCGUCUACGG AGA CUACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCG AACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUAC AGCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGC AAACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACG GAGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUC GCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAU CGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGG AAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCA AGAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGA CAGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGG UCGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAA CUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAA GAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAG UCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUG UUCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGC AGGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCA AGUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAG CUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUU CGUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAAC AGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCA AACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGA CAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGU UCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUAC UUCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAA GGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAG GACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGA GACUAG dCas9 coding sequence encoding SEQ ID NO: 19 using GACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAG CGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGA GCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACACAGC AUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAGCGG AGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAAGAA GAAGAUACACAAGAAGAAAGA ACAGAAUCUGCUACCUGCAG 21 2024224004   30 Sep 2024 minimal uridine codons as listed in Table 3 (no start or stop codons; suitable for inclusion in fusion protein coding setpence) GAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUU CUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACA AGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUCGAC GAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCACCU GAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGA GACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGA GGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAG CGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAUACA ACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGAGUC GACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAG AAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGA AGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGA CUGACACCGAACUUCAAGAGCAACLTUCGACCUGGCAGAAGA CGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACC UGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGAC CUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCU GAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCAC CGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCAC CAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCU GCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGA ACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAA GAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGA CGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACC UGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCG CACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAG ACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAA AGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUC GG ACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGAC AAGA AAG AGCGA AG AA ACAAUCAC ACCGUGGAACUUCGAAG AAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAA AGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGU CCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCU ACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUG AGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAU CGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCA AGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUC GACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGC AAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGG ACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUG GAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGA AAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCG ACGACAAGGUCAUGAAGCAGCUGAAGAGA AGAA GAUAC A CA GGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAG AGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGA GCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCAC GACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACA GGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAA ACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAG ACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAG ACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAA ACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGA AUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCA GAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGA ACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGAC AUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGA CUACGACGUCGACGCAAUCGUCCCGCAGAGCUUCCUGAAGG acgacagcaucgacaacaagguccugacaagaagcgacaag 2024224004   30 Sep 2024 AACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGU CAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAA AGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCA GAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAU C A AG A G AC A GC UG GUCG AA AC AAG A CAG AU CAC AAAG CA CG UCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGAC GAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACU GAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGU UCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACAC GACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAA GAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACU ACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAA CAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAG CAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAA ACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGA GAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGC AACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCG UCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAA AGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAG A A AG A A GG A C UGGG AC CC GAAGAAGUACGGAGGAUUCG ACA GCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUC GAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACU GCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGA ACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUC AAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUU CGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAG G A G A A C UGC A G A A G GG A A AC G A AC UG GC AC UG CCG AG C A AG UACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCU GAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCG UCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAG AUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAA CCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACA AGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUC ACAC UGACA AACCUGGGAGCACCGGCAGCAUUCAAGUACUU CGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGG AAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGA CUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGA CGGAGGAGGAAGC Amino acid sequence of Cas9 with two nuclear localization signals as the C-terminal amino acids MDKKYSIGLDIGTNSVGWAVJTDEYXVPSKKFKVLGNTDRHSIK KNLJGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNE MAKVDDSFFHRLEESFLVEEDKKHERI-IPiFGNrVDEVAYI-IEKYPT IYHLRKKLVDSTDKABLRLIYLALAHMIKERGHFL1EGDLNPDNS DVDKLFJQLVQTYNQLFEENPTNASGVDAKAJLSARLSKSRRLENL IAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDT YDDDLDNLLAQlGDQYADLFLAAKNLSDAiLLSDlLRVNTElTKA PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA GYIDGGASQEEFYKF1KPILEKMDGTEELLVKLNREDLLRKQRTF DNGSIPHQ1H LGELHAILRRQEDF YPFLKDNREKf EK [LTFRIPYYV GPLARGNSRFAWMTRKSEETJTPWNFEEVVDKGASAQSFIERMT NFDKNLPNEKVLPKI-1SLLYEYFTVYNELTKVKYVTEGMRKPAFL SGEQKKAIVDLLFKTNRKVTVKQLKEDYFKK1ECFDSVEISGVED RFNASLGTYl-IDLLKJJKDKDFLDNEENEDlLEDIVLTLTLFEDREMl EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSG KT1LDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH EI-HANLAGSPAJKKG1LQTVKVVDELVKVMGRHKPENIVIEMARE NQTTQKGQKNSRERMKRIEEGIKELGSO1LKEHPVENTOLQNEKL 22 2024224004   30 Sep 2024 YLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKL1TQRKFD NLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKY DENDKLJREVKV1TLKSKLVSDFRKPFQFYK.VREJNNYHHAHDA YLNAWGTAL1KKYPKLESEFVYGDYK.VYDVRKMTAKSEQE1GK ATAKYFFYSNTMNFFKTE1TLANGEIRKRPLIETNGETGEIVWDKG RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLrAR KKDWDPKKYGGFDSPTVAYSVLWAKVEKGKSKKLKSVKELLG 1TIMERSSFEKNP1DFLEAKGYKEVKKDLIIKLPKYSLFELENGRKR MLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPI REQAEN1IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI HQS1TGLYETR1DLSQLGGD GSGSPKKK.RKVDGSPKKKRKVDSG Cas9 mRNA ORF encoding SEQ ID NO: 22 using minimal uridine codons as listed in Table 3, with start and stop codons AUGGACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAAA CAGCGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCC CGAGCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACAC AGCAUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAG CGGAGAAACAGCAGAAGCAACAAGACUGAAGAG AACAGCAA GAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUG CAGGAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAG CUUCUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAG ACAAGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUC GACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCA CCUGAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACC UGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUC AGAGGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAA CAGCGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAU ACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGA GUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAG CAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAA AGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUG GGACUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGA AGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACG ACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCA GACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCU GCUGAGCGACAUCCUGAGAGU CAACACAGAAAUCACAAAGG CACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACAC CACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCA GCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCA AGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAG GAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAU GGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAG ACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUC CCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGA AGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGA AAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACG UCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUG ACA AGAA AG AGCG A AGAA AC AAUC ACACCGUGGAACUUCGA AGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCG aaagaaugacaaacuucgacaagaaccugccgaacgaaaag GUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGU CUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAA UGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCA AUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGU 23 2024224004   30 Sep 2024 CAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCU UCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAAC GCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAA GGACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCC UGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGA GAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUU CGACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACA CAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUC AGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAA GAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCC ACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCA CAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGC AAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGC AGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGA AGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGGAAGAGA AAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAA GAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGG CAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCA GAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAG ACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGC GACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAG GACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAA GAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCG UCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCA AAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGC AGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCA UCAA GAGACAGCUGGUCGAAAC AAGACAGA UCA CAAAGCAC GUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGA CGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACAC UGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAG UUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACA CGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCA AGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGAC UACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGA ACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACA GCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCA AACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGG AGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCG CAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUC GUCAAGAAGACAGAAGUCCAGACAGGAG GAU UCA GCAA GGA AAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAA GAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGAC AGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGU CGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAAC UGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAG AACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGU CAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGU UCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCA GGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAG C AA GUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGC UGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUC GUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACA GAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAA ACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGAC AAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUU CACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACU UCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAG 2024224004   30 Sep 2024 GAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGG ACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAG ACGGAAGCGGAAGCCCGAAGAAGAAGAGAAAGGUCGACGGA AGCCCGAAGAAGAAGAGAAAGGUCGACAGCGGAUAG Cas'? coding sequence encoding SEQ ID NO: 23 using minimal uridine codons as listed in Table 3 (no start or stop codons; suitable for inclusion in fission protein coding sequence) GACAAGAAGUACAGCAUCGGACUGGACAUCGGAACAAACAG CGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGA GCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACACAGC AUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAGCGG AGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAAGAA GAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAG GAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUU CUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACA AGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUCGAC GAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCACCU GAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGA GACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGA GGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAG CGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAUACA ACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGAGUC GACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAG A A GA CUGG A A AA CC UGA U CGC AC AG CUGC CG GGAGA AAAGA AGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGA CUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGA CGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACC UGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGAC CUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCU GAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCAC CGCUGAGCGCAAGCAUGAUCA AGAGAUACGACGAACACCAC CAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCU GCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGA ACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAA GAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGA CGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACC UGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCG CACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAG ACAGGAAGAC U UCUACCCGU UCCUGAAGGACAACAGAGA A A AGAUCGAAAAGA UCCUGACAUUCAGAAUCCCGUACUACG UC GGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGAC AAGAAAGAGCGAAGAAACAAUCACAC CG UG GAA C UUCGAAG A A GUCG UCG AC A A GGG A G CA AG CGC A CAGA GCU UCAU CGAA A G AA U G AC A AC UU CG A C AAG A ACC U G C C G A AC G A AA AG GU CCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCU ACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUG AGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAU CGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCA AGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUC GACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGC AAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGG ACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUG GAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGA AAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCG ACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACA GGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAG AGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGA 24 2024224004   30 Sep 2024 GCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCAC GACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCACA GGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAA ACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAG ACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAG ACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAA ACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGA AUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCA GAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGA ACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGAC AUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGA CUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGA CGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAGA ACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGUC AAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAA GCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAG AGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUC AAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGU CGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGACG AAAACGACAAGCUGAUCAGAGAAGUCAAGG UCAUCACA CUG AAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUU CUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACG ACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAG AAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACUA CAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAAC AGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACAGC AACAUCAUGAACUUCUUCAAGACAG AAAUCACACUGGCAAA CGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAG AAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGCA ACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCGU CAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAAA GCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGA AAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAG CCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGG UCG AAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACUG CUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAA CCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCA AGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUC GAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGG AGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAGU ACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCUG AAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGU CGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGA UCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAAC CUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAA GCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCA CACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUC GACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGGA AGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGAC UGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGAC GGAAGCGGAAGCCCGaAGAAGAAGAGAAAGGUCGACGGAAG CCCGAAGAAGAAGAGAAAGGUCGACAGCGGA Amino acid sequence of MDKKYSlGLAIGTNSVGWAViTDEYKVPSKKFKVLGNTDRHSlK KNLJGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNE 25 2024224004   30 Sep 2024 Cas9 nickase with two nuclear localization signals as the C- ter min al amino acids MAKVDDSFFHRLEESFLVEEDKKHERHPiFGNIVDEVAYI-TEKYPT IYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS DVDKLF1QLVQTYNQLFEENPINASGVDAKA1LSARLSKSRRLENL IAQLPGEKKNGLFGNLJALSLGLTPNFKSNFDLAEDAKLQLSK.DT YDDDLDNLLAQTGDQYADLFLAAKNLSDAILLSDILRVNTEITKA PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSK.NGYA GYIDGGASQEEFYKF1KPILEKMDGTEELLVKLNREDLLRKQRTF DNGSIPHQiHLGELHAlLRRQEDm’FLKDNTlEKIEKILTFRIPYYV GPLARGNSRFAWMTRKSEETITPWNFEEWDKGASAQSFIERMT NFDKNLPNEKVLPKI-1SLLYEYFTVYNELTKVKYVTEGMRKPAFL SGEQKKAWDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVED RFNASLGTYHDLLKIlKDKDFLDNEENEDILEDIVLTLTLFEDREMi EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKL1NGIRDKQSG KTJLDFLKSDGFANRNFMQLfl-iDDSLTFKEDIQKAQVSGQGDSLH EH 1ANLA GSPA1KKG1LQT VK VVDELVK VMGRHK PEN IV [EM ARE NQ1TQKGQKNSRERMKRTEEGIKELGSQ1LKEHPVENTQLQNEKL YLYYLQNGRDMYVDQELDtNRLSDYDVDHIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKXMKNYWRQLLNAKLiTQRKFD NLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKY DENDKL1REVKV1TLKSKLVSDFRKDFQFYKVREINNYHHAHDA YLNAVVGTALJKK.YPKLESEFVYGDYKVYDVRKM1AK.SEQEIGK ATAKYFFYSN1MNFFKTE1TLANGEIRKRPLIETNGETGEIVWDKG RDFATVRKVLSMPQVNrVKKTEVQTGGFSKESILPKRNSDKLIAR KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLG ITlMERSSFEKNPiDFLEAKGYKEVKKDLlfKLPKYSLFELENGRKR MLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPI REQAEN1IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI HQS1TGLYETR1DLSQLGGDGSGSPKKKRKVDGSPKKKRKVDSG Cas9 nickase mRNA ORF encoding SEQ ID NO: 25 using minimal uridine codons as listed in Table 3, with start and stop codons AUGGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAA CAGCGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCC CGAGCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACAC AGCAUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAG CGGAGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAA GAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUG CAGGAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAG CUUCUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAG acaagaagcacgaaagacacccgaucuucggaaacaucguc GACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCA ccugagaaagaagcuggucgacagcacagacaaggcagacc UGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUC AGAGGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAA CAGC.GACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAU ACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGA GUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAG CAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAA AGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUG GGACUGACACCGAACUTJCAAGAGCAACUUCGACCUGGCAGA AGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACG ACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCA GACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCU GCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGG CACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACAC CACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCA 26 2024224004   30 Sep 2024 GCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCA AGAACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAG GAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAU GGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAG ACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUC CCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGA AGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGA AAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACG UCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUG ACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGA AGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCG AAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAG GUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGU CUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAA UGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCA AUCGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGU CAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCU UCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAAC GCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAA GGACAAGGAC UUCCUGGACAACGAAGAAAACGAAGACAUCC UGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGA GAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUU CGA C G AC A A GG UC AUG A AGC A GC UG AAG A GAAG AAGA UA CA CAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUC AGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAA GAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCC ACGACGAC AGCCUGACAUUCAAGGAAG AC AUCC AGAA GG CA CAGGUCAGCGGACAGGGAG ACAGCCUGCACGAACACAUCGC AAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGC AGACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGA AGACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGA AAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAA GAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGC CAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCA GAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAG ACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGC GACUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAG GACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAA GAACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCG UCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCA AAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGC AGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCA UCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCAC GUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGA CGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACAC UGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAG UUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACA CGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCA AGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGAC UA CAA GG UC UAC G A CG U CAG AAAGAUGAUCGCAAAGAGCGA ACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUACA GCAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCA AACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGG AGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCG CAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUC GUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGA AAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAA 2024224004   30 Sep 2024 GAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGAC AGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGU CGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAAC UGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAG AACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGU CAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGU UCGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCA GGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAA GUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGC (JGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUC GUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACA GAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAA ACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGAC AAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUU CACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACU UCGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAG GAAG UCCUGGACGCAACACUGA UCCACCA GAGCAUCACAGG ACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAG ACGGAAGCGGAAGCCCGAAGAAGAAGAGAAAGGUCGACGGA A G CCC GA A GAA G A AG A G AA AG GUC G A CAGC GGA UAG Cas') nickase coding sequence encoding SEQ ID NO: 25 using minimal uridine codons as listed in Table 3 (no start or stop codons; suitable for inclusion in fusion protein coding sequence) GACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAG CGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGA GCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACACAGC AUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAGCGG A G AA A C AGC A GA A G CA A CA AG AC UGAAGAGAACAGCAAGAA GAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUGCAG GAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCDU CUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACA AGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUCGAC GAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCACCU GAGAAAGAAGCL'GGUCGACAGCACAGACAAGGCAGACCUGA GACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGA GGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAG CGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAUACA ACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGAGUC GACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAG AAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGA AGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGA CUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGA CGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACC UGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGAC CUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCU GAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCAC CGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACACCAC CAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCU GCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGA ACGGAUACGCAGGAUACAUCGACGGAGGA GCAAGCCAGGAA GAAUUCUACAAGUVCAUCAAGCCGAUCCUGGAAAAGAUGGA CGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACC (JGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCG CACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAG ACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAA AGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUC GGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGAC AAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAG AAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAA 27 2024224004   30 Sep 2024 AGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAGGU CCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCU ACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUG AGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAU CGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCA AGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCUUC GACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGC AAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGG ACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUG GAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGAGA AAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCG ACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACA GGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAG AGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGA GCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCCAC GACGACAGCCUGACA UUCAAGGAAGACAUCCAGAAGGCACA GGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAA ACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAG ACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAG A C AC A A GCC GG A A A A C A UC G U C AUC GAAAUG GC AAGA GAAA A C CA G A C AA C A C A GA A G GG A CA G A AG AACAGCAGAGAAAGA AUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCA GAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGA ACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGAC AUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGA CUACGACGUCGACCACAUCGUCCCGCAGAGCUUCCUGAAGGA CGACAGCAUCGACAACAAGGUCC UGACAAGAAGCGACAAGA ACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGUC AAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAAA GCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCAG AGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAUC AAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACGU CGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGACG AAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACUG AAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGUU CUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACACG ACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAAG AAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACUA CAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAAC AGGAAAUCGGAAAGGC'AACAGCAAAGUACUUCUUCUACAGC AACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAAA CGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGAG AA AC A GG A GAAAUCGUCUGGGACAAGGGAAGAGACUUCGCA ACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCGU CAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAAA GCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAGA AAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACAG CCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUCG AAAAGGGAAAGAGCAAGAAGCUGAAGAGCG UCAA GGAA CU G C U GGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAAGAA CCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUCA AGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUGUUC GAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAGG AGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAGU ACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCUG AAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCGU CGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAGA 2024224004   30 Sep 2024 UCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAAC CUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACAA GCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUCA CACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUUC G A CA C AA C A AUCG AC A G AA AG AG AUA CACAAGCACAAAGGA AGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGAC UGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGAC GGAAGCGGAAGCCCGAAGAAGAAGAGAAAGGUCGACGGAAG CCCGAAGAAGAAGAGAAAGGUCGACAGCGGA Amino acid sequence of dCas9 with two nuciear idealization signals as the C-terminal amino acids MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIK KNL1GALLFDSGETAEATRLKRTARRRYTRRKNRICYLQE1FSNE MAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPT rYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS DVDKLFJQLVQTYNQLFEENPTNASGVDAKAJLSARLSKSRRLENL IAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDT YDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA PLSASM1KRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA GYIDGGASQEEFYKFJKPILEKMDGTEELLVKLNREDLLRKQRTF DNGSIPHQ1H LGELHAILRRQEDF YPFLKDNREKf EK [LTFRIPYYV GPLARGNSRFAWMTRKSEET1TPWNFEEVVDKGASAQSFIERMT NFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL SGEQKKArVDLLFKTNRKVTVKQLKEDYFKKIECFDSVETSGVED RFNASLGTYHDLLKDKDKDFLDNEENEDILEDIVLTLTLFEDREMt EERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSG KT1LDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH EI-DANLAGSPAlKKGlLQTVKVVDELVKVMGRHKPENrviEMARE NQTTQKGQKNSRERMKRIEEGIKELG SQ1LKEHPV ENTQLQNEKL YLYYLQNGRDMYVDQELDTNRLSDYDVDAIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKL1TQRKFD NLTKAERGGLSELDKAGFIKRQLVETRQ1TKHVAQILDSRMNTKY DENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDA YLNAWGTAL1KKYPKLESEFVYGDYK.VYDVRKMTAKSEQEIGK ATAKYFFYSNhWFFKTElTLANGEIRKRPLnETNGETGEmVDKG RDFATVRKVLSMPQVNfVKKTEVQTGGFSKESlLPKRNSDKLlAR KKDWDPKKYGGFDSPTVAYSVLWAKVEKGKSKKLKSVKELLG 1T1MERSSFEKNP1DFLEAKGYKEVKKDL1IKLPKYSLFELENGRKR MLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPI REQAENUHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATU HQS1TGLYETR1DLSQLGGDGSGSPKKKRKVDGSPKKKRKVDSG 28 dCas9 mRNA ORF encoding SEQ ID NO: 28 using minimal uridine codons as listed in Table 3, with start astd stop codons AUGGACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAA CAGCGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCC CGAGCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACAC AGCAUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAG C G G A G AA A C A GC A G AA GC AAC AAG A CUG AAG AG AAC AGC AA GAAGAAGAUACACAAGAAGAAAGAACAGAAUCUGCUACCUG CAGGAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAG CUUCUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAG ACAAGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUC GACGAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCA CCUGAGAAAGAAGCUGGUCGACAGCACAG ACAA GGCAGACC UGAGACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUC AGAGGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAA CAGCGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAU 29 2024224004   30 Sep 2024 ACAACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGA GUCGACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAG CAGAAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAA AGAAGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUG GGACUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGA AGACGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACG ACCUGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCA GACCUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCU GCUGAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGG CACCGCUGAGCGCAAGCAUGAUCAAGAGAUACGACGAACAC CACCAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCA GCUGCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCA AGAACGGA U A CGCAGGAUACAUCGACGGAGGAGCAAGCCAG GAAGAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAU GGACGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAG ACCUGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUC CCGCACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGA AGACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGA AAAGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACG UCGGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUG ACAAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGA AGAAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCG AAAGAAUGACAAACUUCGACAAGAACCUGCCGAACGAAAAG GUCCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGU CUACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAA UGAGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCA A U CG UCGACCUGCUG UUCAAGACAAACAGAAAGGUCACAGU CAAGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCU UCGACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAAC GCAAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAA GGACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCC UGGAAGACAUCGUCCUGACACUGACACUGUUCGAAGACAGA GAAAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUU CGACGACAAGG GCA UGAAGCAGC UGAAGAGAAGAAGAUACA CAGGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUC AGAGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAA GAGCGACGGAUUCGCAAACAGAAACUUCAUGCAGCUGAUCC ACGACGACAGCCUGACAUUCAAGGAAGACAUCCAGAAGGCA CAGGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGC AAACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGC agacagucaaggucgucgacgaacuggucaaggucauggga A G AC A CA A G C CGG AA A AC AUCGUC A UC GAAAUGGCAAGAGA AAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAA GAAUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGC CAGAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCA GAACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAG ACAUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGC GACUACGACGUCGACGCAAUCGUCCCGCAGAGCUUCCUGAA GGACGACAGCAUCGACAACAAGGUCCUGACAAG AAGC GA CA A G AA C A GA GG AA AGAGCGACAACGUCCCGA GCG AAGA AGU C GUCAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGC AAAGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGG CAGAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUC AUCAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCA CGUCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACG ACGAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACA CUGAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCA 2024224004   30 Sep 2024 GUUCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCAC ACGACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUC AAGAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGA CUACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCG AACAGGAAAUCGGAAAGGCAACAGCAAAGUACUUCUUCUAC AGCAACAUCAUGAACUUCL'UCAAGACAGAAAUCACACUGGC AAACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACG GAGAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUC GCAACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAU CGUCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGG AAAGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCA AGAAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGA CAGCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGG UCGAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAA CUGCUGGGAAUCACAAUCAUGGAAAGAAGCAGCUUCGAAAA GAACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAG UCAAGAAGGACCUGAUCAUCAAGCUGCCGAAGUACAGCCUG UVCGAACUGGAAAACGG AAGAAAGAGAAUGCUGGCAAGCGC AGGAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCA AGUACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAG CUGAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUU CGUCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAAC AGAUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCA AACCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGA CAAGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGU UCACACUGACAAACCUGGGAGCACCGGCAGCAUUCAAGUAC UU CGACACAACAA UCGACAGAAAGAGAUACACAAGCACAAA GGAAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAG GACUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGA GAC GGAAGCGGAAGCCCGAAGAAGAAGAGAAAGGUCGACGGAAG CCCGAAGAAGAAGAGAAAGGUCGACAGCGGAUAG dCas? coding sequence encoding SEQ JDNO: 28 using minimal uridine codons as listed in Table 3 (no start or stop codons; suitable for inclusion in fusion protein coding sequence) GACAAGAAGUACAGCAUCGGACUGGCAAUCGGAACAAACAG CGUCGGAUGGGCAGUCAUCACAGACGAAUACAAGGUCCCGA GCAAGAAGUUCAAGGUCCUGGGAAACACAGACAGACACAGC AUCAAGAAGAACCUGAUCGGAGCACUGCUGUUCGACAGCGG AGAAACAGCAGAAGCAACAAGACUGAAGAGAACAGCAAGAA G A AGAUAC AC AAG AAG A A AG A ACAG AAUC UGCUACCUGCAG GAAAUCUUCAGCAACGAAAUGGCAAAGGUCGACGACAGCUU CUUCCACAGACUGGAAGAAAGCUUCCUGGUCGAAGAAGACA AGAAGCACGAAAGACACCCGAUCUUCGGAAACAUCGUCGAC GAAGUCGCAUACCACGAAAAGUACCCGACAAUCUACCAC CU GAGAAAGAAGCUGGUCGACAGCACAGACAAGGCAGACCUGA GACUGAUCUACCUGGCACUGGCACACAUGAUCAAGUUCAGA GGACACUUCCUGAUCGAAGGAGACCUGAACCCGGACAACAG CGACGUCGACAAGCUGUUCAUCCAGCUGGUCCAGACAUACA ACCAGCUGUUCGAAGAAAACCCGAUCAACGCAAGCGGAGUC GACGCAAAGGCAAUCCUGAGCGCAAGACUGAGCAAGAGCAG AAGACUGGAAAACCUGAUCGCACAGCUGCCGGGAGAAAAGA AGAACGGACUGUUCGGAAACCUGAUCGCACUGAGCCUGGGA CUGACACCGAACUUCAAGAGCAACUUCGACCUGGCAGAAGA CGCAAAGCUGCAGCUGAGCAAGGACACAUACGACGACGACC UGGACAACCUGCUGGCACAGAUCGGAGACCAGUACGCAGAC CUGUUCCUGGCAGCAAAGAACCUGAGCGACGCAAUCCUGCU GAGCGACAUCCUGAGAGUCAACACAGAAAUCACAAAGGCAC CGCUGAGCGCAAGCAUGAUCA AGAGAUACGACGAACACCAC 30 2024224004   30 Sep 2024 CAGGACCUGACACUGCUGAAGGCACUGGUCAGACAGCAGCU GCCGGAAAAGUACAAGGAAAUCUUCUUCGACCAGAGCAAGA ACGGAUACGCAGGAUACAUCGACGGAGGAGCAAGCCAGGAA GAAUUCUACAAGUUCAUCAAGCCGAUCCUGGAAAAGAUGGA CGGAACAGAAGAACUGCUGGUCAAGCUGAACAGAGAAGACC UGCUGAGAAAGCAGAGAACAUUCGACAACGGAAGCAUCCCG CACCAGAUCCACCUGGGAGAACUGCACGCAAUCCUGAGAAG ACAGGAAGACUUCUACCCGUUCCUGAAGGACAACAGAGAAA AGAUCGAAAAGAUCCUGACAUUCAGAAUCCCGUACUACGUC GGACCGCUGGCAAGAGGAAACAGCAGAUUCGCAUGGAUGAC AAGAAAGAGCGAAGAAACAAUCACACCGUGGAACUUCGAAG AAGUCGUCGACAAGGGAGCAAGCGCACAGAGCUUCAUCGAA AGAAUGACAAACWCGACAAGAACCUGCCGAACGAAAAGGU CCUGCCGAAGCACAGCCUGCUGUACGAAUACUUCACAGUCU ACAACGAACUGACAAAGGUCAAGUACGUCACAGAAGGAAUG AGAAAGCCGGCAUUCCUGAGCGGAGAACAGAAGAAGGCAAU CGUCGACCUGCUGUUCAAGACAAACAGAAAGGUCACAGUCA AGCAGCUGAAGGAAGACUACUUCAAGAAGAUCGAAUGCWC GACAGCGUCGAAAUCAGCGGAGUCGAAGACAGAUUCAACGC AAGCCUGGGAACAUACCACGACCUGCUGAAGAUCAUCAAGG ACAAGGACUUCCUGGACAACGAAGAAAACGAAGACAUCCUG GAAGACAUCGUCCUGACACUGACACUGUBCGAAGACAGAGA AAUGAUCGAAGAAAGACUGAAGACAUACGCACACCUGUUCG ACGACAAGGUCAUGAAGCAGCUGAAGAGAAGAAGAUACACA GGAUGGGGAAGACUGAGCAGAAAGCUGAUCAACGGAAUCAG AGACAAGCAGAGCGGAAAGACAAUCCUGGACUUCCUGAAGA GC G A CGG A U UC GC A A A C A G A A AC GU CAUG CAGC UGAU CCA C GACGACAGCCUGACAUVCAAGGAAGACAUCCAGAAGGCACA GGUCAGCGGACAGGGAGACAGCCUGCACGAACACAUCGCAA ACCUGGCAGGAAGCCCGGCAAUCAAGAAGGGAAUCCUGCAG ACAGUCAAGGUCGUCGACGAACUGGUCAAGGUCAUGGGAAG ACACAAGCCGGAAAACAUCGUCAUCGAAAUGGCAAGAGAAA ACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAAAGA AUGAAGAGAAUCGAAGAAGGAAUCAAGGAACUGGGAAGCCA GAUCCUGAAGGAACACCCGGUCGAAAACACACAGCUGCAGA ACGAAAAGCUGUACCUGUACUACCUGCAGAACGGAAGAGAC AUGUACGUCGACCAGGAACUGGACAUCAACAGACUGAGCGA CUACGACGUCGACGCAAUCGUCCCGCAGAGCUUCCUGAAGG ACGACAGCAUCGACAACAAGGUCCUGACAAGAAGCGACAAG AACAGAGGAAAGAGCGACAACGUCCCGAGCGAAGAAGUCGU CAAGAAGAUGAAGAACUACUGGAGACAGCUGCUGAACGCAA AGCUGAUCACACAGAGAAAGUUCGACAACCUGACAAAGGCA GAGAGAGGAGGACUGAGCGAACUGGACAAGGCAGGAUUCAU CAAGAGACAGCUGGUCGAAACAAGACAGAUCACAAAGCACG UCGCACAGAUCCUGGACAGCAGAAUGAACACAAAGUACGAC GAAAACGACAAGCUGAUCAGAGAAGUCAAGGUCAUCACACU GAAGAGCAAGCUGGUCAGCGACUUCAGAAAGGACUUCCAGU UCUACAAGGUCAGAGAAAUCAACAACUACCACCACGCACAC GACGCAUACCUGAACGCAGUCGUCGGAACAGCACUGAUCAA GAAGUACCCGAAGCUGGAAAGCGAAUUCGUCUACGGAGACU ACAAGGUCUACGACGUCAGAAAGAUGAUCGCAAAGAGCGAA CA GGAAA UCGGAAA GGCAACAGCAAAGUACUUCUUCUACAG CAACAUCAUGAACUUCUUCAAGACAGAAAUCACACUGGCAA ACGGAGAAAUCAGAAAGAGACCGCUGAUCGAAACAAACGGA GAAACAGGAGAAAUCGUCUGGGACAAGGGAAGAGACUUCGC AACAGUCAGAAAGGUCCUGAGCAUGCCGCAGGUCAACAUCG UCAAGAAGACAGAAGUCCAGACAGGAGGAUUCAGCAAGGAA 2024224004   30 Sep 2024 AGCAUCCUGCCGAAGAGAAACAGCGACAAGCUGAUCGCAAG AAAGAAGGACUGGGACCCGAAGAAGUACGGAGGAUUCGACA GCCCGACAGUCGCAUACAGCGUCCUGGUCGUCGCAAAGGUC GAAAAGGGAAAGAGCAAGAAGCUGAAGAGCGUCAAGGAACU GCUGGGAAUCACAAUCAUGGAAAGAAGCA GCUUC GAAAAGA ACCCGAUCGACUUCCUGGAAGCAAAGGGAUACAAGGAAGUC AAGAA GGACCUGA UCA UCAAGCUGCCGAAGUACAGCCUGUU CGAACUGGAAAACGGAAGAAAGAGAAUGCUGGCAAGCGCAG GAGAACUGCAGAAGGGAAACGAACUGGCACUGCCGAGCAAG UACGUCAACUUCCUGUACCUGGCAAGCCACUACGAAAAGCU GAAGGGAAGCCCGGAAGACAACGAACAGAAGCAGCUGUUCG UCGAACAGCACAAGCACUACCUGGACGAAAUCAUCGAACAG AUCAGCGAAUUCAGCAAGAGAGUCAUCCUGGCAGACGCAAA CCUGGACAAGGUCCUGAGCGCAUACAACAAGCACAGAGACA AGCCGAUCAGAGAACAGGCAGAAAACAUCAUCCACCUGUUC ACAC UGACAAACCUGGGAGCACCGGCAGCAUUCAAGUACUU CGACACAACAAUCGACAGAAAGAGAUACACAAGCACAAAGG AAGUCCUGGACGCAACACUGAUCCACCAGAGCAUCACAGGA CUGUACGAAACAAGAAUCGACCUGAGCCAGCUGGGAGGAGA c GG AA GC GG AAGC C CG A A GA AGAA G A GAA AGGUCGACGGAAG CCCGAAGAAGAAGAGAAAGGUCGACAGCGGA T7 promoter TAATACGACTCACTATA 31 Human betaglobin 5’ UTR ACATTTGCTTCTGACACAACTGTGITCACTAGCAACCTCAAAC AGACACC 32 Human betaglobin 3’ UTR GCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTr CCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCT TGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTG C 33 Human alpha-globin 5’ UTR CATAAACCCTGGCGCGCTCGCGGCCCGGCACTCTTCTGGTCCC CACAGACTCAGAGAGAACCCACC 34 Hitman alpha-giobin 3’ UTR GCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCC CCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTT TGAATAAAGTCTGAGTGGGCGGC 35 Xenoptts laevis be taglobin 5’ UTR AAGCTCAGAATAAACGCTCAACTTTGGCC 36 Xeitopus laevis be taglobin 3’ UTR ACCAGCCTCAAGAACACCCGAATGGAGTCTCTAAGCTACATA ATACCAACTTACACTTTACAAAATGTTGTCCCCCAAAATGTAG CCATTCGTATCTGCTCCTAATAAAAAGAAAGTTTCTTCACATTC T 37 Bovine Growth Hormone 5’ UTR CAGGGTCCTGTGGACAGCTCACCAGCT 38 Bovine Growth Hormone 3’ TTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGA CCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGA GGAAATTGCATCGCA 39 2024224004   30 Sep 2024 UTR Mus musculus hemoglobin alpha, adult chain 1 (Hba-al), 3’UTR GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTC TCCCTTGCACCTGTACCTCTTGGTCTTTGAATaAAGCCTGAGTA GGAAG 40 HSD17B4 5’ UTR TCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGT CGTTGCAGGCCTTATTC 41 G2S2 guide RNA targeting TTR mU*mU*mA*CAGCCACGUCUACAGCAGUUUUAGAmGm CmUmAmGmArnA mAmU mAmGmC AAGUUAAAAUAAGG C UAGUCCG UU AUC AmA mC mUmlfmG mA mAmA mAmAm GmUmGmGmCmA mCmCmGmAmGmUmCmGmGmUmGmC mU*mU*mU*mU 42 Cas9 transcript with 5’ UTR ofHSD, ORF corresponds ng to SEQ ID NO: 4, Kozak sequence, and 3’ UTR of ALB GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGT GTGTCGTTGCAGGCCTTATTCGGATCCGCCACCATGGACAAGA AGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGAT GGGCAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGT TCAAGGTCCTGGGAAACACAGACAGACACAGCATCAAGAAGA ACCTGATCGGAGCACTGCTGTTCGACAGCGGAGAAACAGCAG AAGCAACAAGACTGAAGAGAACAGCAAGAAGAAGATACACA AGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGC AACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTG GAAGAAAGCTTCCTGGTCGAAGAAGACAAGAAGCACGAAAGA CACCCGATCTTCGGAAACATCGTCGACGAAGTCGCATACCACG AAAAGTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCG AC AGC ACA G A CA A GGC AGACCTG AG ACTGATCTACCTGGCAC TGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGG AGACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCAT CCAGCTGGTCCAGACATACAACCAGCTGTTCGAAGAAAACCC GATCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAGCGC AAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCACA GCTGCCGGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGAT CGCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTC G A CC TG GC A G A A G AC GC A A A G CTGC AG C TG A GC A AGG AC A C A TACGACGACGACCTGGACAACCTGCTGGCACAGATCGGAGAC CAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGAC GCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATC ACAAAGGCACCGCTGAGCGCAAGCATGATCAAGAGATACGAC GAACACCACCAGGACCTGACACTGCTGAAGGCAC1GGTCAGA CAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAG AGCAAGAACGGATACGCAGGATACATCGACGGAGGAGCAAGC CAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAG ATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAA GACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATC CCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGA AGACAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAA AAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCG GACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAA GAAAGAGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAG TCGTCGAC A AGGG AGC A AGCGCACA GAGCTTCATCGA AAG A A 43 2024224004   30 Sep 2024 TGACAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGC CGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGA ACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCC GGCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCT GCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGCTGAA GGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGA A ATC AGCGG AGTCG A A GAC AGATTC AACGC AA GCCTGGG AA C ATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTG GACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTG ACACTGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGA CTGAAGACATACGCACACCTGTTCGACGACAAGGTCATGAAG CAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAG CAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAA AGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACA G A AACTTC ATGC AGCTG ATCCACG ACGAC A GCCTGAC ATTC AA GGAAGACATCCAGAAGGCACAGGTCAGCGGACAGGGAGACA GCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCAA TCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAAC TGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCA TCG A A ATGG CA A G AG A A AAC C A GAC AAC ACAGAAGGGACAG AA G A A C AGC A G A G A AA GAATGAAGAG AAT CG AA GAA GGA AT CAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCGA AAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCT GCAGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACAT CAACAGACTGAGCGACTACGACGTCGACCACATCGTCCCGCA GAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGAC AAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGA GCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGC TGCTGAACGC A A AGCTGATC AC ACAG AGAAA GTTCGACA ACC TGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAG GCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATC ACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACA AAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTC ATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGAC TTCCA GTTCTACA AGGTC AGAG A A ATC A AC AA CTACC ACC ACG C ACA CGA CGC ATACCTGA ACGC AGTCGTCGG AACA GCACTGA TCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAG ACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCG AACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACA GCAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAA ACGGAGAAATCAGAAAGAGACCGCTGATCGAAA C A AA CGG A GAAACAGGAGAAATCGTCTGGGAC AAGG GA AGA GAC TT CG C A ACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTC AAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAG CATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCAAGAAA GAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCC GACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAA GGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGG gaatcacaatcatggaaagaagcagcttcgaaaagaacccga TCGACTTCCTGGAAGC AAAGGG ATAC AAGGAAG1C AAGAAGG ACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGA aaacggaagaaagagaatgctggcaagcgcaggagaactgc AGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACT TCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCC CGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACA AGCACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCA GCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCC 2024224004   30 Sep 2024 TG A GC GC AT A C A AC A A GC AC A G AG A C A A GC CG ATC AG AG A A C AGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGG GAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGACA GAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAACAC TGATCCACCAGAGCATCACAGGACTGTACGAAACAAGAATCG ACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAG AAGAAGAGAAAGGTCTAGCTAGCCATCACATTTAAAAGCATC TCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAAT AGCTTATTCATCTCTTnTCTTTTTCGTTGGTGTAAAGCCAACA CCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTT TCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCTCGAG Cas9 transcript with 5’ UTR ofHSD, ORF correspond! ng to SEQ ID NO: 4, and 3 ’ UTR of ALB GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGT GTGTGTCGTTGCAGGCCTTATTCGGATCCATGGACAAGAAGTA CAGCATCGGACTGGACATCGGAACAAACAGCGTCGGATGGGC AGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCAA GGTCCTGGGAAACACAGACAGACACAGCATCAAGAAGAACCT GATCGGAGCACTGCTGTTCGACAGCGGAGAAACAGCAGAAGC AACAAGACTGAAGAGAACAGCAAG AAG AA GAT A CAC AAGA A GAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCAACG AAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAAG AAAGCTTCCTGGTCGAAGAAGACAAGAAGCACGAAAGACACC CGATCTTCGGAAACATCGTCGACGAAGTCGCATACCACGAAA AGTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCGACA GCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCACTGG CACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGAG ACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCATCC AGCTGGTCCAGACATACAACCAGCTGTTCGAAGAAAACCCGA TCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAGCGCAA GACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCACAGC TGCCGGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGATCG CACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTCG ACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACAT ACGACGACGACCTGGACAACCTGCTGGCACAGATCGGAGACC AGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGACG CAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATCA CAAAGGCACCGC1GAGCGCAAGCA1GATCAAGAGAJACGAUG AACACCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGAC AGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAGA GCAAGAACGGATACGC'AGGATACATCGACGGAGGAGCAAGCC AGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAGA TGG A C GG AAC A G A A G A A CTG CTG GT C A A GC TG AAC AG AG A A G ACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATCC CGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGAA GACAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAAA AGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCGG ACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAAG AAAGAGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAGT CGTCGACjAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAAT GACAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGCC GAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGAA CTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCCG GCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCTG CTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGCTGAAG GAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGAA 44 2024224004   30 Sep 2024 ATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAACA TACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGG ACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTGA CACTGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGAC TGAAGACATACGCACACCTGTTCGACGACAAGGTCATGAAGC AGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAGC agaaagctgatcaacggaatcagagacaagcagagcggaaa GACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACAG AAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAAG GAAGACATCCAGAAGGCACAGGTCAGCGGACAGGGAGACAG CCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCAAT CAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAACT GGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCAT CGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAGA AGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAATC AAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCGAA AACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCTG CAGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACATC AACAGACTGAGCGACTACGACGTCGACCACATCGTCCCGCAG AGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGACA A G AA GC G A C A AG AA C A G A GG A A AG AG CG AC AACG TCCCG AG CGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGCT GCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACCT GACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAGG CAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATCA CAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACAA A GTA C GA CG AA A A CG A C AAGCTGATCAGAGAAGTCAAGGTCA TCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGACT TCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACCACGC ACACGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGAT CAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAGA CTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCGA ACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACAG CAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAAAC GGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGAGA AACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCAAC AGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTCAA GAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAGCAT CCTGCCGAAGAGAAACAGCGACAAGCTGATCGCAAGAAAGAA GGACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCCGAC AGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAAGGG AAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGGGAAT CACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGATCGA CTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGGACCT GATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGAAAA CGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGCAGA AGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACTTCC TGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCCCGG AAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACAAGC A CT A C CTGG AC G A AA TC A TC G A AC AG AT C AG C G AA TT C A GC A AGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCCTGA GCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAACAGG CAGAAAACATCATCCACCTGTTCACACTGACAAACCTGGGAGC ACCGGCAGCATTCAAGTACTTCGACACAACAATCGACAGAAA GAGATACACAAGCACAAAGGAAGTCCTGGACGCAACACTGAT CCA CCAGAGCATC ACAGG ACTGTACG AAAC A AGAATCGACCT GAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAGAAGA 2024224004   30 Sep 2024 AGAGAAAGGTCTAGCTAGCCATCACATTTAAAAGCATCTCAGC CTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTT ATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTG TCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCT GTGCTTCAATTAATAAAAAATGGAAAGAACCTCGAG Alternative Cas9 ORI-' with 19.36% U content ATGGATAAGAAGTACTCGATCGGGCTGGATATCGGAACTAATT CCGTGGGTTGGGCAGTGATCACGGATGAATACAAAGTGCCGT CCAAGAAGTTCAAGGTCCTGGGGAACACCGATAGACACAGCA TCAAGAAGAATCTCATCGGAGCCCTGCTGTTTGACTCCGGCGA AACCGCAGAAGCGACCCGGCTCAAACGTACCGCGAGGCGACG CTACACCCGGCGGAAGAATCGCATCTGCTATCTGCAAGAAATC TTTTCGAACGAAATGGCAAAGGTGGACGACAGCTTCTTCCACC GCCTGGAAGAATCTnCCTGGTGGAGGAGGACAAGAAGCATG AACGGCATCCTATCTTTGGAAACATCGTGGACGAAGTGGCGTA CCACGAAAAGTACCCGACCATCTACCATCTGCGGAAGAAGTT GGTTGACTCAACTGACAAGGCCGACCTCAGATTGATCTACTTG GCCCTCGCCCATATGATCAAATTCCGCGGACACTTCCTGATCG aaggcgatctgaaccctgataactccgacgtggataagctgtt CATTCAACTGGTGCAGACCTACAACCAACTGTTCGAAGAAAAC CCAATCAATGCCAGCGGCGTCGATGCCAAGGCCATCCTGTCCG cccggctgtcgaagtcgcggcgcctcgaaaacctgatcgcaca GCTGCCGGGAGAGAAGAAGAACGGACTTTTCGGCAACTTGAT CGCTCTCTCACTGGGACTCACTCCCAATTTCAAGTCCAATTTTG ACCTGGCCGAGGACGCGAAGCTGCAACTCTCAAAGGACACCT ACGACGACGACTTGGACAATTTGCTGGCACAAATTGGCGATCA GTACGCGGATCTGTTCCTTGCCGCTAAGAACCn TCGGACGCA ATCTTGCTGTCCGATATCCTGCGCGTGAACACCGAAATAACCA AAGCGCCGCTTAGCGCCTCGATGATTAAGCGGTACGACGAGC ATCACCAGGATCTCACGCTGCTCAAAGCGCTCGTGAGACAGCA ACTGCCTGAAAAGTACAAGGAGATTTTCTTCGACCAGTCCAAG AATGGGTACGCAGGGTACATCGATGGAGGCGCCAGCCAGGAA GAGrrCTATAAGTTCATCAAGCCAATCCTGGAAAAGATGGACG GAACCGAAGAACTGCTGGTCAAGCTGAACAGGGAGGATCTGC TCCGC A AAC AGAGA ACCTTTGACA ACGG AAGC ATTCCACACC AGATCCATCTGGGTGAGCTGCACGCCATCTTGCGGCGCCAGGA GGACJTriACCCATJCCrCAAGGACAAC'CGGGAAAAGATCGA GAAAATTCTGACGTrCCGCATCCCGTATTACGTGGGCCCACTG GCGCGCGGCAATTCGCGCTTCGCGTGGATGACTAGAAAATCA GAGGAAACCATCACTCCTTGGAATTTCGAGGAAGTTGTGGATA AGGGAGCTTCGGCACAATCCTTCATCGAACGAATGACCAACTT CGACAAGAATCTCCCAAACGAGAAGGTGCTTCCTAAGCACAG CCTCCTTTACGAATACTrCACTGTCTACAACGAACTGACTAAA GTGAAATACGTTACTGAAGGAATGAGGAAGCCGGCCTTTCTG AGCGGAGAACAGAAGAAAGCGATTGTCGATCTGCTGTTCAAG ACCAACCGCAAGGTGACCGTCAAGCAGCTTAAAGAGGACTAC TTCAAGAAGATCGAGTGTTTCGACTCAGTGGAAATCAGCGGA GTGGAGGACAGATTCAACGCTTCGCTGGGAACCTATCATGATC TCCTGAAGATCATCAAGGACAAGGACTTCCTTGACAACGAGG AGAACGAGGACATCCTGGAAGATATCGTCCTGACCTTGACCCT TTTCGAGGATCGCGAGATGATCGAGGAGAGGCTTAAGACCTA CGCTCATCTCTTCGACGATAAGGTCATGAAACAACTCAAGCGC CGCCGGTACACTGGTTGGGGCCGCCTCTCCCGCAAGCTGATCA ACGGTATTCGCGATAAACAGAGCGGTAAAACTATCCTGGATTT CCTCAAATCGGATGGCTTCGCTAATCGTAACTTCATGCAGTTG 45 2024224004   30 Sep 2024 ATCCACGACGACAGCCTGACCTTTAAGGAGGACATCCAGAAA GCACAAGTGAGCGGACAGGGAGACTCACTCCATGAACACATC GCGAATCTGGCCGGTTCGCCGGCGATTAAGAAGGGAATCCTG CAAACTGTGAAGGTGGTGGACGAGCTGGTGAAGGTCATGGGA CGGCACAAACCGGAGAATATCGTGATTGAAATGGCCCGAGAA AACCAGACTACCCAGAAGGGCCAGAAGAACTCCCGCGAAAGG ATGAAGCGGATCGAAGAAGGAATCAAGGAGCTGGGCAGCCAG ATCCTGAAAGAGCACCCGGTGGAAAACACGCAGCTGCAGAAC GAGAAGCTCTACCTGTACTATTTGC.AAAATGGACGGGACATGT ACGTGGACCAAGAGCTGGACATCAATCGGTTGTCTGATTACGA CGTGGACCACATCGTTCCACAGTCCTTTCTGAAGGATGACTCC ATCGATAACAAGGTGTTGACTCGCAGCGACAAGAACAGAGGG AAGTCAGATAATGTGCCATCGGAGGAGGTCGTGAAGAAGATG AAGAATTACTGGCGGCAGCTCCTGAATGCGAAGCTGATTACCC AGAGAAAGTTTGACAATCTCACTAAAGCCGAGCGCGGCGGAC TCTCAGAGCTGGATAAGGCTGGATTCATCAAACGGGAGCTGGT CGAGACTCGGCAGATTACCAAGCACGTGGCGCAGATCCTGGA CTCCCGCATGAACACTAAATACGACGAGAACGATAAGCTCAT CCGGGAAGTGAAGGTGATTACCCTGAAAAGCAAACTTGTGTC GGACTTTCGGAAGGACTTTCAGTTTTACAAAGTGAGAGAAATC AACAACTACCATCACGCGCATGACGCATACCTCAACGCTGTGG TCGGCACCGCCCTGATCAAGAAGTACCCTAAACTTGAATCGGA gtttgtgtacggagactacaaggtctacgacgtgaggaagat GATAGCCAAGTCCGAACAGGAAATCGGGAAAGCAACTGCGAA ATACTTCTTTTACTCAAACATCATGAACTTCTTCAAGACTGAA ATTACGCTGGCCAATGGAGAAATCAGGAAGAGGCCACTGATC G A A A C T A A C GG A G A A A CG GGC G A A ATC GT GTG GG AC A AG G GC AGGGACTTCGCAACTGTTCGCAAAGTGCTCTCTATGCCGCAAG TCAATATTGTGAAGAAAACCGAAGTGCAAACCGGCGGATTTTC AAAGGAATCGATCCTCCCAAAGAGAAATAGCGACAAGCTCAT TGCACGCAAGAAAGACTGGGACCCGAAGAAGTACGGAGGATT CGATTCGCCGACTGTCGCATACTCCGTCCTCGTGGTGGCCAAG GTGGAGAAGGGAAAGAGCAAGAAGCTCAAATCCGTCAAAGA GCTGCTGGGGATTACCATCATGGAACGATCCTCGTTCGAGAAG AACCCGATTGATTTCCTGGAGGCGAAGGGTTACAAGGAGGTG AAGAAGGATCTGATCATCAAACTGCCCAAGTACTCACTGTTCG A ACTGGA a a atggtcgga agcgc ATGCTGGCTTCGGCCGGAG AACTCCAGAAAGGAAATGAGCTGGCCTTGCCTAGCAAGTACG TCAACTrCCTCTATCITGCrrCGCACTACGAGAAACTCAAAGG GTCACCGGAAGATAACGAACAGAAGCAGCTTTTCGTGGAGCA GCACAAGCATTATCTGGATGAAATCATCGAACAAATCTCCGAG ttttcaaagcgcgtgatcctcgccgacgccaacctcgacaaag TCCTGTCGGCCTACAATAAGCATAGAGATAAGCCGATCAGAG AACAGGCCGAGAACATTATCCAC'rrGTTCACCCTGACTAACCT GGGAGCTCCAGCCGCCTTCAAGTACTTCGATACTACTATCGAC CGCAAAAGATACACGTCCACCAAGGAAGTTCTGGACGCGACC CTGATCCACCAAAGCATCACTGGACTCTACGAAACTAGGATCG ATCTGTCGCAGCTGGGTGGCGATGGTGGCGGTGGATCCTACCC atacgacgtgcctgactacgcctccggaggtggtggccccaag AAGAAACGGAAGGTGTGATAG Cas9 transcript with 5’ UTR ofHSD, GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGT GTGTCGTTGCAGGCCTTATTCGGATCTGCCACCATGGATAAGA AGTACTCGATCGGGCTGGATATCGGAACTAATTCCGTGGGTTG GGCAGTGATCACGGATGAATACAAAGTGCCGTCCAAGAAGTT 46 2024224004   30 Sep 2024 ORF corresponds ng to SEQ ID NO: 45, Kozak sequence, and 3’ UTR of ALB C A AGGTC C T GGGG AA C A CC G ATAG A CAC AG CA TC AA GA AG AA TCTCATCGGAGCCCTGCTGTTTGACTCCGGCGAAACCGCAGAA GCGACCCGGCTCAAACGTACCGCGAGGCGACGCTACACCCGG CGGAAGAATCGCATCTGCTATCTGCAAGAAATCTTTTCGAACG AAATGGCAAAGGTGGACGACAGCTTCTTCCACCGCCTGGAAG AATCTTTCCTGGTGGAGGAGGACAAGAAGCATGAACGGCATC CTATCTTTGGAAACATCGTGGACGAAGTGGCGTACCACGAAA AGTACCCGACCATCTACCATCTGCGGAAGAAGTTGGTTGACTC AACTGACAAGGCCGACCTCAGATTGATCTACTTGGCCCTCGCC CATATGATCAAATTCCGCGGACACTTCCTGATCGAAGGCGATC TGAACCCTGATAACTCCGACGTGGATAAGCTGTTCATTCAACT GGTGCAGACCTACAACCAACTGTTCGAAGAAAACCCAATCAA TGCCAGCGGCGTCGATGCCAAGGCCATCCTGTCCGCCCGGCTG TCGAAGTCGCGGCGCCTCGAAAACCTGATCGCACAGCTGCCG GGAGAGAAGAAGAACGGACTrrrCGGCAACTTGATCGCTCTCT CACTGGGACTCACTCCCAATTTCAAGTCCAATTTTGACCTGGC CGAGGACGCGAAGCTGCAACTCTCAAAGGACACCTACGACGA CGAC'TFGGACAATTTGCTGGCACAAATTGGCGATCAGTACGCG GATCTGTTCCTTGCCGCTAAGAACCTTTCGGACGCAATCTTGCT gtccgatatcctgcgcgtgaacaccgaaataaccaaagcgcc GCTTAGCGCCTCGATGATTAAGCGGTACGACGAGCATCACCAG GATCTCACGCTGCTCAAAGCGCTCGTGAGACAGCAACTGCCTG AAAAGTACAAGGAGATTTTCTTCGACCAGTCCAAGAATGGGT ACGCAGGGTACATCGATGGAGGCGCCAGCCAGGAAGAGTTCT ATAAGTTCATCAAGCCAATCCTGGAAAAGATGGACGGAACCG AAGAACTGCTGGTCAAGCTGAACAGGGAGGATCTGCTCCGCA AACAGAGAACCTTTGACAACGGAAGCATTCCACACCAGATCC ATCTGGGTGAGCTGCACGCCATCTTGCGGCGCCAGGAGGACTT TTACCCATTCCTCAAGGACAACCGGGAAAAGATCGAGAAAAT TCTGACGTTCCGCATCCCGTATTACGTGGGCCCACTGGCGCGC GGCAATTCGCGCTTCGCGTGGATGACTAGAAAATCAGAGGAA ACCATCACTCCTTGGAATTTCGAGGAAGTTGTGGATAAGGGAG CTTCGGCACAATCCTTCATCGAACGAATGACCAACTTCGACAA GAATCTCCCAAACGAGAAGGTGCTTCCTAAGCACAGGCTCCTT TACGAATACTTCACTGTCTACAACGAACTGACTAAAGTGAAAT ACGTTACTGAAGGAATGAGGAAGCCGGCCTTTCTGAGCGGAG A ACAG AA G A AAGCG ATTGTCG ATCTGCTGTTCAAG ACCA ACC GCAAGGTGACCGTCAAGCAGCTTAAAGAGGACTACTTCAAGA AGATCGAGTGTTTCGACTCAGTGGAAATCAGCGGAGTGGAGG ACAGATTCAACGCTTCGCTGGGAACCTATCATGATCTCCTGAA GATCATCAAGGACAAGGACTTCCTTGACAACGAGGAGAACGA GGACATCCTGGAAGATATCGTCCTGACCTTGACCCTTTTCGAG GATCGCGAGATGATCGAGGAGAGGCTTAAGACCTACGCTCAT CTCTTCGACGATAAGGTCATGAAACAACTCAAGCGCCGCCGGT ACACTGGTTGGGGCCGCCTCTCCCGCAAGCTGATCAACGGTAT TCGCGATAAACAGAGCGGTAAAACTATCCTGGATTTCCTCAAA TCGGATGGCTTCGCTAATCGTAACTTCATGCAGTTGATCCACG ACGACAGCCTGACCTTTAAGGAGGACATCCAGAAAGCACAAG TGAGCGGACAGGGAGACTCACTCCATGAACACATCGCGAATC TGGCCGGTTCGCCGGCGATTAAGAAGGGAATCCTGCAAACTGT GAAGGTGGTGGACGAGCTGGTGAAGGTCATGGGACGGCACAA ACCGGAGAATATCGTGATTGAAATGGCCCGAGAAAACCAGAC TACCCAGAAGGGCCAGAAGAACTCCCGCGAAAGGATGAAGCG GATCGAAGAAGGAATCAAGGAGCTGGGCAGCCAGATCCTGAA AGAGCACCCGGTGGAAAACACGCAGCTGCAGAACGAGAAGCT CTACCTGTACTATTTGCAAAATGGACGGGACATGTACGTGGAC CAAGAGCTGGACATCAATCGGTTGTCTGATTACGACGTGGACC 2024224004   30 Sep 2024 ACATCGTTCCACAGTCCTTTCTGAAGGATGACTCCATCGATAA CAAGGTGTTGACTCGCAGCGACAAGAACAGAGGGAAGTCAGA TAATGTGCCATCGGAGGAGGTCGTGAAGAAGATGAAGAATTA CTGGCGGCAGCTCCTGAATGCGAAGCTGATTACCCAGAGAAA GTTTGACAATCTCACTAAAGCCGAGCGCGGCGGACTCTCAGAG CTGGATAAGGCTGGATTCATCAAACGGCAGCTGGTCGAGACTC GGCAGATTACCAAGCACGTGGCGCAGATCCTGGACTCCCGCAT GA AC ACTA A ATA CGACGAGA ACG ATAAGCTCATCCGGGA AGT GAAGGTGATTACCCTGAAAAGCAAACTTGTGTCGGACTTTCGG AAGGACTTTCAGTTTTACAAAGTGAGAGAAATCAACAACTACC ATCACGCGCATGACGCATACCTCAACGCTGTGGTCGGCACCGC CCTGATCAAGAAGTACCCTAAACTTGAATCGGAGTTTGTGTAC GGAGACTACAAGGTCTACGACGTGAGGAAGATGATAGCCAAG TCCG AA CAGGA AATCGGG AA AGCAA CTGCGAA ATACTTCTTTT ACTCAAACATCATGAACTTCTTCAAGACTGAAATTACGCTGGC CAATGGAGAAATCAGGAAGAGGCCACTGATCGAAACTAACGG AGAAACGGGCGAAATCGTGTGGGACAAGGGCAGGGACTTCGC AACTGTTCGCAAAGTGCTCTCTATGCCGCAAGTCAATATTGTG AAGAAAACCGAAGTGCAAACCGGC GG ATTTT C AA AG G A ATC G ATCCTCCCAAAGAGAAATAGCGACAAGCTCATTGCACGCAAG AAAGACTGGGACCCGAAGAAGTACGGAGGATTCGATTCGCCG ACTGTCGCATACTCCGTCCTCGTGGTGGCCAAGGTGGAGAAGG GAAAGAGCAAGAAGCTCAAATCCGTC A AAG AG CTGCTGGGGA TTACCATCATGGAACGATCCTCGTTCGAGAAGAACCCGATTGA TTTCCTGGAGGCGAAGGGTTACAAGGAGGTGAAGAAGGATCT GATCATCAAACTGCCCAAGTACTCACTGTTCGAACTGG.AAAAT GGTCG G A A GCGC A TGC T GGCTT CGGCCGGAG AACT CCA G AAA ggaaatgagctggccttgcctagcaagtacgtcaacttcctct ATCTTGCTTCGCACTACGAGAAACTCAAAGGGTCACCGGAAG ATAACGAACAGAAGCAGCTTTTCGTGGAGCAGCACAAGCATT ATCTGGATGAAATCATCGAACAAATCTCCGAGTTTTCAAAGCG CGTGATCCTCGCCGACGCCAACCTCGACAAAGTCCTGTCGGCC TACAATAAGCATAGAGATAAGCCGATCAGAGAACAGGCCGAG AACATTATCCACTrGTTCACCCTGACTAACCTGGGAGCTCCAG CCGCCTTCAAGTACTTCGATACTACTATCGACCGCAAAAGATA CACGTCCACCAAGGAAGTTCTGGACGCGACCCTGATCCACCAA AGCATCACTGGACTCTACGAAACTAGGATCGATCTGTCGCAGC TGGGTGGCGATGGTGGCGGTGGATCCTACCCATACGACGTGCC TGACTACGCCTCCGGAGGTGGTGGCCCCAAGAAGAAACGGAA GGTGTGATAGCTAGCCATCACATTTAAAAGCATCTCAGCCTAC CATGAGA ATA A GAGA A AGA A AATG AAG AT C A A T AGC TT A TT C ATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTA AAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGC TTCAATTAATAAAAAATGGAAAGAACCTCGAG Cas9 transcript with 5’ UTR ofHSD, ORF correspond! ng to SEQ ID NO: 45, and 3 ’ UTR of ALB GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGT GTGTCGTTGCAGGCCTTATTCGGATCTATGGATAAGAAGTACT CGATCGGGCTGGATATCGGAACTAATTCCGTGGGTTGGGCAGT GATCACGGATGAATACAAAGTGCCGTCCAAGAAGTTCAAGGT CCTGGGGAACACCGATAGACACAGCATCAAGAAGAATCTCAT CGGAGCCCTGCTGTTTGACTCCGGCGAAACCGCAGAAGCGAC CCGGCTCAAACGTACCGCGAGGCGACGCTACACCCGGCGGAA GAATCGCATCTGCTATCTGC.AAGAAATC'nTTCGAACGAAATG GCAAAGGTGGACGACAGC'TTCTTCCACCGCCTGGAAGAATCTT TCCTGGTGGA GG AGG AC AAG A AGC ATG AACGGC ATCCTATCT 47 2024224004   30 Sep 2024 TTGGAAACATCGTGGACGAAGTGGCGTACCACGAAAAGTACC CGACCATCTACCATCTGCGGAAGAAGTTGGTTGACTCAACTGA CAAGGCCGACCTCAGATTGATCTACTTGGCCCTCGCCCATATG ATCAAATTCCGCGGACACTTCCTGATCGAAGGCGATCTGAACC CTGATAACTCCGACGTGGATAAGCTGTTCATTCAACTGGTGCA GACCTACAACCAACTGTTCGAAGAAAACCCAATCAATGCCAG CGGCGTCGATGCCAAGGCCATCCTGTCCGCCCGGCTGTCGAAG TCGCGGCGCCTCGAAAACCTGATCGCACAGCTGCCGGGAGAG AAGAAGAACGGACTTTTCGGCAACTTGATCGCTCTCTCACTGG GACTCACTCCCAATTTCAAGTCCAATTTTGACCTGGCCGAGGA CGCGAAGCTGCAACTCTCAAAGGACACCTACGACGACGACTT GGACAATTFGCTGGCACAAATTGGCGATCAGTACGCGGATCTG TTCCTTGCCGCTAAGAACCTTTCGGACGCAATCTTGCTGTCCG ATATCCTGCGCGTGAACACCGAAATAACCAAAGCGCCGCTTA GCGCCTCGATGATTAAGCGGTACGACGAGCATCACCAGGATCT CACGCTGCTCAAAGCGCTCGTGAGACAGCAACTGCCTGAAAA GTACAAGGAGATTTTCTTCGACCAGTCCAAGAATGGGTACGCA GGGTACATCGATGGAGGCGCCAGCCAGGAAGAGTTCTATAAG TTCATCAAGCCAATCCTGGAAAAGATGGACGGAACCGAAGAA CTGCTGGTCAAGCTGAACAGGGAGGATCTGCTCCGCAAACAG AGAACCTTTGACAACGGAAGCATTCCACACCAGATCCATCTGG GTGAGCTGCACGCCATC'TFGCGGCGCCAGGAGGACTTTTACCC ATTCCTCAAGGACAACCGGGAAAAGATCGAGAAAATTCTGAC GTTCCGCATCCCGTATTACGTGGGCCCACTGGCGCGCGGCAAT TCGCGCTTCGCGTGGATGACTAGAAAATCAGAGGAAACCATC ACTCCTTGGAATTTCGAGGAAGTTGTGGATAAGGGAGCTTCGG C AC A ATC C TTC A TCG A A CG A ATG AC C A AC TTCG AC A AG AAT C T CCCAAACGAGAAGGTGCTTCCTAAGCACAGCCTCCTTTACGAA TACTTCACTGTCTACAACGAACTGACTAAAGTGAAATACGTTA CTGAAGGAATGAGGAAGCCGGCCTTTCTGAGCGGAGAACAGA AGAAAGCGATTGTCGATCTGCTGTTCAAGACCAACCGCAAGGT GACCGTCAAGCAGCTTAAAGAGGACTACTTCAAGAAGATCGA GTGTTrCGACTCAGTGGAAATCAGCGGAGTGGAGGACAGATT CAACGCTTCGCTGGGAACCTATCATGATCTCCTGAAGATCATC AAGGACAAGGACTFCCTTGACAACGAGGAGAACGAGGACATC CTGGAAGATATCGTCCTGACCTTGACCCTTTTCGAGGATCGCG AG ATG ATCG AGG AGAGGCTTA AG ACCTA CGCTCATCTCTTCGA CGATAAGGTCATGAAACAACTCAAGCGCCGCCGGTACACTGG TTGGGGCCGCCTCTCCCGCAAGCTGATCAACGGTATTCGCGAT AAACAGAGCGGTAAAACTATCCTGGATTTCCTCAAATCGGATG GCTTCGCTAATCGTAACTTCATGCAGTTGATCCACGACGACAG CCTGACCTTTAAGGAGGACATCCAGAAAGCACAAGTGAGCGG ACAGGGAGACTCACTCCATGAACACATCGCGAATCTGGCCGG TTCGCCGGCGATTAAGAAGGGAATCCTGCAAACTGTGAAGGT GGTGGACGAGCTGGTGAAGGTCATGGGACGGCACAAACCGGA GAATATCGTGATTGAAATGGCCCGAGAAAACCAGACTACCCA GAAGGGCCAGAAGAACTCCCGCGAAAGGATGAAGCGGATCGA AGAAGGAATCAAGGAGCTGGGCAGCCAGATCCTGAAAGAGCA CCCGGTGGAAAACACGCAGCTGCAGAACGAGAAGCTCTACCT GTACTATTTGCAAAATGGACGGGACATGTACGTGGACCAAGA GCTGGACATCAATCGGTTGTCTGATTACGACGTGGACCACATC GTTCC AC A GTCCTTTCTG A AGG ATG A CTCCATCGATAACAAGG TGTTGACTCGCAGCGACAAGAACAGAGGGAAGTCAGATAATG TGCCATCGGAGGAGGTCGTGAAGAAGATGAAGAATTACTGGC GGCAGCTCCTGAATGCGAAGCTGATTACCCAGAGAAAGTTTG AC AATCTC ACTA AAGCCG AGCGCGGCGG ACTCTCAGAGCTGG ATAAGGCTGGATTCATCAAACGGCAGCTGGTCGAGACTCGGC 2024224004   30 Sep 2024 agattaccaagcacgtggcgcagatcctggactcccgcatga ACACTAAATACGACGAGAACGATAAGCTCATCCGGGAAGTGA AGGTGATTACCCTGAAAAGCAAACTTGTGTCGGACTTTCGGAA GGACTTTCAGTTTTACAAAGTGAGAGAAATCAACAACTACCAT CACGCGCATGACGCATACCTCAACGCTGTGGTCGGCACCGCCC TGATCAAGAAGTACCCTAAACTTGAATCGGAGTTTGTGTACGG AG ACTAC A AGGTCTACGA CGTG AGGA AG ATG ATAGCCAA GTC CGAACAGGAAATCGGGAAAGCAACTGCGAAATACTTCTTTTA CTCAAACATCATGAACTTCTTCAAGACTGAAATTACGCTGGCC AATGGAGAAATCAGGAAGAGGCCACTGATCGAAACTAACGGA GAAACGGGCGAAATCGTGTGGGACAAGGGCAGGGACTTCGCA ACTGTTCGCAAAGTGCTCTCTATGCCGCAAGTCAATATTGTGA AGAAAACCGAAGTGCAAACCGGCGGATTTTCAAAGGAATCGA TCCTCCCAAAGAGAAATAGCGACAAGCTCATTGCACGCAAGA AAGACTGGGACCCGAAGAAGTACGGAGGATTCGATTCGCCGA CTGTCGCATACTCCGTCCTCGTGGTGGCCAAGGTGGAGAAGGG AAAGAGCAAGAAGCTCAAATCCGTCAAAGAGCTGCTGGGGAT TACCATCATGGAACGATCCTCGTTCGAGAAGAACCCGATTGAT TTCCTGGAGGCGAAGGGTTACAAGGAGGTGAAGAAGGATCTG ATCATCAAACTGCCCAAGTACTCACTGTTCGAACTGGAAAATG GTCGGAAGCGCATGCTGGCTTCGGCCGGAGAACTCCAGAAAG GAAATGAGCTGGCCTTGCCTAGCAAGTACGTCAACTTCCTCTA TCTTGCTTCGCACTACGAGAAACTCAAAGGGTCACCGGAAGAT AACGAACAGAAGCAGCTTTTCGTGGAGCAGCACAAGGATTAT CTGGATGAAATCATCGAACAAATCTCCGAGTTTTCAAAGCGCG TGATCCTCGCCGACGCCAACCTCGACAAAGTCCTGTCGGCCTA C A AT A AGC AT AG AG AT A AGCCG ATC AG AG A AC AGGCCG AG A A CATTATCCACTTGTTCACCCTGACTAACCTGGGAGCTCCAGCC GCCTTCAAGTACTTCGATACTACTATCGACCGCAAAAGATACA CGTCCACCAAGGAAGTTCTGGACGCGACCCTGATCCACCAAA GCATCACTGGACTCTACGAAACTAGGATCGATCTGTCGCAGCT GGGTGGCGATGGTGGCGGTGGATCCTACCCATACGACGTGCCT GACTACGCCTCCGGAGGTGGTGGCCCCAAGAAGAAACGGAAG GTGTGATAGCTAGCCATCACAITTAAAAGCATCTCAGCCTACC ATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCA TCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAA AAAACATA AATTTCTTTA ATCATTTTGCCTCTTTTCTCTGTGCT TCAATTAATAAAAAATGGAAAGAACCTCGAG Cas9 trans crips comprising Cas9 ORF using codons with generally high expression in humans GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGT GTGTCGTTGCAGGCCTTATTCGGATCCATGCCTAAGAAAAAGC GGAAGGTCGACGGGGATAAGAAGTACTCAATCGGGCTGGATA TCGGAACTAArrCCGTGGGTTGGGCAGTGATCACGGATGAATA CAAAGTGCCGTCCAAGAAGTTCAAGGTCCTGGGGAACACCGA TAGACACAGCATCAAGAAAAATCTCATCGGAGCCCTGCTGTTT GACTCCGGCGAAACCGCAGAAGCGACCCGGCTCAAACGTACC GCGAGGCGACGCTACACCCGGCGGAAGAATCGCATCTGCTAT CTGCAAGAGATCTTTTCGAACGAAATGGCAAAGGTCGACGAC AGCTTCTTCCACCGCCTGGAAGAATCTTTCCTGGTGGAGGAGG ACAAGAAGCATGAACGGCATCCTATCTTTGGAAACATCGTCGA CGAAGTGGCGTACCACGAAAAGTACCCGACCATCTACCATCTG CGGAAGAAGTTGGTTGACTCAACTGACAAGGCCGACCTCAGA TTGATCTACTTGGCCCTCGCCCATATGATCAAATTCCGCGGAC ACTTCCTGATCGAAGGCGATCTGAACCCTGATAACTCCGACGT GGATAAGCTTTTCATTCAACTGGTGCAGACCTACAACCAACTG 48 2024224004   30 Sep 2024 TTCGAAGAAAACCCAATCAATGCTAGCGGCGTCGATGCCAAG GCCATCCTGTCCGCCCGGCTGTCGAAGTCGCGGCGCCTCGAAA ACCTGATCGCACAGCTGCCGGGAGAGAAAAAGAACGGACTTT TCGGCAACTTGATCGCTCTCTCACTGGGACTCACTCCCAATTTC AAGTCCAATTTTGACCTGGCCGAGGACGCGAAGCTGCAACTCT CAAAGGACACCTACGACGACGACTTGGACAATTTGCTGGCAC AAATTGGCGATCAGTACGCGGATCTGTTCCTTGCCGCTAAGAA CCTTTCGGACGCAATCTTGCTGTCCGATATCCTGCGCGTGAAC ACCGAAATAACCAAAGCGCCGCTTAGCGCCTCGATGATTAAG CGGTACGACGAGCATCACCAGGATCTCACGCTGCTCAAAGCG CTCGTGAGACAGCAACTGCCTGAAAAGTACAAGGAGATCTTCT TCGACCAGTCCAAGAATGGGTACGCAGGGTACATCGATGGAG GCGCTAGCCAGGAAGAGTTCTATAAGTTCATCAAGCCAATCCT GGAAAAGATGGACGGAACCGAAGAACTGCTGGTCAAGCTGAA CAGGGAGGATCTGCTCCGGAAACAGAGAACCTTTGACAACGG ATCCATTCCCCACCAGATCCATCTGGGTGAGCTGCACGCCATC TTGCGGCGCCAGGAGGACTTTTACCCATTCCTCAAGGACAACC GGGAAAAGATCGAGAAAATTCTGACGTTCCGCATCCCGTATTA CGTGGGCCCACTGGCGCGCGGCAATTCGCGCTTCGCGTGGATG ACTAGAAAATCAGAGGAAACCATCACTCCTTGGAATTTCGAG GAAGTTGTGGATAAGGGAGCTTCGGCACAAAGCTTCATCGAA CGAATGACCAACTTCGACAAGAATCTCCCAAACGAGAAGGTG cttcctaagcacagcctcctttacgaatacttcactgtctacaa CGAACTGACTAAAGTGAAATACGTTACTGAAGGAATGAGGAA GCCGGCCTTTCTGTCCGGAGAACAGAAGAAAGCAATTGTCGAT CTGCTGTTCAAGACCAACCGCAAGGTGACCGTCAAGCAGCTTA AAGAGGACTACTTCAAGAAGATCGAGTGTTTCGACTCAGTGG AAATCAGCGGGGTGGAGGACAGATTCAACGCTTCGCTGGGAA CCTATCATGATCTCCTGAAGATCATCAAGGACAAGGACTTCCT TGACAACGAGGAGAACGAGGACATCCTGGAAGATATCGTCCT GACCTTGACCCTTTTCGAGGATCGCGAGATGATCGAGGAGAG GCTTAAGACCTACGCTCATCTCTTCGACGATAAGGTCATGAAA CAACTCAAGCGCCGCCGGTACACTGGTTGGGGCCGCCTCTCCC GCAAGCTGATCAACGGTATTCGCGATAAACAGAGCGGTAAAA CTATCCTGGAT1TCCTCAAATCGGATGGCTTCGCTAATCGTAA CTTCATGCAATTGATCCACGACGACAGCCTGACCTTTAAGGAG GACATCCAAAAAGCACAAGTGTCCGGACAGGGAGACTCACTC CATGAACACATCGCGAATCTGGCCGGTTCGCCGGCGATTAAGA AGGGAATTCTGCAAACTGTGAAGGTGGTCGACGAGCTGGTGA AGGTCATGGGACGGCACAAACCGGAGAATATCGTGATTGAAA TGGCCCGAGAAAACCAGACTACCCAG A A GGGCCAGA AAAA C T CCC GC G AA A GG AT GA A G CGG ATCGAAGAAGG AA TCAAGGAG CTGGGCAGCCAGATCCTGAAAGAGCACCCGGTGGAAAACACG CAGCTGCAGAACGAGAAGCTCTACCTGTACTATTTGCAAAATG GACGGGACATGTACGTGGACCAAGAGCTGGACATCAATCGGT TGTCTGATTACGACGTGGACCACATCGTTCCACAGTCCTTTCTG AAGGATGACTCGATCGATAACAAGGTGTTGACTCGCAGCGAC AAGAACAGAGGGAAGTCAGATAATGTGCCATCGGAGGAGGTC GTGAAGAAGATGAAGAATTACTGGCGGCAGCTCCTGAATGCG AAGCTGATTACCCAGAGAAAGTTTGACAATCTCACTAAAGCCG AGCGCGGCGGACTCTCAGAGCTGGATAAGGCTGGATTCATCA AACGGCAGCTGGTCGAGACTCGGCAGATTACCAAGCACGTGG CGCAGATCTTGGACTCCCGCATGAACACTAAATACGACGAGA ACGATAAGCTCATCCGGGAAGTGAAGGTGATTACCCTGAAAA GCAAACTTGTGTCGGACTTTCGGAAGGACTTTCAGTTTTACAA AGTGAGAGAAATCAACAACTACCATCACGCGCATGACGCATA CCTCAACGCTGTGGTCGGTACCGCCCTGATCAAAAAGTACCCT 2024224004   30 Sep 2024 AAACTTGAATCGGAGTTTGTGTACGGAGACTACAAGGTCTACG ACGTGAGGAAGATGATAGCCAAGTCCGAACAGGAAATCGGGA AAGCAACTGCGAAATACTTCTTTTACTCAAACATCATGAACTT TTTCAAGACTGAAATTACGCTGGCCAATGGAGAAATCAGGAA GAGGCCACTGATCGAAACTAACGGAGAAACGGGCGAAATCGT GTGGGACAAGGGCAGGGACTTCGCAACTGTTCGCAAAGTGCT CTCTATGCCGCAAGTCAATATTGTGAAGAAAACCGAAGTGCA AACCGGCGGATTTTCAAAGGAATCGATCCTCCCAAAGAGAAA TAGCGACAAGCTCATTGCACGCAAGAAAGACTGGGACCCGAA GAAGTACGGAGGATTCGATTCGCCGACTGTCGCATACTCCGTC CTCGTGGTGGCCAAGGTGGAGAAGGGAAAGAGCAAAAAGCTC AAATCCGTCAAAGAGCTGCTGGGGATTACCATCATGGAACGA TCCTCGTTCGAGAAGAACCCGATTGATTTCCTCGAGGCGAAGG GTTACAAGGAGGTGAAGAAGGATCTGATCATCAAACTCCCCA AGTACTCACTGTTCGAACTGGAAAATGGTCGGAAGCGCATGCT GGCTTCGGCCGGAGAACTCCAAAAAGGAAATGAGCTGGCCTT GCCTAGCAAGTACGTCAACTTCCTCTATCTTGCTTCGCACTACG AAAAACTCAAAGGGTCACCGGAAGATAACGAACAGAAGCAGC TTTTCGTGGAGCAGCACAAGCATTATCTGGATGAAATCATCGA ACAAATCTCCGAGTTTTCAAAGCGCGTGATCCTCGCCGACGCC AACCTCGACAAAGTCCTGTCGGCCTACAATAAGCATAGAGAT AAGCCGATCAGAGAACAGGCCGAGAACATTATCCACTTGTTC ACCCTGACTAACCTGGGAGCCCCAGCCGCCTTCAAGTACTTCG ATACTACTATCGATCGCAAAAGATACACGTCCACCAAGGAAG TTCTGGACGCGACCCTGATCCACCAAAGCATCACTGGACTCTA CGAAACTAGGATCGATCTGTCGCAGCTGGGTGGCGATTGATAG TCT A G CC ATC A C ATT TAAAAGCATCTCAGCCTACCATGAG AAT AAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTT CTTTTTCGTTGGTGTA AAGCC AAC ACCCTGTCTAA AA AACATA AATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAAT AAAAAATGGAAAGAACCTCGAG Cas9 transcript comprising Kozak sequence with Cas!) ORF using codons with generally high expression in humans GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGT GTGTCGTTGCAGGCCTTATTCGGATCCGCCACCATGCCTAAGA AAAAGCGGAAGGTCGACGGGGATAAGAAGTACTCAATCGGGC TGGATATCGGAACTAATTCCGTGGGTTGGGCAGTGATCACGGA IGAATACAAACrrGCCGTCeAAGAAGlTCAAGGTCCTGGGGAA CACCGATAGACACAGCATCAAGAAAAATCTCATCGGAGCCCT GCTGTTTGACTCCGGCGAAACCGCAGAAGCGACCCGGCTCAA ACGTACCGCGAGGCGACGCTACACCCGGCGGAAGAATCGCAT CTGCTATCTGCAAGAGATCTTTTCGAACGAAATGGCAAAGGTC GACGACAGCTTCTTCCACCGCCTGGAAGAATCTTTCCTGGTGG AGGAGGACAAGAAGCATGAACGGCATCCTATCTTIGGAAACA TCGTCGACGAAGTGGCGTACCACGAAAAGTACCCGACCATCT ACCATCTGCGGAAGAAGTTGGTTGACTCAACTGACAAGGCCG ACCTCAGATTGATCTACTTGGCCCTCGCCCATATGATCAAATT CCGCGGACACTTCCTGATCGAAGGCGATCTGAACCCTGATAAC TCCGACGTGGATAAGCTTTTCATTCAACTGGTGCAGACCTACA ACCAACTGTTCGAAGAAAACCCAATCAATGCTAGCGGCGTCG ATGCCAAGGCCATCCTGTCCGCCCGGCTGTCGAAGTCGCGGCG CCTCG AAA ACCTGATCGC ACAGCTGCCGGG AGAG AAAAA GA A CGGACTTTTCGGCAACTTGATCGCTCTCTCACTGGGACTCACTC CCAATTTCAAGTCCAATTTTGACCTGGCCGAGGACGCGAAGCT GCAACTCTCAAAGGACACCTACGACGACGACTTGGACAATTTG CTGGCACAAATTGGCGATCAGTACGCGGATCTGTTCCTTGCCG 49 2024224004   30 Sep 2024 CTAAGAACCTTTCGGACGCAATCTTGCTGTCCGATATCCTGCG CGTGAACACCGAAATAACCAAAGCGCCGCTTAGCGCCTCGAT GATTAAGCGGTACGACGAGCATCACCAGGATCTCACGCTGCTC AAAGCGCTCGTGAGACAGCAACTGCCTGAAAAGTACAAGGAG atcttcttcgaccagtccaagaatgggtacgcagggtacatcg ATGGAGGCGCTAGCCAGGAAGAGTTCTATAAGTTCATCAAGC CAATCCTGGAAAAGATGGACGGAACCGAAGAACTGCTGGTCA AGCTGAACAGGGAGGATCTGCTCCGGAAACAGAGAACCTTTG ACAACGGATCCATTCCCCACCAGATCCATCTGGGTGAGCTGCA CGCCATCTTGCGGCGCCAGGAGGACTTTTACCCATTCCTCAAG GACAACCGGGAAAAGATCGAGAAAATTCTGACGTTCCGCATC CCGTATrACGTGGGCCCACTGGCGCGCGGCAATTCGCGCTTCG CGTGGATGACTAGAAAATCAGAGGAAACCATCACTCCTTGGA ATTTCGAGGAAGTTGTGGATAAGGGAGCTTCGGCACAAAGCTT CATCGAACGAATGACCAACTTCGACAAGAATCTCCCAAACGA GAAGGTGCTTCCTAAGCACAGCCTCCTTTACGAATACTTCACT GTCTACAACGAACTGACTAAAGTGAAATACGTTACTGAAGGA ATGAGGAAGCCGGCCTTTCTGTCCGGAGAACAGAAGAAAGCA ATTGTCGATCTGCTGTTCAAGACCAACCGCAAGGTGACCGTCA AGCAGCTTAAAGAGGACTACTTCAAGAAGATCGAGTGTTTCG ACTCAGTGGAAATCAGCGGGGTGGAGGACAGATTCAACGCTT CGCTGGGAACCTATCATGATCTCCTGAAGATCATCAAGGACAA GGACTTCCTTGACAACGAGGAGAACGAGGACATCCTGGAAGA TATCGTCCTGACCTTGACCCTTTTCGAGGATCGCGAGATGATC GAGGAGAGGCTTAAGACCTACGCTCATCTCTTCGACGATAAGG TCATGAAACAACTCAAGCGCCGCCGGTACACTGGTTGGGGCC GC CTCTCC CGC A AGC T GA TCA AC GG T ATT C GCG AT AA AC AG A G CGGTAAAACTATCCTGGATTTCCTCAAATCGGATGGCTTCGCT AATCGTAACTTCATGCAATTGATCCACGACGACAGCCTGACCT TTAAGGAGGACATCCAAAAAGCACAAGTGTCCGGACAGGGAG ACTCACTCCATGAACACATCGCGAATCTGGCCGGTTCGCCGGC GATTAAGAAGGGAATTCTGCAAACTGTGAAGGTGGTCGACGA GCTGGTGAAGGTCATGGGACGGCACAAACCGGAGAATATCGT GATTGAAATGGCCCGAGAAAACCAGACTACCCAGAAGGGCCA GAAAAACTCCCGCGAAAGGATGAAGCGGATCGAAGAAGGAAT CAAGGAGCTGGGCAGCCAGATCCTGAAAGAGCACCCGGTGGA AAACACGCAGCTGCAGAACGAGAAGCTCTACCTGTACTATTTG CAAAATGGACGGGACATGTACGTGGACCAAGAGCTGGACATC AATCGGTTGTCTGATTACGACGTGGACCACATCGTTCCACAGT CCTTTCTGAAGGATGACTCGATCGATAACAAGGTGTTGACTCG CAGCGACAAGAACAGAGGGAAGTCAGATAATGTGCCATCGGA GGAGGTCGTGAAGAAGATGAAGAATTACTGGCGGCAGCTCCT GAATGCGAAGCTGATTACCCAGAGAAAGTTTGACAATCTCACT AAAGCCGAGCGCGGCGGACTCTCAGAGCTGGATAAGGCTGGA TTCATCAAACGGCAGCTGGTCGAGACTCGGCAGATTACCAAGC ACGTGGCGCAGATCTTGGACTCCCGCATGAACACTAAATACGA CGAGAACGATAAGCTCATCCGGGAAGTGAAGGTGATTACCCT GAAAAGCAAACTTGTGTCGGACTTTCGGAAGGACTTTCAGTTT TACAAAGTGAGAGAAATCAACAACTACCATCACGCGCATGAC GCATACCTCAACGCTGTGGTCGGTACCGCCCTGATCAAAAAGT ACCCTAAACTTGAATCGGAGTTTGTGTACGGAGACTACAAGGT CTACGACGTGAGGAAGATGATAGCCAAGTCCGAACAGGAAAT CGGGAAAGCAACTGCGAAATACTTCTTTTACTCAAACATCATG AACTTTTTCAAGACTGAAATTACGCTGGCCAATGGAGAAATCA GGAAGAGGCCACTGATCGAAACTAACGGAGAAACGGGCGAA ATCGTGTGGGACAAGGGCAGGGACTTCGCAACTGTTCGCAAA GTGCTCTCTATGCCGCAAGTCAATATTGTGAAGAAAACCGAAG 2024224004   30 Sep 2024 tgcaaaccggcggattttcaaaggaatcgatcctcccaaaga GAAATAGCGACAAGCTCATTGCACGCAAGAAAGACTGGGACC CGAAGAAGTACGGAGGATTCGATTCGCCGACTGTCGGATACTC CGTCCTCGTGGTGGCCAAGGTGGAGAAGGGAAAGAGCAAAAA GCTCAAATCCGTCAAAGAGCTGCTGGGGATTACCATCATGGAA CGATCCTCGTTCGAGAAGAACCCGATTGATTTCCTCGAGGCGA AGGGTTACAAGGAGGTGAAGAAGGATCTGATCATCAAACTCC CCAAGTACTCACTGTTCGAACTGGAAAATGGTCGGAAGCGCAT GCTGGCTTCGGCCGGAGAACTCCAAAAAGGAAATGAGCTGGC CTTGCCTAGCAAGTACGTCAACTTCCTCTATCTTGCTTCGCACT ACGAAAAACTCAAAGGGTCACCGGAAGATAACGAACAGAAGC AGCTTTTCGTGGAGCAGCACAAGCAITATCTGGATGAAATCAT CGAACAAATCTCCGAGTTTTCAAAGCGCGTGATCCTCGCCGAC GCCAACCTCGACAAAGTCCTGTCGGCCTACAATAAGCATAGA GATAAGCCGATCAGAGAACAGGCCGAGAACATTATCCACTTG nCACCCTGACTAACCTGGGAGCCCCAGCCGCCTTCAAGTACT TCGATACTACTATCGATCGCAAAAGATACACGTCCACCAAGGA AG1TCTGGACGCGACCCTGATCCACCAAAGCATCACTGGACTC TACGAAACTAGGATCGATCTGTCGCAGCTGGGTGGCGATTGAT AGTCTAGCCATCACATTTAAAAGCATCTCAGCCTACCATGAGA ATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTCTTT TTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACA TAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTA ATAAAAAATGGAAAGAACCTCGAG Cas9 ORF with sptice junctions removed; 12.75% U content ATGG A C A A G A A GT AC A G C ATC GG ACTGG AC AT CGG A AC A A A C AGCGTCGGATGGGCAGTCATCACAGACGAATACAAGGTCCCG AGCAAGAAGTTCAAGGTCCTGGGAAACACAGACAGACACAGC ATCAAGAAGAACCTGATCGGAGCACTGCTGTTCGACAGCGGA GAAACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAAG AAGATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGA AATCTTCAGCAACGAAATGGCAAAGGTCGACGACAGCTTCTTC CACcggCTGGAAGAAAGCTTCCTGGTCGAAGAAGACAAGAAGC ACGAAAGACACCCGATCTTCGGAAACATCGTCGACGAAGTCG C ATACCA CGA AA AGTACCCG ACA ATCTACCA CCTGAG A AAGA AGCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCT ACCTGGCACTGGCACACATGA1CAAG nCAGAGGACAC'iTCC i' GATCGAAGGAGACCTGAACCCGGACAACAGCGACGTCGACAA GCTGTTCATCCAGCTGGTCCAGACATACAACCAGCTGTTCGAA GAAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGGCAATC CTGAGCGCAAGACTGAGCAAGAGCAGAA GACTGGAA AACCT G ATCGCACAGCTGCCGGGAGAAAAGAAGAACGGACTGTTCGGA AACCTGATCGCACTGAGCCTGGGACTGACACCGAACTTCAAG AGCAACTTCGACCTGGCAGAAGACGCAAAGCTGCAGCTGAGC AAGGACACATACGACGACGACCTGGACAACCTGCTGGCACAG ATCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAAC CTGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAAC ACAGAAATCACAAAGGCACCGCTGAGCGCAAGCATGATCAAG AGATACGACGAACACCACCAGGACCTGACACTGCTGAAGGCA CTGGTCAGACAGCAGCTGCCGGAAAAGTACAAGGAAATCTTC TTCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGA GGAGCAAGCCAGGAAGAATTCTACAAGITCATCAAGCCGATC CTGGAAAAGATGGACGGAACAGAAGAACTGCTGGTCAAGCTG AACAGAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAAC GGAAGCATCCCGCACCAGATCCACCTGGGAGAACTGCACGCA 50 2024224004   30 Sep 2024 ATCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGAC AACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCG TACTACGTCGGACCGCTGGCAAGAGGAAACAGCAGATTCGCA TGGATGACAAGAAAGAGCGAAGAAACAATCACACCGTGGAAC TTCGAAGAAGTCGTCGACAAGGGAGCAAGCGCACAGAGCTTC ATCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAA AAGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAG TCTACAACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAA TGAGAAAGCCGGCATTCCTGAGCGGAGAACAGAAGAAGGCAA TCGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTCA AGCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTCG ACAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAA GCCTGGGAACATACCACGACCTGCTGAAGATCATCAAGGACA AGGACTTCCTGGACAACGAAGAAAACGAAGACATCCTGGAAG ACATCGTCCTGACACTGACACTGTTCGAAGACAGAGAAATGAT CGAAGAAAGACTGAAGACATACGCACACCTGTTCGACGACAA GGTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGG AAGACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCA GAGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGACGGATT CGCAAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCT GACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCGGACA GGGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAG CCCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGT CGACGAACTGGTCAAGGTCATGGGAAGACACAAGCCGGAAAA CATCGTCATCGAAATGGCAAGAGAAAACCAGACAACACAGAA GGGACAGAAGAACAGCAGAGAAAGAATGAAGAGAATCGAAG AAGGAATCAAGGAACTGGGAAGCCAGATCCTGAAGGAACACC CGGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGT ACTACCTGCAaA ACGG A AG AG AC ATGTACGTCGACCA GGA>ACT GGACATCAACAGACTGAGCGACTACGACGTCGACCACATCGT CCCGCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGT CCTGACAAGAAGCGACA.AGAACAGAGGAA.AGAGCGACA.ACG TCCCGAGCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGA GACAGCTGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCG ACAACCTGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTG GACAAGGCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGA CAGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATG AACACAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTC AAGGTCATCACACTGAAGAGCAAGCTGGTCAGCGAC1TCAGA AAGGACTTCCAGTTCTACAAGGTCAGAGAAATCAACAACTAC CACCACGCACACGACGCATACCTGAACGCAGTCGTCGGAACA GCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTC TACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCA AAGAGCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTC TTCTACAGCAACATCATGAACTTCTTCAAGACAGAAATCACAC TGGCAAACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAA ACGGAGAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGAC TTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAAC ATCGTCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAG G A AA GC ATC CTGCC G A AG AG AA ACAGCGACAAGCTGATCGCA AGAAAGAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGAC AGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCG AAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTG CTGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAAC CCGATCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAG AAGGACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAAC TGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAA 2024224004   30 Sep 2024 CTGCAGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTC AACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGA AGCCCGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAG CACAAGCACTACCTGGACGAAATCATCGAACAGATCAGCGAA TTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAG GTCCTGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGA GAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAAC CTGGGAGCACCGGCAGCATTCAAGTACTTCGACACAACAATC GACAGAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCA ACACTGATCCACCAGAGCATCACAGGACTGTACGAAACAAGA ATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCG AAGAAGAAGAGAAAGGTCTAG Cas9 transcript with 5’ UTR ofHSD. ORF correspond! ng to SEQ ID NO: 50, Kozak sequence, and 3’ UTR of ALB GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGT GTGTCGTTGCAGGCCTTATTCGGATCCGCCACCATGGACAAGA AGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGAT GGGCAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGT TC A A G GTCCTGG G A A A C A C A G AC A G AC A C AGC ATC AAG A AGA ACCTGATCGGAGCACTGCTGTTCGACAGCGGAGAAACAGCAG AA GC A AC AA GAC TG AA GAGAACAGCAAGAAGAAGATACA C A AGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGC AACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACcggCTGG AAGAAAGCTTCCTGGTCGAAGAAGACAAGAAGCACGAAAGAC ACCCGATCTTCGGAAACATCGTCGACGAAGTCGCATACCACGA AAAGTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCGA CAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCACT GGCAC ACA TGATC AAGTTC AGAGGAC ACTTCCTG ATCGAAGG AGACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCAT CCAGCTGGTCC AGAC ATAC A ACCAGCTGTTCGAAGAAA ACCC GATCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAGCGC AAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCACA GCTGCCGGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGAT CGCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTC GACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACA TACGACGACGACCTGGACAACCTGCTGGCACAGATCGGAGAC CAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGAC GCAATCCTGCrGAGCGACAlCCTGAGAGTCAACACAGAAATC ACAAAGGCACCGCTGAGCGCAAGCATGATCAAGAGATACGAC GAACACCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGA CAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAG AGCAAGAACGGATACGCAGGATACATCGACGGAGGAG C AA G C CAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAG ATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAA GACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATC CCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGA AGACAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAA AAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCG GACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAA GAAAGAGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAG TCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAA TGACAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGC CGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGA ACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCC GGCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCT GCTGTTC A AG AC AA A C AG AA AGGTCAC AGTC AAGC AGCTGAA 51 2024224004   30 Sep 2024 GGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGA AATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAAC ATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTG GACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTG ACACTGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGA CTGAAGACATACGCACACCTGTTCGACGACAAGGTCATGAAG CAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAG C A GA A AGCTGA TCA A CGG AATC AG AG AC AAGCAGAGCGG A A AGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACA GAAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAA GGAAGACATCCAGAAGGCACAGGTCAGCGGACAGGGAGACA GCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCAA TCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAAC TGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCA TCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAG AAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAAT CAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCGA AAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCT GCAaAACGGAAGAGACATGTACGTCGACCAGGAACTGGACAT CAACAGACTGAGCGACTACGACGTCGACCACATCGTCCCGCA GAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGAC AAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGA GCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGC TGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACC TGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAG GCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATC A C A A A G C A C GTCG C A C AG AT CCTGGACAGCAGAATGAACACA AAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTC ATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGAC TTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACCACG CACACGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGA TCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAG ACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCG AACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACA GCAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAA ACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGA GAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCA ACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTC AAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAG CATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCAAGAAA GAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCC GACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAA GGGA AAGAGCAAGA AGCTGA AGAGCGTCAAGGAACTGCTGG GAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGA TCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGG ACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGA AAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGC AGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACT TCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCC CGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACA AGCACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCA GCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCC TGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAAC AGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGG GAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGACA GAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAACAC TGATCCACCAGAGCATCAC AGG ACTGTACGA AACA AGAATCG 2024224004   30 Sep 2024 acctgagccagctgggaggagacggaggaggaagcccgaag AAGAAGAGAAAGGTCTAGCTAGCCATCACATTTAAAAGCATC TCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAAT AGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACA CCCTGTC'TAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTT tctctgtgcttcaattaataaaaaatggaaagaacctcgag Cas9 ORF with minimal uridine codons frequently used in humam is general; 12.75% U content ATGGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAAC AGCGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCC AGCAAGAAGTTCAAGGTGCTGGGCAACACCGACAGACACAGC ATCAAGAAGAACCTGATCGGCGCCCTGCTGTTCGACAGCGGC GAGACCGCCGAGGCCACCAGACTGAAGAGAACCGCCAGAAGA AGATACACCAGAAGAAAGAACAGAATCTGCTACCTGCAGGAG ATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCC ACAGACTGGAGGAGAG CTT CCTGGTGGAGGAGGAC AA GA AG C ACGAGAGACACCCCATCTTCGGCAACATCGTGGACGAGGTGG CCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGA AGCTGGTGGACAGCACCGACAAGGCCGACCTGAGACTGATCT ACCTGGCCCTGGCCCACATGATCAAGTTCAGAGGCCACTTCCT GATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAA GCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAG GAGAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATC CTGAGCGCCAGACTGAGCAAGAGCAGAAGACTGGAGAACCTG ATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGC AACCTGATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGA GCAACTTCGACCTGGCCGAGGACGCCAAGCTGCAGCTGAGCA AGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGA TCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAAGAACCT GAGCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACAC CGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGAG ATACGACGAGCACCACCAGGACCTGACCCTGCTGAAGGCCCT GGTGAGACAGCAGCTGCCCGAGAAGTACAAGGAGATCTTCTT CGACCAGAGCAAGAACGGCTACGCCGGCTACATCGACGGCGG CGCCAGCCAGGAGGAGTTCTACAAGTTCATCAAGCCCATCCTG GAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAAC AGAGAGGACCTGCTGAGAAAGCAGAGAACCTTCGACAACGGC AGCAJCCCCCACCAGA'i CCACCTGGGCGAGC i GCACGCCATCC TGAGAAGACAGGAGGACTTCTACCCCTTCCTGAAGGACAACA GAGAGAAGATCGAGAAGATCCTGACCTTCAGAATCCCCTACT ACGTGGGCCCCCTGGCCAGAGGCAACAGCAGATTCGCCTGGA TGACCAGAAAGAGCGAGGAGACCATCACCCCCTGGAACTTCG AGGAGGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCG AGAGAATGACCAACTTCGACAAGAACCTGCCCAACGAGAAGG TGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTA CAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGAG AAAGCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCGT GGACCTGCTGTTCAAGACCAACAGAAAGGTGACCGTGAAGCA GCTGAAGGAGGACTACTTCAAGAAGATCGAGTGCTTCGACAG CGTGGAGATCAGCGGCGTGGAGGACAGATTCAACGCCAGCCT GGGCACCTACCACGACCTGCTGAAGATCATCAAGGACAAGGA CTTCCTGG AC A ACG AGG AG A ACG AGG AC ATCCTGG AGGA CAT CGTGCTGACCCTGACCCTGTTCGAGGACAGAGAGATGATCGA GGAGAGACTGAAGACCTACGCCCACCTGTTCGACGACAAGGT GATGAAGCAGCTGAAGAGAAGAAGATACACCGGCTGGGGCAG ACTG AGC AG AA A GCTG ATC A ACGGC ATC AGAG ACAAGCAG AG 52 2024224004   30 Sep 2024 CGGCAAGACCATCCTGGACTTCCTGAAGAGCGACGGCTTCGCC AACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCT TCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCG ACAGCCTGCACGAGCACATCGCCAACCTGGCCGGCAGCCCCG CCATCAAGAAGGGCATCCTGCAGACCGTGAAGGTGGTGGACG AGCTGGTGAAGGTGATGGGCAGACACAAGCCCGAGAACATCG TGATCGAGATGGCCAGAGAGAACCAGACCACCCAGAAGGGCC AGAAGAACAGCAGAGAGAGA ATGAAGAGAATCGAGGAGGGC ATCAAGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTG GAGAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTAC CTGCAGAACGGCAGAGACATGTACGTGGACCAGGAGCTGGAC ATCAACAGACTGAGCGACTACGACGTGGACCACATCGTGCCC CAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTG ACCAGAAGCGACAAGAACAGAGGCAAGAGCGACAACGTGCC CAGCGAGGAGGTGGTGAAGAAGATGAAGAACTACTGGAGACA GCTGCTGAACGCCAAGCTGATCACCCAGAGAAAGTTCGACAA CCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAGCTGGACAA GGCCGGCTTCATCAAGAGACAGCTGGTGGAGACCAGACAGAT caccaagcacgtggcccagatcctggacagcagaatgaacac C A AGT AC G A CG A G A A C G A C A AGC T GAT CAGAGAGGTGAAGGT G ATC ACCCTG AA GAGC A AGCTGGTG AGCGACTTC AGAA AGGA CTTCCAGTTCT AC AA GGTG AG AG AG ATCA AC AACTACC ACC AC GCCCACGACGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGA TCAAGAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCG ACTACAAGGTGTACGACGTGAGAAAGATGATCGCCAAGAGCG AGCAGGAGATCGGCAAGGCCACCGCCAAGTACTTCTTCTACA GCAACATCATGAACTTCTTCAAGACCGAGATCACCCTGGCCAA CGGCGAGATCAGAAAGAGACCCCTGATCGAGACCAACGGCGA GACCGGCGAGATCGTGTGGGACAAGGGCAGAGACTTCGCCAC CGTGAGAAAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAA GAAGACCGAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCAT CCTGCCCAAGAGAAACAGCGACAAGCTGATCGCCAGAAAGAA GGACTGGGACCCCAAGAAGTACGGCGGCTTCGACAGCCCCAC CGTGGCCTACAGCGTGCTGGTGGTGGCCAAGGTGGAGAAGGG CAAGAGCAAGAAGCTGAAGAGCGTGAAGGAGCTGCTGGGCAT CACCATCATGGAGAGAAGCAGCTTCGAGAAGAACCCCATCGA CTTCCTGGAGGCCAAGGGCTACAAGGAGGTGAAGAAGGACCT GATCATCAAGCTGCCCAAGTACAGCCTGTTCGAGCTGGAGAAC GGCAGAAAGAGAATGCTGGCCAGCGCCGGCGAGCTGCAGAAG GGCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACTTCCTGT ACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGG ACAACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCACT ACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCAGCAAGA GAGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCTGAGCG CCTACAACAAGCACAGAGACAAGCCCATCAGAGAGCAGGCCG AGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCCCC CGCCGCCTTCAAGTACTTCGACACCACCATCGACAGAAAGAG ATACACCAGCACCAAGGAGGTGCTGGACGCCACCCTGATCCA CCAGAGCATCACCGGCCTGTACGAGACCAGAATCGACCTGAG C C AGC TGGG CGGC G A C GGC GGC GGC AG CC CC A AGAA GA AG A G AAAGGTGTGA Cas9 transcript with 5’ UTR GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGT GTGTCGTTGCAGGCCTTATTCGGATCCGCCACCATGGACAAGA AGTACAGCATCGGCCTGGACATCGGCACCAACAGCGTGGGCT 53 2024224004   30 Sep 2024 ofHSD, ORF corresponds ng to SEQ ID NO: 52, Kozak sequence, and 3’ UTR of ALB GGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAGT TCAAGGTGCTGGGCAACACCGACAGACACAGCATCAAGAAGA ACCTGATCGGCGCCCTGCTGTTCGACAGCGGCGAGACCGCCGA GGCCACCAGACTGAAGAGAACCGCCAGAAGAAGATACACCAG AAGAAAGAACAGAATCTGCTACCTGCAGGAGATCTTCAGCAA CGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGA GGAGAGCTTCCTGGTGGAGGAGGACAAGAAGCACGAGAGACA CCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAG AAGTACCCCACCATCTACCACCTGAGAAAGAAGCTGGTGGAC AGCACCGACAAGGCCGACCTGAGACTGATCTACCTGGCCCTG GCCCACATGATCAAGTTCAGAGGCCACTTCCTGATCGAGGGCG ACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCC AGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAGAACCCCA TCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGAGCGCCA GACTGAGCAAGAGCAGAAGACTGGAGAACCTGATCGCCCAGC TGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCTGATCG CCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGA CCTGGCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTA CGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCA GTACGCCGACCTGTTCCTGGCCGCCAAGAACCTGAGCGACGCC ATCCTGCTGAGCGAC ATCCTG AG AGTGA AC ACCGAGATCA CC AAGGCCCCCCTGAGCGCCAGCATGATCAAGAGATACGACGAG CACCACCAGGACCTGACCCTGCTGAAGGCCCTGGTGAGACAG CAGCTGCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGC AAGAACGGCTACGCCGGCTACATCGACGGCGGCGCCAGCCAG GAGGAGTTCTACAAGTTCATCAAGCCCATCCTGGAGAAGATG G A CGGC AC CG A GG A GC TGC TG GTG A AGCT GAACAGAGAGGAC CTGCTGAGAAAGCAGAGAACCTTCGACAACGGCAGCATCCCC CACCAGATCCACCTGGGCGAGCTGCACGCCATCCTGAGAAGA CAGGAGGACTTCTACCCCTTCCTGAAGGACAACAGAGAGAAG ATCGAGAAGATCCTGACCTTCAGAATCCCCTACTACGTGGGCC CCCTGGCCAGAGGCAACAGCAGATTCGCCTGGATGACCAGAA AGAGCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGGTGG TGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGAGAATGA CCAACTTCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCA AGCAC A GCCTGCTGTA CG AGTACTTC ACCGTGTAC AACGAGCT GACCAAGGTGAAGTACGTGACCGAGGGCATGAGAAAGCCCGC CTTCCTGAGCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCT GTTCAAGACCAACAGAAAGGTGACCGTGAAGCAGCTGAAGGA GGACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGAT CAGCGGCGTGGAGGACAGATTCAACGCCAGCCTGGGCACCTA CCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGA CAACGAGGAGAACGAGGACATCCTGGAGGACATCGTGCTGAC CCTGACCCTGTTCGAGGACAGAGAGATGATCGAGGAGAGACT GAAGACCTACGCCCACCTGTTCGACGACAAGGTGATGAAGCA GCTGAAGAGAAGAAGATACACCGGCTGGGGCAGACTGAGCAG AAAGCTGATCAACGGCATCAGAGACAAGCAGAGCGGCAAGAC CATCCTGGACTTCCTGAAGAGCGACGGCTTCGCCAACAGAAAC TTCATGCAGCTGATCCACGACGACAGCCTGACCTTCAAGGAGG A C ATC CAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTG C ACGAGCACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGA AGGGCATCCTGCAGACCGTGAAGGTGGTGGACGAGCTGGTGA AGGTGATGGGCAGACACAAGCCCGAGAACATCGTGATCGAGA TGGCCAGAGAGAACCAGACCACCCAG.AAGGGCCAGAAGAAC AGCAGAGAGAGAATGAAGAGAATCGAGGAGGGCATCAAGGA GCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAACAC CCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAA 2024224004   30 Sep 2024 CGGCAGAGACATGTACGTGGACCAGGAGCTGGACATCAACAG ACTGAGCGACTACGACGTGGACCACATCGTGCCCCAGAGCTTC CTGAAGGACGACAGCATCGACAACAAGGTGCTGACCAGAAGC GACAAGAACAGAGGCAAGAGCGACAACGTGCCCAGCGAGGA GGTGGTGAAGAAGATGAAGAACTACTGGAGACAGCTGCTGAA CGCCAAGCTGATCACCCAGAGAAAGTTCGACAACCTGACCAA GGCCGAGAGAGGCGGCCTGAGCGAGCTGGACAAGGCCGGCTT CATCAAGAGACAGCTGGTGGAGACCAGACAGATCACCAAGCA CGTGGCCCAGATCCTGGACAGCAGAATGAACACCAAGTACGA CGAGAACGACAAGCTGATCAGAGAGGTGAAGGTGATCACCCT GAAGAGCAAGCTGGTGAGCGACTTCAGAAAGGACTTCCAGTT CTACAAGGTGAGAGAGATCAACAACTACCACCACGCCCACGA CGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGATCAAGAA GTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCGACTACAA GGTGTACGACGTGAGAAAGATGATCGCCAAGAGCGAGCAGGA GATCGGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATC ATGAACTTCTTCAAGACCGAGATCACCCTGGCCAACGGCGAG ATCAGAAAGAGACCCCTGATCGAGACCAACGGCGAGACCGGC GAGATCGTGTGGGACAAGGGCAGAGACTTCGCCACCGTGAGA AAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAAGAAGACC GAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCATCCTGCCC AAGAGAAACAGCGACAAGCTGATCGCCAGAAAGAAGGACTG GGACCCCAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGC CTACAGCGTGCTGGTGGTGGCCAAGGTGGAGAAGGGGAAGAG CAAGAAGCTGAAGAGCGTGAAGGAGCTGCTGGGCATCACCAT CATGGAGAGAAGCAGCTTCGAGAAGAACCCCATCGACTTCCT GG AGGC C A A GGGCT A C A A GG A GG TG A AG A AG G A CCTG ATC AT CAAGCTGCCCAAGTACAGCCTGTTCGAGCTGGAGAACGGCAG AAAGAGAATGCTGGCCAGCGCCGGCGAGCTGCAGAAGGGCAA CGAGCTGGCCCTGCCCAGCAAGTACGTGAACTTCCTGTACCTG GCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGGACAAC GAGCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTG GACGAGATCATCGAGCAGATCAGCGAGTTCAGCAAGAGAGTG ATCCTGGCCGACGCCAACCTGGACAAGGTGCTGAGGGCCTAC AACAAGCACAGAGACAAGCCCATCAGAGAGCAGGGCGAGAA CATCATCCACCTGTTCACCCTGACCAACCTGGGCGCCCCCGCC GCCTTCA A GTACTTCGAC ACC ACC ATCG A C AGA A AGAG ATAC ACCAGCACCAAGGAGGTGCTGGACGCCACCCTGATCCACCAG AGCATCACCGGCCTGTACGAGACCAGAATCGACCTGAGCCAG CTGGGCGGCGACGGCGGCGGCAGCCCCAAGAAGAAGAGAAA GGTGTGACTAGCCATCACATTTAAAAGCATCTCAGCCTACCAT GAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATC TCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAA AACATAAArrrCTn'AATCArrrCGCCTCTTITCTCTGTGCTTCA ATTAATAAAAAATGGAAAGAACCTCGAG Cas9 ORF with minimal uridine codons infrequently used in hsjmans in general; ATGGACAAAAAATACAGCATAGGGCTAGACATAGGGACGAAC AGCGTAGGGTGGGCGGTAATAACGGACGAATACAAAGTACCG AGCAAAAAATTCAAAGTACTAGGGAACACGGACCGACACAGC ATAAAAAAAAACCTAATAGGGGCGCTACTATTCGACAGCGGG GAAACGGCGGAAGCGACGCGACTAAAACGAACGGCGCGACG ACGATACACGCGACGAAAAAACCGAATATGCTACCTACAAGA AATATTCAGCAACGAAATGGCGAAAGTAGACGACAGCTFCTT CCACCGACTAGAAGAAAGCTTCCTAGTAGAAGAAGACAAAAA AC ACG A ACG ACA CCCG ATATTCGGG A AC ATA GTAG ACGA AGT 54 2024224004   30 Sep 2024 12.75% U content AGCGTACCACGAAAAATACCCGACGATATACCACCTACGAAA AAAACTAGTAGACAGCACGGACAAAGCGGACCTACGACTAAT ATACCTAGCGCTAGCGCACATGATAAAATTCCGAGGGCACTTC CTAATAGAAGGGGACCTAAACCCGGACAACAGCGACGTAGAC aaactattcatacaactagtacaaacgtacaaccaactattcg AAGAAAACCCGATAAACGCGAGCGGGGTAGACGCGAAAGCG ATACTAAGCGCGCGACTAAGCAAAAGCCGACGACTAGAAAAC CTAATAGCGCAACTACCGGGGGAAAAAAAAAACGGGCTATTC GGGAACCTAATAGCGCTAAGCCTAGGGCTAACGCCGAACTTC AAAAGCAACTTCGACCTAGCGGAAGACGCGAAACTACAACTA AGCAAAGACACGTACGACGACGACCTAGACAACCTACTAGCG C A AATAGGGGACC AATA CGCGGACCTATTCCT AGCGGCGA AA AACCTAAGCGACGCGATACTACTAAGCGACATACTACGAGTA AACACGGAAATAACGAAAGCGCCGCTAAGCGCGAGCATGATA AAACGATACGACGAACACCACCAAGACCTAACGCTACTAAAA GCGCTAGTACGACAACAACTACCGGAAAAATACAAAGAAATA TTCTTCGACCAAAGCAAAAACGGGTACGCGGGGTACATAGAC GGGGGGGCGAGCCAAGAAGAATTCTACAAATTCATAAAACCG ATACTAGAAAAAATGGACGGGACGGAAGAACTACTAGTAAAA ctaaaccgagaagacctactacgaaaacaacgaacgttcgac AACGGGAGCATACCGCACCAAATACACCTAGGGGAACTACAC GCGATACTACGACGACAAGAAGACTTCTACCCGTTCCTAAAAG acaaccgagaaaaaatagaaaaaatactaacgttccgaatac CGTACTACGTAGGGCCGCTAGCGCGAGGGAACAGCCGATTCG CGTGGATGACGCGAAAAAGCGAAGAAACGATAACGCCGTGGA ACTTCGAAGAAGTAGTAGACAAAGGGGCGAGCGCGCAAAGCT TC AT AG A ACG A ATG ACG A AC TT C G AC A AAA A CCT ACCG AA C G AAAAAGTACTACCGAAACACAGCCTACTATACGAATACTTCAC GGTATAC AACGA ACTA ACG AA AGT AA AATACGTAACGGA AGG G ATGCGA AA ACCGGCGTTCCTAAGCGGGG AACAAA AA AAAGC GATAGTAGACCTACTATTCAAAACGAACCGAAAAGTAACGGT AAAACAACTAAAAGAAGACTACTTCAAAAAAATAGAATGCTT CGACAGCGTAGAAATAAGCGGGGTAGAAGACCGATTCAACGC GAGCCTAGGGACGTACCACGACCTACTAAAAATAATAAAAGA CAAAGACTTCCTAGACAACGAAGAAAACGAAGACATACTAGA AGACATAGTACTAACGCTAACGCTATTCGAAGACCGAGAAAT GATAGAAGAACGACTAAAAACGTACGCGCACCTATTCGACGA CAAAGTAATGAAACAACTAAAACGACGACGATACACGGGGTG GGGGCGACTAAGCCGAAAACTAATAAACGGGATACGAGACAA A C A A A GC GG G A A A A CG AT AC T AG A CTT CC T A AA AA GCG A CG G GTTCGCGAACCGAAACTTCATGCAACTAATACACGACGACAG CCTAACGTTCAAAGAAGACATACAAAAAGCGCAAGTAAGCGG GCAAGGGGACAGCCTACACGAACACATAGCGAACCTAGCGGG GAGCCCGGCGATAAAAAAAGGGATACTACAAACGGTAAAAGT AGTAGACGAACTAGTAAAAGTAATGGGGCGACACAAACCGGA AAACATAGTAATAGAAATGGCGCGAGAAAACCAAACGACGCA AAAAGGGCAAAAAAACAGCCGAGAACGAATGAAACGAATAG AAGAAGGGATAAAAGAACTAGGGAGCCAAATACTAAAAGAA CACCCGGTAGAAAACACGCAACTACAAAACGAAAAACTATAC CT A TACT A CCT AC AAA ACGGGCGAGACATGTACGTAG ACC AA GAACTAGACATAAACCGACTAAGCGACTACGACGTAGACCAC ATAGTACCGCAAAGCTTCCTAAAAGACGACAGCATAGACAAC AAAGTACTAACGCGAAGCGACAAAAACCGAGGGAAAAGCGA CAACGTACCGAGCGAAGAAGTAGTAAAAAAAATGAAAAACTA CTGGCGACAACTACTAAACGCGAAACTAATAACGCAACGAAA ATTCGACAACCTAACGAAAGCGGAACGAGGGGGGCTAAGCGA ACTAGACAAAGCGGGGTTCATAAAACGACAACTAGTAGAAAC 2024224004   30 Sep 2024 GC GA C A AA TAA CG AA A C AC G T AGC G CAAATAC TAGA CAGC C G AATGAACACGAAATACGACGAAAACGACAAACTAATACGAGA AGTAAAAGTAATAACGCTAAAAAGCAAACTAGTAAGCGACTT CCGAAAAGACTTCCAATTCTACAAAGTACGAGAAATAAACAA CTACCACCACGCGCACGACGCGTACCTAAACGCGGTAGTAGG GACGGCGCTAATAAAAAAATACCCGAAACTAGAAAGCGAATT CGTATACGGGGACTACAAAGTATACGACGTACGAAAAATGAT AGCGAAAAGCGAACAAGAAATAGGGAAAGCGACGGCGAAAT ACTTCTTCTACAGCAACATAATGAACTTCTTCAAAACGGAAAT AACGCTAGCGAACGGGGAAATACGAAAACGACCGCTAATAGA AACGAACGGGGAAACGGGGGAAATAGTATGGGACAAAGGGC GAGACTTCGCGACGGTACGAAAAGTACTAAGCATGCCGCAAG TAAACATAGTAAAAAAAACGGAAGTACAAACGGGGGGGTTCA GCAAAGAAAGCATACTACCGAAACGAAACAGCGACAAACTAA TAGCGCGAAAAAAAGACTGGGACCCGAAAAAATACGGGGGGT TCGACAGCCCGACGGTAGCGTACAGCGTACTAGTAGTAGCGA AAGTAGAAAAAGGGAAAAGCAAAAAACTAAAAAGCGTAAAA GAACTACTAGGGATAACGATAATGGAACGAAGCAGCTTCGAA AAAAACCCGATAGAC TT CCTAGAAGCG AA AG GGT A C A AA GA A GT A A A A A A A GA CCT A AT A AT A A A A CTACC G A AAT A C AGC CT A TTCGAACTAGAAAACGGGCGAAAACGAATGCTAGCGAGCGCG GGGG AACTACA AA AAGGGA ACGA ACT AGCGCTACCG AGC A AA TACGTAAACTTCCTATACCTAGCGAGCCACTACGAAAAACTAA AAGGGAGCCCGGAAGACAACGAACAAAAACAACTATTCGTAG AACAACACAAACACTACCTAGACGAAATAATAGAACAAATAA GCGAATTCAGCAAACGAGTAATACTAGCGGACGCGAACCTAG A C AA A GTA C TA A G CGCGTAC AACAAACACCGAGACAAACCGA TACG AGA AC AAGCGG AA AACATAATACACCTA TTC ACGCTAA CGAACCTAGGGGCGCCGGCGGCGTTCAAATACTTCGACACGA CGATAGACCGAAAACGATACACGAGCACGAAAGAAGTACTAG ACGCGACGCTAATACACCAAAGCATAACGGGGCTATACGAAA CGCGAATAGACCTAAGCCAACTAGGGGGGGACGGGGGGGGG AGCCCG AA AA A A A AA CG AA AAGTATG A Cas9 transcript with 5’ UTR ofHSD. ORF correspond! ng to SEQ ID NO: 54, Kozak sequence, aad 3’ UTR of ALB GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGT GTGTCGTTGCAGGCCTTATTCGGATCCGCCACCATGGACAAAA AATACAGCATAGGGCTAGACATAGGGACGAACAGCGTAGGGT GGGCGGTAATAACGGACGAATACAAAGTACCGAGCAAAAAAT TCAAAGTACTAGGGAACACGGACCGACACAGCATA A A A A A AA, ACCTAATAGGGGCGCTACTATTCGACAGCGGGGAAACGGCGG AAGCGACGCGACTAAAACGAACGGCGCGACGACGATACACGC GACGAAAAAACCGAATATGCTACCTACAAGAAATATTCAGCA ACGAAATGGCGAAAGTAGACGACAGCTTCTTCCACCGACTAG AAGAAAGCTTCCTAGTAGAAGAAGACAAAAAACACGAACGAC ACCCGATATTCGGGAACATAGTAGACGAAGTAGCGTACCACG AAAAATACCCGACGATATACCACCTACGAAAAAAACTAGTAG ACAGCACGGACAAAGCGGACCTACGACTAATATACCTAGCGC TAGCGCACATGATAAAATTCCGAGGGCACTTCCTAATAGAAG GGGACCTAAACCCGGACAACAGCGACGTAGACAAACTATTCA TACAACTAGTACAAACGTACAACCAACTATTCGAAGAAAACC CGATAAACGCGAGCGGGGTAGACGCGAAAGCGATACTAAGCG CGCGACTAAGCAAAAGCCGACGACTAGAAAACCTAATAGCGC AACTACCGGGGGAAAAAAAAAACGGGCTATTCGGGAACCTAA TAGCGCTAAGCCTAGGGCTAACGCCGAACTTCAAAAGCAACTT CGACCTAGCGGAAGACGCGAAACTACAACTAAGCAAAGACAC 55 2024224004   30 Sep 2024 GTACGACGACGACCTAGACAACCTACTAGCGCAAATAGGGGA CCAATACGCGGACCTATTCCTAGCGGCGAAAAACCTAAGCGA CGCGATACTACTAAGCGACATACTACGAGTAAACACGGAAAT AACGAAAGCGCCGCTAAGCGCGAGCATGATAAAACGATACGA CGAACACCACCAAGACCTAACGCTACTAAAAGCGCTAGTACG ACAACAACTACCGGAAAAATACAAAGAAATATTCTTCGACCA AAGCAAAAACGGGTACGCGGGGTACATAGACGGGGGGGCGA GCCAAGA AG A ATTCTA CAA ATTCATAAAACCGATACTAGAA A AAATGGACGGGACGGAAGAACTACTAGTAAAACTAAACCGAG AAGACCTACTACGAAAACAACGAACGTTCGACAACGGGAGCA TACCGCACCAAATACACCTAGGGGAACTACACGCGATACTAC GACGACAAGAAGACTTCTACCCGTTCCTAAAAGACAACCGAG AAAAAATAGAAAAAATACTAACGTTCCGAATACCGTACTACG TAGGGCCGCTAGCGCGAGGGAACAGCCGATTCGCGTGGATGA CGCGAAAAAGCGAAGAAACGATAACGCCGTGGAACTTCGAAG AAGTAGTAGACAAAGGGGCGAGCGCGCAAAGCTTCATAGAAC GAATGACGAACTTCGACAAAAACCTACCGAACGAAAAAGTAC TACCG A AACACAGCCTACTATACGAATA CTT CACGGTATAC AA CG A A CT A A C GA A A GT A A A AT AC GT AAC GGAAGGGATGCGA AA ACCGGCGTTCCTAAGCGGGGAACAAAAAAAAGCGATAGTAGA CCTACTATTCAAA ACGA ACCGAAAAGTAACGGTAAAAC AACT AAAAGAAGACTACTTCAAAAAAATAGAATGCTTCGACAGCGT AGAAATAAGCGGGGTAGAAGACCGATTCAACGCGAGCCTAGG GACGTACCACGACCTACTAAAAATAATAAAAGACAAAGACTT CCTAGACAACGAAGAAAACGAAGACATACTAGAAGACATAGT ACTAACGCTAACGCTATTCGAAGACCGAGAAATGATAGAAGA A C G A C T A A A A A CGT A C GC GC A CC T ATTCG AC G AC A AAGTA AT GAAACAACTAAAACGACGACGATACACGGGGTGGGGGCGACT AAGCCGAAAACTAATAAACGGGATACGAGACAAACAAAGCG GGAAAACGATACTAGACTTCCTAAAAAGCGACGGGTTCGCGA ACCGAAACTTCATGCAACTAATACACGACGACAGCCTAACGTT CAAAGAAGACATACAAAAAGCGCAAGTAAGCGGGCAAGGGG ACAGCCTACACGAACACATAGCGAACCTAGCGGGGAGCCCGG CGATAAAAAAAGGGATACTACAAACGGTAAAAGTAGTAGACG AACTAGTAAAAGTAATGGGGCGACACAAACCGGAAAACATAG TAATAGAAATGGCGCGAGAAAACCAAACGACGCAAAAAGGG caaaaaaacagccgagaacgaatgaaacgaatagaagaagg GATAAAAGAACTAGGGAGCCAAATACTAAAAGAACACCCGGT AGAAAACACGCAACTACAAAACGAAAAACTATACCTATACTA CCTACAAAACGGGCGAGACATGTACGTAGACCAAGAACTAGA cataaaccgactaagcgactacgacgtagaccacatagtacc gcaaagcttcctaaaagacgacagcatagacaacaaagtact AA CGC GA A G C GA C A AAAACCGAGGG AAA A GCGA CAAC GTA C cgagcgaagaagtagtaaaaaaaatgaaaaactactggcgac aactactaaacgcgaaactaataacgcaacgaaaattcgaca acctaacgaaagcggaacgaggggggctaagcgaactagaca aagcggggttcataaaacgacaactagtagaaacgcgacaaa taacgaaacacgtagcgcaaatactagacagccgaatgaaca CG AA A T A CG AC G A AA A C GAC AA AC T AATAC GA GAAG TAAA A G TAATAACGCTAAAAAGCAAACTAGTAAGCGACTTCCGAAAAG ACTTCCAATTCTACAAAGTACGAGAAATAAACAACTACCACCA CGCGCACGACGCGTACCTAaACGCGGTAGTAGGGACGGCGCT AATAAAAAAATACCCGAAACTAGAAAGCGAATTCGTATACGG GGACTACAAAGTATACGACGTACGAAAAATGATAGCGAAAAG CGAACAAGAAATAGGGAAAGCGACGGCGAAATACTTCTTCTA C A GC A ACA TAATGAACTTCTTC A AA ACGG AA ATA ACGCTAGC GAACGGGGAAATACGAAAACGACCGCTAATAGAAACGAACG 2024224004   30 Sep 2024 GGGAAACGGGGGAAATAGTATGGGACAAAGGGCGAGACTTCG CGACGGTACGAAAAGTACTAAGCATGCCGCAAGTAAACATAG TAAAAAAAACGGAAGTACAAACGGGGGGGTTCAGCAAAGAA AGCATACTACCGAAACGAAACAGCGACAAACTAATAGCGCGA A A A A A A G A C TGGG AC CC G A A A A A AT A CG GG GG GTT CG AC A GC CCGACGGTAGCGTACAGCGTACTAGTAGTAGCGAAAGTAGAA AAAGGGAAAAGCAAAAAACTAAAAAGCGTAAAAGAACTACT AGGGATAACGATAATGGAACGAAGCAGCTTCGAAAAAAACCC GATAGACTTCCTAGAAGCGAAAGGGTACAAAGAAGTAAAAAA AGACCTA.aTAATAAAACTACCGAAATACAGCCTATTCGA.ACTA GAAAACGGGCGAAAACGAATGCTAGCGAGCGCGGGGGAACT ACAAAAAGGGAACGAACTAGCGCTACCGAGCAAATACGTAAA CTTCCTATACCTAGCGAGCCACTACGAAAAACTAAAAGGGAG CCCGGAAGACAACGAACAAAAACAACTATTCGTAGAACAACA CAAACACTACCTAGACGAAATAATAGAACAAATAAGCGAATT CAGCAAACGAGTAATACTAGCGGACGCGAACCTAGACAAAGT ACTAAGCGCGTACAACAAACACCGAGACAAACCGATACGAGA ACAAGCGGAAAACATAATACACCTATTCACGCTAACGAACCT AGGGGCGCCGGCGGCGTTCAAATACTTCGACACGACGATAGA CCGAAAACGATACACGAGCACGAAAGAAGTACTAGACGCGAC GCTAATACACCAAAGCATAACGGGGCTATACGAAACGCGAAT AGACCTAAGCCAACTAGGGGGGGACGGGGGGGGGAGCCCGA A A A A A A A A CG A A A A GT AT G ACT AGC CATC ACA TTT AA AAG C A TCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCA ATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAA CACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCT TTT CTCTGTGCTTCAA TT A AT A A A A A AT G G A AAG AACCT CG AG Cas9 transcript with AGG as first three nucleotides for use with CleanCap™, 5’ UTR of HSD, ORF corresponds ng to SEQ ID NO: 4, Kozak sequence, and 3’ UTR of ALB AGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGT GTGTCGTTGCAGGCCTTATTCGGATCCGCCACCATGGACAAGA AGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGAT GGGCAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGT TCAAGGTCCTGGGAAACACAGACAGACACAGCATCAAGAAGA ACCTGATCGGAGCACTGCTGTTCGACAGCGGAGAAACAGCAG AAGCAACAAGACTGAAGAGAACAGCAAGAAGAAGATACACA AGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGC AACGAAATGGCAAAGGTCGACGACAGCT1CT1CCACAGACTG GAAGAAAGCTTCCTGGTCGAAGAAGACAAGAAGCACGAAAGA CACCCGATCTTCGGAAACATCGTCGACGAAGTCGCATACCACG AAAAGTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCG ACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCAC TGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGG AGACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCAT CCAGCTGGTCCAGACATACAACCAGCTGTTCGAAGAAAAC.ee GATCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAGCGC AAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCACA GCTGCCGGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGAT CGCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTC G A CC TG GC A GA A G AC G CAA AGCTGCAGCTGAGCAAGGACACA TACGACGACGACCTGGACAACCTGCTGGCACAGATCGGAGAC CAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGAC GCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATC ACAAAGGCACCGCTGAGCGCAAGCATGATCAAGAGATACGAC GAACACCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGA CAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAG 56 2024224004   30 Sep 2024 A G CA A G AA CG GA T A CGC AGG ATAC ATC G AC GG AGGA G C AA G C CAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAG ATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAA GACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATC CCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGA AGACAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAA AAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCG GACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAA GAAAGAGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAG TCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAA TGACAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGC CGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGA ACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCC GGCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCT GCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGCTGAA GGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGA AATC AGCGG AGTCG AA GACAGATTC AACGC AAGCCTGGG AA C ATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTG GACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTG ACACTGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGA CTGAAGACATACGCACACCTGTTCGACGACAAGGTCATGAAG CAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAG CAGAAAGCTGATCAACGGAATCAGAGAC AAGC AG A GCGG A A AGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACA GAAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAA GGAAGACATCCAGAAGGCACAGGTCAGCGGACAGGGAGACA GC CTG CA CG AA C A CA TC GC AA AC CTGGC AGGAAGCCCGGCAA TCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAAC TGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCA TCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAG AAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAAT CAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCGA AAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCT GCAGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACAT CAACAGACTGAGCGACTACGACGTCGACCACATCGTCCCGCA GAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGAC AAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGA GCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGC TGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACC TGACAAAGGCAGAGAGAGGAGGACTGAGCG AACTGG A C A AG GCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATC ACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACA AAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTC ATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGAC TICCA GTTCTACA AGGTC AGAGA AATC AAC AA CTACC ACC ACG CACACGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGA TCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAG ACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCG AACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACA GCAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAA ACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGA GAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCA ACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTC AAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAG C ATCCTGCCG A AGAG AA ACAGCG ACA AGCTGATCGCAAG AA A GAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCC GACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAA 2024224004   30 Sep 2024 GG G A A AG A GC A A G A AGC TG A AG AGC GTC A A GG AAC TGCTGG GAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGA TCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGG ACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGA AAACGGAAGAAAGAGAATGCTGGCAAGCGCAGG AGAA CTGC AGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACT TCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCC CGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACA AGCACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCA GCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCC TGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAAC AGGCAGAAAACATCATCCACCTG'ITCACACTGACAAACCTGG GAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGACA GAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAACAC TGATCCACCAGAGCATCACAGGACTGTACGAAACAAGAATCG ACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAG AAGAAGAGAAAGGTCTAGCTAGCCATCACATTTAAAAGCATC TCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAAT AGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACA CCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTT TCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCTCGAG Cas9 transcript with 5’ UTR from CMV, ORF correspond! ng to SEQ ID NO: 4, Kozak sequence, and 3’ UTR of ALB GGGCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCC ATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAAC GGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACTCACC GTCCTTGACACGGCCACCATGGACAAGAAGTACAGCATCGGA CTGGACATCGGAACAAACAGCGTCGGATGGGCAGTCATCACA GACGAATACAAGGTCCCGAGCAAGAAGTTCAAGGTCCTGGGA AACACAGACAGACACAGCATCAAGAAGAACCTGATCGGAGCA CTGCTGTTCGACAGCGGAGAAACAGCAGAAGCAACAAGACTG AAGAGAACAGCAAGAAGAAGATACACAAGAAGAAAGAACAG AATCTGCTACCTGCAGGAAATCTTCAGCAACGAAATGGCAAA GGTCGACGACAGCTTCTTCCACAGACTGGAAGAAAGCTTCCTG GTCGAAGAAGACAAGAAGCACGAAAGACACCCGATCTTCGGA AACATCGTCGACGAAGTCGCATACCACGAAAAGTACCCGACA ATCTACCACCTGAGAAAGAAGCTGGTCGACAGCACAGACAAG GCAGACCTGAGACTGATCTACCTGGCACTGGCACACATGATCA AGTTCAGAGGACACTTCCTGATCGAAGGAGACCTGAACCCGG ACAACAGCGACGTCGACAAGCTGTTCATCCAGCTGGTCCAGAC ATACAACCAGCTGTTCGAAGAAAACCCGATCAACGCAAGCGG A G TCG AC GC A A A G GC A A TC CTG AG CGC AAG ACT GA GC AA GAG CAGAAGACTGGAAAACCTGATCGCACAGCTGCCGGGAGAAAA G A AG AA CGG ACTGTTCGGA AACCTGATCGC ACTG AGCCTGGG ACTGACACCGAACTTCAAGAGCAACTTCGACCTGGCAGAAGA CGCAAAGCTGCAGCTGAGCAAGGACACATACGACGACGACCT GGACAACCTGCTGGCACAGATCGGAGACCAGTACGCAGACCT GTTCCTGGCAGCAAAGAACCTGAGCGACGCAATCCTGCTGAG CGACATCCTGAGAGTCAACACAGAAATCACAAAGGCACCGCT G A GC G CA A G C ATG ATC AA G A GATA CG AC GAA CACCACCA GGA CCTGACACTGCTGAAGGCACTGGTCAGACAGCAGCTGCCGGA AAAGTACAAGGAAATCTTCTTCGACCAGAGCAAGAACGGATA CGCAGGATACATCGACGGAGGAGCAAGCCAGGAAGAATTCTA CAAGTTCATCAAGCCGATCCTGGAAAAGATGGACGGAACAGA AGAACTGCTGGTCAAGCTGAACAGAGAAGACCTGCTGAGAAA GCAGAGAACATTCGACAACGGAAGCATCCCGCACCAGATCCA 57 2024224004   30 Sep 2024 CCTGGGAGAACTGCACGCAATCCTGAGAAGACAGGAAGACTT CTACCCGTTCCTGAAGGACAACAGAGAAAAGATCGAAAAGAT CCTGACATTCAGAATCCCGTACTACGTCGGACCGCTGGCAAGA GGAAACAGCAGATTCGCATGGATGACAAGAAAGAGCGAAGA AACAATCACACCGTGGAACTTCGAAGAAGTCGTCGACAAGGG AGCAAGCGCACAGAGCTTCATCGAAAGAATGACAAACTTCGA CAAGAACCTGCCGAACGAAAAGGTCCTGCCGAAGCACAGCCT GCTGTACGAATACTTCACAGTCTACAACGAACTGACAAAGGTC AAGTACGTCACAGAAGGAATGAGAAAGCCGGCATTCCTGAGC GGAGAACAGAAGAAGGCAATCGTCGACCTGCTGTTCAAGACA AACAGAAAGGTCACAGTCAAGCAGCTGAAGGAAGACTACTTC AAGAAGATCGAATGCTTCGACAGCGTCGAAATCAGGGGAGTC GAAGACAGATTCAACGCAAGCCTGGGAACATACCACGACCTG CTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAAGAA AACGAAGACATCCTGGAAGACATCGTCCTGACACTGACACTGT TCGAAGACAGAGAAATGATCGAAGAAAGACTGAAGACATACG CACACCTGTTCGACGACAAGGTCATGAAGCAGCTGAAGAGAA GAAGATACACAGGATGGGGAAGACTGAGCAGAAAGCTGATCA ACGGAATCAGAGACAAGCAGAGCGGAAAGACAATCCTGGACT TCCTGAAGAGCGACGGATTCGCAAACAGAAAC T TCATGCAGC TGATCCACGACGACAGCCTGACATTCAAGGAAGACATCCAGA AGGCACAGGTCAGCGGACAGGGAGACAGCCTGCACGAACACA TCGCAAACCTGGCAGGAAGCCCGGCAATCAAGAAGGGAATCC TGCAGACAGTCAAGGTCGTCGACGAACTGGTCAAGGTCATGG GAAGACACAAGCCGGAAAACATCGTCATCGAAATGGGAAGAG AAAACCAGACAACACAGAAGGGACAGAAGAACAGCAGAGAA AGAATGAAGAGAATCGAAGAAGGAATCAAGGAACTGGGAAG CCAG ATCCTG AA GG AAC ACCCGGTCG A AAACAC AC A GCTGCA GAACGAAAAGCTGTACCTGTACTACCTGCAGAACGGAAGAGA CATGTACGTCGACCAGGAACTGGACATCAACAGACTGAGCGA CTACGACGTCGACCACATCGTCCCGCAGAGCTTCCTGAAGGAC GACAGCATCGACAACAAGGTCCTGACAAGAAGCGACAAGAAC AGAGGAAAGAGCGACAACGTCCCGAGCGAAGAAGTCGTCAAG AAGATGAAGAACTACTGGAGACAGCTGCTGAACGCAAAGCTG ATCACACAGAGAAAGTTCGACAACCTGACAAAGGCAGAGAGA GGAGGACTGAGCGAACTGGACAAGGCAGGATTCATCAAGAGA CAGCTGGTCGAAACAAGACAGATCACAAAGCACGTCGCACAG ATCCTGGACAGCAGAATGAACACAAAGTACGACGAAAACGAC AAGCTGATCAGAGAAGTCAAGGTCATCACACTGAAGAGCAAG CTGGTCAGCGACTTCAGAAAGGACTTCCAGTTCTACAAGGTCA GAGAAATCAACAACTACCACCACGCACACGACGCATACCTGA ACGCAGTCGTCGGAACAGCACTGATCAAGAAGTACCCGAAGC TGGAAAGCGAATTCGTCTACGGAGACTACAAGGTCTACGACG TCAGAAAGATGATCGCAAAGAGCGAACAGGAAATCGGAAAG GCAACAGCAAAGTACTTC'nCTACAGCAACATCATGAACTI’CT TCAAGACAGAAATCACACTGGCAAACGGAGAAATCAGAAAGA GACCGCTGATCGAAACAAACGGAGAAACAGGAGAAATCGTCT GGGACAAGGGAAGAGACTTCGCAACAGTCAGAAAGGTCCTGA GCATGCCGCAGGTCAACATCGTCAAGAAGACAGAAGTCCAGA CAGGAGGATTCAGCAAGGAAAGCATCCTGCCGAAGAGAAACA GCGACAAGCTGATCGCAAGAAAGAAGGACTGGGACCCGAAGA AGTACGGAGGATTCGACAGCCCGACAGTCGCATACAGCGTCC TGGTCGTCGCAAAGGTCGAAAAGGGAAAGAGCAAGAAGCTGA AGAGCGTCAAGGAACTGCTGGGAATCACAATCATGGAAAGAA GCAGCTTCGAAAAGAACCCGATCGACTTCCTGGAAGCAAAGG GATACAAGGAAGTCAAGAAGGACCTGATCATCAAGCTGCCGA AGTACAGCCTGTTCGAACTGGAAAACGGAAGAAAGAGAATGC 2024224004   30 Sep 2024 TGGC A AGCG CA GG AG A A CTGCAG A AG GG AAAC G AAC TGGC A C TGCCGAGCAAGTACGTCAACTTCCTGTACCTGGCAAGCCACTA CGAAAAGCTGAAGGGAAGCCCGGAAGACAACGAACAGAAGC AGCTGTTCGTCGAACAGCACAAGCACTACCTGGACGAAATCAT CGAACAGATCAGCGAATTCAGCAAGAGAGTCATCCTGGCAGA CGCAAACCTGGACAAGGTCCTGAGCGCATACAACAAGCACAG AGACAAGCCGATCAGAGAACAGGCAGAAAACATCATCCACCT GTTCACACTGACAAACCTGGGAGCACCGGCAGCATTCAAGTA CTTCGACACAACAATCGACAGAAAGAGATACACAAGCACAAA GGAAGTCCTGGACGCAACACTGATCCACCAGAGCATCACAGG ACTGTACGAAACAAGAATCGACCTGAGCCAGCTGGGAGGAGA CGGAGGAGGAAGCCCGAAGAAGAAGAGAAAGGTCTAGCTAG CCATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAG AAAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTT CGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTC TTTAATCA'HTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAA ATGGAAAGAACCTCGAG Cas9 transcript with 5’ UTR from HBB, ORF corresponds ng to SEQ ID NO: 4, Kozak sequence, and 3’ UTR of HBB GG Ga ca ttt gc Etc tgac aca ac tgt gt tc act agca ac etc aa ac agac acc ggat c igcc acc AT GGACAAGAAGTACAGCATCGGACTGGACATCGGAACAAACAG CGTCGGATGGGCAGTCATCACAGACGAATACAAGGTCCCGAG CAAGAAGTTCAAGGTCCTGGGAAACACAGACAGACACAGCAT CAAGAAGAACCTGATCGGAGCACTGCTGTTCGACAGCGGAGA AACAGCAGAAGCAACAAGACTGAAGAGAACAGCAAGAAGAA GATACACAAGAAGAAAGAACAGAATCTGCTACCTGCAGGAAA TCTTCAGCAACGAAATGGCAAAGGTCGACGACAGCTTCTTCCA CAGACTGGAAGAAAGCTTCCTGGTCGAAGAAGACAAGAAGCA CGAAAGACACCCGATCTTCGGAAACATCGTCGACGAAGTCGC ATA CC ACG A AA AGTA CCCG AC AATCTACCACCTGAGA AAGA A GCTGGTCGACAGCACAGACAAGGCAGACCTGAGACTGATCTA CCTGGCACTGGCACACATGATCAAGTTCAGAGGACACTTCCTG ATCGAAGGAGACCTGAACCCGGACAACAGCGACGTCGACAAG CTGTTCATCCAGCTGGTCCAGACATACAACCAGCTGTTCGAAG AAAACCCGATCAACGCAAGCGGAGTCGACGCAAAGGCAATCC TGAGCGCAAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGA TCGCACAGCTGCCGGGAGAAAAGAAGAACGGACTGTTCGGAA ACCrGATCGCAClGAGCCTGGGACiGACACCGAACTTCAAGA GCAACTTCGACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCA AGGACACATACGACGACGACCTGGACAACCTGCTGGCACAGA TCGGAGACCAGTACGCAGACCTGTTCCTGGCAGCAAAGAACC TGAGCGACGCAATCCTGCTGAGCGACATCCTGAGAGTCAACA CAGAAATCACAAAGGCACCGCTGAGCGCAAGCATGATCAAGA GATACGACGAACACCACCAGGACCTGACACTGCTGAAGGCAC TGGTCAGACAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCT TCGACCAGAGCAAGAACGGATACGCAGGATACATCGACGGAG GAGCAAGCCAGGAAGAATTCTACAAGTTCATCAAGCCGATCC TGGAAAAGATGGACGGAACAGAAGAACTGCTGGTCAAGCTGA ACAGAGAAGACCTGCTGAGAAAGCAGAGAACATTCGACAACG GAAGCATCCCGCACCAGATCCACCTGGGAGAAC1GCACGCAA TCCTGAGAAGACAGGAAGACTTCTACCCGTTCCTGAAGGACA ACAGAGAAAAGATCGAAAAGATCCTGACATTCAGAATCCCGT ACTACGTCGGACCGCTGGCAAGAGGAAACAGCAGATTCGCAT GGATGACAAGAAAGAGCGAAGAAACAATCACACCGTGGAACT TCGAAGAAGTCGTCGACAAGGGAGCAAGCGCACAGAGCTTCA TCGAAAGAATGACAAACTTCGACAAGAACCTGCCGAACGAAA 58 2024224004   30 Sep 2024 AGGTCCTGCCGAAGCACAGCCTGCTGTACGAATACTTCACAGT CTACAACGAACTGACAAAGGTCAAGTACGTCACAGAAGGAAT GAGAAAGCCGGCATTCCTGAGCGGAGAACAGAAGAAGGCAAT CGTCGACCTGCTGTTCAAGACAAACAGAAAGGTCACAGTCAA GCAGCTGAAGGAAGACTACTTCAAGAAGATCGAATGCTTCGA CAGCGTCGAAATCAGCGGAGTCGAAGACAGATTCAACGCAAG CCTGGGAACATACCACGACCTGCTGAAGATCATCAAGGACAA GGACTTCCTGGACAACGAAGAAAACGAAGACATCCTGGAAGA CATCGTCCTGACACTGACACTGTTCGAAGACAGAGAAATGATC GAAGAAAGACTGAAGACATACGCACACCTGTTCGACGACAAG GTCATGAAGCAGCTGAAGAGAAGAAGATACACAGGATGGGGA AGACTGAGCAGAAAGCTGATCAACGGAATCAGAGACAAGCAG AGCGGAAAGACAATCCTGGACTTCCTGAAGAGCGACGGATTC GCAAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTG ACATTCAAGGAAGACATCCAGAAGGCACAGGTCAGCGGACAG GGAGACAGCCTGCACGAACACATCGCAAACCTGGCAGGAAGC CCGGCAATCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTC GACGAACTGGTCAAGGTCATGGGAAGACACAAGCCGGAAAAC ATC GT C A TCG A A ATG GC A AG AG A A A AC C AG AC AAC AC AG AA. G GGACAGAAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGA A G G A AT C A A G G A A CTGG G A AGCC AG ATC CTG AAGG AAC AC C C GGTCGAAAACACACAGCTGCAGAACGAAAAGCTGTACCTGTA CTACCTGCAGAACGGAAGAGACATGTACGTCGACCAGGAACT GGACATCAACAGACTGAGCGACTACGACGTCGACCACATCGT CCCGCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGT CCTGACAAGAAGCGACAAGAACAGAGGAAAGAGCGACAACG TCC CG AGCG A A G A AG TC GTC A AG A AG ATG A AG AAC TACT GG A GACAGCTGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCG AC AACCTGACAA AGGCAGAG AG AGGAGG ACTGAGCGA ACTG GACAAGGCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGA CAGATCACAAAGCACGTCGCACAGATCCTGGACAGCAGAATG AACACAAAGTACGACGAAAACGACAAGCTGATCAGAGAAGTC AAGGTCATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGA AAGGACTTCCAGTTCTACAAGGTCAGAGAAATCAACAACTAC CACCACGCACACGACGCATACCTGAACGCAGTCGTCGGAACA GCACTGATCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTC TACGGAGACTACAAGGTCTACGACGTCAGAAAGATGATCGCA AAGAGCGAACAGGAAATCGGAAAGGCAACAGCAAAGTACTTC TTCTACAGCAACATCATGAACTTCTTCAAGACAGAAATCACAC TGGCAAACGGAGAAATCAGAAAGAGACCGCTGATCG AA AC A A A C GG A G A A A C AGG A G A A ATC G TC TGG G A C A AG G G AA G AG A C TTCGCAACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAAC ATCGTCAAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAG GAAAGCATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCA AGAAAGAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGAC AGCCCGACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCG AAAAGGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTG CTGGGAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAAC CCGATCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAG AAGGACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAAC TGGAAAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAA CTGC AGA AGGG A A ACG A A CTGGC ACTGCCG AGC AAGTACGTC AACTTCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGA AGCCCGGAAGACAACGAACAGAAGCAGCTGTTCGTCG.AACAG CACAAGCACTACCTGGACGAAATCATCGAACAGATCAGCGAA TTCAGCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAG GTCCTGAGCGC ATAC A AC A AGC AC AG AGAC AA GCCG ATCAGA 2024224004   30 Sep 2024 GAACAGGCAGAAAACATCATCCACCTGTTCACACTGACAAAC CTGGGAGCACCGGCAGCATTCAAGTACTTCGACACAACAATC GACAGAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCA ACACTGATCCACCAGAGCATCACAGGACTGTACGAAACAAGA ATCGACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCG A A G A A GA A G A G A A A G GTCT A Get agcgctc gc til ct tgc t glee a a n t cia i ta aa gg! t c c ttt gtt cc eta agte c a act ac taa a c tgggggat a ft atgaa gggc ct tga gc at etgga tl cig c cta at aaa a aac at Ila ft i t ca t tgee tc gag Cas9 transcript with 5’ UTR from XBG, ORF correspond! ng to SEQ H) NO: 4, Kozak sequence, and 3 ’ UTR ut'XBG G G Ga a getea gaa t aaa cgctc aa ct 11 ggc egga t ct gc ca cC ATGG AC AA GA AGT ACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGATGGG CAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCA AGGTCCTGGGAAACACAGACAGACACAGCATCAAGAAGAACC TGATCGG AGCACTGCTGTTCGACAGCGGAG A AAC AGCAG AA G C AAC AAG A CTG A A GAG AAC AG CAAGAAG AAGA TACA C AAGA AGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCAAC GAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAA GAAAGCTTCCTGGTCGAAGAAGAC AAG AAG C ACG AA AG AC A C CCGATCTTCGGAAACATCGTCGACGAAGTCGCATACCACGAA AAGTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCGAC AGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCACTG GCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGA GACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCATC CAGCTGGTCCAGACATACAACCAGCTGTTCGAAGAAAACCCG ATCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAGCGCA A G AC TG A GC A A G A GC A G A AG A CTGG A A A A CCTG ATC GCAC A G CTGCCGGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGATC GCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTC GACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACA TACGACGACGACCTGGACAACCTGCTGGCACAGATCGGAGAC CAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGAC GCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATC ACAAAGGCACCGCTGAGCGCAAGCATGATCAAGAGATACGAC GAACACCACCAGGACCTGACACTGCTGAAGGCACTGGTCAGA CAGCAGCTGCCGG AA AAGTACA AGG AAATCTTCTTCGA CCAG AGCAAGAACGGATACGCAGGATACATCGACGGAGGAGCAAGC CAGGAAGAAT IC 1 ACAAGTi CA 1CAAGCCGA1CC1GGAAAAG ATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAA GACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATC CCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGA AGACAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAA AAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCG GACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAA GAAAGAGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAG TCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAA TGACAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGC CGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGA A CTG A C A A A GG TC A A GT A C GTC AC AG AAG G A ATG A G A AA GC C GGCATTCCTGAGCGGAGiAACAGAAGAAGGCAATCGTCGACCT GCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGCTGAA GGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGA AATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAAC ATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTG GACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTG ACACTGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGA 59 2024224004   30 Sep 2024 CTGAAGACATACGCACACCTGTTCGACGACAAGGTCATGAAG CAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAG CAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAA AGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACA GAAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAA GGAAGACATCCAGAAGGCACAGGTCAGCGGACAGGGAGACA GCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCAA TCA AG AAGGG AATCCTGCAG AC AGTC AA GGTCGTCGACGAAC TGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCA TCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAG AAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAAT CAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCGA AAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCT GCAGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACAT CAACAGACTGAGCGACTACGACGTCGACCACATCGTCCCGCA GAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGAC AAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGA GCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGC TGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACC TGACAAAGGCAGAGAGAGGAGGACTGAGCG AACTGGA C A AG GCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATC ACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACA AAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTC ATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGAC TTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACCACG CACACGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGA TC A A G A A GT AC CC G A A GC TGG A A A GC G AATT CGTCTACGGAG ACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCG AACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACA GCAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAA ACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGA GAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCA acagtcagaaaggtcctgagcatgccgcaggtcaacatcgtc AAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAG CATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCAAGAAA GAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCC GACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAA GGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGG GAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGA TCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGG ACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGA AAACGGAAGAAAGAGAATGCTGGCAAGCGCAGG AG AA CTGC AGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACT TCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCC CGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACA AGCACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCA GCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCC TGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAAC AGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGG GAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGACA GAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAACAC TGA TCCACCAG AGC ATCAC AGG ACTGTACGA AACAAGAATCG ACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAG .AA GA A G AG A .AA GGTCTAGctagcaccagccicaagaacacccgaalggagictciaa gc!acataaraccaac1tacacf.Hacaaaa!gHg(i:ccccaaaa!g(agcca!lcg!ak:igc(cclaa!a aaaagaaagsftcttcacaitctctcgag 2024224004   30 Sep 2024 Cas9 transcript with AGG as first three nucleotides for use with CleanCap™, 5’ UTR from XBG, ORF corresponds ng to SEQ H) NO: 4, Kozak sequence, and 3’ UTR ofXBG A G Ga agcscagaa i a aa cgctc aa clt i ggc cgga i ct gc ca cC A TGG AC AA G A AGT ACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGATGGG CAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGTTCA AGGTCCTGGGAAACACAGACAGACACAGCATCAAGAAGAACC TGA TCGG AGCACTGCTGTTCGAC AGCGGAGA AACAGCAG AA G CAACAAGACTGAAGAGAACAGCAAGAAGAAGATACACAAGA AGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGCAAC GAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTGGAA GAAAGCTTCCTGGTCGAAGAAGACAAGAAGCACGAAAGACAC CCGATCTTCGGAAACATCGTCGACGAAGTCGCATACCACGAA AAGTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCGAC AGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCACTG GCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGGA GACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCATC C A GCTGGTCCAGA CATACAA CC AGCTGTTCGA AG AAA ACC C G ATCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAGCGCA AGACTGAGCAAGAGCAGAAGACTGG A A A A CCTG ATC GCAC A G CTGCCGGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGATC GCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTC G A CC TG GC A GA A G AC GC AA AG CTGC AG C TG AGCAAGG ACA CA TACGACGACGACCTGGACAACCTGCTGGCACAGATCGGAGAC CAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGAC GCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATC ACAAAGGCACCGCTGAGCGCAAGCATGATCAAGAGATACGAC G A AC A CC A C C AGG AC C T GAC A CTG CTGAAGGCACTGGTCAGA CAGCAGCTGCCGGAAAAGTACAAGGAAATCTTCTTCGACCAG AGCAAGAACGGATACGCAGGATACATCGACGGAGGAGCAAGC CAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAG ATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAA GACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATC CCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGA AGACAGGAAGACTTCTACCCGTTCCTGAAGGACAACAGAGAA AAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCG GACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAA GAAAGAGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAG rCGJCGACAAGGGAGCAAGCGCACAGAGCnCAlCGAAAGAA TGACAAACITCGACAAGAACCTGCCGAACGAAAAGGTCCTGC CGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGA A CTG A C A A A GG TC A A GT A CGTC AC AG AAG G A ATG A G A AA GC C GGCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCT GCTGTTCAAGACAAACAGAAAGGTCACAGTCAAGCAGCTGAA GGAAGACTACTTCAAGAAGATCGAATGCTTCGACAGCGTCGA AATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAAC ATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTG GACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTG ACACTGACACTGTTCGAAGACAGAGAAATGATCGAAGAAAGA CTGAAGACATACGCACACCTGTTCGACGACAAGGTCATGAAG CAGCTGAAGAGtLAGAAGATACACAGGATGGGGAAGACTGAG CAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAA AGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACA GAAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAA GGAAGACATCCAGAAGGCACAGGTCAGCGGACAGGGAGACA GCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCAA TCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAAC 60 2024224004   30 Sep 2024 TGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCA TCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAG AAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAAT CAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCGA AAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCT GCAGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACAT CAACAGACTGAGCGACTACGACGTCGACCACATCGTCCCGCA GAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGAC AAGAAGCGACAAGAACAGAGGAAAGAGCGACAACGTCCCGA GCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGC TGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACC TGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAG GCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATC ACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACA AAGTACGACGAAAACGACAAGCTGATCAGAGAAGTCAAGGTC ATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGAC TTCCAGTTCTACAAGGTCAGAGAAATCAACAACTACCACCACG CACACGACGCATACCTGAACGCAGTCGTCGGAACAGCACTGA TCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAG ACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCG AA C A GG AA AT CGG A AA G GC AA C AGC A AAGT ACT TC TT C TACA GCAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAA ACGGAGAAATCAGAAAGAGACCGCTGATCGAAA C AAA CGG A GAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCA ACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTC AAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAG C ATCCTGCCG A AG AG A A AC AGCG AC A AGCTG ATCGC A AG AA A GAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCC GACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAA GGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGG GAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGA TCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGG ACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGA AAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGC AGAAGGGAAACGAACTGGCACTGCCGAGCAAGTACGTCAACT TCCTGTACCTGGCAAGCCACTACGAAAAGCTGAAGGGAAGCC CGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACA AGCACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCA GCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCC TG A GC GC A T A C A AC A A GC AC A G AG A C A A GC CG ATC AG AG AA c AGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGG GAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGACA GAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAACAC TGATCCACCAGAGCATCACAGGACTGTACGAAACAAGAATCG ACCTGAGCCAGCTGGGAGGAGACGGAGGAGGAAGCCCGAAG .AA GA A G AG A .AA GG TCT A Get ag ca cca gc etc aa ga ac ac ccgaa 1 gga gt ct c iaa gciacasaaiaccaaciiacacuiacaaaaigugicccccaaaasgiagccaucgtascigcicciaata aaaagaaagdttcttcacattdctcgag Cas9 transcript with AGG as first three nite Sent ides for use with AGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGT GTGTCGTTGCAGGCCTTATTCGGATCCGCCACCATGGACAAGA AGTACAGCATCGGACTGGACATCGGAACAAACAGCGTCGGAT GGGCAGTCATCACAGACGAATACAAGGTCCCGAGCAAGAAGT TCAAGGTCCTGGGAAACACAGACAGACACAGCATCAAGAAGA ACCTGATCGGAGCACTGCTGTTCGACAGCGGAGAAACAGCAG 61 2024224004   30 Sep 2024 CleanCap™, 5’ UTR from HSD, ORF correspond! ng to SEQ ID NO: 4, Kozak sequence, aad3’ UTR of ALB A A GC A AC A A G A C TG A A G AG A AC AGC AAG AAGAAGATACACA AGAAGAAAGAACAGAATCTGCTACCTGCAGGAAATCTTCAGC AACGAAATGGCAAAGGTCGACGACAGCTTCTTCCACAGACTG GAAGAAAGCTTCCTGGTCGAAGAAGACAAGAAGCACGAAAGA CACCCGATCTTCGGAAACATCGTCGACGAAGTCGCATACCACG AAAAGTACCCGACAATCTACCACCTGAGAAAGAAGCTGGTCG ACAGCACAGACAAGGCAGACCTGAGACTGATCTACCTGGCAC TGGCACACATGATCAAGTTCAGAGGACACTTCCTGATCGAAGG AGACCTGAACCCGGACAACAGCGACGTCGACAAGCTGTTCAT CCAGCTGGTCCAGACATACAACCAGCTGTTCGAAGAAAACCC GATCAACGCAAGCGGAGTCGACGCAAAGGCAATCCTGAGCGC AAGACTGAGCAAGAGCAGAAGACTGGAAAACCTGATCGCACA GCTGCCGGGAGAAAAGAAGAACGGACTGTTCGGAAACCTGAT CGCACTGAGCCTGGGACTGACACCGAACTTCAAGAGCAACTTC GACCTGGCAGAAGACGCAAAGCTGCAGCTGAGCAAGGACACA TACGACGACGACCTGGACAACCTGCTGGCACAGATCGGAGAC CAGTACGCAGACCTGTTCCTGGCAGCAAAGAACCTGAGCGAC GCAATCCTGCTGAGCGACATCCTGAGAGTCAACACAGAAATC ACAAAGGCACCGCTGAGCGCAAGCATGATCAAGAGATACGAC gaacaccaccaggacctgacactgctgaaggcactggtcaga CAGC AGCTGCCGGAAAAGTACA aggaaatcttcttcgaccag AGCAAGAACGGATACGCAGGATACA TCGACGGAGGAGCAAGC CAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAG ATGGACGGAACAGAAGAACTGCTGGTCAAGCTGAACAGAGAA GACCTGCTGAGAAAGCAGAGAACATTCGACAACGGAAGCATC CCGCACCAGATCCACCTGGGAGAACTGCACGCAATCCTGAGA A G AC A G GA A G AC TT CT ACCCG TT CCTGAAGGACAACAGAGAA AAGATCGAAAAGATCCTGACATTCAGAATCCCGTACTACGTCG GACCGCTGGCAAGAGGAAACAGCAGATTCGCATGGATGACAA GAAAGAGCGAAGAAACAATCACACCGTGGAACTTCGAAGAAG TCGTCGACAAGGGAGCAAGCGCACAGAGCTTCATCGAAAGAA TGACAAACTTCGACAAGAACCTGCCGAACGAAAAGGTCCTGC CGAAGCACAGCCTGCTGTACGAATACTTCACAGTCTACAACGA ACTGACAAAGGTCAAGTACGTCACAGAAGGAATGAGAAAGCC GGCATTCCTGAGCGGAGAACAGAAGAAGGCAATCGTCGACCT GCTGTTC A AG AC AA A C AG AA AGGTCAC AGTC AAGC AGCTGAA GG AA G A CTACTTCA AG AAG ATCG A ATGCTTCGAC AGCGTCG A AATCAGCGGAGTCGAAGACAGATTCAACGCAAGCCTGGGAAC ATACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTG GACAACGAAGAAAACGAAGACATCCTGGAAGACATCGTCCTG ACACTGACACTGTTCGAAGACAGAGAAATGATCG A AG AA> AG A CTGAAGACATACGCACACCTGTTCGACGACAAGGTCATGAAG CAGCTGAAGAGAAGAAGATACACAGGATGGGGAAGACTGAG CAGAAAGCTGATCAACGGAATCAGAGACAAGCAGAGCGGAA AGACAATCCTGGACTTCCTGAAGAGCGACGGATTCGCAAACA GAAACTTCATGCAGCTGATCCACGACGACAGCCTGACATTCAA GGAAGACATCCAGAAGGCACAGGTCAGCGGACAGGGAGACA GCCTGCACGAACACATCGCAAACCTGGCAGGAAGCCCGGCAA TCAAGAAGGGAATCCTGCAGACAGTCAAGGTCGTCGACGAAC TGGTCAAGGTCATGGGAAGACACAAGCCGGAAAACATCGTCA TCGAAATGGCAAGAGAAAACCAGACAACACAGAAGGGACAG AAGAACAGCAGAGAAAGAATGAAGAGAATCGAAGAAGGAAT CAAGGAACTGGGAAGCCAGATCCTGAAGGAACACCCGGTCGA AAACACACAGCTGCAGAACGAAAAGCTGTACCTGTACTACCT GCAGAACGGAAGAGACATGTACGTCGACCAGGAACTGGACAT CAACAGACTGAGCGACTACGACGTCGACCACATCGTCCCGCA GAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTCCTGAC 2024224004   30 Sep 2024 A A G A A G CG A C A A G A AC A G AGG A A AG AG CG A C A AC GTC CCG A GCGAAGAAGTCGTCAAGAAGATGAAGAACTACTGGAGACAGC TGCTGAACGCAAAGCTGATCACACAGAGAAAGTTCGACAACC TGACAAAGGCAGAGAGAGGAGGACTGAGCGAACTGGACAAG GCAGGATTCATCAAGAGACAGCTGGTCGAAACAAGACAGATC ACAAAGCACGTCGCACAGATCCTGGACAGCAGAATGAACACA A AGTACG ACGAA A ACG A ca AGCTG atca gaga agtc aaggtc ATCACACTGAAGAGCAAGCTGGTCAGCGACTTCAGAAAGGAC ttccagttctacaaggtcagagaaatcaacaactaccaccacg CACACGACGCATACCTGAACGCAGTCGTCGGAACAGGACTGA TCAAGAAGTACCCGAAGCTGGAAAGCGAATTCGTCTACGGAG ACTACAAGGTCTACGACGTCAGAAAGATGATCGCAAAGAGCG AACAGGAAATCGGAAAGGCAACAGCAAAGTACTTCTTCTACA GCAACATCATGAACTTCTTCAAGACAGAAATCACACTGGCAA ACGGAGAAATCAGAAAGAGACCGCTGATCGAAACAAACGGA GAAACAGGAGAAATCGTCTGGGACAAGGGAAGAGACTTCGCA ACAGTCAGAAAGGTCCTGAGCATGCCGCAGGTCAACATCGTC AAGAAGACAGAAGTCCAGACAGGAGGATTCAGCAAGGAAAG CATCCTGCCGAAGAGAAACAGCGACAAGCTGATCGCAAGAAA GAAGGACTGGGACCCGAAGAAGTACGGAGGATTCGACAGCCC GACAGTCGCATACAGCGTCCTGGTCGTCGCAAAGGTCGAAAA GGGAAAGAGCAAGAAGCTGAAGAGCGTCAAGGAACTGCTGG GAATCACAATCATGGAAAGAAGCAGCTTCGAAAAGAACCCGA TCGACTTCCTGGAAGCAAAGGGATACAAGGAAGTCAAGAAGG ACCTGATCATCAAGCTGCCGAAGTACAGCCTGTTCGAACTGGA AAACGGAAGAAAGAGAATGCTGGCAAGCGCAGGAGAACTGC A G A A GG G A A A CG A A CTG GC ACTGC C G AGC AAGT ACG T C AA CT TCCTGTACCTGGC AA GCCACT ACGA A AAGCTG AA GGG A AG CC CGGAAGACAACGAACAGAAGCAGCTGTTCGTCGAACAGCACA AGCACTACCTGGACGAAATCATCGAACAGATCAGCGAATTCA GCAAGAGAGTCATCCTGGCAGACGCAAACCTGGACAAGGTCC TGAGCGCATACAACAAGCACAGAGACAAGCCGATCAGAGAAC AGGCAGAAAACATCATCCACCTGTTCACACTGACAAACCTGG GAGCACCGGCAGCATTCAAGTACTTCGACACAACAATCGACA GAAAGAGATACACAAGCACAAAGGAAGTCCTGGACGCAACAC TGATCCACCAGAGCATCACAGGACTGTACGAAACA AGAATCG acctgagccagctgggaggagacggaggaggaagcccgaag AAGAAGAGAAAGGTCTAGCTAGCCATCACATTTAAAAGCATC TCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAAT agcttattcatctctttttctttttcgttggtgtaaagccaaca ccctgtctaaaaaacataaatttctttaatcattttgcctcttt tctctgtgcttcaattaataaaaaatggaaagaacctcgag 30 / 30 / 39 poiy-A sequence AAAA.AAAA.AAAA / UlAAA-AAAAAAAAAAAAAGCGAAAAAAA aaaaaaaaaaaaaaaaaaaaaaaccgaaaaaaaaaaaaaaa AAAAAAAAAAAAAAAAAAAAAAAA 62 poly-A 100 sequence AA AA AAA A AA AA AAAA AA AA A AAAA AAAA AA AA AA AAAAA AA AAAAAA AA AA AA AA AA AAA AAA A AAA AAA AAAA AAAA A AAAA AAA A.AAAA A 63 G209 guide RNA mC*mC*mA*GUCCAGCGAGGCAAAGGGUUUUAGAGCUAGAAA UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAA AGUGGCACCGAGUCGGUGCmU*mU*mU*U 64 ORF encoding ATGGCAGCATTCAAGCCGAACTCGATCAACTACATCCTGGGAC TGGACATCGGAATCGCATCGGTCGGATGGGCAATGGTCGAAA 6S 2024224004   30 Sep 2024 Neisseria meningitidis Cas9 TCGACGAAGAAGAAAACCCGATCAGACTGATCGACCTGGGAG TCAGAGTCTTCGAAAGAGCAGAAGTCCCGAAGACAGGAGACT CGCTGGCAATGGCAAGAAGACTGGCAAGATCGGTCAGAAGAC TGACAAGAAGAAGAGCACACAGACTGCTGAGAACAAGAAGA CTGCTGAAGAGAGAAGGAGTCCTGCAGGCAGCAAACTTCGAC GAAAACGGACTGATCAAGTCGCTGCCGAACACACCGTGGCAG CTGAGAGCAGCAGCACTGGACAGAAAGCTGACACCGCTGGAA TGGTCGGCAGTCCTGCTGCACCTGATCAAGCACAGAGGATACC TGTCGCAGAGAAAGAACGAAGGAGAAACAGCAGACAAGGAA CTGGGAGCACTGCTGAAGGGAGTCGCAGGAAACGCACACGCA CTGCAGACAGGAGACTTCAGAACACCGGCAGAACTGGCACTG AACAAGTTCGAAAAGGAATCGGGACACATCAGAAACCAGAGA TCGGACTACTCGCACACATTCTCGAGAAAGGACCTGCAGGCA GAACTGATCCTGCTGTTCGAAAAGCAGAAGGAATTCGGAAAC CCGCACGTCTCGGGAGGACTGAAGGAAGGAATCGAAACACTG CTGATGACACAGAGACCGGCACTGTCGGGAGACGCAGTCCAG AAGATGCTGGGACACTGCACATTCGAACCGGCAGAACCGAAG GCAGCAAAGAACACATACACAGCAGAAAGATTCATCTGGCTG ACAAAGCTGAACAACCTGAGAATCCTGGAACAGGGATCGGAA A G AC CG CTG AC A G AC A C AG A A AG AG C AAC ACTG ATG G ACG AA CCGTA CAGAAA GTC G A AGC TG AC ATAC GCACAGGC AAGA A A G CTGCTGGGACTGGAAGACACAGCATTCTTCAAGGGACTGAGA tacggaaaggacaacgcagaagcatcgacactgatggaaatg AAGGCATACCACGCAATCTCGAGAGCACTGGAAAAGGAAGGA CTGAAGGACAAGAAGTCGCCGCTGAACCTGTCGCCGGAACTG caggacgaaatcggaacagcattctcgctgttcaagacagac G A AG A C ATC AC A G G A A G AC TG A AG G AC A GA AT CCAGCCGGAA ATCCTGGAAGCACTGCTGAAGCACATCTCGTTCGACAAGTTCG TCC A G ATCTCGCTG A AGGC ACTG AG AAGAATCGTCCCGCTGAT GGAACAGGGAAAGAGATACGACGAAGCATGCGCAGAAATCTA CGGAGACCACTACGGAAAGAAGAACACAGAAGAAAAGATCT ACCTGCCGCCGATCCCGGCAGACGAAATCAGAAACCCGGTCG TCCTG AG AG CACTGTCGC A G GC AA G A AAGGTCATCAACG G AG TCGTCAGAAGATACGGATCGCCGGCAAGAATCCACATCGAAA CAGCAAGAGAAGTCGGAAAGTCGTTCAAGGACAGAAAGGAA ATCGAAAAGAGACAGGAAGAAAACAGAAAGGACAGAGAAAA GGCAGCAGCAAAGTTCAGAGAATACTTCCCGAACTTCGTCGG AGAACCGAAGTCGAAGGACATCCTGAAGCTGAGACTGTACGA ACAGCAGCACGGAAAGTGCCTGTACTCGGGAAAGGAAATCAA CCTGGGAAGACTGAACGAAAAGGGATACGTCGAAATCGACCA CGCACTGCCGTTCTCGAGAACATGGGACGACTCGTTCAACAAC AAGGTCCTGGTCCTGGGATCGGAAAACCAGAACAAGGGAAAC CAGACACCGTACGAATACTTCAACGGAAAGGACAACTCGAGA GAATGGCAGGAATTCAAGGCAAGAGTCGAAACATCGAGATTC CCGAGATCGAAGAAGCAGAGAATCCTGCTGCAGAAGTTCGAC GAAGACGGATTCAAGGAAAGAAACCTGAACGACACAAGATAC GTCAACAGATTCCTGTGCCAGrTCGTCGCAGACAGAATGAGAC TGACAGGAAAGGGAAAGAAGAGAGTCTTCGCATCGAACGGAC AGATCACAAACCTGCTGAGAGGATTCTGGGGACTGAGAAAGG TCAGAGCAGAAAACGACAGACACCACGCACTGGACGCAGTCG TCGTCGCATGCTCGACAGTCGCAATGCAGCAGAAGATCACAA GATTCGTCAGATACAAGGAAATGAACGCATTCGACGGAAAGA CAATCGACAAGGAAACAGGAGAAGTCCTGCACCAGAAGACAC ACTTCCCGCAGCCGTGGGAATTCTTCGCACAGGAAGTCATGAT CAGAGTCTTCGGAAAGCCGGACGGAAAGCCGGAATTCGAAGA AGCAGACACACTGGAAAAGCTGAGAACACTGCTGGCAGAAAA GCTGTCGTCGAGACCGGAAGCAGTCCACGAATACGTCACACC 2024224004   30 Sep 2024 gctgttcgtctcgagagcaccgaacagaaagatgtcgggaca GGGACACATGGAAACAGTCAAGTCGGCAAAGAGACTGGACGA AGGAGTCTCGGTCCTGAGAGTCCCGCTGACACAGCTGAAGCTG AAGGACCTGGAAAAGATGGTCAACAGAGAAAGAGAACCGAA GCTGT ACG A A GC AC TG A A G GC A AG AC TGG A A G C AC AC A AG G A CGACCCGGCAAAGGCATTCGCAGAACCGTTCTACAAGTACGA CAAGGCAGGAAACAGAACACAGCAGGTCAAGGCAGTCAGAGT CGAACAGGTCCAGAAGACAGGAGTCTGGGTCAGAAACCACAA CGGAATCGCAGACAACGCAACAATGGTCAGAGTAGACGTCTT CGAAAAGGGAGACAAGTACTACCTGGTCCCGATCTACTCGTG GCAGGTCGCAAAGGGAATCCTGCCGGACAGAGCAGTCGTCCA GGGAAAGGACGAAGAAGACTGGCAGCTGATCGACGACTCGTT CAACTTCAAGTTCTCGCTGCACCCGAACGACCTGGTCGAAGTC ATCACAAAGAAGGCAAGAATGTTCGGATACTTCGCATCGTGCC ACAGAGGAACAGGAAACATCAACATCAGAATCCACGACCTGG ACCACAAGATCGGAAAGAACGGAATCCTGGAAGGAATCGGAG TCAAGACAGCACTGTCGTTCCAGAAGTACCAGATCGACGAACT GGGAAAGGAAATCAGACCGTGCAGACTGAAGAAGAGACCGCC GGTC A G A TC CGG A A A G A G A A C AGC AG AC GG AT CGG A ATT CG A ATC GC CG A A G A A G A A G A G A A A GG TC GA ATGA ORF encoding Neisseria meningitidis Cas9 (no start or stop codons; suitable for inclusion in fusion protein coding sequence) GCAGCATTCAAGCCGAACTCGATCAACTACATCCTGGGACTGG ACATCGGAATCGCATCGGTCGGATGGGCAATGGTCGAAATCG ACGAAGAAGAAAACCCGATCAGACTGATCGACCTGGGAGTCA GAGTCTTCGAAAGAGCAGAAGTCCCGAAGACAGGAGACTCGC TGGCAATGGCAAGAAGACTGGCAAGATCGGTCAGAAGACTGA CAAGAAGAAGAGCACACAGACTGCTGAGAACAAGAAGACTGC TGAAGAGAGAAGGAGTCCTGCAGGCAGCAAACTTCGACGAAA ACGGACTGATCAAGTCGCTGCCGAACACACCGTGGCAGCTGA GAGCAGCAGCACTGGACAGAAAGCTGACACCGCTGGAATGGT CGGCAGTCCTGCTGCACCTGATCAAGCACAGAGGATACCTGTC GC AG A GA A A GA A C G AA GG AG AA AC AGC AGA CAAG GAAC TG G GAGCACTGCTGAAGGGAGTCGCAGGAAACGCACACGCACTGC AGACAGGAGACTTCAGAACACCGGCAGAACTGGCACTGAACA AGTTCGAAAAGGAATCGGGACACATCAGAAACCAGAGATCGG ACTACTCGCACACATTCTCGAGAAAGGACCTGCAGGCAGAAC TGATCCTGCTGTTCGAAAAGCAGAAGGAATTCGGAAACCCGC ACGTCTCGGGAGGACTGAAGGAAGGAATCGAAACACTGCTGA TGACACAGAGACCGGCACTGTCGGGAGACGCAGTCCAGAAGA TGCTGGGACACTGCACATTCGAACCGGCAGAACCGAAGGCAG CAAAGAACACATACACAGCAGAAAGATTCATCTGGCTGACAA AGCTGAACAACCTGAGAATCCTGGAACAGGGATCGGAAAGAC CGCTGACAGACACAGAAAGAGCAACACTGATGGACGAACCGT ACAGAAAGTCGAAGCTGACATACGCACAGGCAAGAAAGCTGC TGGGACTGGAAGACACAGCATTCTTCAAGGGACTGAGATACG GAAAGGACAACGCAGAAGCATCGACACTGATGGAAATGAAGG CATACCACGCAATCTCGAGAGCACTGGAAAAGGAAGGACTGA AGGACAAGAAGTCGCCGCTGAACCTGTCGCCGGAACTGCAGG ACGAAATCGGAACAGCATTCTCGCTGTTCAAGACAGACGAAG ACATCACAGGAAGACTGAAGGACAGAATCCAGCCGGAAATCC TGGAAGCACTGCTGAAGCACATCTCGTTCGACAAGTTCGTCCA GATCTCGCTGAAGGCACTGAGAAGAATCGTCCCGCTGATGGA ACAGGGAAAGAGATACGACGAAGCATGCGCAGAAATCTACGG AGACCACTACGGAAAGAAGAACACAGAA GA AAA GATCTACCT GCCGCCGATCCCGGCAGACGAAATCAGAAACCCGGTCGTCCT GAGAGCACTGTCGCAGGCAAGAAAGGTCATCAACGGAGTCGT CAGAAGATACGGATCGCCGGCAAGAATCCACATCGAAACAGC 66 2024224004   30 Sep 2024 A A G A G A AG TC GG A A AG TCG TT CAAGGACAGAAAGGA AAT C G A AAAGAGACAGGAAGAAAACAGAAAGGACAGAGAAAAGGCAG CAGCAAAGTTCAGAGAATACTTCCCGAACTTCGTCGGAGAACC GAAGTCGAAGGACATCCTGAAGCTGAGACTGTACGAACAGCA GCACGGAAAGTGCCTGTACTCGGGAAAGGAAATCAACCTGGG AAGACTGAACGAAAAGGGATACGTCGAAATCGACCACGCACT GCCGTTCTCGAGAACATGGGACGACTCGTTCAACAACAAGGTC CTGGTCCTGGGATCGGAAAACCAGAACAAGGGAAACCAGACA CCGTACGAATACTTCAACGGAAAGGACAACTCGAGAGAATGG CAGGAATTCAAGGCAAGAGTCGAAACATCGAGATTCCCGAGA TCGAAGAAGCAGAGAATCCTGCTGCAGAAGTTCGACGAAGAC GGATTCAAGGAAAGAAACCTGAACGACACAAGATACGTCAAC AGATTCCTGTGCCAGTTCGTCGCAGACAGAATGAGACTGACAG GAAAGGGAAAGAAGAGAGTCTTCGCATCGAACGGACAGATCA CAAACCTGCTGAGAGGATTCTGGGGACTGAGAAAGGTCAGAG CAGAAAACGACAGACACCACGCACTGGACGCAGTCGTCGTCG CATGCTCGACAGTCGCAATGCAGCAGAAGATCACAAGATTCG TCAGATACAAGGAAATGAACGCATTCGACGGAAAGACAATCG ACAAGGAAACAGGAGAAGTCCTGCACCAGAAGACACACTTCC CGCAGCCGTGGGAATTCTTCGCACAGGAAGTCATGATCAGAGT CTTCGGAAAGCCGGACGGAAAGCCGGAATTCGAAGAAGCAGA CACACTGGAAAAGCTGAGAACACTGCTGGCAGAAAAGCTGTC GTCGAGACCGGAAGCAGTCCACGAATACGTCACACCGCTGTTC GTCTCGAGAGCACCGAACAGAAAGATGTCGGGACAGGGACAC ATGGAAACAGTCAAGTCGGCAAAGAGACTGGACGAAGGAGTC TCGGTCCTGAGAGTCCCGCTGACACAGCTGAAGCTGAAGGAC C TGG A A A A G ATGGTC A AC AG AG A A A G AG A ACCG AAGCT GT A C GAAGCACTGAAGGCAAGACTGGAAGCACACAAGGACGACCCG GCAAAGGCATTCGCAGAACCGTTCTACAAGTACGACAAGGCA GGAAACAGAACACAGCAGGTCAAGGCAGTCAGAGTCGAACAG GTCCAGAAGACAGGAGTCTGGGTCAGAAACCACAACGGAATC GCAGACAACGCAACAATGGTCAGAGTAGACGTCTTCGAAAAG GGAGACAAGTACTACCTGGTCCCGATCTACTCGTGGCAGGTCG CAAAGGGAATCCTGCCGGACAGAGCAGTCGTCCAGGGAAAGG ACGAAGAAGACTGGCAGCTGATCGACGACTCGTTCAACTTCA AGTTCTCGCTGCACCCG A ACG ACCTGGTCG AAGTCATCACA AA GAAGGCAAGAATGTTCGGATACTTCGCATCGTGCCACAGAGG AACAGGAAACATCAACATCAGAATCCACGACCTGGACCACAA GATCGGAAAGAACGGAATCCTGGAAGGAATCGGAGTCAAGAC AGCACTGTCGTTCCAGAAGTACCAGATCGACGAACTGGGAAA GGAAATCAGACCGTGCAGACTGAAGAAGAGACCGCCGGTCAG ATCCGGAAAGAGAACAGCAGACGGATCGGAATTCGAATCGCC G A AG AA G A A G AG AA A GG TCG A A Transcript comprising SEQ ID NO: 6S (encoding Neisseria meningitidis Cas9) GGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGGATCC GCCACCATGGCAGCATTCAAGCCGAACTCGATCAACTACATCC TGGGACTGGACATCGGAATCGCATCGGTCGGATGGGCAATGG TCG AA AT C G AC G AAG A A GA AA AC C C GAT CAGACTGATCGACC TGGGAGTCAGAGTCTTCGAAAGAGCAGAAGTCCCGAAGACAG GAGACTCGCTGGCAATGGCAAGAAGACTGGCAAGATCGGTCA GAAGACTGACAAGAAGAAGAGCACACAGACTGCTGAGAACA AGAAGACTGCTGAAGAGAGAAGGAGTCCTGCAGGCAGCAAAC TTCGACGAAAACGGACTGATCAAGTCGCTGCCGAACACACCG TGGC'AGC'TGAGAGCAGCAGCACTGGACAGAAAGCTGACACCG CTGGAATGGTCGGCAGTCCTGCTGCACCTGATCAAGCACAGAG GATACCTGTCGCAGAGAAAGAACGAAGGAGAAACAGCAGAC AAGGAACTGGGAGCACTGCTGAAGGGAGTCGCAGGAAACGCA 67 2024224004   30 Sep 2024 CACGCACTGCAGACAGGAGACTTCAGAACACCGGCAGAACTG GCACTGAACAAGTTCGAAAAGGAATCGGGACACATCAGAAAC CAGAGATCGGACTACTCGCACACATTCTCGAGAAAGGACCTG CAGGCAGAACTGATCCTGCTGTTCGAAAAGCAGAAGGAATTC GGAAACCCGCACGTCTCGGGAGGACTGAAGGAAGGAATCGAA ACACTGCTGATGACACAGAGACCGGCACTGTCGGGAGACGCA GTCCAGAAGATGCTGGGACACTGCACATTCGAACCGGCAGAA CCG AAGGC AGC AA AG A AC AC AT ACACAGC AGAA AGATTCATC TGGCTGACAAAGCTGAACAACCTGAGAATCCTGGAACAGGGA TCGGAAAGACCGCTGACAGACACAGAAAGAGCAACACTGATG GACGAACCGTACAGAAAGTCGAAGCTGACATACGCACAGGCA AGAAAGCTGCTGGGACTGGAAGACACAGCATTCTTCAAGGGA CTGAGATACGGAAAGGACAACGCAGAAGCATCGACACTGATG GAAATGAAGGCATACCACGCAATCTCGAGAGCACTGGAAAAG GAAGGACTGAAGGACAAGAAGTCGCCGCTGAACCTGTCGCCG GAACTGCAGGACGAAATCGGAACAGCATTCTCGCTGTTCAAG ACAGACGAAGACATCACAGGAAGACTGAAGGACAGAATCCAG CCGGAAATCCTGGAAGCACTGCTGAAGCACATCTCGTTCGACA AGTTCGTCCAGATCTCGCTGAAGGCACTGAGAAGAATCGTCCC GCTGATGGAACAGGGAAAGAGATACGACG AAGC AT GC GC AG A AAT C T A C G G A G A CC A C T A C GG AA AG AAG A A C AC AG AAG A A A AGATCTACCTGCCGCCGATCCCGGCAGACGAAATCAGAAACC CGGTCGTCCTGAGAGCACTGTCGCAGGCAAGAAAGGTCATCA ACGGAGTCGTCAGAAGATACGGATCGCCGGCAAGAATCCACA TCGAAACAGCAAGAGAAGTCGGAAAGTCGTTCAAGGACAGAA AGGAAATCGAAAAGAGACAGGAAGAAAACAGAAAGGACAGA GAAAAGGCAGCAGCAAAGTTCAGAGAATACTTCCCGAACTTC GTCGGAGAACCGAAGTCGAAGGACATCCTGAAGCTGAGACTG TACGAACAGCAGCACGGAAAGTGCCTGTACTCGGGAAAGGAA ATCAACCTGGGAAGACTGAACGAAAAGGGATACGTCGAAATC GACCACGCACTGCCGTTCTCGAGAACATGGGACGACTCGTTCA ACAACAAGGTCCTGGTCCTGGGATCGGAAAACCAGAACAAGG GAAACCAGACACCGTACGAATACTTCAACGGAAAGGACAACT CGAGAGAATGGCAGGAATTCAAGGCAAGAGTCGAAACATCGA GATTCCCGAGATCGAAGAAGCAGAGAATCCTGCTGCAGAAGT TCGACGAAGACGGATTCAAGGAAAGAAACCTGAACGACACAA GATACGTCAACAGATTCCTGTGCCAGTTCGTCGCAGACAGAAT GAGACTGACAGGAAAGGGAAAGAAGAGAGTCTTCGCATCGAA CGGACAGATCACAAACCTGCTGAGAGGATTCTGGGGACTGAG AAAGGTCAGAGCAGAAAACGACAGACACCACGCACTGGACGC AGTCGTCGTCGCATGCTCGACAGTCGCAATGCAGCAGAAGATC ACAAGATTCGTCAGATACAAGGAAATGAACGCATTCGACGGA A A G A C AA TC G A C A AGG A AA.C A GG AG AAGTC CT GC ACC AG AA G ACACACTTCCCGCAGCCGTGGGAATTCTTCGCACAGGAAGTCA TGATC AG AGTCTTCGGA AAGCCGGACGGA AAGCCGG AATTCG AAGAAGCAGACACACTGGAAAAGCTGAGAACACTGCTGGCAG AAAAGCTGTCGTCGAGACCGGAAGCAGTCCACGAATACGTCA CACCGCTGTTCGTCTCGAGAGCACCGAACAGAAAGATGTCGG GACAGGGACACATGGAAACAGTCAAGTCGGCA AAGA G ACTGG ACGAAGGAGTCTCGGTCCTGAGAGTCCCGCTGACACAGCTGA AGCTGAAGGACCTGGAAAAGATGGTCAACAGAGAAAGAGAA CCGAAGCTGTACGAAGCACTGAAGGCAAGACTGGAAGCACAC AAGGACGACCCGGCAAAGGCATTCGCAGAACCGTTCTACAAG TACGACAAGGCAGGAAACAGAACACAGCAGGTCAAGGCAGTC AGAGTCGAACAGGTCCAGAAGACAGGAGTCTGGGTCAGAAAC CACAACGG AATCGCAGACA ACGC AACAATGGTCAGAGTAGAC GTCTTCGAAAAGGGAGACAAGTACTACCTGGTCCCGATCTACT 2024224004   30 Sep 2024 CGTGGCAGGTCGCAAAGGGAATCCTGCCGGACAGAGCAGTCG TCCAGGGAAAGGACGAAGAAGACTGGCAGCTGATCGACGACT CGTTCAACTTCAAGTTCTCGCTGCACCCGAACGACCTGGTCGA AGTCATCACAAAGAAGGCAAGAATGTTCGGATACTTCGCATC GTGCCACAGAGGAACAGGAAACATCAACATCAGAATCCACGA CCTGGACCACAAGATCGGAAAGAACGGAATCCTGGAAGGAAT CGGAGTCAAGACAGCACTGTCGTTCCAGAAGTACCAGATCGA CGAACTGGGAAAGGAAATCAGACCGTGCAGACTGAAGAAGAG ACCGCCGGTCAGATCCGGAAAGAGAACAGCAGACGGATCGGA ATTCGAATCGCCGAAGAAGAAGAGAAAGGTCGAATGATAGCT AGCTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTC GACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCC CCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTT TCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGT GTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGG GGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGG TGGGCTCTATGG Amino acid secpence of Neisseria meningitidis Cas9 MAAFKPNSINYILGLDIGIASVGWAMVEIDEEENPIRLIDLGVRVF ERAEVPKTGDSLAMARRLARSVRRLTRRRAHRLLRTRRLLKREG VLQAANFDENGL1KSLPNTPWQLRAAALDRKLTPLEWSAVLLHLI KHRGYLSQRKNEGETADKELGALLKGVAGNAHALQTGDFRTPA ELALNKFEKESGHIRNQRSDYSHTFSRKDLQAELILLFEKQKEFGN PHVSGGLKEG! ETLLMTQRPALSGDAVQKM LGHCTFEPAEPKAA KNTYTAERF1WLTKLNNLRJLEQGSERPLTDTERATLMDEPYRKS KLTYAQARKLLGLEDTAFFKGLRYGKDNAEASTLMEMKAYHAI SRALEKEGLKDKKSPLNLSPELQDE1GTAFSLFKTDEDITGRLKDR IQPElLEALLKHISFDKFVQISLKALRRfVPLMEQGKRYDEACAEtY GDHYGKKNTEEKIYLPPIPADEIRNPWLRALSQARKVfNGWRR YGSPAR1HJETAREVGKSFKDRKE1EKRQEENRKDREKAAAKFRE YFPNFVGEPKSKDJLKLRLYEQQHGKCLYSGKEfNLGRLNEKGYV EIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDN SREWQEFKARVETSRFPRSKKQRJLLQKFDEDGFK.ERNLNDTRYV NRFLCQFVADRMRLTGKGKKRVFASNGQ1TNLLRGFWGLRKVR AENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAFDGKTID KETGEVLHQKTH FPQPWEFFAQEVMIRVFGKPDGKPEFEEADTLE KLRTLLAEKLSSRPEAVI-1EYVTPLFVSRAPNRKMSGQGHMETVK. SAKRLDEGVSVLRVPLTQLKLKDLEK.MVNREREPKLYEALKARL EAHKDDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQKTGVW VRNHNGJADNATMVRVDVFEKGDKYYLVP1YSWQVAKGJLPDR AVVQGKDEEDWQLIDDSFNFKFSLHPNDLVEVITKKARMFGYFA SCHRGTGNINIRIHDLDHKIGKNG1LEGIGVKTALSFQKYQ1DELG KEI R PCRLKKR PPVRSGKRT ADGSEFESPKKKRKVE 68 G390 guide RNA mG*mC*mC*GAGUCUGGAGAGCUGCAGUUUUAGAmGmCmUm A m Gm A mA mA m U mAm GmC A AG UUAAAAU AAGGCUAGUC CGIJ UAUCAmAinCmUinUmGmAmAmAmAmAmGmUmGmGniCniAmC mC mG mA m Gm U m Cm Gm G m U mG m Cm U * m U * m U * m U 69 G502 guide RNA mA*mC*mA*CAAAUACCAGUCCAGCGGULrUUAGAmGmCmUm A m Gm A mA mA m U mAm Gm C A AG U U A AAAU A AGGCU AGUCCGU UAUCAmAinCmUinUmGmAmAmAmAmAmGmUniGmGmCniAmC mC mG mA m Gm U m Cm Gm G m U mG m Cm U * m U * m U * m U 70 G509 guide RNA mA*mA*mA*GUUCUAGAUGCCGUCCGGUUUUAGAmGmCmUm Am Gm A mA mA m U mAmGm C AAG UU AA AAU AA GG C U AG UC CG U UAUCAmAinCmUinUmGrnAmAmAmAmAmGmUniGmGmCmAmC mC mG mA. m Gm U m Cm Gm G m U mG m Cm U * m U * m U * m U 71 2024224004   30 Sep 2024 G534 guide RNA mA*mC*mG*CAAAUAUCAGUCCAGCGGUUUUAGAmGmCmUm AmGmA mAtnAmUmAmGmC AAGUUA A AAUAA GGCUA GUCCGU U A UC A mA mCm UmUm G mA mA m A m A m A mG m U mGmGmC mA mC mC mG mA m Gm U m CmGm G m U mC m Cm U * m U * m U * m U 72 * = PS linkage; 'm' = 2’-O-Me nucleotide Monse G000282 NGS primer sequences Forward primer: CACTCTTTCCCTACACGACGCTCTTCCGATCTGTTTTGTTCCAGAGTCTATCACCG Reverse primer: GGAGTTCAGACGTGTGCTCTTCCGATCTACACGAATAAGAGCAAATGGGAAC Rat GD00390 NGS primer sequences Forward Primer: CACTCTTTCCCTACACGACGCTCTTCCGATCTTGCATTTCATGAGACCGAAAACA Reverse Primer: GGAGTTCAGACGTGTGCTCTTCCGATCTGCTACAGTAGAGCTGTACATAAAACTT GFP sequence: TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGAC GGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGC GTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCA GATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAG GAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGA AGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATG TGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGT AAAACGACGGCCAGTGAATTCTAATACGACTCACTATAGGGTCCCGCAGTCGGC GTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTTGCAGGCCTTATTCGGATCCAT GGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCT GGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCG ATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCC CGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGC CGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAA GGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACC CGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAG GGCATCGACTTCAAGGAGGACGGCA ACATCCTGGGGCACAAGCTGGAGTACAAC TACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAG GTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGAC CACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAAC 2024224004   30 Sep 2024 CACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGAT CACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACG AGCTGTACAAGTAATAGGAATTATGCAGTCTAGCCATCACATTTAAAAGCATCTC AGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTC TTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTT TAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCTC GAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA A A A A A A A A AAA A A AA AA A A A AA A A A A A A A A A A A A A A A A A AAA A A AAA A A A AT CTAGACTTAAGCTTGATGAGCTCTAGCTTGGCGTAATCATGGTCATAGCTGTTTC CTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCAT AAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTG CGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAA TCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTC GCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCAC TCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAAC ATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCT GGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCA AGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCT GGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGT CCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTAT CTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCG TTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGT AAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGC GAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTAC ACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAA AAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTT ttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcc TTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGG ATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAA AATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTA CCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCA TAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATC tggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagattta TCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACT TTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTT CGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTC ACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGA GTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGA TCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACT GCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGT ACTCAACC A AGTC ATTCTG AG AATAGTGT ATGCGGCG ACC G AGTTGCTCTTG CCC GGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCAT CATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGA TCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTT CACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGG GAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTA TTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTT 182 2024224004   29 Sep 2026 AGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTG acgtct aagaaaccatt attatc atgacatta acct ata aaaataggcgtatcac GAGGCCCTTTCGTCG

[0287] Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

[0288] Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

Claims

1. A lipid nanoparticle (LNP) composition comprising:an RNA component, wherein the RNA component comprises (i) an mRNA encodingan RNA-guided DNA-binding agent and (ii) a gRNA nucleic acid; anda lipid component, wherein the lipid component comprises:about 45-60 mol-% amine lipid;about 8-10 mol-% neutral lipid; andabout 2.5-4 mol-% PEG lipid,wherein the remainder of the lipid component is helper lipid, the N / P ratio of the LNP composition is 5-7, andthe amine lipid is Lipid A or an acetal analog of Lipid A, wherein Lipid A is represented by2.     The LNP composition of claim 1, wherein the N / P ratio is about 6.

3. The LNP composition of any one of the preceding claims, wherein the lipidcomponent comprises about 9 mol-% neutral lipid.

4. The LNP composition of any one of the preceding claims wherein the lipid component comprises about 3 mol-% PEG lipid.

5. The LNP composition of any one of the preceding claims, wherein the lipid component comprises about 27-39.5 mol-% helper lipid.

6. The LNP composition of claim 5, wherein the lipid component comprises about 38 mol-% helper lipid.

7. The LNP composition of any one of claims 1-6, wherein the lipid component comprises about 50 mol-% amine lipid, about 9 mol-% neutral lipid, about 38 mol-% helper lipid, and about 3 mol-% PEG lipid.2024224004   29 Sep 20268. The LNP composition of any one of the preceding claims, wherein the neutral lipid is a phospholipid.

9. The LNP composition of claim 8, wherein the neutral lipid is DSPC.

10. The LNP composition of any one of the preceding claims, wherein the PEG lipidcomprises dimyristoylglycerol (DMG) or a PEG-2k.

11. The LNP composition of claim 10, wherein the PEG lipid is a PEG2k-DMG.

12. The LNP composition of any one of the preceding claims, wherein the helper lipid is asteroid, sterol, or alkyl resorcinol.

13. The LNP composition of any one of the preceding claims, wherein the lipid component comprises Lipid A, DSPC, cholesterol, and PEG2k-DMG.

14. The LNP composition of any one of the preceding claims, wherein the RNA-guided DNA-binding agent is a Class 2 Cas nuclease.

15. The LNP composition of claim 14, wherein the RNA-guided DNA-binding agent is a Cas9 nuclease.

16. The LNP composition of any one of the preceding claims, wherein the mRNA is a modified mRNA.

17. The LNP composition of claim 16, wherein the mRNA comprises:an open reading frame encoding the RNA-guided DNA-binding agent, wherein the open reading frame has a uridine content ranging from its minimum uridine content to 150% of the minimum uridine content; oran open reading frame encoding an RNA-guided DNA-binding agent, wherein the open reading frame has a uridine dinucleotide content ranging from its minimum uridine dinucleotide content to 150% of the minimum uridine dinucleotide content.

18. The LNP composition of any one of the preceding claims, wherein the gRNA and Class 2 nuclease mRNA are present in a ratio ranging from about 2:1, 1:1, or 1:2 by weight.

19. A lipid nanoparticle (LNP) composition comprising:2024224004   29 Sep 2026an RNA component, wherein the RNA component comprises (i) an mRNA encoding an RNA-guided DNA-binding agent and (ii) a gRNA nucleic acid; anda lipid component, wherein the lipid component comprises:about 45-60 mol-% Lipid A, wherein Lipid A is represented by the following structural formulaabout 27-39.5 mol-% cholesterol;about 8-10 mol-% DSPC; andabout 2.5-4 mol-% PEG2k-DMG,wherein the N / P ratio of the LNP composition is 5-7.

20. The LNP composition of claim 19, wherein the RNA-guided DNA-binding agent is a Cas9, and the gRNA nucleic acid is an sgRNA.

21. A lipid nanoparticle (LNP) composition comprising:an RNA component, wherein the RNA component comprises (i) an mRNA encodingan RNA-guided DNA-binding agent and (ii) a gRNA nucleic acid; anda lipid component, wherein the lipid component comprises:about 50 mol-% Lipid A, wherein Lipid A is represented by the following structural formulaabout 38 mol-% cholesterol;about 9 mol-% DSPC; andabout 3 mol-% PEG2k-DMG,2024224004   29 Sep 2026wherein the N / P ratio of the LNP composition is 5-7.

22. The LNP composition of claim 21, wherein the RNA-guided DNA-binding agent is a Cas9, and the gRNA nucleic acid is an sgRNA.

23. A method of gene editing, comprising contacting a cell with an LNP composition of any one of claims 1-22.

24. The method of claim 23, wherein the cell is a liver cell.

25. The method of claim 23 or 24, wherein the method further comprises introducing atemplate nucleic acid into the cell.

26. Use of an LNP composition of any one of claims 1-22 for the manufacture of a medicament for gene editing in a cell.

27. The use of claim 26, wherein the cell is a liver cell.

28. The use of claim 26 or claim 27, wherein the gene editing in a cell further comprisesintroducing a template nucleic acid into the cell.

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