Compositions and methods for expressing factor IX for hemophilia B therapy - Patents.com

Abstract

Nucleic acid constructs and compositions are provided that allow for the insertion and / or expression of a FIX coding sequence at a target genomic locus, such as the endogenous ALB locus. The nucleic acid constructs and compositions can be used in methods of introducing an F9 nucleic acid into a cell, integrating an F9 nucleic acid into a target genomic locus, expressing FIX in a cell, and treating hemophilia B or FIX deficiency in a subject.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Application No. 63 / 272,324, filed October 27, 2021, U.S. Application No. 63 / 306,037, filed February 2, 2022, and U.S. Application No. 63 / 369,864, filed July 29, 2022, each of which is incorporated by reference herein in its entirety for all purposes.

[0002] Reference to sequence listings submitted as text files via EFS Web The sequence listing set forth in file 057766-586369.xml is 516 kilobytes, was created on October 26, 2022, and is incorporated herein by reference. [Background technology]

[0003] Hemophilia B is a rare congenital genetic disorder characterized by clotting factor IX (FIX) deficiency or absence, resulting in a bleeding diathesis. It is caused by inherited or spontaneous X-linked recessive mutations in the factor 9 (F9) gene, which result in a missing or defective FIX protein. The global incidence of hemophilia B is approximately 1 in 25,000 male births. The most recent worldwide hemophilia survey, dated 2019, reported 4,093 patients living with hemophilia B in the United States and a total of 31,997 patients with hemophilia B worldwide. World Federation of Hemophilia, "Report on the Annual Global Survey 2019," World Federation of Hemophilia (2020).

[0004] Due to an X-linked recessive mutation, hemophilia B primarily affects males, although female carriers may also be affected. The bleeding tendency in hemophilia B is related to the measured concentration of the factor and is classified as mild, moderate, or severe. In severe and moderately severe cases of hemophilia B, frequent spontaneous bleeding episodes are the most common symptom. Such bleeding episodes can occur in muscles and joints, causing pain and limited movement, and if left untreated, can lead to long-term joint damage with synovitis, arthropathy, and muscle weakness.

[0005] The current standard of care in the United States is the prevention and treatment of bleeding episodes with clotting factor concentrates (CFCs), either recombinant or derived from blood donor plasma. However, allergic reactions can occur in 3-4% of CFC infusions, and approximately 5% of patients develop FIX-neutralizing antibodies after repeated administration. (Srivastava et al., "WFH Guidelines for the Management of Hemophilia," Haemophilia 26.6:1-158 (2020)) Factor replacement is also cumbersome (requiring frequent intravenous administration), costly, reduces but does not eliminate bleeding episodes, and still leads to the progression of joint disease and disability. Summary of the Invention

[0006] Compositions comprising a nucleic acid construct comprising a first Factor IX protein coding sequence or the reverse complement of a first Factor IX protein coding sequence, cells comprising the compositions, and methods of using such nucleic acid constructs, such as methods for introducing a Factor IX nucleic acid into a cell, methods for integrating a Factor IX nucleic acid construct into a target gene in a cell, methods for expressing Factor IX in a cell, methods for treating Factor IX deficiency in a subject, methods for treating hemophilia B in a subject, and methods for preventing or inhibiting spontaneous bleeding in a subject with hemophilia B, are provided.

[0007] In one aspect, a composition is provided comprising a nucleic acid construct comprising a first Factor IX protein coding sequence or the reverse complement of a first Factor IX protein coding sequence. In some such compositions, the first Factor IX protein coding sequence is (I) at least 95% identical to any one of SEQ ID NOs: 166, 165, 164, and 167-171, optionally wherein the Factor IX protein coding sequence is CpG-depleted and codon-optimized, and optionally wherein the Factor IX protein is not a hyperactive Factor IX variant, or (II) at least 99% identical to any one of SEQ ID NOs: 159, 160, and 161, and either (i) CpG-depleted, (ii) modified to mutate one or more cryptic splice donor sequences, or (iii) CpG-depleted and modified to mutate one or more cryptic splice donor sequences, and optionally wherein the Factor IX protein is not a hyperactive Factor IX variant.

[0008] In some such compositions, the first Factor IX protein coding sequence is (I) at least 99% identical to SEQ ID NO: 166 and CpG-depleted and codon-optimized, optionally wherein the Factor IX protein is not a hyperactive Factor IX variant; (II) at least 99% identical to SEQ ID NO: 165 and CpG-depleted and codon-optimized, optionally wherein the Factor IX protein is not a hyperactive Factor IX variant; or (III) at least 99% identical to SEQ ID NO: 159, CpG-depleted and modified to mutate one or more cryptic splice donor sequences, optionally wherein the Factor IX protein is not a hyperactive Factor IX variant.

[0009] In some such compositions, the first Factor IX protein coding sequence is (I) at least 99% identical to SEQ ID NO: 166, CpG-depleted, and codon-optimized, optionally wherein the Factor IX protein is not a hyperactive Factor IX variant, or (II) at least 99% identical to SEQ ID NO: 159, CpG-depleted, and modified to mutate one or more cryptic splice donor sequences, optionally wherein the Factor IX protein is not a hyperactive Factor IX variant.

[0010] In some such compositions, the first Factor IX protein coding sequence is at least 99% identical to SEQ ID NO: 166, CpG-depleted, and codon-optimized, and optionally the Factor IX protein is not a hyperactive Factor IX variant. In some such compositions, the first Factor IX protein coding sequence is at least 99% identical to SEQ ID NO: 165, CpG-depleted, and codon-optimized, and optionally the Factor IX protein is not a hyperactive Factor IX variant. In some such compositions, the first Factor IX protein coding sequence is at least 99% identical to SEQ ID NO: 159, CpG-depleted, and modified to mutate one or more cryptic splice donor sequences, and optionally the Factor IX protein is not a hyperactive Factor IX variant. In some such compositions, the first Factor IX protein coding sequence has all but one CpG dinucleotide removed or is completely CpG-depleted. In some such compositions, the first Factor IX protein coding sequence encodes a Factor IX protein that is at least 99% identical to SEQ ID NO: 195, and optionally, the Factor IX protein is not a hyperactive Factor IX variant. In some such compositions, the first Factor IX protein coding sequence encodes a Factor IX protein comprising SEQ ID NO: 195. In some such compositions, the first Factor IX protein coding sequence comprises SEQ ID NO: 166, 165, or 159. In some such compositions, the first Factor IX protein coding sequence consists of SEQ ID NO: 166, 165, or 159. In some such compositions, the first Factor IX protein coding sequence comprises SEQ ID NO: 166 or 159. In some such compositions, the first Factor IX protein coding sequence consists of SEQ ID NO: 166 or 159. In some such compositions, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. In some such compositions, the first Factor IX protein coding sequence consists of SEQ ID NO: 166.In some such compositions, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. In some such compositions, the first Factor IX protein coding sequence consists of SEQ ID NO: 165. In some such compositions, the first Factor IX protein coding sequence comprises SEQ ID NO: 159. In some such compositions, the first Factor IX protein coding sequence consists of SEQ ID NO: 159.

[0011] In some such compositions, the nucleic acid construct comprises a splice acceptor upstream of the first Factor IX protein coding sequence. In some such compositions, the nucleic acid construct comprises a polyadenylation signal downstream of the first Factor IX protein coding sequence. In some such compositions, the nucleic acid construct comprises a splice acceptor upstream of the first Factor IX protein coding sequence and the nucleic acid construct comprises a polyadenylation signal downstream of the first Factor IX protein coding sequence. In some such compositions, the nucleic acid construct does not comprise a homology arm. In some such compositions, the nucleic acid construct comprises a homology arm. In some such compositions, the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein.

[0012] In some such compositions, the nucleic acid construct is a bidirectional construct. In some such compositions, the nucleic acid construct comprises a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, or the nucleic acid construct comprises a second Factor IX protein coding sequence and the reverse complement of the first Factor IX protein coding sequence. In some such compositions, the first Factor IX protein coding sequence and the second Factor IX protein coding sequence are different but encode the same Factor IX protein sequence, and optionally, the Factor IX protein is not a hyperactive Factor IX variant.

[0013] In some such compositions, (I) the first Factor IX protein coding sequence is at least 99% identical to SEQ ID NO: 159 and is (i) CpG-depleted, (ii) modified to mutate one or more cryptic splice donor sequences, or (iii) CpG-depleted and modified to mutate one or more cryptic splice donor sequences, optionally wherein the Factor IX protein is not a hyperactive Factor IX variant; and (II) the second Factor IX protein coding sequence is at least 95% identical to SEQ ID NO: 166 and is CpG-depleted and codon-optimized, optionally wherein the Factor IX protein is not a hyperactive Factor IX variant.

[0014] In some such compositions, (I) the first Factor IX protein coding sequence is at least 99% identical to SEQ ID NO: 159 and is (i) CpG-depleted, (ii) modified to mutate one or more cryptic splice donor sequences, or (iii) CpG-depleted and modified to mutate one or more cryptic splice donor sequences, optionally wherein the Factor IX protein is not a hyperactive Factor IX variant; and (II) the second Factor IX protein coding sequence is at least 95% identical to SEQ ID NO: 165 and is CpG-depleted and codon-optimized, optionally wherein the Factor IX protein is not a hyperactive Factor IX variant.

[0015] In some such compositions, (I) a first Factor IX protein coding sequence is at least 99% identical to SEQ ID NO: 159, is CpG-depleted, and has been modified to mutate one or more cryptic splice donor sequences, optionally wherein the Factor IX protein is not a hyperactive Factor IX variant; and (II) a second Factor IX protein coding sequence is at least 99% identical to SEQ ID NO: 166, is CpG-depleted, and has been codon-optimized, optionally wherein the Factor IX protein is not a hyperactive Factor IX variant.

[0016] In some such compositions, (I) a first Factor IX protein coding sequence is at least 99% identical to SEQ ID NO: 159, is CpG-depleted, and has been modified to mutate one or more cryptic splice donor sequences, optionally wherein the Factor IX protein is not a hyperactive Factor IX variant; and (II) a second Factor IX protein coding sequence is at least 99% identical to SEQ ID NO: 165, is CpG-depleted, and has been codon-optimized, optionally wherein the Factor IX protein is not a hyperactive Factor IX variant.

[0017] In some such compositions, the first Factor IX protein coding sequence has all but one CpG dinucleotide removed or is completely CpG-depleted, and the second Factor IX protein coding sequence has all but one CpG dinucleotide removed or is completely CpG-depleted.

[0018] In some such compositions, the first Factor IX protein coding sequence encodes a Factor IX protein that is at least 99% identical to SEQ ID NO: 195, optionally wherein the Factor IX protein is not a hyperactive Factor IX variant, and the second Factor IX protein coding sequence encodes a Factor IX protein that is at least 99% identical to SEQ ID NO: 195, optionally wherein the Factor IX protein is not a hyperactive Factor IX variant. In some such compositions, the first Factor IX protein coding sequence encodes a Factor IX protein comprising SEQ ID NO: 195, and the second Factor IX protein coding sequence encodes a Factor IX protein comprising SEQ ID NO: 195.

[0019] In some such compositions, (I) the first Factor IX protein coding sequence comprises SEQ ID NO: 159 and (II) the second Factor IX protein coding sequence comprises SEQ ID NO: 166. In some such compositions, (I) the first Factor IX protein coding sequence consists of SEQ ID NO: 159 and (II) the second Factor IX protein coding sequence consists of SEQ ID NO: 166.

[0020] In some such compositions, (I) the first Factor IX protein coding sequence comprises SEQ ID NO: 159 and (II) the second Factor IX protein coding sequence comprises SEQ ID NO: 165. In some such compositions, (I) the first Factor IX protein coding sequence consists of SEQ ID NO: 159 and (II) the second Factor IX protein coding sequence consists of SEQ ID NO: 165.

[0021] In some such compositions, the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, a first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, or the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, a second Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the first Factor IX protein coding sequence, and the reverse complement of the second splice acceptor. In some such compositions, the first Factor IX protein coding sequence and the second Factor IX protein coding sequence are different but encode the same Factor IX protein sequence, and the first polyadenylation signal and the second polyadenylation signal are different.

[0022] In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms.

[0023] In some such compositions, the nucleic acid construct comprises SEQ ID NO: 210, or 180, or a reverse complement thereof.

[0024] In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms.

[0025] In some such compositions, the nucleic acid construct comprises SEQ ID NO: 209, or 179, or a reverse complement thereof.

[0026] In some such compositions, the nucleic acid construct is single-stranded DNA or double-stranded DNA. In some such compositions, the nucleic acid construct is single-stranded DNA. In some such compositions, the nucleic acid construct is in a nucleic acid vector or lipid nanoparticle. In some such compositions, the nucleic acid construct is in a nucleic acid vector. In some such compositions, the nucleic acid vector is a viral vector. In some such compositions, the nucleic acid vector is an adeno-associated viral (AAV) vector, and optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprising, consisting essentially of, or consisting of SEQ ID NO: 198, optionally the ITRs at each end comprising, consisting essentially of, or consisting of SEQ ID NO: 198, or optionally the ITRs at at least one end comprising, consisting essentially of, or consisting of SEQ ID NO: 196, optionally the ITRs at each end comprising, consisting essentially of, or consisting of SEQ ID NO: 196. In some such compositions, the AAV vector is a single-stranded AAV (ssAAV) vector. In some such compositions, the AAV vector is derived from an AAV8 vector, an AAV3B vector, an AAV5 vector, an AAV6 vector, an AAV7 vector, an AAV9 vector, an AAVrh.74 vector, or an AAVhu.37 vector. In some such compositions, the AAV vector is a recombinant AAV8 (rAAV8) vector. In some such compositions, the AAV vector is a single-stranded rAAV8 vector.

[0027] In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and a reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159; The nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, the nucleic acid construct does not comprise homology arms, the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 198, optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 198, or optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 196, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 196.

[0028] In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and a reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159; The nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, the nucleic acid construct does not comprise homology arms, the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 198, optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 198, or optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 196, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 196.

[0029] In some such compositions, the nucleic acid construct is a unidirectional construct. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. In some such compositions, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0030] In some such compositions, the nucleic acid construct is a unidirectional construct. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. In some such compositions, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0031] In some such compositions, the nucleic acid construct is single-stranded or double-stranded DNA. In some such compositions, the nucleic acid construct is single-stranded DNA. In some such compositions, the nucleic acid construct is within a nucleic acid vector or lipid nanoparticle. In some such compositions, the nucleic acid construct is within a nucleic acid vector. In some such compositions, the nucleic acid vector is a viral vector. In some such compositions, the nucleic acid vector is an adeno-associated viral (AAV) vector, and optionally, the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally wherein the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, optionally wherein the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally wherein the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally wherein the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 196. In some such compositions, the AAV vector is a single-stranded AAV (ssAAV) vector. In some such compositions, the AAV vector is derived from an AAV8 vector, an AAV3B vector, an AAV5 vector, an AAV6 vector, an AAV7 vector, an AAV9 vector, an AAVrh.74 vector, or an AAVhu.37 vector. In some such compositions, the AAV vector is a recombinant AAV8 (rAAV8) vector. In some such compositions, the AAV vector is a single-stranded rAAV8 vector.

[0032] In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO:159 or SEQ ID NO:166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, and optionally wherein the nucleic acid construct comprises a polyadenylation signal at each end. and optionally an ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally an ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally an ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally an ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 196. In some such compositions, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. In some such compositions, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0033] In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO:159 or SEQ ID NO:165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, and optionally wherein the nucleic acid construct comprises a polyadenylation signal at each end. and optionally an ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally an ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally an ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally an ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 196. In some such compositions, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. In some such compositions, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0034] Some such compositions further comprise a nuclease agent that targets a nuclease target site in the target gene. In some such compositions, the target gene is an albumin gene, and optionally, the albumin gene is a human albumin gene. In some such compositions, the nuclease agent comprises (a) a zinc finger nuclease (ZFN), (b) a transcription activator-like effector nuclease (TALEN), or (c) (i) a Cas protein or a nucleic acid encoding a Cas protein, and (ii) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets the guide RNA target sequence, and the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence. In some such compositions, the nuclease agent comprises (a) a Cas protein or a nucleic acid encoding a Cas protein, and (b) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets a guide RNA target sequence in intron 1 of the albumin gene, and the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence. In some such compositions, the albumin gene is a human albumin gene.

[0035] In some such compositions, (I) the DNA-targeting segment comprises at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of the sequence set forth in any one of SEQ ID NOs: 30-61, optionally wherein the DNA-targeting segment comprises at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of the sequence set forth in any one of SEQ ID NOs: 36, 30, 33, and 41; and / or (II) the DNA-targeting segment is at least 90% or at least 95% identical to the sequence set forth in any one of SEQ ID NOs: 30-61, optionally wherein the DNA-targeting segment is at least 90% or at least 95% identical to the sequence set forth in any one of SEQ ID NOs: 36, 30, 33, and 41. In some such compositions, the DNA-targeting segment comprises any one of SEQ ID NOs: 30-61, optionally wherein the DNA-targeting segment comprises any one of SEQ ID NOs: 36, 30, 33, and 41. In some such compositions, the DNA-targeting segment consists of any one of SEQ ID NOs: 30-61, and optionally, the DNA-targeting segment consists of any one of SEQ ID NOs: 36, 30, 33, and 41. In some such compositions, the guide RNA comprises any one of SEQ ID NOs: 62-125, and optionally, the guide RNA comprises any one of SEQ ID NOs: 68, 100, 62, 94, 65, 97, 73, and 105.

[0036] In some such compositions, (I) the DNA-targeting segment comprises at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of SEQ ID NO: 36, and / or (II) the DNA-targeting segment is at least 90% or at least 95% identical to SEQ ID NO: 36. In some such compositions, the DNA-targeting segment comprises SEQ ID NO: 36. In some such compositions, the DNA-targeting segment consists of SEQ ID NO: 36. In some such compositions, the guide RNA comprises SEQ ID NO: 68 or 100.

[0037] In some such compositions, (I) the DNA-targeting segment comprises at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of SEQ ID NO: 30, and / or (II) the DNA-targeting segment is at least 90% or at least 95% identical to SEQ ID NO: 30. In some such compositions, the DNA-targeting segment comprises SEQ ID NO: 30. In some such compositions, the DNA-targeting segment consists of SEQ ID NO: 30. In some such compositions, the guide RNA comprises SEQ ID NO: 62 or 94.

[0038] In some such compositions, (I) the DNA-targeting segment comprises at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of SEQ ID NO: 33, and / or (II) the DNA-targeting segment is at least 90% or at least 95% identical to SEQ ID NO: 33. In some such compositions, the DNA-targeting segment comprises SEQ ID NO: 33. In some such compositions, the DNA-targeting segment consists of SEQ ID NO: 33. In some such compositions, the guide RNA comprises SEQ ID NO: 65 or 97.

[0039] In some such compositions, (I) the DNA-targeting segment comprises at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides of SEQ ID NO: 41, and / or (II) the DNA-targeting segment is at least 90% or at least 95% identical to SEQ ID NO: 41. In some such compositions, the DNA-targeting segment comprises SEQ ID NO: 41. In some such compositions, the DNA-targeting segment consists of SEQ ID NO: 41. In some such compositions, the guide RNA comprises SEQ ID NO: 73 or 105.

[0040] In some such compositions, the composition comprises a guide RNA in the form of RNA. In some such compositions, the guide RNA comprises at least one modification. In some such compositions, at least one modification comprises a 2'-O-methyl modified nucleotide. In some such compositions, at least one modification comprises an internucleotide phosphorothioate bond. In some such compositions, at least one modification comprises a modification in one or more of the first five nucleotides at the 5' end of the guide RNA. In some such compositions, at least one modification comprises a modification in one or more of the last five nucleotides at the 3' end of the guide RNA. In some such compositions, at least one modification comprises a phosphorothioate bond between the first four nucleotides at the 5' end of the guide RNA. In some such compositions, at least one modification comprises a phosphorothioate bond between the last four nucleotides at the 3' end of the guide RNA. In some such compositions, at least one modification comprises a 2'-O-methyl modified nucleotide in the first three nucleotides at the 5' end of the guide RNA. In some such compositions, at least one modification comprises 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA. In some such compositions, at least one modification comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) a 2'-O-methyl modified nucleotide in the first three nucleotides at the 5' end of the guide RNA, and (iv) a 2'-O-methyl modified nucleotide in the last three nucleotides at the 3' end of the guide RNA.

[0041] In some such compositions, the guide RNA is a single guide RNA (sgRNA). In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 100, and the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA.

[0042] In some such compositions, the guide RNA is a single guide RNA (sgRNA). In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 94, and the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA.

[0043] In some such compositions, the guide RNA is a single guide RNA (sgRNA). In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 97, and the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA.

[0044] In some such compositions, the guide RNA is a single guide RNA (sgRNA). In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 105, and the guide RNA comprises (i) phosphorothioate linkages between the first four nucleotides at the 5' end of the guide RNA, (ii) phosphorothioate linkages between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA.

[0045] In some such compositions, the Cas protein is a Cas9 protein. In some such compositions, the Cas protein is or is derived from a Streptococcus pyogenes Cas9 protein. In some such compositions, the Cas protein comprises the sequence set forth in SEQ ID NO: 11. In some such compositions, the nucleic acid encoding the Cas protein is codon-optimized for expression in mammalian or human cells. In some such compositions, the composition comprises a nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein. In some such compositions, the mRNA encoding the Cas protein comprises at least one modification. In some such compositions, the mRNA encoding the Cas protein is modified to include modified uridines at one or more or all uridine positions. In some such compositions, the modified uridine is pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine. In some such compositions, the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine. In some such compositions, the modified uridine is pseudouridine. In some such compositions, the mRNA encoding the Cas protein is fully substituted with pseudouridine. In some such compositions, the mRNA encoding the Cas protein comprises a 5' cap. In some such compositions, the mRNA encoding the Cas protein comprises a poly(A) tail. In some such compositions, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 12. In some such compositions, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 225. In some such compositions, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226.

[0046] In some such compositions, the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail. In some such compositions, the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine, comprises a 5' cap, and comprises a poly(A) tail.

[0047] In some such compositions, the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 225, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail. In some such compositions, the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 225, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine, comprises a 5' cap, and comprises a poly(A) tail.

[0048] In some such compositions, the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail. In some such compositions, the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine, comprises a 5' cap, and comprises a poly(A) tail.

[0049] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 68 or 100; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 12. In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0050] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 68 or 100; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 12. In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0051] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 62 or 94; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 12. In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0052] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 62 or 94; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 12. In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0053] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 65 or 97; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 12. In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0054] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 65 or 97; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 12. In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0055] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 73 or 105; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 12. In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0056] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 73 or 105; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 12. In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0057] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 100, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail. In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 100, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridines, comprises a 5' cap, and comprises a poly(A) tail.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0058] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 100, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail. In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 100, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridines, comprises a 5' cap, and comprises a poly(A) tail.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0059] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 94, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail. In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 94, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridines, comprises a 5' cap, and comprises a poly(A) tail.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0060] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 94, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail. In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 94, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridines, comprises a 5' cap, and comprises a poly(A) tail.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0061] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 97, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail. In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 97, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridines, comprises a 5' cap, and comprises a poly(A) tail.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0062] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 97, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail. In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 97, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridines, comprises a 5' cap, and comprises a poly(A) tail.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0063] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 105, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail. In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 105, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridines, comprises a 5' cap, and comprises a poly(A) tail.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0064] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 105, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail. In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 105, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridines, comprises a 5' cap, and comprises a poly(A) tail.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0065] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 68 or 100; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 225. In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0066] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 68 or 100; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 225. In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0067] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 62 or 94; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 225. In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0068] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 62 or 94; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 225. In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0069] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 65 or 97; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 225. In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0070] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 65 or 97; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 225. In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0071] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 73 or 105; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 225. In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0072] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 73 or 105; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 225. In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0073] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 100, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 225, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail. In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 100, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 225, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridines, comprises a 5' cap, and comprises a poly(A) tail.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0074] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 100, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 225, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail. In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 100, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 225, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridines, comprises a 5' cap, and comprises a poly(A) tail.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0075] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 94, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 225, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail. In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 94, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 225, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridines, comprises a 5' cap, and comprises a poly(A) tail.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0076] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 94, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 225, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail. In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 94, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 225, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridines, comprises a 5' cap, and comprises a poly(A) tail.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0077] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 97, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 225, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail. In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 97, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 225, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridines, comprises a 5' cap, and comprises a poly(A) tail.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0078] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 97, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 225, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail. In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 97, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 225, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridines, comprises a 5' cap, and comprises a poly(A) tail.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0079] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 105, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 225, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail. In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 105, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 225, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridines, comprises a 5' cap, and comprises a poly(A) tail.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0080] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 105, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 225, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail. In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 105, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 225, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridines, comprises a 5' cap, and comprises a poly(A) tail.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0081] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 68 or 100; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226. In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0082] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 68 or 100; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226. In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0083] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 62 or 94; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226. In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0084] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 62 or 94; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226. In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0085] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 65 or 97; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226. In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0086] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 65 or 97; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226. In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0087] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 73 or 105; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226. In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0088] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 73 or 105; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226. In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0089] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 100, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail. In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 100, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridines, comprises a 5' cap, and comprises a poly(A) tail.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0090] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 100, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail. In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 100, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridines, comprises a 5' cap, and comprises a poly(A) tail.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0091] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 94, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail. In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 94, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridines, comprises a 5' cap, and comprises a poly(A) tail.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0092] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 94, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail. In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 94, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridines, comprises a 5' cap, and comprises a poly(A) tail.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0093] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 97, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail. In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 97, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridines, comprises a 5' cap, and comprises a poly(A) tail.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0094] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 97, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail. In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 97, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridines, comprises a 5' cap, and comprises a poly(A) tail.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0095] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 105, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail. In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 105, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridines, comprises a 5' cap, and comprises a poly(A) tail.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0096] In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 105, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail. In some such compositions, the composition comprises a guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 105, wherein the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; the composition comprises a nucleic acid encoding a Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 226, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridines, comprises a 5' cap, and comprises a poly(A) tail.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and the reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO: 159 or SEQ ID NO: 165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0097] In some such compositions, the Cas protein or a nucleic acid encoding the Cas protein and the guide RNA or one or more DNAs encoding the guide RNA are associated with a lipid nanoparticle. In some such compositions, the lipid nanoparticle comprises a cationic lipid, a neutral lipid, a helper lipid, and a stealth lipid. In some such compositions, the cationic lipid is lipid A. In some such compositions, the neutral lipid is DSPC. In some such compositions, the helper lipid is cholesterol. In some such compositions, the stealth lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2k-DMG). In some such compositions, the cationic lipid is lipid A, the neutral lipid is DSPC, the helper lipid is cholesterol, and the stealth lipid is PEG2k-DMG. In some such compositions, the lipid nanoparticles comprise four lipids in the following molar ratio: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.

[0098] In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprising SEQ ID NO: 68 or 100, the composition comprises a nucleic acid encoding a Cas protein, the nucleic acid comprising an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprising the sequence set forth in SEQ ID NO: 12, and the guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and a reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159; The nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, the nucleic acid construct does not comprise homology arms, the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 198, optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 198, or optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 196, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 196.In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO:159 or SEQ ID NO:166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, and optionally wherein the nucleic acid construct comprises a polyadenylation signal at each end. It is flanked on the ends by inverted terminal repeats (ITRs), and optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 196. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0099] In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprising SEQ ID NO: 68 or 100, the composition comprises a nucleic acid encoding a Cas protein, the nucleic acid comprising an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprising the sequence set forth in SEQ ID NO: 12, and the guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and a reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159; The nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, the nucleic acid construct does not comprise homology arms, the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 198, optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 198, or optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 196, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 196.In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO:159 or SEQ ID NO:165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, and optionally wherein the nucleic acid construct comprises a polyadenylation signal at each end. It is flanked on the ends by inverted terminal repeats (ITRs), and optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 196. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0100] In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprising SEQ ID NO: 62 or 94, the composition comprises a nucleic acid encoding a Cas protein, the nucleic acid comprising an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprising the sequence set forth in SEQ ID NO: 12, and the guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and a reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159; The nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, the nucleic acid construct does not comprise homology arms, the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 198, optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 198, or optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 196, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 196.In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO:159 or SEQ ID NO:166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, and optionally wherein the nucleic acid construct comprises a polyadenylation signal at each end. It is flanked on the ends by inverted terminal repeats (ITRs), and optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 196. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0101] In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprising SEQ ID NO: 62 or 94, the composition comprises a nucleic acid encoding a Cas protein, the nucleic acid comprising an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprising the sequence set forth in SEQ ID NO: 12, and the guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and a reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159; The nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, the nucleic acid construct does not comprise homology arms, the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 198, optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 198, or optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 196, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 196.In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO:159 or SEQ ID NO:165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, and optionally wherein the nucleic acid construct comprises a polyadenylation signal at each end. It is flanked on the ends by inverted terminal repeats (ITRs), and optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 196. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0102] In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprising SEQ ID NO: 65 or 97, the composition comprises a nucleic acid encoding a Cas protein, the nucleic acid comprising an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprising the sequence set forth in SEQ ID NO: 12, and the guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and a reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159; The nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, the nucleic acid construct does not comprise homology arms, the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 198, optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 198, or optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 196, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 196.In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO:159 or SEQ ID NO:166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, and optionally wherein the nucleic acid construct comprises a polyadenylation signal at each end. It is flanked on the ends by inverted terminal repeats (ITRs), and optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 196. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0103] In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprising SEQ ID NO: 65 or 97, the composition comprises a nucleic acid encoding a Cas protein, the nucleic acid comprising an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprising the sequence set forth in SEQ ID NO: 12, and the guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and a reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159; The nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, the nucleic acid construct does not comprise homology arms, the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 198, optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 198, or optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 196, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 196.In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO:159 or SEQ ID NO:165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, and optionally wherein the nucleic acid construct comprises a polyadenylation signal at each end. It is flanked on the ends by inverted terminal repeats (ITRs), and optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 196. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0104] In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprising SEQ ID NO: 73 or 105, the composition comprises a nucleic acid encoding a Cas protein, the nucleic acid comprising an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprising the sequence set forth in SEQ ID NO: 12, and the guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and a reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159; The nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, the nucleic acid construct does not comprise homology arms, the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 198, optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 198, or optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 196, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 196.In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO:159 or SEQ ID NO:166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, and optionally wherein the nucleic acid construct comprises a polyadenylation signal at each end. It is flanked on the ends by inverted terminal repeats (ITRs), and optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 196. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0105] In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprising SEQ ID NO: 73 or 105, the composition comprises a nucleic acid encoding a Cas protein, the nucleic acid comprising an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprising the sequence set forth in SEQ ID NO: 12, and the guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and a reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159; The nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, the nucleic acid construct does not comprise homology arms, the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 198, optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 198, or optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 196, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 196.In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO:159 or SEQ ID NO:165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, and optionally wherein the nucleic acid construct comprises a polyadenylation signal at each end. It is flanked on the ends by inverted terminal repeats (ITRs), and optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 196. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0106] In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprising SEQ ID NO: 100, the guide RNA comprising (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA, and the composition comprises a nucleic acid encoding a Cas protein, the nucleic acid being , comprising an mRNA encoding a Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail, wherein the guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 100, the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA, and the composition is a Cas The guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and a reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159; The nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, the nucleic acid construct does not comprise homology arms, the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 198, optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 198, or optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 196, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 196.In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO:159 or SEQ ID NO:166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, and optionally wherein the nucleic acid construct comprises a polyadenylation signal at each end. It is flanked on the ends by inverted terminal repeats (ITRs), and optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 196. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0107] In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprising SEQ ID NO: 100, the guide RNA comprising (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA, and the composition comprises a nucleic acid encoding a Cas protein, the nucleic acid being , comprising an mRNA encoding a Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail, wherein the guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 100, the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA, and the composition is a Cas The guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and a reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159; The nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, the nucleic acid construct does not comprise homology arms, the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 198, optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 198, or optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 196, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 196.In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO:159 or SEQ ID NO:165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, and optionally wherein the nucleic acid construct comprises a polyadenylation signal at each end. It is flanked on the ends by inverted terminal repeats (ITRs), and optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 196. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0108] In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprising SEQ ID NO: 94, the guide RNA comprising (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA, and the composition comprises a nucleic acid encoding a Cas protein, the nucleic acid being , comprising an mRNA encoding a Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail, wherein the guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 94, the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA, and the composition is a Cas tag. The guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and a reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159; The nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, the nucleic acid construct does not comprise homology arms, the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 198, optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 198, or optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 196, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 196.In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO:159 or SEQ ID NO:166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, and optionally wherein the nucleic acid construct comprises a polyadenylation signal at each end. It is flanked on the ends by inverted terminal repeats (ITRs), and optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 196. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0109] In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprising SEQ ID NO: 94, the guide RNA comprising (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA, and the composition comprises a nucleic acid encoding a Cas protein, the nucleic acid being , comprising an mRNA encoding a Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail, wherein the guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 94, the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA, and the composition is a Cas tag. The guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and a reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159; The nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, the nucleic acid construct does not comprise homology arms, the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 198, optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 198, or optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 196, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 196.In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO:159 or SEQ ID NO:165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, and optionally wherein the nucleic acid construct comprises a polyadenylation signal at each end. It is flanked on the ends by inverted terminal repeats (ITRs), and optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 196. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0110] In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprising SEQ ID NO: 97, the guide RNA comprising (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA, and the composition comprises a nucleic acid encoding a Cas protein, the nucleic acid being , comprising an mRNA encoding a Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail, wherein the guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 97, the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA, and the composition is a Cas tag. The guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and a reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159; The nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, the nucleic acid construct does not comprise homology arms, the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 198, optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 198, or optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 196, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 196.In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO:159 or SEQ ID NO:166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, and optionally wherein the nucleic acid construct comprises a polyadenylation signal at each end. It is flanked on the ends by inverted terminal repeats (ITRs), and optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 196. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0111] In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprising SEQ ID NO: 97, the guide RNA comprising (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA, and the composition comprises a nucleic acid encoding a Cas protein, the nucleic acid being , comprising an mRNA encoding a Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail, wherein the guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 97, the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA, and the composition is a Cas tag. The guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and a reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159; The nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, the nucleic acid construct does not comprise homology arms, the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 198, optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 198, or optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 196, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 196.In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO:159 or SEQ ID NO:165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, and optionally wherein the nucleic acid construct comprises a polyadenylation signal at each end. It is flanked on the ends by inverted terminal repeats (ITRs), and optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 196. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0112] In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprising SEQ ID NO: 105, the guide RNA comprising (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA, and the composition comprises a nucleic acid encoding a Cas protein, the nucleic acid being , comprising an mRNA encoding a Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail, wherein the guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 105, the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA, and the composition is a Cas The guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and a reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159; The nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, the nucleic acid construct does not comprise homology arms, the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 198, optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 198, or optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 196, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 196.In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO:159 or SEQ ID NO:166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, and optionally wherein the nucleic acid construct comprises a polyadenylation signal at each end. It is flanked on the ends by inverted terminal repeats (ITRs), and optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 196. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0113] In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprising SEQ ID NO: 105, the guide RNA comprising (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA, and the composition comprises a nucleic acid encoding a Cas protein, the nucleic acid being , comprising an mRNA encoding a Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 12, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail, wherein the guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 105, the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA, and the composition is a Cas The guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and a reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159; The nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, the nucleic acid construct does not comprise homology arms, the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 198, optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 198, or optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 196, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 196.In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO:159 or SEQ ID NO:165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, and optionally wherein the nucleic acid construct comprises a polyadenylation signal at each end. It is flanked on the ends by inverted terminal repeats (ITRs), and optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 196. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0114] In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprising SEQ ID NO: 68 or 100, the composition comprises a nucleic acid encoding a Cas protein, the nucleic acid comprising an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprising the sequence set forth in SEQ ID NO: 225, and the guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and a reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159; The nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, the nucleic acid construct does not comprise homology arms, the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 198, optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 198, or optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 196, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 196.In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO:159 or SEQ ID NO:166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, and optionally wherein the nucleic acid construct comprises a polyadenylation signal at each end. It is flanked on the ends by inverted terminal repeats (ITRs), and optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 196. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0115] In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprising SEQ ID NO: 68 or 100, the composition comprises a nucleic acid encoding a Cas protein, the nucleic acid comprising an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprising the sequence set forth in SEQ ID NO: 225, and the guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and a reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159; The nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, the nucleic acid construct does not comprise homology arms, the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 198, optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 198, or optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 196, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 196.In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO:159 or SEQ ID NO:165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, and optionally wherein the nucleic acid construct comprises a polyadenylation signal at each end. It is flanked on the ends by inverted terminal repeats (ITRs), and optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 196. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0116] In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprising SEQ ID NO: 62 or 94, the composition comprises a nucleic acid encoding a Cas protein, the nucleic acid comprising an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprising the sequence set forth in SEQ ID NO: 225, and the guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol % lipid A, about 9 mol % DSPC, about 38 mol % cholesterol, and about 3 mol % PEG2k-DMG.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and a reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159; The nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, the nucleic acid construct does not comprise homology arms, the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 198, optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 198, or optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 196, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 196.In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO:159 or SEQ ID NO:166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, and optionally wherein the nucleic acid construct comprises a polyadenylation signal at each end. It is flanked on the ends by inverted terminal repeats (ITRs), and optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 196. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0117] In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprising SEQ ID NO: 62 or 94, the composition comprises a nucleic acid encoding a Cas protein, the nucleic acid comprising an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprising the sequence set forth in SEQ ID NO: 225, and the guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol % lipid A, about 9 mol % DSPC, about 38 mol % cholesterol, and about 3 mol % PEG2k-DMG.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and a reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159; The nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, the nucleic acid construct does not comprise homology arms, the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 198, optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 198, or optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 196, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 196.In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO:159 or SEQ ID NO:165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, and optionally wherein the nucleic acid construct comprises a polyadenylation signal at each end. It is flanked on the ends by inverted terminal repeats (ITRs), and optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 196. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0118] In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprising SEQ ID NO: 65 or 97, the composition comprises a nucleic acid encoding a Cas protein, the nucleic acid comprising an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprising the sequence set forth in SEQ ID NO: 225, and the guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol % lipid A, about 9 mol % DSPC, about 38 mol % cholesterol, and about 3 mol % PEG2k-DMG.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and a reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159; The nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, the nucleic acid construct does not comprise homology arms, the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 198, optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 198, or optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 196, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 196.In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO:159 or SEQ ID NO:166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, and optionally wherein the nucleic acid construct comprises a polyadenylation signal at each end. It is flanked on the ends by inverted terminal repeats (ITRs), and optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 196. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0119] In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprising SEQ ID NO: 65 or 97, the composition comprises a nucleic acid encoding a Cas protein, the nucleic acid comprising an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprising the sequence set forth in SEQ ID NO: 225, and the guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol % lipid A, about 9 mol % DSPC, about 38 mol % cholesterol, and about 3 mol % PEG2k-DMG.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and a reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159; The nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, the nucleic acid construct does not comprise homology arms, the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 198, optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 198, or optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 196, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 196.In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO:159 or SEQ ID NO:165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, and optionally wherein the nucleic acid construct comprises a polyadenylation signal at each end. It is flanked on the ends by inverted terminal repeats (ITRs), and optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 196. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0120] In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprising SEQ ID NO: 73 or 105, the composition comprises a nucleic acid encoding a Cas protein, the nucleic acid comprising an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprising the sequence set forth in SEQ ID NO: 225, and the guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and a reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159; The nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, the nucleic acid construct does not comprise homology arms, the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 198, optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 198, or optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 196, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 196.In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO:159 or SEQ ID NO:166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, and optionally wherein the nucleic acid construct comprises a polyadenylation signal at each end. It is flanked on the ends by inverted terminal repeats (ITRs), and optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 196. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0121] In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprising SEQ ID NO: 73 or 105, the composition comprises a nucleic acid encoding a Cas protein, the nucleic acid comprising an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprising the sequence set forth in SEQ ID NO: 225, and the guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and a reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159; The nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, the nucleic acid construct does not comprise homology arms, the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 198, optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 198, or optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 196, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 196.In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO:159 or SEQ ID NO:165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, and optionally wherein the nucleic acid construct comprises a polyadenylation signal at each end. It is flanked on the ends by inverted terminal repeats (ITRs), and optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 196. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0122] In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprising SEQ ID NO: 100, the guide RNA comprising (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA, and the composition comprises a nucleic acid encoding a Cas protein, the nucleic acid being , comprising an mRNA encoding a Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 225, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail, wherein the guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 100, the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA, and the composition is a Cas tag. The guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and a reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159; The nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, the nucleic acid construct does not comprise homology arms, the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 198, optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 198, or optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 196, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 196.In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO:159 or SEQ ID NO:166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, and optionally wherein the nucleic acid construct comprises a polyadenylation signal at each end. It is flanked on the ends by inverted terminal repeats (ITRs), and optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 196. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0123] In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprising SEQ ID NO: 100, the guide RNA comprising (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA, and the composition comprises a nucleic acid encoding a Cas protein, the nucleic acid being , comprising an mRNA encoding a Cas protein, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 225, wherein the mRNA encoding the Cas protein is fully substituted with pseudouridine or N1-methyl-pseudouridine, optionally N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail, wherein the guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 100, the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA, and the composition is a Cas tag. The guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and a reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:165, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:165 and the second Factor IX protein coding sequence comprises SEQ ID NO:159; The nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, the nucleic acid construct does not comprise homology arms, the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 198, optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 198, or optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 196, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 196.In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO:159 or SEQ ID NO:165, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, and optionally wherein the nucleic acid construct comprises a polyadenylation signal at each end. It is flanked on the ends by inverted terminal repeats (ITRs), and optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 196. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 165. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0124] In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprising SEQ ID NO: 94, the guide RNA comprising (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA, and the composition comprises a nucleic acid encoding a Cas protein, the nucleic acid comprising The guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 94, the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA, and the composition is a Cas tag. The guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In some such compositions, the nucleic acid construct is a bidirectional construct comprising a first Factor IX protein coding sequence and a reverse complement of a second Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of the second splice acceptor, wherein (i) the first Factor IX protein coding sequence comprises SEQ ID NO:159 and the second Factor IX protein coding sequence comprises SEQ ID NO:166, or (ii) the first Factor IX protein coding sequence comprises SEQ ID NO:166 and the second Factor IX protein coding sequence comprises SEQ ID NO:159; The nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, the nucleic acid construct does not comprise homology arms, the nucleic acid construct is in a single-stranded rAAV8 vector, and optionally the nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 198, optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 198, or optionally the ITRs at at least one end comprise, consist essentially of, or consist of SEQ ID NO: 196, and optionally the ITRs at each end comprise, consist essentially of, or consist of SEQ ID NO: 196.In some such compositions, the nucleic acid construct is a unidirectional construct comprising a first Factor IX protein coding sequence, wherein the nucleic acid construct comprises, from 5' to 3', a splice acceptor, the first Factor IX protein coding sequence, and a polyadenylation signal, wherein the first Factor IX protein coding sequence comprises SEQ ID NO:159 or SEQ ID NO:166, wherein the nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein, wherein the nucleic acid construct does not comprise homology arms, wherein the nucleic acid construct is in a single stranded rAAV8 vector, and optionally wherein the nucleic acid construct comprises a polyadenylation signal at each end. It is flanked on the ends by inverted terminal repeats (ITRs), and optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 198, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 198, or optionally the ITR at at least one end comprises, consists essentially of, or consists of SEQ ID NO: 196, and optionally the ITR at each end comprises, consists essentially of, or consists of SEQ ID NO: 196. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 166. Optionally, the first Factor IX protein coding sequence comprises SEQ ID NO: 159.

[0125] In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprising SEQ ID NO: 94, the guide RNA comprising (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA, and the composition comprises a nucleic acid encoding a Cas protein, the nucleic acid comprising The guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In some such compositions, the albumin gene is a human albumin gene, the composition comprises a guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 94, the guide RNA comprises (i) a phosphorothioate linkage between the first four nucleotides at the 5' end of the guide RNA, (ii) a phosphorothioate linkage between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA, and the composition is a Cas tag. The guide RNA and the mRNA encoding the Cas protein are associated with lipid nanoparticles comprising lipid A, DSPC, cholesterol, and PEG2k-DMG, optionally in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.In...

Claims

Claim 1: A composition comprising a nucleic acid construct comprising a first factor IX protein coding sequence or a reverse complement of said first factor IX protein coding sequence, wherein said first factor IX protein coding sequence is: (I) at least 95% identical to any one of the sequences set forth in SEQ ID NOs: 166, 165, 164, and 167-171; or (II) at least 99% identical to the sequence set forth in any one of SEQ ID NOs: 159, 160, and 161; composition.

2. The nucleic acid construct comprises a bidirectional nucleic acid construct comprising the first factor IX protein coding sequence and the reverse complement of the second factor IX protein coding sequence, or a bidirectional nucleic acid construct comprising the second factor IX protein coding sequence and the reverse complement of the first factor IX protein coding sequence; the first Factor IX protein coding sequence encodes a Factor IX protein comprising the sequence set forth in SEQ ID NO:195, and the second Factor IX protein coding sequence encodes a Factor IX protein comprising the sequence set forth in SEQ ID NO:195; (I) the first Factor IX protein coding sequence is at least 99% identical to the sequence set forth in SEQ ID NO: 159 and the second Factor IX protein coding sequence is at least 99% identical to the sequence set forth in SEQ ID NO: 166; or (II) the first Factor IX protein coding sequence is at least 99% identical to the sequence set forth in SEQ ID NO: 166, and the second Factor IX protein coding sequence is at least 99% identical to the sequence set forth in SEQ ID NO:

159. The composition of claim 1.

3. The composition described in claim 2, wherein the bidirectional nucleic acid construct does not contain a homology arm.

4. The composition described in claim 2, wherein the bidirectional nucleic acid construct does not include a promoter that drives expression of the factor IX protein.

5. The composition described in claim 2, wherein the first factor IX protein coding sequence has all but one CpG dinucleotides removed or is completely CpG-depleted, and the second factor IX protein coding sequence has all but one CpG dinucleotides removed or is completely CpG-depleted. (I) the first Factor IX protein coding sequence comprises the sequence set forth in SEQ ID NO:159 and the second Factor IX protein coding sequence comprises the sequence set forth in SEQ ID NO:166; or (II) The composition of claim 2, wherein the first Factor IX protein coding sequence comprises the sequence set forth in SEQ ID NO: 166 and the second Factor IX protein coding sequence comprises the sequence set forth in SEQ ID NO:

159. (I) the first Factor IX protein coding sequence consists of the sequence set forth in SEQ ID NO:159 and the second Factor IX protein coding sequence consists of the sequence set forth in SEQ ID NO:166; or (II) The composition of claim 2, wherein the first Factor IX protein coding sequence consists of the sequence set forth in SEQ ID NO: 166 and the second Factor IX protein coding sequence consists of the sequence set forth in SEQ ID NO:

159.

8. The bidirectional nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of a second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of a second splice acceptor; or The composition of claim 2, wherein the bidirectional nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the second Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of the second polyadenylation signal, the reverse complement of the first Factor IX protein coding sequence, and the reverse complement of the second splice acceptor.

9. The composition described in claim 8, wherein the first factor IX protein coding sequence and the second factor IX protein coding sequence are different but encode the same factor IX protein sequence, and the first polyadenylation signal and the second polyadenylation signal are different.

10. The bidirectional nucleic acid construct comprises, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of a second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of a second splice acceptor; the first Factor IX protein coding sequence comprises the sequence set forth in SEQ ID NO: 159 and the second Factor IX protein coding sequence comprises the sequence set forth in SEQ ID NO: 166, or the first Factor IX protein coding sequence comprises the sequence set forth in SEQ ID NO: 166 and the second Factor IX protein coding sequence comprises the sequence set forth in SEQ ID NO: 159; the bidirectional nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein; the bidirectional nucleic acid construct does not contain homology arms; The composition of claim 2.

11. The composition described in claim 2, wherein the bidirectional nucleic acid construct comprises the sequence set forth in SEQ ID NO: 210 or 180, or their reverse complements.

12. The composition described in claim 2, wherein the bidirectional nucleic acid construct is within a nucleic acid vector, and the nucleic acid vector is a viral vector.

13. The composition described in claim 12, wherein the nucleic acid vector is an adeno-associated virus (AAV) vector.

14. The composition of claim 13, wherein the bidirectional nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), and wherein the ITR at at least one end comprises the sequence set forth in SEQ ID NO:198, or the ITR at at least one end comprises the sequence set forth in SEQ ID NO:

196.

15. The composition described in claim 13, wherein the AAV vector is a single-stranded rAAV8 vector.

16. The bidirectional nucleic acid construct comprising, from 5' to 3', a first splice acceptor, the first Factor IX protein coding sequence, a first polyadenylation signal, the reverse complement of a second polyadenylation signal, the reverse complement of the second Factor IX protein coding sequence, and the reverse complement of a second splice acceptor; the first Factor IX protein coding sequence comprises the sequence set forth in SEQ ID NO: 159 and the second Factor IX protein coding sequence comprises the sequence set forth in SEQ ID NO: 166, or the first Factor IX protein coding sequence comprises the sequence set forth in SEQ ID NO: 166 and the second Factor IX protein coding sequence comprises the sequence set forth in SEQ ID NO: 159; the bidirectional nucleic acid construct does not comprise a promoter driving expression of the Factor IX protein; the bidirectional nucleic acid construct does not comprise a homology arm; The bidirectional nucleic acid construct is in a single-stranded rAAV8 vector. The composition of claim 2.

17. The composition of claim 16, wherein the bidirectional nucleic acid construct is flanked at each end by inverted terminal repeats (ITRs), and the ITRs at each end comprise the sequence set forth in SEQ ID NO:198, or the ITRs at each end comprise the sequence set forth in SEQ ID NO:

196.

18. A composition described in any one of claims 1 to 17, further comprising a nuclease agent that targets a nuclease target site in a target gene, wherein the target gene is an albumin gene, and the albumin gene is a human albumin gene.

19. The nuclease agent, (a) a Cas9 protein or a nucleic acid encoding the Cas9 protein; (b) a guide RNA or one or more DNAs encoding the guide RNA; wherein the guide RNA comprises a DNA targeting segment that targets a guide RNA target sequence in intron 1 of the human albumin gene, and the guide RNA binds to and targets the Cas9 protein to the guide RNA target sequence.

19. The composition of claim 18. (I) the DNA targeting segment comprises the sequence set forth in SEQ ID NO: 36, or the guide RNA comprises the sequence set forth in SEQ ID NO: 68 or 100, and the guide RNA comprises at least one modification; and (II) the nucleic acid encoding the Cas9 protein comprises an mRNA encoding the Cas9 protein; 20. The composition of claim 19.

21. The at least one modification in (I) comprises (i) phosphorothioate linkages between the first four nucleotides at the 5' end of the guide RNA, (ii) phosphorothioate linkages between the last four nucleotides at the 3' end of the guide RNA, (iii) 2'-O-methyl modified nucleotides in the first three nucleotides at the 5' end of the guide RNA, and (iv) 2'-O-methyl modified nucleotides in the last three nucleotides at the 3' end of the guide RNA; 21. The composition of claim 20, wherein the mRNA encoding the Cas9 protein in (II) comprises the sequence set forth in SEQ ID NO: 226, 225, or 12, and wherein the mRNA encoding the Cas9 protein is fully substituted with N1-methyl-pseudouridine, comprises a 5' cap, and comprises a poly(A) tail.

22. The composition described in claim 19, wherein the Cas9 protein or the nucleic acid encoding the Cas9 protein, and the guide RNA or the one or more DNAs encoding the guide RNA are associated with lipid nanoparticles.

23. The composition of claim 22, wherein the lipid nanoparticles comprise a cationic lipid, a neutral lipid, a helper lipid, and a stealth lipid.

24. The composition described in claim 23, wherein the cationic lipid is lipid A, the neutral lipid is DSPC, the helper lipid is cholesterol, and the stealth lipid is PEG2k-DMG.

25. The composition of claim 24, wherein the lipid nanoparticles comprise four lipids in the following molar ratios: about 50 mol% lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.

26. A cell comprising a composition described in any one of claims 1 to 17.

27. (I) the nucleic acid construct is integrated into intron 1 of the endogenous albumin locus, and Factor IX protein is expressed from the endogenous albumin locus; and / or (II) the cell is a human cell, and / or (III) the cells are liver cells, and the liver cells are hepatocytes; and / or (IV) the cells are neonatal cells; The cell of claim 26.

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