PRIME EDITING OF SINGLE BASE MUTATIONS IN ALPHA-1 ANTITRYPSIN DEFICIENCY

1 . A prime editing guide RNA (PEgRNA) comprising:

a spacer that is complementary to a search target sequence on a first strand of a human SERPINA1 gene,

a primer binding site sequence (PBS) at least partially complementary to the spacer,

an editing template that comprises a region of complementarity to an editing target sequence on a second strand of the SERPINA1 gene, and

a gRNA core that associates with a prime editor comprising a DNA binding domain and a DNA polymerase domain,

wherein the PBS comprises or consists of the sequence GCACGGC (SEQ ID NO: 2) and the editing template comprises or consists of the sequence

(SEQ ID NO: 25)

UUUCUCGUCGAUGGUCAGCACAGCCUUAU

2 . The PEgRNA of claim 1 , wherein

the spacer sequence comprises or consists of the sequence

(SEQ ID NO: 1)

UCCCCUCCAGGCCGUGCAUA;

the gRNA core is between the spacer and the editing template;

the editing template comprises an intended nucleotide edit compared to the SERPINA1 gene;

the PEgRNA guides the prime editor to incorporate the intended nucleotide edit into the SERPINA1 gene when contacted with the SERPINA1 gene;

the prime editor synthesizes a single stranded DNA encoded by the editing template, wherein the single stranded DNA replaces the editing target sequence and results in incorporation of the intended nucleotide edit into a region corresponding to the editing target in the SERPINA1 gene;

the search target sequence is complementary to a protospacer sequence in the SERPINA1 gene, and wherein the protospacer sequence is adjacent to a search target adjacent motif (PAM) in the SERPINA1 gene;

the PAM comprises NGG;

the PEgRNA results in incorporation of the intended nucleotide edit in the PAM when contacted with the SERPINA1 gene;

wherein the PBS is about 2 to about 20 base pairs in length;

the PBS is about 8 to about 16 base pairs in length;

the PBS comprises or consists of the sequence GCACGGCC (SEQ ID NO: 3), GCACGGCCU (SEQ ID NO: 4), GCACGGCCUG (SEQ ID NO: 5), GCACGGCCUGG (SEQ ID NO: 6), GCACGGCCUGGA (SEQ ID NO: 7), GCACGGCCUGGAG (SEQ ID NO: 8), GCACGGCCUGGAGG (SEQ ID NO: 9), or GCACGGCCUGGAGGG (SEQ ID NO: 10);

wherein the editing template is about 4 to 30 base pairs in length;

the editing template is about 10 to 30 base pairs in length; and/or

the editing template comprises or consists of the sequence

(SEQ ID NO: 11)

GCAGCUUCAGUCCCUUUCUCGUCGAUGGUCAGCACAGCCUUAU,

(SEQ ID NO: 12)

CAGCUUCAGUCCCUUUCUCGUCGAUGGUCAGCACAGCCUUAU,

(SEQ ID NO: 13)

AGCUUCAGUCCCUUUCUCGUCGAUGGUCAGCACAGCCUUAU,

(SEQ ID NO: 14)

GCUUCAGUCCCUUUCUCGUCGAUGGUCAGCACAGCCUUAU,

(SEQ ID NO: 15)

CUUCAGUCCCUUUCUCGUCGAUGGUCAGCACAGCCUUAU,

(SEQ ID NO: 16)

UUCAGUCCCUUUCUCGUCGAUGGUCAGCACAGCCUUAU,

(SEQ ID NO: 17)

UCAGUCCCUUUCUCGUCGAUGGUCAGCACAGCCUUAU,

(SEQ ID NO: 18)

CAGUCCCUUUCUCGUCGAUGGUCAGCACAGCCUUAU,

(SEQ ID NO: 19)

AGUCCCUUUCUCGUCGAUGGUCAGCACAGCCUUAU,

(SEQ ID NO: 20)

GUCCCUUUCUCGUCGAUGGUCAGCACAGCCUUAU,

(SEQ ID NO: 21)

UCCCUUUCUCGUCGAUGGUCAGCACAGCCUUAU,

(SEQ ID NO: 22)

CCCUUUCUCGUCGAUGGUCAGCACAGCCUUAU,

(SEQ ID NO: 23)

CCUUUCUCGUCGAUGGUCAGCACAGCCUUAU,

(SEQ ID NO: 24)

CUUUCUCGUCGAUGGUCAGCACAGCCUUAU,

or

(SEQ ID NO: 25)

UUUCUCGUCGAUGGUCAGCACAGCCUUAU;

the PEgRNA results in incorporation of intended nucleotide edit about 0 to 27 base pairs downstream of the 5′ end of the PAM when contacted with the SERPINA1 gene;

the intended nucleotide edit comprises a single nucleotide substitution compared to the region corresponding to the editing target in the SERPINA1 gene;

the intended nucleotide edit comprise an insertion compared to the region corresponding to the editing target in the SERPINA1 gene;

the intended nucleotide edit comprises a deletion compared to the region corresponding to the editing target in the SERPINA1 gene;

the editing target sequence comprises a mutation associated with alpha-1 antitrypsin deficiency (A1AD);

the editing template comprises a wild type SERPINA1 gene sequence;

the PEgRNA results in correction of the mutation when contacted with the SERPINA1 gene;

the PEgRNA comprises or consists of any one of the sequences recited in Table 1;

the PEgRNA comprises at least one chemical modification;

the at least one chemical modification is selected from the group consisting of a 2′-O-methyl (2′-OMe) modification, a 2′-deoxy (2′-H) modification, a 2′-fluoro (2′-F) modification, a 2′-methoxyethyl (2′-MOE) modification, a 2′-amino (2′—NH2) modification, a 2′-arabinosyl (2′-arabino) modification, a 2′-F-arabinosyl (2′-F-arabino) modification, and a locked nucleic acid (LNA) modification;

the at least one chemical modification comprises an internucleotide linkage modification;

the at least one internucleotide linkage modification comprises a phosphonoacetate (PACE) modification; and/or

the PEgRNA comprises or consists of any one of the sequences recited in Table 1.

3 .- 28 . (canceled)

29 . A PEgRNA system comprising the PEgRNA according to claim 1 and further comprising a nick guide RNA (ngRNA), wherein the ngRNA comprises an ng spacer that is complementary to a second search target sequence in the SERPINA1 gene.

30 . The PEgRNA system of claim 29 , wherein the second search target sequence is on the second strand of the SERPINA1 gene, optionally wherein

the ngRNA comprises a spacer sequence selected from the group consisting of:

(SEQ ID NO: 26)

GAAGCAGAGACACGUUGUA,

(SEQ ID NO: 27)

GUCAGCACAGCCUUAUGCA,

(SEQ ID NO: 28)

GAAAGGGACUGAAGCUGCU,

(SEQ ID NO: 29)

CCUCGGGGGGGAUAGACAU,

(SEQ ID NO: 30)

UGAUCCCAGGCCUCGAGCA,

(SEQ ID NO: 31)

ACGUUGUAAGGCUGAUCCC,

(SEQ ID NO: 32)

AAAGGGACUGAAGCUGCUG,

(SEQ ID NO: 33)

GGUAUGGCCUCUAAAAACA,

(SEQ ID NO: 34)

CCCAUGUCUAUCCCCCCCG,

(SEQ ID NO: 35)

GCCUCGAGCAAGGCUCACG,

(SEQ ID NO: 36)

GGUUUGUUGAACUUGACCU,

(SEQ ID NO: 37)

CCUUAUGCACGGCCUGGAG,

(SEQ ID NO: 38)

AGAAAGGGACUGAAGCUGC,

(SEQ ID NO: 39)

CACAGCCUUAUGCACGGCC,

(SEQ ID NO: 40)

GGGGGGAUAGACAUGGGUA,

(SEQ ID NO: 41)

GUUUGUUGAACUUGACCUC,

(SEQ ID NO: 42)

UGCUGACCAUCGACAAGAA,

(SEQ ID NO: 43)

UUGUUGAACUUGACCUCGG,

(SEQ ID NO: 44)

GCCUUAUGCACGGCCUGGA,

(SEQ ID NO: 45)

UUUGUUGAACUUGACCUCG,

(SEQ ID NO: 46)

GUUGAACUUGACCUCGGGG,

(SEQ ID NO: 47)

CUCUGCUUCUCUCCCCUCC,

(SEQ ID NO: 48)

UGAGCCUUGCUCGAGGCCU,

(SEQ ID NO: 49)

AGCCUUAUGCACGGCCUGG,

(SEQ ID NO: 50)

ACCUCGGGGGGGAUAGACA,

(SEQ ID NO: 51)

UCAGUCCCUUUCUUGUCGA,

(SEQ ID NO: 52)

UGUUGAACUUGACCUCGGG,

(SEQ ID NO: 53)

CCCCUCCAGGCCGUGCAUA,

(SEQ ID NO: 54)

GUGAGCCUUGCUCGAGGCC,

(SEQ ID NO: 55)

GCUGACCAUCGACAAGAAA,

(SEQ ID NO: 56)

GCUGGGGCCAUGUUUUUAG,

(SEQ ID NO: 57)

UGCUGACCAUCGACGAGAA,

(SEQ ID NO: 58)

UCAGUCCCUUUCUCGUCGA,

and/or

(SEQ ID NO: 59)

GCUGACCAUCGACGAGAAA.

31 . (canceled)

32 . A PEgRNA system comprising the PEgRNA according to claim 1 , and a ngRNA wherein the ngRNA comprises an ng spacer that is complementary to a second search target sequence in the SERPINA1 gene.

33 . A prime editing complex comprising: (i) the PEgRNA of claim 1 or a PEgRNA system comprising the PEgRNA according to claim 1 , and a the ngRNA wherein the ngRNA comprises an ng spacer that is complementary to a second search target sequence in the SERPINA1 gene; and (ii) a prime editor comprising a DNA binding domain and a DNA polymerase domain.

34 . The prime editing complex of claim 33 , wherein

the DNA binding domain is a CRISPR associated (Cas) protein domain;

the Cas protein domain has nickase activity;

the Cas protein domain is a Cas9;

the Cas9 comprises a mutation in an HNH domain;

the Cas9 comprises a H840A mutation in the HNH domain;

the Cas9 comprises the sequence of SEQ ID NO: 60;

the Cas9 comprises a mutation at one or more amino acids positions of R765, K848, K855, K959, K961, K968, K974, or R976 relative to SEQ ID NO: 60;

the Cas9 comprises one or more mutations of R765A, K848A, K855A, K959A, K961A, K968A, K974A, or R976A relative to SEQ ID NO: 60;

the Cas9 comprises or consists of any one of the sequences of SEQ ID NO: 61 to 72;

the DNA polymerase domain is a reverse transcriptase;

the reverse transcriptase is a retrovirus reverse transcriptase; and/or

the reverse transcriptase is a Moloney murine leukemia virus (M-MLV) reverse transcriptase.

35 .- 45 . (canceled)

46 . The prime editing complex of claim 34 , wherein the reverse transcriptase comprises the sequence of SEQ ID NO: 75 (TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVS IKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQ GTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPK TPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTA PALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLR MVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTD RVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGGGSKRTADGSEFE), optionally wherein

the DNA polymerase and the DNA binding domain are fused or linked to form a fusion protein,

the DNA polymerase and the programmable DNA binding domain are linked by a linker comprising an amino acid sequence of SGGSEAAAKEAAAKEAAAKEAAAKSGGS (SEQ ID NO: 277),

the fusion protein comprises the sequence of SEQ ID NO: 77

(MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR

HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR

LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH

MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSR

RLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLL

AQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALV

RQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL

RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNS

RFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFT

VYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS

VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT

YAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQL

IHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHK

PENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYY

LQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEE

VVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVA

QILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA

VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT

LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIL

PKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIM

ERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP

SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDK

VLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ

SITGLYETRIDLSQLGGDSGGSEAAAKEAAAKEAAAKEAAAKSGGSTLNIEDEYRLHETS

KEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGI

KPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPY

NLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGF

KNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLG

YRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGF

CRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELF

VDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGK

LTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATL

LPLPEEGLQHNCLDILAEAHGGGSKRTADGSEFE);

the fusion protein comprises a nuclear localization signal (NLS);

the NLS comprises an amino acid sequence of PKKKRKV (SEQ ID NO: 282),

the fusion protein is encoded by the polynucleotide sequence:

(SEQ ID NO: 78)

ATGAAACGGACAGCCGACGGAAGCGAGTTCGAGTCACCAAAGAAGAAGCGGAAAG

TCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCC

GTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACAC

CGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCG

AAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACG

GAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGG

ACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAG

CACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAA

GTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCG

ACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCC

TGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAG

CTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGT

GGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATC

TGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCC

CTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGC

CAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCC

AGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCC

ATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAG

CGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAG

CTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCA

AGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAG

TTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCT

GAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCC

ACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTAC

CCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCC

CTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAA

AGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCT

TCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGA

GAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCT

GACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCG

AGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTG

AAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAAT

CTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAA

AATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAG

ATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTG

AAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAG

ATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGC

AGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAAC

TTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGC

CCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCA

GCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTG

AAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGA

ACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGA

AGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAAC

ACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATAT

GTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACGCTA

TCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGA

AGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGA

AGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAG

TTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGG

CTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGA

TCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAA

GTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAG

TTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAA

CGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCG

TGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAG

GAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTC

AAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGAC

AAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGC

GGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACA

GGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGC

CAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGG

CCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAG

AGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAA

TCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCA

TCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTG

GCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGT

GAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATA

ATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATC

GAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAA

AGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGA

ATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTT

TGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCA

CCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGC

TGGGAGGTGACTCCGGCGGCAGCGAGGCCGCCGCCAAGGAAGCCGCCGCCAAGGA

AGCCGCTGCCAAGGAGGCCGCTGCTAAAAGCGGCGGATCTACCCTGAACATCGAGG

ACGAGTACAGGCTGCACGAGACCAGCAAGGAGCCCGACGTGAGCCTGGGCAGCAC

CTGGCTGAGCGATTTCCCTCAGGCTTGGGCCGAGACCGGCGGCATGGGCCTGGCCGT

GCGGCAGGCCCCCCTGATTATCCCCCTGAAGGCCACCAGCACCCCCGTGAGCATCA

AGCAGTACCCAATGTCCCAGGAGGCCAGGCTGGGCATCAAGCCTCACATCCAGAGG

CTGCTGGACCAGGGCATCCTGGTGCCATGCCAGTCCCCCTGGAACACCCCTCTGCTG

CCCGTGAAGAAGCCTGGCACCAACGACTACCGGCCCGTGCAGGACCTGAGAGAAGT

GAACAAGCGGGTGGAGGACATCCACCCAACCGTGCCCAACCCTTACAACCTGCTGT

CCGGCCTGCCCCCCAGCCACCAGTGGTACACCGTGCTGGACCTGAAGGACGCCTTCT

TCTGCCTGAGACTGCACCCCACCTCTCAGCCCCTGTTCGCCTTCGAGTGGCGCGACC

CCGAGATGGGCATCAGCGGCCAGCTGACCTGGACCAGACTGCCACAGGGCTTTAAG

AATAGCCCAACCCTGTTTAACGAGGCCCTGCACAGGGACCTGGCCGACTTCAGGAT

CCAGCACCCCGACCTGATTCTGCTGCAGTACGTGGACGACCTGCTGCTGGCCGCTAC

CAGCGAGCTGGACTGCCAGCAGGGCACCAGAGCCCTGCTGCAGACCCTGGGCAACC

TGGGCTACAGAGCCAGCGCCAAGAAGGCCCAGATCTGTCAGAAGCAGGTGAAGTAT

CTGGGCTACCTGCTGAAGGAAGGCCAGAGATGGCTGACCGAGGCCAGAAAGGAGA

CTGTGATGGGCCAGCCCACCCCCAAGACCCCCAGGCAGCTGCGGGAGTTCCTGGGC

AAGGCCGGCTTTTGCAGACTGTTTATCCCTGGCTTCGCCGAGATGGCCGCCCCACTG

TACCCTCTGACCAAGCCTGGCACCCTGTTTAACTGGGGCCCCGACCAGCAGAAGGCC

TACCAGGAGATCAAGCAGGCCCTGCTGACCGCCCCCGCCCTGGGCCTGCCCGACCT

GACCAAGCCTTTCGAGCTGTTCGTGGACGAGAAGCAGGGATACGCCAAAGGCGTGC

TGACCCAGAAGCTGGGCCCCTGGCGGAGGCCCGTGGCCTACCTGAGCAAAAAACTG

GACCCTGTGGCCGCCGGCTGGCCCCCATGCCTGCGGATGGTGGCCGCCATCGCTGTG

CTGACCAAGGACGCCGGCAAGCTGACCATGGGCCAGCCCCTGGTGATCCTGGCCCC

TCACGCCGTGGAGGCTCTGGTGAAGCAGCCTCCAGACAGGTGGCTGTCCAACGCCA

GGATGACCCACTACCAGGCCCTGCTGCTGGACACCGACCGGGTGCAGTTCGGCCCT

GTGGTGGCCCTGAACCCCGCCACCCTGCTGCCTCTGCCAGAGGAGGGCCTGCAGCA

CAACTGCCTGGACATCCTGGCCGAGGCCCACGGCGGCGGCTCCAAACGCACCGCCG

ACGGGAGCGAGTTCGAGCCCAAGAAGAAGAGGAAAGTCTAA;

and/or

the polynucleotide sequence is an mRNA.

47 .- 53 . (canceled)

54 . A PEgRNA system comprising:

i) a prime editing guide RNA (PEgRNA) comprising:

a spacer that is complementary to a search target sequence on a first strand of a human SERPINA1 gene,

a primer binding site sequence (PBS) at least partially complementary to the spacer,

an editing template that comprises a region of complementarity to an editing target sequence on a second strand of the SERPINA1 gene, and

a gRNA core that associates with a prime editor comprising a DNA binding domain and a DNA polymerase domain; and

ii) a nick guide RNA (ngRNA), wherein the ngRNA comprises an ng spacer that is complementary to a second search target sequence in the SERPINA1 gene,

wherein the ngRNA comprises a spacer sequence selected from the group consisting of:

(SEQ ID NO: [[##]]26)

GAAGCAGAGACACGUUGUA,

(SEQ ID NO: [[##]]27)

GUCAGCACAGCCUUAUGCA,

(SEQ ID NO: [[##]]28)

GAAAGGGACUGAAGCUGCU,

(SEQ ID NO: [[##]]29)

CCUCGGGGGGGAUAGACAU,

(SEQ ID NO: [[##]]30)

UGAUCCCAGGCCUCGAGCA,

(SEQ ID NO: [[##]]31)

ACGUUGUAAGGCUGAUCCC,

(SEQ ID NO: [[##]]32)

AAAGGGACUGAAGCUGCUG,

(SEQ ID NO: [[##]]33)

GGUAUGGCCUCUAAAAACA,

(SEQ ID NO: [[##]]34)

CCCAUGUCUAUCCCCCCCG,

(SEQ ID NO: [[##]]35)

GCCUCGAGCAAGGCUCACG,

(SEQ ID NO: [[##]]36)

GGUUUGUUGAACUUGACCU,

(SEQ ID NO: [[##]]37)

CCUUAUGCACGGCCUGGAG,

(SEQ ID NO: [[##]]38)

AGAAAGGGACUGAAGCUGC,

(SEQ ID NO: [[##]]39)

CACAGCCUUAUGCACGGCC,

(SEQ ID NO: [[##]]40)

GGGGGGAUAGACAUGGGUA,

(SEQ ID NO: [[##]]41)

GUUUGUUGAACUUGACCUC,

(SEQ ID NO: [[##]]42)

UGCUGACCAUCGACAAGAA,

(SEQ ID NO: [[##]]43)

UUGUUGAACUUGACCUCGG,

(SEQ ID NO: [[##]]44)

GCCUUAUGCACGGCCUGGA,

(SEQ ID NO: [[##]]45)

UUUGUUGAACUUGACCUCG,

(SEQ ID NO: [[##]]46)

GUUGAACUUGACCUCGGGG,

(SEQ ID NO: [[##]]47)

CUCUGCUUCUCUCCCCUCC,

(SEQ ID NO: [[##]]48)

UGAGCCUUGCUCGAGGCCU,

(SEQ ID NO: [[##]]49)

AGCCUUAUGCACGGCCUGG,

(SEQ ID NO: [[##]]50)

ACCUCGGGGGGGAUAGACA,

(SEQ ID NO: [[##]]51)

UCAGUCCCUUUCUUGUCGA,

(SEQ ID NO: [[##]]52)

UGUUGAACUUGACCUCGGG,

(SEQ ID NO: [[##]]53)

CCCCUCCAGGCCGUGCAUA,

(SEQ ID NO: [[##]]54)

GUGAGCCUUGCUCGAGGCC,

(SEQ ID NO: [[##]]55)

GCUGACCAUCGACAAGAAA,

(SEQ ID NO: [[##]]56)

GCUGGGGCCAUGUUUUUAG,

(SEQ ID NO: [[##]]57)

UGCUGACCAUCGACGAGAA,

(SEQ ID NO: [[##]]58)

UCAGUCCCUUUCUCGUCGA,

and/or

(SEQ ID NO: [[##]]59)

GCUGACCAUCGACGAGAAA.

55 . The PEgRNA system of claim 54 , wherein

the PBS comprises or consists of the sequence GCACGGC (SEQ ID NO: 2) and the editing template comprises or consists of the sequence UUUCUCGUCGAUGGUCAGCACAGCCUUAU (SEQ ID NO: 25); and/or,

the spacer sequence comprises or consists of the sequence

(SEQ ID NO: 1)

UCCCCUCCAGGCCGUGCAUA

56 . (canceled)

57 . A prime editing guide RNA (PEgRNA) comprising:

a spacer that is complementary to a search target sequence on a first strand of a human SERPINA1 gene,

a primer binding site sequence (PBS) at least partially complementary to the spacer,

an editing template that comprises a region of complementarity to an editing target sequence on a second strand of the SERPINA1 gene, and

a gRNA core that associates with a prime editor comprising a DNA binding domain and a DNA polymerase domain,

wherein the editing template comprises or consists of the sequence

(SEQ ID NO: [[##]]283)

UUUCUCAUCGAUGGUCAGCACAGCCUUAU

or

(SEQ ID NO: [[##]]284)

CUUUCUCAUCGAUGGUCAGCACAGCCUUAU.

58 . The PEgRNA of claim 57 , wherein

the PBS comprises or consists of the sequence GCACGGC (SEQ ID NO: 2); and/or,

the spacer sequence comprises or consists of the sequence

(SEQ ID NO: 1)

UCCCCUCCAGGCCGUGCAUA

59 . (canceled)

60 . A lipid nanoparticle (LNP) or ribonucleoprotein (RNP) comprising the prime editing complex of claim 33 , or a component thereof.

61 . A lipid nanoparticle (LNP) composition comprising i) the PEgRNA of claim 1 , ii) a ngRNA, and iii) a polynucleotide encoding the prime editor.

62 . The LNP composition of claim 61 , wherein

the PERNA, the ngRNA, and the polynucleotide encoding the prime editor are each encapsulated in separate LNPs;

the PEgRNA, the ngRNA, and the polynucleotide encoding the prime editor are encapsulated in a single LNP

the mass ratio of the polynucleotide encoding the prime editor to the combination of the PERNA and the ngRNA (total gRNA content) is about 0.1:1 to about 3.0:1;

the mass ratio of the polynucleotide encoding the prime editor to the combination of the PERNA and the ngRNA (total gRNA content) is about 0.5:1;

the mass ratio of the PERNA to the ngRNA is about 1:1 to about 25:1; and/or

the mass ratio of the PEgRNA to the ngRNA is about 19:1.

63 .- 67 . (canceled)

68 . A polynucleotide encoding the PEgRNA of claim 1 optionally wherein

the polynucleotide is a mRNA;

the polynucleotide is operably linked to a regulatory element; and/or

the regulatory element is an inducible regulatory element.

69 .- 71 . (canceled)

72 . A vector comprising the polynucleotide of claim 68 , optionally wherein the vector is an AAV vector.

73 . (canceled)

74 . An isolated cell comprising the PERNA of claim 1 , the PEgRNA system comprising the PEgRNA of claim 1 , the prime editing complex comprising the PEgRNA of claim 1 , the LNP or RNP comprising the PEgRNA of claim 1 , the polynucleotide encoding the PERNA of claim 1 , or the vector comprising a polynucleotide encoding the PEgRNA of claim 1 , optionally wherein

the cell is a human cell;

the cell is a hepatocyte, a hepatic stellate cell, a Kupffer cell, or a liver sinusoidal endothelial cell; and/or

wherein the cell is a hepatocyte or a hepatic stellate cell.

75 .- 77 . (canceled)

78 . A pharmaceutical composition comprising (i) the PEgRNA of of claim 1 , the PEgRNA system comprising the PEgRNA of claim 1 , the prime editing complex comprising the PEgRNA of claim 1 , the LNP or RNP comprising the PERNA of claim 1 , the polynucleotide encoding the PERNA of claim 1 , the vector of claim comprising a polynucleotide encoding the PEgRNA, or the cell comprising PEgRNA of claim 1 ; and (ii) a pharmaceutically acceptable carrier.

79 . A method for

editing a SERPINA1 gene, the method comprising contacting the SERPINA1 gene with (i) the PERNA of claim 1 or the PEgRNA system comprising a PEgRNA of claim 1 and (ii) a prime editor comprising a DNA binding domain and a DNA polymerase domain, wherein the PEgRNA or the PERNA system directs the prime editor to incorporate the intended nucleotide edit in the SERPINA1 gene, thereby editing the SERPINA1 gene;

editing an SERPINA1 gene, the method comprising contacting the SERPINA1 gene with the prime editing complex comprising a PEgRNA of claim 1 , wherein the PERNA directs the prime editor to incorporate the intended nucleotide edit in the SERPINA1 gene, thereby editing the SERPINA1 gene,

optionally wherein

the method comprising contacting a cell with the LNP composition comprising a PEgRNA of claim 1 ;

the prime editor synthesizes a single stranded DNA encoded by the editing template, wherein the single stranded DNA replaces the editing target sequence and results in incorporation of the intended nucleotide edit into a region corresponding to the editing target in the SERPINA1 gene;

the SERPINA1 gene is in a cell;

the cell is a mammalian cell;

the cell is a human cell;

the cell is a hepatocyte or a hepatic stellate cell;

the cell is in a subject;

the subject is a human;

the cell is from a subject having A1AD;

further comprising administering the cell to the subject after incorporation of the intended nucleotide edit;

a cell or a population of cells is generated by said method; and/or

treating A1AD in a subject in need thereof, the method comprising administering to the subject (i) the PEgRNA of claim 1 or the PEgRNA system comprising a PEgRNA of claim 1 and (ii) a prime editor comprising a DNA binding domain and a DNA polymerase domain, wherein the PERNA directs the prime editor to incorporate the intended nucleotide edit in the SERPINA1 gene in the subject, thereby treating A1AD in the subject optionally wherein the subject is a human;

the method comprising administering to the subject the prime editing complex comprising a PEgRNA of claim 1 , the LNP or RNP comprising a PEgRNA of claim 1 , or the pharmaceutical composition comprising a PERNA of claim 1 , wherein the PERNA directs the prime editor to incorporate the intended nucleotide edit in the SERPINA1 gene in the subject, thereby treating A1AD in the subject optionally wherein the subject is a human; and/or

treating A1AD in a subject in need thereof, the method comprising administering to the subject the LNP composition comprising a PEgRNA of claim 1 , thereby treating A1AD in the subject, optionally wherein the subject is a human.

80 .- 96 . (canceled)