Compositions and methods for targeting pcsk9

Description

"Compositions and Methods for Targeting PCSK9" CROSS-REFERENCE TO RELATED REQUESTS

[001] This request claims priority and the benefit of the Requests U.S. Provisional Applications Nos. 63 / 492,978, filed March 29, 2023, and 63 / 505,888, filed June 2, 2023, the contents of which are incorporated herein by reference in their entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[002] The contents of the electronic sequence listing (SCRB_058_02WO_SeqList_ST26.xml; Size: 141,865,746 bytes; and Creation Date: March 26, 2024) are incorporated herein by reference in their entirety. BACKGROUND

[003] In mammals, cholesterol is transported within lipoproteins through emulsification. Lipoprotein particles are classified based on their density: low-density lipoproteins (LDL), very low-density lipoproteins (VLDL), high-density lipoproteins (HDL), and chylomicrons. Surface LDL receptors are internalized during cholesterol absorption. A cell with abundant cholesterol will have its LDL receptor synthesis blocked to prevent new cholesterol in LDL particles from being absorbed. Conversely, LDL receptor synthesis is promoted when a cell is cholesterol-deficient. When the process is not regulated, excess LDL particles will travel in the blood without being absorbed by an LDL receptor. LDL particles in the blood are oxidized and taken up by macrophages, which then become fatty and form foam cells.These foam cells can become trapped in the walls of blood vessels and contribute to the formation of atherosclerotic plaques, which is one of the main causes. Petition 870250104944, dated 11 / 17 / 2025, page 6 / 689 2 / 579 of heart attacks, strokes, and other serious medical problems.

[004] Liver protein proprotein convertase subtilisin / kexin type 9 (PCSK9) is a secreted, globular, autoactivating serine protease that binds to the low-density lipoprotein receptor (LDL-R) during LDL particle endocytosis, preventing the recycling of LDL-R to the cell surface and leading to reduced LDL cholesterol clearance. PCSK9 binds to LDL-R (via the EGF-A domain), preventing the conformational change of the receptor-ligand complex, which redirects LDL-R to the lysosome instead. Because the receptor for low-density lipoprotein (LDL) particles normally transports thousands of fat molecules (including cholesterol) per particle within the extracellular fluid, blocking or inhibiting PCSK9 function to increase LDL-R-mediated clearance of LDL cholesterol may reduce LDL particle concentrations.PCSK9 is primarily expressed in the liver, intestine, kidney, and central nervous system, but it is also highly expressed in arterial walls, such as endothelium, smooth muscle cells, and macrophages, with a local effect that may regulate vascular homeostasis and atherosclerosis.

[005] PCSK9 is a member of the proprotein convertase (PC) family, and its gene is mutated in ~2% to 3% of individuals with familial hipsterolemia (FH) (Sepideh Mikaeeli, S., et al. Functional analysis of natural PCSK9 mutants in modern and archaic humans. FEBS J. August 6, 2019. doi: 10.1111 / febs.15036). Researchers have identified several PCSK9 mutations that cause an inherited form of high cholesterol (hypercholesterolemia). These mutations alter a single amino acid in the PCSK9 protein. Researchers describe the mutations responsible for hipsterolemia as gain-of-function mutations because they appear to increase the protein's activity. Petition 870250104944, dated 11 / 17 / 2025, page 7 / 689 3 / 579 PCSK9, or giving the protein a new and atypical function (Blesa, S., et al. A New PCSK9 Gene Promoter Variant Affects Gene Expression and Causes Autosomal Dominant Hypercholesterolemia. J. Clin. Endocrinol. & Metab. 93:3577(2008)). The hyperactive PCSK9 protein substantially reduces the number of low-density lipoprotein receptors on the surface of liver cells. With fewer receptors to remove low-density lipoproteins from the blood, people with gain-of-function mutations in the PCSK9 gene have very high blood cholesterol levels. Autosomal dominant hypercholesterolemia (ADH) is a genetic disorder characterized by increased levels of low-density lipoprotein (LDL) cholesterol, leading to a high risk of premature cardiovascular disease. Approximately 10 mutations in PCSK9 have been identified as a cause of the disease in different populations.All known mutations in PCSK9 that cause hyposterolemia produce an increase in the enzymatic activity of this protease (Bleasa, S., 2008). Furthermore, mutations in PCSK9 can lead to autosomal dominant familial hypobetalipoproteinemia, which can result in hepatic steatosis, cirrhosis, and other disorders.

[006] The emergence of CRISPR / Cas systems and the programmable nature of these minimal systems have facilitated their use as a versatile technology for genomic manipulation and engineering. However, current methods for generating protective PCSK9 variants and loss-of-function mutants in vivo have been ineffective due to the large number of cells that need to be modified to modulate cholesterol levels. Other concerns involve off-target effects, genome instability, or oncogenic modifications that may be caused by genome editing, as well as a lack of safe delivery modalities for protein repression systems. Additionally, in certain disease indications, gene silencing or repression is preferable to gene editing. Petition 870250104944, dated 11 / 17 / 2025, page 8 / 689 4 / 579 ca. The ability to process catalytically inactive CRISPR nucleases, such as Cas9 and CasX, has been demonstrated (WO2020247882A1 and US20200087641A1, incorporated by reference herein), making these systems an attractive platform for generating fusion proteins with repressor domains capable of silencing genes. Although certain repressor systems have already been described, there is still a need for additional repressor systems that have been optimized and / or offer improvements over previous generation gene repressor systems, such as those based on Cas9, for use in a variety of therapeutic, diagnostic, and research applications. Thus, the need for improved compositions and methods to regulate PCSK9 remains. SUMMARY

[007] The present invention provides systems comprising or encoding fusion proteins comprising DNA-linked and DNA-binding repressor domains used in the repression and / or epigenetic modification of target nucleic acid sequences of the proprotein convertase subtilisin / kexin type 9 (PCSK9) gene. In some respects, the fusion protein comprises a DNA-binding protein comprising a catalytically killed CRISPR protein, such as a Class 2, Type V CRISPR protein, and a guide nucleic acid comprising a targeting sequence complementary to the target nucleic acid sequence of the PCSK9 gene. The guide proteins and nucleic acids can be modified for passive entry into target cells and are useful in a variety of methods for PCSK9 repression, which methods are also provided.The present invention also provides vectors and lipid nanoparticles (LNPs) that encode or encapsulate fusion proteins and nucleic acid guide components for the delivery of the systems to cells. Petition 870250104944, dated 11 / 17 / 2025, page 9 / 689 5 / 579 transcriptional repression of the target nucleic acid sequence of PCSK9. The present invention also provides methods for modifying a target nucleic acid sequence of the PCSK9 gene. The present invention also provides methods for treating individuals with a PCSK9-related disease. In some embodiments, the compositions and methods are useful in individuals with a metabolic disorder, such as, but not limited to, familial hypercholesterolemia, familial hypobetalipoproteinemia, or elevated cholesterol levels.

[008] In other respects, this document provides systems comprising PCSK9 repressor systems, or vectors comprising or encoding PCSK9 repressor systems for use in the manufacture of a medicament for the treatment of a PCSK9-related disease in an individual in need.

[009] Other features and advantages of certain embodiments of the present invention will become more fully apparent in the following description of the embodiments and figures thereof and of the claims. Incorporation by Reference

[0010] All publications, patents and patent applications mentioned in this descriptive report are incorporated herein by reference to the same extent as if each individual publication, patent or patent application were specifically and individually indicated as incorporated by reference. The contents of WO 2020 / 247882, WO 2020 / 247883, WO 2021 / 113772, WO2022 / 120095, WO 2022 / 125843, WO 2022 / 261150, WO 2022 / 261149, WO 2023 / 049872, WO 2023 / 049742 and WO 2023 / 240162, which disclose CasX variants and gRNA variants, and methods for distributing them, and WO2021 / 142342 which discloses compositions and methods for modifying PCSK9 are incorporated herein by reference in their entirety. Petition 870250104944, dated 11 / 17 / 2025, page 10 / 689 6 / 579 BRIEF DESCRIPTION OF THE FIGURES

[0011] Understanding the features and advantages of the present invention will be facilitated by reference to the following figures:

[0012] FIG. 1 illustrates schematic diagrams of various configurations of repressor fusion proteins with the DNMT3A ADD domain incorporated. D3A ADD, D3A CD, and D3L ID denote the DNMT3A ADD domain, the DNMT3A catalytic domain, and the DNMT3L interaction domain, respectively. L1-L3 are ligands. NLS is the nuclear localization signal.

[0013] FIG. 2 is a bar graph showing the quantification of PCSK9 levels secreted at 6, 18, and 36 days post-transfection in Huh7 cells lipo-infected with mRNA encoding CasX 676, dXR1, or LTRP5-ADD-ZIM3 when paired with the indicated targeting gRNAs, as described in Example 1. PCSK9 levels secreted were normalized to total cell count. Untreated and unexposed cells served as an experimental control.

[0014] FIG. 3 is a bar graph showing the quantification of normalized secreted PCSK9 levels 4 days after transfection in HepG2 cells lipo-infected with mRNA encoding CasX 676, dXR1, or LTRP5-ADD-ZIM3 when paired with the indicated targeting gRNAs, as described in Example 2. Secreted PCSK9 levels were normalized to total cell count. Untreated and unexposed cells served as experimental controls.

[0015] FIG. 4 is a bar graph showing the quantification of normalized secreted PCSK9 levels 4 days after transfection in Huh7 cells lipo-infected with mRNA encoding CasX 676, dXR1, or LTRP5-ADD-ZIM3 when paired with the indicated targeting gRNAs, as described in Example 2. The levels Petition 870250104944, dated 11 / 17 / 2025, page 11 / 689 7 / 579 of secreted PCSK9 cells were normalized to the total cell count. Untreated and unexposed cells served as experimental controls.

[0016] FIG. 5 is a bar graph showing the quantification of normalized secreted PCSK9 levels 4 days after transfection in Hep3B cells lipo-infected with mRNA encoding CasX 676, dXR1, or LTRP5-ADD-ZIM3 when paired with the indicated targeting gRNAs, as described in Example 2. Secreted PCSK9 levels were normalized to total cell count. Untreated and unexposed cells served as experimental controls.

[0017] FIG. 6 is a bar graph showing the quantification of PCSK9 levels secreted at 4, 14, and 27 days post-transfection in Huh7 cells lipo-infected with mRNA encoding CasX 676, dXR1, or LTRP5-ADD-ZIM3 when paired with the indicated targeting gRNAs, as described in Example 2. The quantification of secreted PCSK9 levels is shown relative to the levels secreted and detected in untreated and unexposed cells at the time point on day 4.

[0018] FIG. 7 is a schematic of the gRNA framework variant. 174 (SEQ ID NO: 1744), as described in Example 3. Structural reasons are highlighted.

[0019] FIG. 8 is a schematic of the gRNA framework variant. 235 (SEQ ID NO: 1745), as described in Example 3. The highlighted structural motifs are the same as in FIG. 7. The differences between variant 174 and variant 235 are in the extended stem motif and several single nucleotide changes (indicated with asterisks). Variant 316 retains the shorter extended stem of variant 174, but incorporates the four substitutions found in the 235 framework.

[0020] FIG. 9 is a schematic of the gRNA framework variant. Petition 870250104944, dated 11 / 17 / 2025, p. 12 / 689 8 / 579 316 (SEQ ID NO: 1746), as described in Example 3. The highlighted structural motifs are the same as in FIG. 7. Variant 316 retains the shorter extended rod of variant 174 (FIG. 7), but incorporates the four substitutions found in framework 235 (FIG. 8).

[0021] FIG. 10 is a diagram illustrating versions 1-3 of chemical modifications made to the gRNA 235 scaffold variant, as described in Example 3. Structural motifs are highlighted. Standard ribonucleotides are represented as open circles and 2'OMe-modified ribonucleotides are represented as black circles. Phosphorothioate linkages are indicated with * below or beside the linkage. For the v2 profile, the addition of three 3' uracils (3'UUU) is denoted with “U”s in the relevant circles.

[0022] FIG. 11 is a diagram illustrating versions 4-6 of chemical modifications made to the gRNA 235 scaffold variant, as described in Example 3. Structural motifs are highlighted. Standard ribonucleotides are represented as open circles and 2'OMe-modified ribonucleotides are represented as black circles. Phosphorothioate linkages are indicated with * below or beside the linkage.

[0023] FIG. 12 is a diagram illustrating versions 7-9 of chemical modifications made to the gRNA 316 scaffold variant, as described in Example 3. Structural motifs are highlighted. Standard ribonucleotides are represented as open circles and 2'OMe-modified ribonucleotides are represented as black circles. Phosphorothioate linkages are indicated with * below or beside the linkage.

[0024] FIG. 13 is a diagram illustrating versions 1-3 of chemical modifications made to the gRNA 316 scaffold variant, as described in Example 3. Structural motifs are highlighted. Standard ribonucleotides are represented as open circles. Petition 870250104944, dated 11 / 17 / 2025, page 13 / 689 9 / 579 and ribonucleotides modified by 2'OMe are represented as black circles. Phosphorothioate linkages are indicated with an asterisk (*) below or next to the linkage.

[0025] FIG. 14 is a diagram illustrating versions 4-6 of chemical modifications made to the gRNA 316 scaffold variant, as described in Example 3. Structural motifs are highlighted. Standard ribonucleotides are represented as open circles and 2'OMe-modified ribonucleotides are represented as black circles. Phosphorothioate linkages are indicated with * below or beside the linkage.

[0026] FIG. 15 is a graph displaying a correlation between the indel rate (represented as an edit fraction) at the PCSK9 locus, as measured by next-generation sequencing (NGS) (x-axis), and levels of secreted PCSK9 (ng / mL) detected by enzyme-linked immunosorbent assay (ELISA) (y-axis) in HepG2 cells lipo-infected with CasX 491 mRNA and PCSK9-targeted gRNAs containing the indicated scaffold variant and spacer combination, as described in Example 3.

[0027] FIG. 16 is a graph illustrating the quantification of the percent knockout of B2M in HepG2 cells cotransfected with 100 ng of CasX 491 mRNA and with the indicated doses of end-modified (v1) or unmodified (v0) B2M-targeted gRNAs with a 7.37 spacer, as described in Example 3. The editing level was determined by flow cytometry as the cell population with loss of HLA complex surface presentation due to successful editing at the B2M locus.

[0028] FIG. 17 is a graph representing the results of an editing assay measured as the indel rate detected by NGS at the human B2M locus in HepG2 cells treated with the indicated doses of LNPs formulated with CasX 491 mRNA and the gRNA targeted to Petition 870250104944, dated 11 / 17 / 2025, page 14 / 689 10 / 579 B2M indicated, as described in Example 3.

[0029] FIG. 18 is a graph illustrating the quantification of the percentage knockout of B2M in HepG2 cells treated with the indicated doses of LNPs formulated with CasX 491 mRNA and the indicated B2M-targeted gRNA, as described in Example 3. The editing level was determined by flow cytometry as a population of cells that did not have surface presentation of the HLA complex due to successful editing at the B2M locus.

[0030] FIG. 19 is a graph representing the results of an editing assay measured as indel rate detected by NGS at the mouse ROSA26 locus in Hepa1-6 cells treated with the indicated doses of LNPs formulated with CasX 676 mRNA 2 and the ROSA26-targeted gRNA indicated with the v1 or v5 modification profile, as described in Example 3.

[0031] FIG. 20 is a graph illustrating the quantification of percent edit measured as indel rate detected by NGS at the ROSA26 locus in mice treated with LNPs formulated with CasX 676 mRNA 2 and the chemically modified ROSA26-targeted gRNA indicated, as described in Example 3.

[0032] FIG. 21 is a bar graph showing the results of an editing assay measured as the indel rate detected by NGS at the mouse PCSK9 locus in mice treated with LNPs formulated with CasX 676 mRNA 1 and the indicated chemically modified PCSK9-targeted gRNA, as described in Example 3. Untreated mice served as experimental controls.

[0033] FIG. 22 is a diagram of the secondary structure of guide RNA scaffold 235 (SEQ ID NO: 1745), showing the regions with CpG motifs, as described in Example 6. CpG motifs in (1) the pseudo-node stem, (2) the scaffold stem, (3) the bubble of Petition 870250104944, dated 11 / 17 / 2025, p. 15 / 689 11 / 579 extended stem, (4) extended step and (5) handle on extended stem are marked on the structure.

[0034] FIG. 23 is a diagram of the CpG-reducing mutations that were introduced into each of the five regions in the coding sequence of the guide RNA scaffold, as described in Example 6.

[0035] FIG. 24 provides the results of an editing experiment in which AAV vectors with various reduced or CpG-depleted guide RNA scaffolds were used to edit the B2M locus in induced neurons, as described in Example 6. The AAV vectors were administered at a multiplicity of infection (MOI) of 4e3. The bars show the mean ± SD of two replicates per sample. “No Tx” indicates a non-transduced control and “NT” indicates a control with a non-targeted spacer.

[0036] FIG. 25 provides the results of an editing experiment in which AAV vectors with various reduced or CpG-depleted guide RNA scaffolds were used to edit the B2M locus in induced neurons, as described in Example 6. The AAV vectors were administered at an MOI of 3e3. The bars show the mean ± SD of two replicates per sample. “No Tx” indicates a non-transduced control.

[0037] FIG. 26 provides the results of an editing experiment in which AAV vectors with various reduced or CpG-depleted guide RNA scaffolds were used to edit the B2M locus in induced neurons, as described in Example 6. The AAV vectors were administered at an MOI of 1e3. The bars show the mean ± SD of two replicates per sample. “No Tx” indicates a non-transduced control.

[0038] FIG. 27 provides the results of an editing experiment in which AAV vectors with various reduced or CpG-depleted guide RNA scaffolds were used to edit the B2M locus. Petition 870250104944, dated 11 / 17 / 2025, p. 16 / 689 12 / 579 in induced neurons, as described in Example 6. AAV vectors were delivered at an MOI of MOI = 3e2. Bars show the mean ± SD of two replicates per sample. “No Tx” indicates a non-transduced control.

[0039] FIG. 28 illustrates schematic diagrams of five configurations of long-term repressor protein (LTRP, also referred to in this document as repressor fusion proteins) with repressor molecules bound to catalytically killed CasX. D3A and D3L denote DNA methyltransferase 3 alpha (DNMT3A) and DNMT3A-like protein (DNMT3L), respectively. L1-L4 are ligands. NLS is the nuclear localization signal.

[0040] FIG. 29 illustrates the schematics of the various LTRP architectures. 5, where additional DNMT3A domains were incorporated, as described in Example 7. The additional DNMT3A domains were the DNMT3A ADD domain (D3A ADD) and the DNMT3A PWWP (Pro-Trp-Trp-Pro motif) domain (D3A PWWP). D3A endo encodes an endogenous sequence that occurs between the PWWP and ADD domains of DNMT3A. D3A CD and D3L ID denote the catalytic domain of DNMT3A and the interaction domain of DNMT3L, respectively. L1-L3 are ligands. NLS is the nuclear localization signal. See Table 13 for LTRP sequences.

[0041] FIG. 30 presents the results of a time-course experiment comparing the B2M repression activities (represented as a percentage of HLA-negative cells) of the indicated LTRP-ZIM3 and its variants with B2M-targeting gRNA using spacer 7.37, as described in Example 7. Data are presented as mean with standard deviation, N=3. CD = catalytic domain of DNMT3A.

[0042] FIG. 31 presents the results of the same time course experiment shown in FIG. 30, but shows re-activities. Petition 870250104944, dated 11 / 17 / 2025, page 17 / 689 13 / 579 B2M pressure of the indicated LTRP-ZIM3 variants with B2M targeting gRNA using a 7.160 spacer, as described in Example 7. Data are presented as mean with standard deviation, N=3.

[0043] FIG. 32 presents the results of the same time-course experiment shown in FIG. 30, but shows B2M repression activities of the indicated LTRP-ZIM3 variants with B2M-targeting gRNA using spacer 7.165, as described in Example 7. Data are presented as mean with standard deviation, N=3.

[0044] FIG. 33 presents the results of the same time-course experiment shown in FIG. 30, but shows B2M repression activities of the LTRP-ZIM3 variants indicated with a non-targeting gRNA, as described in Example 7. Data are presented as mean with standard deviation, N=3.

[0045] FIG. 34 is a violin plot of percent CpG methylation for CpG sites downstream of the transcription start site of the VEGFA locus for each LTRP-ZIM3 variant indicated for the three B2M targeting gRNAs and non-targeting gRNAs, as described in Example 7.

[0046] FIG. 35 is a scatter plot showing the relative activity (mean percentage of HLA-negative cells on day 21 for spacer 7.160) versus specificity (percentage of off-target CpG methylation at the VEGFA locus quantified on day 7 for spacer 7.160) for the indicated LTRP5-ZIM3 variants, as described in Example 7.

[0047] FIG. 36 illustrates the general architectural schemes of LTRP molecules with the ADD domain for the LTRP 1, 4, and 5 configurations tested in Example 8. ADD of D3A, CD of D3A, and ID of D3L denote the ADD domain of DNMT3A, the catalytic domain of Petition 870250104944, dated 11 / 17 / 2025, page 18 / 689 14 / 579 DNMT3A and the interaction domain of DNMT3L, respectively, as described in Example 8. L1-L4 are ligands. NLS is the nuclear localization signal. See Table 11 for LTRP sequences.

[0048] FIG. 37 presents the results of a time-course experiment comparing the B2M repression activities (represented as a percentage of HLA-negative cells) of LTRPs with the ZIM3-KRAB domain in configuration 1, 4, or 5 with or without the DNMT3A ADD domain when paired with the B2M-targeting gRNA with spacer 7160, as described in Example 8. Data are presented as mean with standard deviation, N=3. NT is a gRNA with a non-targeting spacer.

[0049] FIG. 38 is a graph showing the results of the same time-course experiment shown in FIG. 37, but illustrates the repression activities of B2M for LTRP 5 with the ZNF10 or ZIM3-KRAB domain, with or without the DNMT3A ADD domain, paired with the B2M targeting gRNA with spacer 7.160, as described in Example 8. Data are presented as mean with standard deviation, N=3. NT is a gRNA with a non-targeting spacer.

[0050] FIG. 39 is a graph showing the results of the same time-course experiment shown in FIG. 37, but illustrates the repression activities of B2M for LTRP5-ZIM3 with or without the ADD domain of DNMT3A paired with a B2M-targeting gRNA with the indicated spacers, as described in Example 8. Data are presented as mean with standard deviation, N=3. NT is a gRNA with a non-targeting spacer.

[0051] FIG. 40 is a graph illustrating the results of B2M repression activities on day 27 after transfection for LTRPs with the ZNF10 or ZIM3-KRAB domain in configuration 1 with or without the DNMT3A ADD domain for the indicated gRNAs, as Petition 870250104944, dated 11 / 17 / 2025, page 19 / 689 15 / 579 described in Example 8. The data are presented as mean with standard deviation, N=3. NT is a gRNA with a non-directional spacer.

[0052] FIG. 41 is a graph illustrating the results of B2M repression activities on day 27 after transfection for LTRPs with the ZNF10 or ZIM3-KRAB domain in configuration 4 with or without the DNMT3A ADD domain for the indicated gRNAs, as described in Example 8. Data are presented as mean with standard deviation, N=3. NT is a gRNA with a non-targeting spacer.

[0053] FIG. 42 is a graph illustrating the results of B2M repression activities on day 27 after transfection for LTRPs with the ZNF10 or ZIM3-KRAB domain in configuration 5 with or without the DNMT3A ADD domain for the indicated gRNAs, as described in Example 8. Data are presented as mean with standard deviation, N=3. NT is a gRNA with a non-targeting spacer.

[0054] FIG. 43 is a graph illustrating the results of bisulfite sequencing used to determine off-target methylation at the VEGFA locus on day 5 post-transfection for LTRPs with the ZNF10 or ZIM3-KRAB domain in configuration 1 with or without the DNMT3A ADD domain for the indicated gRNAs, as described in Example 8. Data are presented as the mean percentage of CpG methylation for CpG sites near the VEGFA locus; the standard error of the mean is also presented; N=3. NT is a gRNA with a non-targeting spacer.

[0055] FIG. 44 is a graph illustrating the results of bisulfite sequencing used to determine off-target methylation at the VEGFA locus on day 5 after transfection for LTRPs with the ZNF10 or ZIM3-KRAB domain in configuration 4 with or without the Petition 870250104944, dated 11 / 17 / 2025, page 20 / 689 16 / 579 DNMT3A ADD domain for the indicated gRNAs, as described in Example 8. Data are presented as the mean percentage of CpG methylation for CpG sites near the VEGFA locus; the standard error of the mean is also presented; N=3. NT is a gRNA with a non-targeting spacer.

[0056] FIG. 45 is a graph illustrating the results of bisulfite sequencing used to determine off-target methylation at the VEGFA locus on day 5 post-transfection for LTRPs with the ZNF10 or ZIM3-KRAB domain in configuration 5 with or without the DNMT3A ADD domain for the indicated gRNAs, as described in Example 8. Data are presented as the mean percentage of CpG methylation for CpG sites near the VEGFA locus; the standard error of the mean is also presented; N=3. NT is a gRNA with a non-targeting spacer.

[0057] FIG. 46 is a scatter plot showing the relative activity (mean percentage of HLA-negative cells on day 27) versus specificity (percentage of off-target CpG methylation at the VEGFA locus quantified on day 5) for LTRP molecules with the ZIM3-KRAB domain with configurations 1, 4, and 5, for B2M-targeting gRNA with spacer 7160, as described in Example 8.

[0058] FIG. 47 is a scatter plot showing the relative activity (mean percentage of HLA-negative cells on day 27) versus specificity (percentage of off-target CpG methylation at the VEGFA locus quantified on day 5) for LTRP molecules with the ZNF10-KRAB domain with configurations 1, 4, and 5, for B2M-targeting gRNA with spacer 7160, as described in Example 8.

[0059] FIG. 48 is a scatter plot showing relative activity (average percentage of HLA-negative cells on day 27) Petition 870250104944, dated 11 / 17 / 2025, page 21 / 689 17 / 579 versus specificity (percentage of off-target CpG methylation at the VEGFA locus quantified on day 5) for LTRP molecules with the ZIM3-KRAB domain with configurations 1, 4, and 5, for B2M targeting gRNA with spacer 7.37, as described in Example 8.

[0060] FIG. 49 is a scatter plot showing the relative activity (mean percentage of HLA-negative cells on day 27) versus specificity (percentage of off-target CpG methylation at the VEGFA locus quantified on day 5) for LTRP molecules with the ZNF10-KRAB domain with configurations 1, 4, and 5, for B2M-targeting gRNA with spacer 7.37, as described in Example 8.

[0061] FIG. 50 is a scatter plot showing the relative activity (mean percentage of HLA-negative cells on day 27) versus specificity (percentage of off-target CpG methylation at the VEGFA locus quantified on day 5) for LTRP molecules with the ZIM3-KRAB domain with configurations 1, 4, and 5, targeting B2M gRNA with spacer 7165, as described in Example 8.

[0062] FIG. 51 is a scatter plot showing the relative activity (mean percentage of HLA-negative cells on day 27) versus specificity (percentage of off-target CpG methylation at the VEGFA locus quantified on day 5) for LTRP molecules with the ZNF10-KRAB domain with configurations 1, 4, and 5, for B2M-targeting gRNA with spacer 7165, as described in Example 8.

[0063] FIG. 52 is a graph that illustrates the percentage of cells HEK293T cells, transfected with a plasmid encoding the indicated CasX or LTRP:gRNA construct, expressed B2M six days after treatment with the DNMT1 inhibitor 5-azadC at concentrations Petition 870250104944, dated 11 / 17 / 2025, page 22 / 689 18 / 579 variables, as described in Example 9.

[0064] FIG. 53 is a graph juxtaposing the quantification of B2M repression in HEK293T cells transfected with a plasmid encoding the indicated CasX or LTRP:gRNA construct and cultured for 58 days, with the quantification of B2M reactivation by treatment of transfected cells with 5-azadC, as described in Example 9.

[0065] FIG. 54 illustrates the scheme of an LTRP5 molecule without the ADD domain of DNMT3A, as described in Example 11. CD of D3A and ID of D3L denote the catalytic domain of DNMT3A and the interaction domain of DNMT3L, respectively. “L1”, “L2”, “L3A”, and “L3B” are ligands. NLS is the nuclear localization signal. “RD1” denotes a repressor domain.

[0066] FIG. 55 is a bar graph illustrating the results of a time-course experiment comparing the levels of B2M repression (represented as the mean percentage of HLA-negative cells) in HEK293T cells transfected with the LTRP5 variant plasmids containing ligand sets 1-11, as described in Example 13. The data for each time point (day 8, day 15, and day 45) are superimposed and are presented as mean with standard deviation, N=3. A non-directed (NT) spacer was used as an experimental control.

[0067] FIG. 56 is a bar graph illustrating the results of a time-course experiment comparing the level of target 1 knockdown (represented as a percentage of total cells with target 1 knockdown) of LTRP5 variants containing ligand sets 1-11 measured on HEK293T, as described in Example 13. The data for each time point (day 8, day 15, and day 45) are superimposed and are presented as mean with standard deviation, N=3. Petition 870250104944, dated 11 / 17 / 2025, page 23 / 689 19 / 579

[0068] FIG. 57 is a bar graph illustrating the results of a time-course experiment comparing the level of target 2 knockdown (represented as a percentage of total target 2 knockdown cells) of LTRP5 variants containing ligand sets 1-11 measured on HEK293T, as described in Example 13. The data for each time point (day 8, day 15, and day 45) are superimposed and are presented as mean with standard deviation, N=3. A non-directed (NT) spacer was used as an experimental control.

[0069] FIG. 58 is a bar graph illustrating the results of a time-course experiment comparing the levels of B2M repression (represented as the mean percentage of HLA-negative cells) in HEK293T cells transfected with the LTRP5 variant plasmids containing ligand sets 12-28, as described in Example 13. The data for each time point (day 7 and day 17) are superimposed and are presented as mean with standard deviation, N=3. A non-directed (NT) spacer was used as an experimental control. A non-directed (NT) spacer was used as an experimental control.

[0070] FIG. 59 is a bar graph illustrating the results of a time-course experiment comparing the level of target 1 knockdown (represented as a percentage of total target 1 knockdown cells) of LTRP5 variants containing ligand sets 12-28 measured on HEK293T, as described in Example 13. The data for each time point (day 7 and day 17) are superimposed and are presented as mean with standard deviation, N=3. A non-directed (NT) spacer was used as an experimental control.

[0071] FIG. 60 is a bar graph illustrating the results of a time-course experiment comparing the level of re Petition 870250104944, dated 11 / 17 / 2025, page 24 / 689 20 / 579 target 2 pressure (represented as a percentage of total cells with target 2 knockdown) of LTRP5 variants containing ligand sets 12-28 measured on HEK293T, as described in Example 13. Data for each time point (day 7 and day 17) are superimposed and are presented as mean with standard deviation, N=3. A non-directed (NT) spacer was used as an experimental control.

[0072] FIG. 61 illustrates schemes of various configurations of LTRP molecules with the incorporation of the DNMT3A ADD. D3A ADD, D3A CD, and D3L ID denote the DNMT3A ADD domain, the DNMT3A catalytic domain, and the DNMT3L interaction domain, respectively. “L1”, “L2”, “L3A”, “L3B”, and “L4” are ligands. NLS is the nuclear localization signal. RD1 denotes a repressor domain, and RD1a and RD1b represent repressor domain variants.

[0073] FIG. 62 is a violin plot with each point representing the mean % methylation in individual CpGs. Median methylation is indicated by dashed lines, with the upper and lower quartiles indicated by dotted lines. Methylation of the transcription start site (TSS) proximal DNA was measured from homogenized gDNA extracted from liver by enzymatic methylation sequencing (EMseq) of amplicons from N=3 mice sacrificed on days 7, 14, and 42 after treatment, as described in Example 14.

[0074] FIG. 63 is a violin plot with each point representing the mean % methylation in individual CpGs. Median methylation is indicated by dashed lines, with the upper and lower quartiles indicated by dotted lines. Methylation of the transcription start site (TSS) proximal DNA was measured from homogenized gDNA extracted from liver by enzymatic methylation sequencing (EM-seq) of amplicons from N=3 sacrificed mice in Petition 870250104944, dated 11 / 17 / 2025, page 25 / 689 21 / 579 day 7 after treatment, as described in example 14. DETAILED DESCRIPTION

[0075] Although exemplary embodiments have been shown and described in this document, it will be apparent to those skilled in the art that such embodiments are provided only by way of example. Numerous variations, alterations, and substitutions will now occur to those skilled in the art without departing from the spirit of the description. Thus, it is understood that various alternatives to the embodiments specifically described in this document may be employed in the practice of the embodiments of the description.

[0076] Unless otherwise defined, all technical and scientific terms used in this document have the same meaning commonly understood by those skilled in the art to which this description pertains. Although methods and materials similar or equivalent to those described in this document may be used in the practice or testing of the present embodiments, suitable methods and materials are described below. In case of conflict, the descriptive report of the patent, including definitions, shall prevail. Furthermore, the materials, methods, and examples disclosed in this document are for illustrative purposes only and are not intended to be limiting. Numerous variations, changes, and substitutions may now occur to those skilled in the art without departing from the spirit of the description and the claims. Definitions

[0077] As used in this descriptive report and the accompanying claims, the singular forms a, an, and the include plural references unless the content clearly indicates otherwise. Thus, for example, a reference to a host cell includes two or more such host cells, a reference to a dCasX protein includes one or more dCasX proteins, a reference to a nucleic acid sequence includes one or more nucleic acid sequences Petition 870250104944, dated 11 / 17 / 2025, p. 26 / 689 22 / 579 nucleic acid and the like.

[0078] As used in this document, the term about is understood by persons skilled in the art and may vary to some extent depending on the context in which it is used. If there are uses of the term that are not clear to those skilled in the art, given the context in which the term about is used, about will mean up to plus or minus 10% of the specific term.

[0079] As will be understood by one skilled in the art, for all and any purposes, all intervals disclosed in this document also include any and all possible subintervals and combinations of subintervals thereof. Furthermore, as will be understood by one skilled in the art, an interval includes each individual member. Thus, for example, a group with 1-3 members refers to groups with 1, 2, or 3 members. Similarly, a group with 1-5 members refers to groups with 1, 2, 3, 4, or 5 members, and so forth.

[0080] The term combinations of them includes all possible combinations of elements to which the term refers.

[0081] The term exemplar, as used in this document, refers to an example or illustration and is not intended to imply any preference or value.

[0082] The term CasX protein, as used in this document, refers to a family of proteins including, for example, all naturally occurring CasX proteins (reference CasX), as well as sequence-modified CasX proteins, such as dCasX, possessing one or more enhanced characteristics compared to a CasX protein from which it was derived, described in more detail below in this document.

[0083] The terms polynucleotide and nucleic acid, used interchangeably in this document, refer to a poly form Petition 870250104944, dated 11 / 17 / 2025, p. 27 / 689 23 / 579 meric nucleotides of any length, ribonucleotides or deoxyribonucleotides. Thus, the terms polynucleotide and nucleic acid encompass single-stranded DNA; double-stranded DNA; multi-stranded DNA; single-stranded RNA; double-stranded RNA; multi-stranded RNA; genomic DNA; cDNA; DNA-RNA hybrids; and a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases.

[0084] Hybridizable or complementary are used interchangeably to mean that a nucleic acid (e.g., RNA, DNA) comprises a nucleotide sequence that enables it to bind non-covalently, i.e., form Watson-Crick base pairs and / or G / U base pairs, annularly, or hybridize, to another nucleic acid in an antiparallel and sequence-specific manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under appropriate in vitro and / or in vivo conditions of temperature and ionic strength of the solution. It is understood that the sequence of a polynucleotide need not be 100% complementary to its target nucleic acid to be specifically hybridizable; it may have at least about 70%, at least about 80%, or at least about 90%, or at least about 95% sequence identity and still hybridize with the target nucleic acid.Furthermore, a polynucleotide can hybridize in one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop or hairpin structure, a protrusion, bubble, and the like). Thus, one skilled in the art will understand that although the individual bases within a sequence may not be complementary to another sequence, the sequence as a whole is still considered complementary. Petition 870250104944, dated 11 / 17 / 2025, page 28 / 689 24 / 579

[0085] A gene, for the purposes of the present invention, includes a region of DNA that encodes a gene product (e.g., a protein or RNA), as well as all regions of DNA that regulate the production of the gene product, whether such regulatory element sequences are adjacent to coding and / or transcribed sequences. Therefore, a gene may include accessory element sequences including, but not necessarily limited to, promoter sequences, terminators, translation regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix binding sites, and locus control regions. Coding sequences encode a gene product by transcription or transcription and translation; the coding sequences of the description may comprise fragments and need not contain a complete open reading frame.A gene can include both the strand that is transcribed and the complementary strand containing the anticodons.

[0086] The term downstream refers to a nucleotide sequence that is located 3' to a reference nucleotide sequence. In certain embodiments, downstream nucleotide sequences refer to sequences that follow the transcription start site. For example, the translation initiation codon of a gene is located downstream of the transcription start site.

[0087] The term upstream refers to a nucleotide sequence that is located 5' to a reference nucleotide sequence. In certain embodiments, upstream nucleotide sequences refer to sequences that are located on the 5' side of a coding region or transcription start site. For example, most promoters are located upstream of the transcription start site. Petition 870250104944, dated 11 / 17 / 2025, p. 29 / 689 25 / 579

[0088] The term adjacent to in relation to polynucleotide or amino acid sequences refers to sequences that are close to or adjacent to each other in a polynucleotide or polypeptide. One skilled in the art will appreciate that two sequences may be considered adjacent to each other and yet encompass a limited amount of intervening sequence, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides or amino acids.

[0089] The term regulatory element is used interchangeably in this document with the term regulatory sequence and is intended to include promoters, enhancers, and other expression regulatory elements. It will be understood that the choice of the appropriate regulatory element will depend on the encoded component to be expressed (e.g., protein or RNA) or whether the nucleic acid comprises multiple components that require different polymerases or is not intended to be expressed as a fusion protein.

[0090] The term accessory element is used interchangeably in this document with the term accessory sequence, and is intended to include coding and non-coding sequences that enhance the expression, nucleic acid trafficking, or function of an mRNA or protein and include, inter alia, polyadenylation signals (poly(A) signal), enhancer elements, introns, posttranscriptional regulatory elements (PTREs), nuclear localization signals (NLS), deaminases, DNA glycosylase inhibitors, additional promoters, factors that stimulate CRISPR-mediated homology-directed repair (e.g., cis or trans), autocleavage sequences, and fusion domains, for example, a fusion domain fused to a CRISPR protein. It will be understood that the choice of the appropriate accessory element or elements will depend on the coding component to be expressed (e.g., protein or RNA) or whether the nucleic acid comprises multiple components that require different Petition 870250104944, dated 11 / 17 / 2025, p. 30 / 689 26 / 579 polymerases are either not intended to be expressed as a fusion protein.

[0091] The term promoter refers to a DNA sequence that contains a transcription start site and additional sequences to facilitate polymerase binding and transcription. Exemplary eukaryotic promoters include elements such as a TATA box and / or B recognition element (BRE) and assist or promote the transcription and expression of an associated transcribable polynucleotide sequence and / or gene (or transgene). A promoter may be produced synthetically or may be derived from a known or naturally occurring promoter sequence or other promoter sequence. A promoter may be proximal or distal to the gene to be transcribed. A promoter may also include a chimeric promoter comprising a combination of two or more heterologous sequences to confer certain properties.A promoter of the present invention may include variants of promoter sequences that are similar in composition to, but not identical to, other known promoter sequences or those provided in this document. A promoter may be classified according to criteria related to the expression pattern of a coding or transcriptable sequence or gene operationally linked to the promoter, such as constitutive, developmental, tissue-specific, inducible, etc. A promoter may also be classified according to its strength. As used in the context of a promoter, strength refers to the transcription rate of the gene controlled by the promoter. A strong promoter means that the transcription rate is high, while a weak promoter means that the transcription rate is relatively low.

[0092] A promoter in the description may be a Polymerase II (Pol II) promoter. Polymerase II transcribes all coding proteins and many non-coding genes. A representative promoter Petition 870250104944, dated 11 / 17 / 2025, page 31 / 689 27 / 579 of Pol II includes a core promoter, which is a sequence of about 100 base pairs surrounding the transcription start site and serves as a binding platform for the Pol II polymerase and associated general transcription factors. The promoter may contain one or more core promoter elements, such as the TATA box, BRE, Initiator (INR), motif ten element (MTE), downstream core promoter element (DPE), downstream core element (DCE), although core promoters without these elements are known in the art. All Pol III promoters are envisioned as being within the scope of the present invention.

[0093] A promoter of the description may be a Polymerase III (Pol III) promoter. Pol III transcribes DNA to synthesize small ribosomal RNAs, such as 5S rRNA, tRNAs, and other small RNAs. Representative Pol III promoters use internal control sequences (sequences within the transcribed section of the gene) to support transcription, although upstream elements such as the TATA box are also sometimes used. All Pol III promoters are envisioned as being within the scope of the present invention.

[0094] The term enhancer refers to regulatory DNA sequences that, when bound to specific proteins called transcription factors, regulate the expression of an associated gene. Enhancers may be located in the gene's intron, or 5' or 3' of the gene's coding sequence. Enhancers may be proximal to the gene (i.e., within a few tens or hundreds of base pairs (bp) of the promoter), or they may be located distal to the gene (i.e., thousands of bp, hundreds of thousands of bp, or even millions of bp away from the promoter). A single gene may be regulated by more than one enhancer, all of which are envisioned as being within the scope of the present invention.

[0095] As used in this document, a regulatory element Petition 870250104944, dated 11 / 17 / 2025, p. 32 / 689 28 / 579 post-transcriptional activity (PTRE), such as a hepatitis PTRE, refers to a DNA sequence that, when transcribed, creates a tertiary structure capable of exhibiting post-transcriptional activity to enhance or promote the expression of an associated gene operationally linked to it.

[0096] Operationally linked means with reference to a juxtaposition of two or more components (such as sequence elements), in which the components are arranged so that both components function normally and allow the possibility that at least one of the components may mediate a function that is exerted on at least one of the other components; for example, a promoter and an encoding sequence. One skilled in the art will appreciate that the two components need not be physically linked to be operationally linked.

[0097] In the context of the present invention and in relation to a gene, repress, repression, “suppressing”, inhibition of gene expression, negative regulation and silencing are used interchangeably in this document to refer to the inhibition or blocking of the transcription of a gene or a portion thereof. Consequently, the repression of a gene can result in a decrease in the production of a gene product. Examples of gene repression processes that decrease transcription include, among others, those that inhibit the formation of a transcription initiation complex, those that decrease the rate of transcription initiation, those that decrease the rate of transcription elongation, those that decrease the processivity of transcription and those that antagonize transcriptional activation (e.g., by blocking the binding of a transcriptional activator). Gene repression can constitute, for example, prevention of activation as well as inhibition of expression below an existing level.Transcriptional repression includes both reversible inactivation. Petition 870250104944, dated 11 / 17 / 2025, page 33 / 689 29 / 579 as to irreversible gene transcription; the latter may result from epigenetic modification of the gene.

[0098] “Repressor” or repressor domain are used interchangeably to refer to polypeptide factors that act as regulatory elements in DNA that inhibit, repress, or block DNA transcription, resulting in the repression of gene expression. In the context of the present invention, the binding of a repressor domain to a DNA-binding protein can, when bound to the target nucleic acid, prevent the transcription of a promoter or otherwise inhibit the expression of a gene.Without wishing to be bound by the theory, it is believed that transcriptional repressors can function through a variety of mechanisms, including physically blocking the passage of RNA polymerase by steric hindrance, altering the post-translational modification state of the polymerase, modifying the epigenetic state of nascent RNA, changing the epigenetic state of DNA through methylation, changing the epigenetic state of DNA through histone deacetylation or modulation of nucleosome remodeling, or preventing enhancer-promoter interactions, thereby leading to gene silencing or a reduction in the level of gene expression.

[0099] Long-term repressor fusion protein or LTRP is used interchangeably in this document with repressor fusion protein and refers to a fusion protein comprising a DNA-binding protein (or DNA-binding domain of a protein) fused to one or more domains capable of repressing the transcription of a target nucleic acid sequence. Optionally, the long-term repressor fusion proteins of the description may contain additional elements, such as linkers between any of the fusion protein domains, nuclear localization signals, nuclear export signals, as well as additional protein domains that Petition 870250104944, dated 11 / 17 / 2025, page 34 / 689 30 / 579 confer additional activities on the long-term repressor fusion protein.

[00100] As used in this document, an LTRP:gRNA system is a system for transcriptional repression and comprises a long-term repressor fusion protein comprising a catalytically killed CRISPR protein and one or more bound repressor domains and a guide nucleic acid (gRNA) that binds to the catalytically killed CRISPR protein. For clarity, the system also includes any coding DNA, RNA, or vectors and the like that may be used to produce the long-term repressor fusion proteins and gRNA components of the system.

[00101] As used in this document, a “catalytically dead DNA-binding protein” refers to a protein, or domain of a protein, capable of binding to DNA but unable to cut or cleave DNA. As used in this document, a catalytically dead CRISPR protein refers to a CRISPR protein lacking endonuclease activity. Those skilled in the art will appreciate that a CRISPR protein can be catalytically dead and still capable of performing additional protein functions, such as DNA binding. Similarly, a catalytically dead CasX refers to a CasX protein lacking endonuclease activity but still capable of performing additional protein functions, such as DNA binding.

[00102] Recombinant, as used in this document, means that a specific nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, and / or ligation steps, resulting in a construct with a coding or non-coding structural sequence distinguishable from endogenous nucleic acids found in natural systems. Generally, DNA sequences that Petition 870250104944, dated 11 / 17 / 2025, page 35 / 689 31 / 579 encoding the structural coding sequence can be assembled from cDNA fragments and short oligonucleotide linkers, or from a series of synthetic oligonucleotides, to provide a synthetic nucleic acid that is capable of being expressed from a recombinant transcriptional unit contained within a cell or in a cell-free transcription and translation system. Such sequences can be provided in the form of an uninterrupted open reading frame by internal untranslated sequences, or introns, which are typically present in eukaryotic genes. Genomic DNA comprising the relevant sequences can also be used in the formation of a recombinant gene or transcriptional unit.Untranslated DNA sequences may be present 5' or 3' from the open reading frame, where such sequences do not interfere with the manipulation or expression of coding regions and may in fact act to modulate the production of a desired product by various mechanisms (see enhancers and promoters above).

[00103] The term recombinant polynucleotide or recombinant nucleic acid refers to one that does not occur naturally; for example, it is produced by the artificial combination of two separate sequence segments through human intervention. This artificial combination is often accomplished by any means of chemical synthesis, or by the artificial manipulation of isolated nucleic acid segments; for example, by genetic manipulation techniques. This is usually done to replace a codon with a redundant codon that codes for the same amino acid or a conservative amino acid, while typically introducing or removing a sequence recognition site. Alternatively, it is performed to join nucleic acid segments of desired functions to generate a desired combination of functions. This artificial combination is often accomplished by any means of chemical synthesis, or by Petition 870250104944, dated 11 / 17 / 2025, p. 36 / 689 32 / 579 artificial manipulation of isolated segments of nucleic acids; for example, by genetic manipulation techniques.

[00104] Similarly, the term recombinant polypeptide or recombinant protein refers to a polypeptide or protein that does not occur naturally; for example, it is made by the artificial combination of two separate amino acid sequence segments through human intervention. Thus, for example, a protein comprising a heterologous amino acid sequence is recombinant.

[00105] As used in this document, lipid nanoparticle or “LNP” refers to a particle with at least one dimension on the order of nanometers (e.g., 1–1,000 nm) comprising one or more lipids (e.g., cationic lipids, non-cationic lipids, auxiliary phospholipids, and PEG-modified lipids), as well as cholesterol. The specific components of LNP are described in more detail below. Lipid nanoparticles may be included in a formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA), to a target site of interest (e.g., cell, tissue, organ, tumor, and the like). The lipid nanoparticles of the description may comprise a nucleic acid. Such lipid nanoparticles typically comprise neutral lipids, charged lipids, steroids, and polymer-conjugated lipids.The active agent or therapeutic agent, such as a nucleic acid, can be encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space surrounded by part or all of the lipid portion of the lipid nanoparticle, thus protecting it from enzymatic degradation or other undesirable effects induced by the organism's or host cells' mechanisms, for example, an adverse immune response.

[00106] As used in this document, encapsulated in lipids refers to a lipid nanoparticle that provides an agent Petition 870250104944, dated 11 / 17 / 2025, page 37 / 689 33 / 579 active or therapeutic agent, such as a nucleic acid (e.g., mRNA, gRNA, or both mRNA and gRNA), with complete encapsulation, partial encapsulation, or both. The nucleic acid (e.g., mRNA) may be fully encapsulated within the lipid nanoparticle.

[00107] As used in this document, lipoprotein, such as VLDL, LDL, and HDL, refers to a group of proteins found in serum, plasma, and lymph that function in the transport of lipids. The chemical composition of each lipoprotein differs, for example, in that HDL has a higher protein-to-lipid ratio, while VLDL has a lower protein-to-lipid ratio.

[00108] As used in this document, atherosclerosis means a hardening of the arteries affecting large and medium-sized arteries and is characterized by the presence of fatty deposits. The fatty deposits are called atheromas or plaques, which consist mainly of cholesterol and other fats, calcium, and scar tissue and damage the lining of the arteries.

[00109] As used in this document, coronary heart disease (CHD) means a narrowing of the small blood vessels that supply blood and oxygen to the heart, which is often a result of atherosclerosis.

[00110] As used in this document, dyslipidemia refers to a disorder of lipid and / or lipoprotein metabolism, including overproduction or deficiency of lipids and / or lipoproteins. Dyslipidemias can be manifested by elevated lipids, such as chylomicrons, cholesterol, and triglycerides, as well as lipoproteins, such as low-density lipoprotein (LDL) cholesterol.

[00111] As used in this document, high-density lipoprotein-C or HDL-C means cholesterol associated with high-density lipoprotein particles. The concentration of HDL-C in serum (or Petition 870250104944, dated 11 / 17 / 2025, page 38 / 689 34 / 579 plasma) is typically quantified in mg / dL or nmol / L. “Serum HDL-C” and “plasma HDL-C” mean HDL-C in serum and plasma, respectively.

[00112] As used in this document, low-density lipoprotein cholesterol (LDL-C) means cholesterol transported in low-density lipoprotein particles. The concentration of LDL-C in serum (or plasma) is typically quantified in mg / dL or nmol / L. Serum LDL-C and plasma LDL-C mean LDL-C in serum and plasma, respectively.

[00113] As used in this document, hypercholesterolemia means a condition characterized by elevated cholesterol or circulating (plasma) cholesterol, LDL cholesterol, and VLDL cholesterol, in accordance with the guidelines of the National Cholesterol Education Program (NCEP) Expert Panel Report on Detection, Evaluation and Treatment of Elevated Cholesterol in Adults (see, Arch. Int. Med. 148: 36 (1988)).

[00114] As used in this document, hyperlipidemia or hyperlipemia is a condition characterized by elevated serum lipids or circulating (plasma) lipids. This condition manifests as an abnormally high concentration of fats. The lipid fractions in circulating blood are cholesterol, low-density lipoproteins, very low-density lipoproteins, chylomicrons, and triglycerides. The Fredrickson classification of dyslipidemias is based on the pattern of TG and cholesterol-rich lipoprotein particles, as measured by electrophoresis or ultracentrifugation, and is commonly used to characterize primary causes of dyslipidemias, such as hypertriglyceridemia.

[00115] As used in this document, triglyceride or TG means a lipid or neutral fat consisting of glycerol combined with three fatty acid molecules. Petition 870250104944, dated 11 / 17 / 2025, page 39 / 689 35 / 579

[00116] As used in this document, hypertriglyceridemia means a condition characterized by elevated triglyceride levels. Its etiology includes primary factors (i.e., genetic causes) and secondary factors (other underlying causes such as diabetes, metabolic syndrome / insulin resistance, obesity, physical inactivity, cigarette smoking, excessive alcohol consumption, and a diet very high in carbohydrates) or, most often, a combination of both.

[00117] As used in this document, diabetes mellitus or diabetes is a syndrome characterized by disordered metabolism and abnormally high blood sugar (hyperglycemia) resulting from insufficient insulin levels or reduced insulin sensitivity.The characteristic symptoms are excessive urine production (polyuria) due to high blood glucose levels, excessive thirst and increased fluid intake (polydipsia) in an attempt to compensate for the increased urine output, blurred vision due to the high effects of blood glucose on the optics of the eye, unexplained weight loss, and lethargy.

[00118] As used in this document, diabetic dyslipidemia or type 2 diabetes with dyslipidemia means a condition characterized by type 2 diabetes, reduced HDL-C, elevated triglycerides (TG), and elevated small, dense LDL particles.

[00119] As used in this document, the term contact means establishing a physical connection between two or more entities. For example, the contact of a target nucleic acid sequence with a guide nucleic acid means that the target nucleic acid sequence and the guide nucleic acid are made to share a physical connection; for example, they can hybridize if the sequences share sequence similarity.

[00120] Dissociation constant, or Kd, are used interchangeably and signify the affinity between a ligand L and a protein P; that is, how tightly a ligand binds to a protein. Petition 870250104944, dated 11 / 17 / 2025, p. 40 / 689 36 / 579 in particular. It can be calculated using the formula Kd=[L] [P] / [LP], where [P], [L] and [LP] represent molar concentrations of the protein, ligand and complex, respectively.

[00121] The term knock-down, as used in this document, refers to a reduction in the expression of a gene or its gene products. As a result of a gene knock-down, the activity or function of the protein may be attenuated, or protein levels may be reduced or eliminated.

[00122] A polynucleotide or polypeptide has a certain percentage of sequence similarity or sequence identity with another polynucleotide or polypeptide, meaning that when aligned, this percentage of bases or amino acids is the same and in the same relative position when comparing the two sequences. Sequence similarity (sometimes referred to as percent similarity, percent identity, or homology) can be determined in several different ways. To determine sequence similarity, sequences can be aligned using methods and computer programs known in the art, including BLAST, available worldwide at ncbi.nlm.nih.gov / BLAST. The percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined using any convenient method.Exemplary methods include BLAST programs (basic local alignment search tools) and PowerBLAST programs (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649656) or using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison, Wis.); for example, using default settings, which uses the Smith and Waterman algorithm (Adv. Appl. Math., 1981, 2, 482-489). Petition 870250104944, dated 11 / 17 / 2025, page 41 / 689 37 / 579

[00123] The terms polypeptide and protein are used interchangeably in this document and refer to a polymeric form of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified or derived amino acids, and polypeptides with modified peptide backbone structures. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence.

[00124] A vector or expression vector is a replicon, such as a plasmid, phage, virus, or cosmid, to which another segment of DNA is attached, i.e., an expression cassette, in order to cause the replication or expression of another segment of DNA in a cell.

[00125] The term naturally occurring or unmodified or wild type, as used in this document, as applied to a nucleic acid, a polypeptide, a cell or an organism, refers to a nucleic acid, polypeptide, cell or organism that is found in nature.

[00126] As used in this document, a mutation refers to an insertion, deletion, substitution, duplication, or inversion of one or more amino acids or nucleotides compared to a wild-type or reference amino acid sequence or to a wild-type or reference nucleotide sequence.

[00127] As used in this document, the term isolate is intended to describe a polynucleotide, polypeptide, or cell that is in an environment different from that in which the polynucleotide, polypeptide, or cell naturally occurs. An isolated genetically modified host cell may be present in a mixed population of genetically modified host cells.

[00128] A host cell, as used in this document, denotes a eukaryotic cell, a prokaryotic cell, or a cell Petition 870250104944, dated 11 / 17 / 2025, page 42 / 689 38 / 579 la of a multicellular organism (e.g., a cell line) grown as a single-cell entity, wherein eukaryotic or prokaryotic cells are used as recipients of a nucleic acid (e.g., an AAV vector) and include the progeny of the original cell that has been genetically modified by the nucleic acid. It is understood that the progeny of a single cell may not necessarily be completely identical in morphology or in the complement of genomic or total DNA as the original precursor, due to natural, accidental, or deliberate mutation. A recombinant host cell (also referred to as a genetically modified host cell) is a host cell into which a heterologous nucleic acid, e.g., an AAV vector, has been introduced.

[00129] The term conservative amino acid substitution refers to the interchangeability in proteins of amino acid residues with similar side chains. For example, a group of amino acids with aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids with aliphatic hydroxyl side chains consists of serine and threonine; a group of amino acids with amide-containing side chains consists of asparagine and glutamine; a group of amino acids with aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; a group of amino acids with basic side chains consists of lysine, arginine, and histidine; and a group of amino acids with sulfur-containing side chains consists of cysteine ​​and methionine. Exemplary conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

[00130] As used in this document, treatment or treat are used interchangeably in this document and refer to an approach to achieving beneficial or desired results, including Petition 870250104944, dated 11 / 17 / 2025, page 43 / 689 39 / 579 including, but not limited to, a therapeutic benefit and / or a prophylactic benefit. By therapeutic benefit is meant eradication or improvement of the underlying disorder or disease being treated. A therapeutic benefit may also be obtained by eradicating or improving one or more of the symptoms or an improvement in one or more clinical parameters associated with the underlying disease, such that an improvement is observed in the individual, notwithstanding that the individual may still be afflicted by the underlying disorder.

[00131] The terms therapeutically effective amount and therapeutically effective dose, as used in this document, refer to an amount of a drug or biological agent, alone or as part of a composition, that is capable of having any detectable and beneficial effect on any symptom, aspect, measured parameter or characteristic of a disease state or condition when administered in one or repeated doses to an individual, such as a human or an experimental animal. Such an effect need not be absolute to be beneficial.

[00132] As used in this document, administer means a method for giving a dosage of a compound (e.g., a composition from the description) or a composition (e.g., a pharmaceutical composition) to an individual.

[00133] An individual is a mammal. Mammals include, among others, domesticated animals, non-human primates, humans, dogs, rabbits, mice, rats, and other rodents.

[00134] The term low-density lipoprotein (LDL) refers to one of five major lipoprotein groups, from least dense (lowest weight-to-volume ratio particles) to most dense (highest weight-to-volume ratio particles): chylomicrons, very low-density lipoproteins (VLDL), low-density lipoproteins (LDL), intermediate-density lipoproteins (IDL), and high-density lipoproteins. Petition 870250104944, dated 11 / 17 / 2025, page 44 / 689 40 / 579 density (HDL). Proteins transfer lipids (fats) around the body in the extracellular fluid, thus facilitating the transfer of fats into the cell body via receptor-mediated endocytosis. An LDL particle is approximately 220-275 angstroms in diameter.

[00135] Low-density lipoprotein (LDL) receptor refers to an 839-amino acid receptor protein (after removal of the 21-amino acid signal peptide) that mediates the endocytosis of cholesterol-rich LDL particles. It is a cell surface receptor that recognizes the apoprotein B100 and apoE proteins found in chylomicron remnants and VLDL (IDL) remnants, resulting in the binding and endocytosis of LDL cholesterol. This process occurs in all nucleated cells, but primarily in the liver, which removes approximately 70% of LDL from circulation. The human LDLR gene is described in part in the NCBI database (ncbi.nlm.nih.gov) as Reference Sequence NG_009060.1, which is incorporated by reference in this document.

[00136] All publications, patents and patent applications mentioned in this descriptive report are incorporated herein by reference to the same extent as if each individual publication, patent or patent application were specifically and individually indicated as incorporated by reference. I. General Methods

[00137] Unless otherwise indicated, the practice of the present invention employs conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which can be found in standard textbooks such as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Pro Petition 870250104944, dated 11 / 17 / 2025, page 45 / 689 41 / 579 Thein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Handbook of Immunology Methods (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), whose publications are incorporated herein by reference.

[00138] When a range of values ​​is provided, it is understood that outcomes are included and that each intervening value, up to the tenth of a unit of the lower limit, unless the context clearly indicates otherwise, between the upper and lower limits of that range and any other stated or intervening value in that stated range, is encompassed. The upper and lower limits of those smaller ranges may be independently included in the smaller ranges and are also encompassed, subject to any limit specifically excluded in the stated range. When the stated range includes one or both limits, ranges excluding one or both included limits will also be included.

[00139] Unless otherwise defined, all technical and scientific terms used in this document have the same meaning commonly understood by those skilled in the art to which this description pertains. All publications mentioned in this document are incorporated herein by reference to disclose and describe the methods and / or materials in relation to which the publications are cited.

[00140] It will be appreciated that certain features of the description, which are, for clarity, described in the context of separate modalities, may also be provided in combination in a single modality. In other cases, several features of the description, which are, for brevity, described in the context of a single modality, may also be provided. Petition 870250104944, dated 11 / 17 / 2025, page 46 / 689 42 / 579 can be supplied separately or in any appropriate subcombination. It is intended that all combinations of the embodiments belonging to the description are specifically covered by the present invention and are disclosed in this document as if each combination were individually and explicitly disclosed. Furthermore, all subcombinations of the various embodiments and elements thereof are also specifically covered by the present invention and are disclosed in this document as if each and every subcombination were individually and explicitly disclosed in this document. II. Systems for Epigenetic Modification and Repression of PCSK9 Genes

[00141] In a first aspect, the present invention provides systems comprising or encoding a long-term repressor fusion protein (“LTRP”) comprising a DNA-binding protein and repressor domains capable of binding to a target nucleic acid sequence of a PCSK9 gene targeted for transcriptional repression, silencing, and / or epigenetic modification. The present invention also provides systems comprising or encoding the LTRP. In some cases, the system is designed to repress the transcription of the PCSK9 gene in eukaryotic cells with a mutation.

[00142] As used in this document, a system is used interchangeably with composition. The description also provides nucleic acids that encode the systems provided in this document. Also provided in this document are methods for producing the systems, as well as methods for using the systems, including methods of gene repression and / or epigenetic modification and methods for treating PCSK9-related diseases. For clarity, the term “system” also includes any coding DNA, Petition 870250104944, dated 11 / 17 / 2025, page 47 / 689 43 / 579 RNA or vectors and similar substances that can be used to produce the repressor fusion proteins and gRNA components of the system.

[00143] In some embodiments, the DNA-binding proteins for use in the long-term repressor fusion proteins of the description comprise zinc finger (ZF) or TALE (transcription activator-type effector) proteins, or DNA-binding domains thereof, also referred to in this document as a DNA-binding protein, which bind to, but do not cleave, the target nucleic acid. The DNA-binding domain of a TALE is composed of a tandem array of 33-34 customizable amino acid (aa) monomers that can theoretically be assembled to recognize any genetic sequence following a recognition code of a base-pair binding repeat (Jain, S., et al. TALE outperforms Cas9 in editing heterochromatin target sites. Nat. Commun. 12:606 (2021)). The specificity of TALEs for binding to DNA arises from two polymorphic amino acids, the so-called repeated variable diresidues (RVDs) located at positions 12 and 13 of a repeating unit.By rearranging the repeats, the DNA-binding specificities of TALE can be altered at will. Zinc finger proteins are transcription factors, where each finger recognizes 3-4 DNA bases. By mixing and matching these finger modules, ZFs can be customized to the sequence to be targeted. Exemplary ZFs that are capable of binding to the PCSK9 gene are described in WO2018049009A2.

[00144] In some embodiments, the DNA-binding protein for use in the long-term repressor fusion protein is a catalytically killed Class 1 or Class 2 CRISPR protein. Catalytically killed CRISPR proteins are also referred to in the art as catalytically inactive CRISPR proteins. In one embodiment, the Class 2 Type II protein is a catalytically killed Cas9. In Petition 870250104944, dated 11 / 17 / 2025, page 48 / 689 44 / 579 In another embodiment, the Class 2 CRISPR protein is selected from the group consisting of a Type II, Type V, or Type VI protein. In one embodiment, the Class 2 Type V protein is selected from the group consisting of Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12f, Cas12g, Cas12h, Cas12i, Cas12j, Cas12k, Cas14, and / or CasΦ, in each case catalytically killed by specific mutations, as described in this document. CRISPR-based systems further comprise a guide nucleic acid, for example, a guide ribonucleic acid (gRNA) with a targeting sequence complementary to the target sequence of a PCSK9 gene to be ligated and repressed by a fusion protein complex (the CRISPR protein and bound repressor domains) and the gRNA.

[00145] In some embodiments, the present invention provides systems comprising or encoding a long-term repressor fusion protein comprising a catalytically killed CasX CRISPR protein and bound repressor domains and a guide ribonucleic acid (gRNA) comprising a targeting sequence complementary to a target nucleic acid sequence of a PCSK9 gene targeted for transcriptional repression, silencing, or downregulation, as well as nucleic acids encoding the long-term repressor fusion protein and / or gRNA. In some embodiments, the system comprises a long-term repressor fusion protein and a gRNA as described as a gene repressor pair (“LTRP:gRNA system”) that are capable of forming a ribonucleoprotein (RNP) complex and binding a PCSK9 target nucleic acid. In other cases, the description provides nucleic acid systems encoding the long-term repressor fusion protein and the gRNA.In still other cases, the description provides systems consisting of a gRNA and an mRNA that encodes the fusion protein. Petition 870250104944, dated 11 / 17 / 2025, page 49 / 689 45 / 579 long-term repressor for use in certain particle formulations (e.g., an LNP) described in this document.

[00146] This document also provides methods for producing long-term repressor fusion proteins and gRNA, as well as methods for using LTRP:gRNA systems, including methods for gene repression and / or epigenetic modification of a PCSK9 gene and methods for treating a PCSK9-related disease or disorder. The DNA-binding proteins (e.g., dCasX) and bound repressor domains, and the gRNA components, of the LTRP:gRNA systems and their features, as well as the delivery modalities and methods of using the systems for the repression, downregulation, or silencing of a PCSK9 gene are described in more detail below.

[00147] In some embodiments, the description provides systems specifically designed to repress or silence the transcription of the PCSK9 gene. In some cases, the system is designed to repress the transcription of the PCSK9 gene in eukaryotic cells with a gain-of-function mutation. In some cases, the system is designed to repress the transcription of the wild-type PCSK9 gene in eukaryotic cells. Alternatively, the system is designed to repress the transcription of a mutant allele of the PCSK9 gene in eukaryotic cells. Generally, any portion of the PCSK9 gene can be targeted using the programmable systems and methods provided in this document, described in more detail in this document.

[00148] The PCSK9 gene encodes proprotein type 9 subtilisin / kexin convertase (“PCSK9”), a protein that binds to the receptor for low-density lipoprotein (LDL) particles for the transport of LDL into the cell. The PCSK9 gene encompasses the sequence spanning chr1:55,039,476-55,064,853 of the human genome (GRCh38 / hg38) (the notation refers to chromosome 1 (chr1)). Petition 870250104944, dated 11 / 17 / 2025, page 50 / 689 46 / 579 starting at 55,039,476 bp to 55,064,853 bp on chromosome 1 (Homo sapiens Updated Annotation Release 109.20190905, GRCh38.p13) (NCBI). The human PCSK9 gene is described in part in the NCBI database (ncbi.nlm.nih.gov) as Reference Sequence NG_009061.1, which is incorporated by reference in this document. The PCSK9 locus has 12 exons that produce a 3636 bp mRNA encoding a 692-amino acid protein that, after its synthesis, undergoes an autocatalytic cleavage reaction that cuts the prodomain, resulting in an activated protein with 540 amino acids. The prodomain remains bound to catalytic and resistin-like domains, probably because the prodomain serves as a chaperone and facilitates folding and secretion (Seidah, NG et al., Proc Natl Acad Sci USA 100(3):928 (2003)). The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation (Seidah NG, et al.).This protein, also called neural apoptosis-regulated convertase, is a serine protease belonging to the protease K subtilase subfamily.

[00149] The human PCSK9 gene (HGNC:20001) encodes a protein (Q8NBP7) with the sequence MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKDPWRLPGTYVVVLKEETHLSQSERTARRLQAQAA RRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQSIPWNLERITPPRYRADEYQPPDGGSLVEVYLLDTSIQSDHREIEGRVMVTDFE NVPEEDGTRFHRQASKCDSHGTHLAGVVSGRDAGVAKGASMRSLRVLNCQGKGTVSGTLIGLEFIRKSQLVQPVGPLVVLLPLAGGYSRVLNAACQRLARAGV VLVTAAGNFRDDACLYSPASAPEVITVGATNAQDQPVTLGTLGTNFGRCVDLFAPGEDIIGASSDCSTCFVSQSGTSQAAAHVAGIAAMMLSAEPEPetition 870250104944, dated 11 / 17 / 2025, page 51 / 689 47 / 579 LTLAELRQRLIHFSAKDVINEAWFPEDQRVLTPNLVAALPPSTHGAGWQLFCRTVWSAHSGPTRMATAVARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGT RVHCHQQGHVLTGCSSHWEVEDLGTHKPPVLRPRGQPNQCVGHREASIHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAYAVDNTCVVRSRDVSTTGSTSEGAVTAVAICCRSRHLAQASQELQ (SEQ ID NO: 1823). III. Catalytically dead proteins for use in repressive systems

[00150] In some embodiments, the DNA-binding proteins for use in the long-term repressor fusion proteins described are zinc finger (ZF) or TALE (transcription activator-type effector) proteins that can bind to, but not cleave, a PCSK9 target nucleic acid.

[00151] In some embodiments, the DNA-binding protein is a catalytically killed Class 1 or Class 2 CRISPR protein. In some embodiments, the DNA-binding protein is a Class 2 Type II CRISPR protein. In one embodiment, the Class 2 Type II protein is a catalytically killed Cas9. In another embodiment, the Class 2 CRISPR protein is selected from the group consisting of a Type II, Type V, or Type VI protein. In one embodiment, the Class 2 Type V CRISPR protein is selected from the group consisting of Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12f, Cas12g, Cas12h, Cas12i, Cas12j, Cas12k, Cas14, and / or CasΦ, in each case catalytically killed by specific mutations, as described in this document. In another embodiment, the Class 2 Type V CRISPR protein is a catalytically killed CasX protein. Petition 870250104944, dated 11 / 17 / 2025, p. 52 / 689 48 / 579

[00152] The CasX proteins described, including dCasX, comprise the following domains: a non-target strand-binding domain (NTSB), a target strand-loading domain (TSL), a helical domain I, a helical domain II, an oligonucleotide-binding domain (OBD), and a RuvC domain, and in some cases, domains may be further divided into subdomains, as listed in Table 1.

[00153] In the context of the present invention, CasX for use in systems is catalytically killed (dCasX); achieved by mutations introduced at selected locations in the RuvC sequence, described below. a. Reference CasX Proteins

[00154] The description provides naturally occurring CasX proteins (referred to in this document as a reference CasX protein) that have been subsequently modified to create the manipulated dCasX described. For example, reference CasX proteins can be isolated from naturally occurring prokaryotes such as Deltaproteobacteria, Planctomycetes, or Candidatus Sungbacteria species. A reference CasX protein (referred to interchangeably in this document as a reference CasX polypeptide) is a Class 2 Type V CRISPR / Cas endonuclease belonging to the CasX protein family (referred to interchangeably as Cas12e) that interacts with a guide RNA to form a ribonucleoprotein (RNP) complex.

[00155] In some cases, a reference CasX protein is isolated or derived from Deltaproteobacteria and comprises a sequence with SEQ ID NO: 1.

[00156] In some cases, a reference CasX protein is isolated or derived from Planctomycetes and comprises a sequence with SEQ ID NO: 2. Petition 870250104944, dated 11 / 17 / 2025, p. 53 / 689 49 / 579

[00157] In some cases, a reference CasX protein is isolated or derived from Candidatus Sungbacteria and comprises a sequence with SEQ ID NO: 3. b. Catalytically killed Class 1 or Class 2 CRISPR proteins

[00158] In long-term repressor fusion proteins and gene repressor systems comprising the same as described, the catalytically killed Class 1 or Class 2 CRISPR protein is catalytically killed in the sense that it is unable to cleave DNA, but retains the ability to bind to a target nucleic acid when complexed with a guide RNA (gRNA). The present invention provides catalytically killed variants of Class 1 or Class 2 CRISPR proteins, wherein the catalytically killed variants comprise multiple modifications in selected domains. The present invention provides catalytically killed CasX variants (referred to interchangeably in this document as dCasX variant or dCasX variant protein), wherein the catalytically killed CasX variants comprise multiple modifications in the RuvC domain relative to the catalytically killed versions of reference CasX proteins comprising sequences of SEQ ID NOS:1-3 (described above).In some embodiments, a catalytically dead reference CasX protein comprises substitutions at residues 672, 769, and / or 935 with reference to SEQ ID NO: 1. In some embodiments, a catalytically dead reference CasX protein comprises substitutions at D672A, E769A, and / or D935A with reference to SEQ ID NO: 1. In other embodiments, a catalytically dead reference CasX protein comprises substitutions at amino acids 659, 756, and / or 922 with reference to SEQ ID NO: 2. In some embodiments, a catalytically dead reference CasX protein comprises substitutions at D659A, E756A, and / or D922A. Petition 870250104944, dated 11 / 17 / 2025, p. 54 / 689 50 / 579 with reference to SEQ ID NO: 2. A RuvC domain exemplifying dCasX from the description comprises amino acids 661-824 and 935-986 of SEQ ID NO: 1, or amino acids 648-812 and 922-978 of SEQ ID NO: 2, with one or more amino acid modifications relative to said RuvC cleavage domain sequence, wherein the dCasX variant exhibits one or more enhanced features compared to the reference dCasX. In further embodiments, a catalytically killed CasX variant protein comprises deletions of all or part of the RuvC domain of the reference CasX protein. It will be understood that the same substitutions or deletions as before can be similarly introduced into known CasX variants in the art, resulting in a dCasX variant (see, for example, WO2022120095A1 and US11,560,555, incorporated by reference herein, for illustrative sequences).

[00159] In some embodiments, the long-term repressor fusion protein comprising a dCasX variant with linked repressor domains exhibits at least one enhanced feature compared to the long-term repressor fusion protein comprising the reference dCasX protein with comparable linked repressor domains. All dCasX variants that enhance one or more functions or features of the long-term repressor fusion protein comprising the dCasX variant protein with linked repressor domains compared to a long-term repressor fusion protein comprising the reference dCasX protein are predicted to be within the scope of the description. In some embodiments, the modification is a mutation in one or more amino acids of the reference dCasX other than those rendering the dCasX catalytically dead.For example, dCasX variants may comprise one or more amino acid substitutions, insertions, deletions, or swapped domains. Petition 870250104944, dated 11 / 17 / 2025, page 55 / 689 51 / 579 any combination thereof, relative to a reference dCasX protein sequence. Any amino acid may be substituted for any other amino acid in the substitutions described in this document. The substitution may be a conservative substitution (e.g., a basic amino acid is substituted for another basic amino acid). The substitution may be a non-conservative substitution (e.g., a basic amino acid is substituted for an acidic amino acid or vice versa). For example, a proline in a reference dCasX protein may be substituted for any of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, or valine to generate a variant dCasX protein of the description. In some embodiments, the variant dCasX exhibits an enhanced feature compared to a reference dCasX.Enhanced features exemplifying variant dCasX modalities include, among others, improved variant folding, increased binding affinity to the target nucleic acid, improved ability to utilize a wider spectrum of PAM sequences in the repression and / or transcriptional binding of the target nucleic acid, improved unwinding of the target DNA, increased target strand loading, increased non-target DNA binding, improved protein stability, increased ability to complex with gRNA, increased binding affinity to gRNA, improved stability of the protein:gRNA complex (RNP), and, with repressor domains bound and when complexed as an RNP, increased repressor activity, improved repressor specificity for the target nucleic acid, decreased off-target repression, increased percentage of a eukaryotic genome that can be efficiently repressed and / or epigenetically modified. In some modalities, a feature... Petition 870250104944, dated 11 / 17 / 2025, p. 56 / 689 The improved 52 / 579 variant of dCasX is at least about 1.1 to about 100,000 times better than the reference dCasX protein. In some modalities, an enhanced feature of the dCasX variant is at least approximately 1.1 to approximately 10,000 times better, at least approximately 1.1 to approximately 1,000 times better, at least approximately 1.1 to approximately 500 times better, at least approximately 1.1 to approximately 400 times better, at least approximately 1.1 to approximately 300 times better, at least approximately 1.1 to approximately 200 times better, at least approximately 1.1 to approximately 100 times better, at least approximately 1.1 to approximately 50 times better, at least approximately 1.1 to approximately 40 times better, at least approximately 1.1 to approximately 30 times better, at least approximately 1.1 to approximately 20 times better, at least approximately 1.1 to approximately 10 times better, at least less than approximately 1.1 to approximately 9 times better. better, at least about 1.1 to about 8 times better, at least about 1.1 to approximately 7 times better, at least approximately 1.1 to approximately 6 times better, at least approximately 1.1 to approximately 5 times better, at least approximately 1.1 to approximately 4 times better, at least approximately 1.1 to approximately 3 times better, at least approximately 1.1 to approximately 2 times better, at least approximately 1.1 to approximately 1.5 times better, at least approximately 1.5 to approximately 3 times better, at least approximately 1.5 to approximately 4 times better, at least approximately 1.5 to approximately 5 times better, at least approximately 1.5 to approximately 10 times better, at least approximately 5 to approximately 10 times better, at least approximately 10 to approximately 20 times better, at least 10 to approximately 30 times better, at least 10 to approximately 50 times better, or at least 10 to approximately 100 times better than the reference dCasX protein. In some modalities,An enhanced variant of dCasX is at least 10 to about 1000 times better than the reference dCasX protein. Further description of... Petition 870250104944, dated 11 / 17 / 2025, p. 57 / 689 53 / 579 enhanced features are described below in this document.

[00160] In other embodiments, the modification is a substitution of one or more domains of the reference dCasX with one or more domains from a different CasX. In some embodiments, the insertion includes the insertion of part or all of a domain from a different CasX protein. Mutations can occur in any one or more domains of the dCasX variant and may include, for example, deletion of part or all of one or more domains, or one or more amino acid substitutions, deletions, or insertions in any domain. The domains of dCasX proteins include the non-target strand-binding domain (NTSB), the target strand-loading domain (TSL), the helical domain I, the helical domain II, the oligonucleotide-binding domain (OBD), and the RuvC DNA cleavage domain, which may further comprise subdomains described below.

[00161] In some embodiments, the dCasX variant protein comprises between 800 and 1100 amino acids or between 900 and 1000 amino acids.

[00162] The long-term repressor fusion protein comprising a dCasX variant and the linked repressor domains described has an enhanced ability to efficiently bind to the target nucleic acid when complexed with a gRNA as an RNP, utilizing and binding to a PAM TC motif, including PAM sequences selected from TTC, ATC, GTC, or CTC, compared to an RNP of a fusion protein comprising the reference dCasX protein and a gRNA in a comparable assay system. In the foregoing, the PAM sequence is located at least 1 nucleotide 5' to the non-target strand of the protospacer with identity to the gRNA targeting sequence.

[00163] In some modalities, an RNP comprising the Petition 870250104944, dated 11 / 17 / 2025, page 58 / 689 54 / 579 A long-term repressor fusion protein comprising a dCasX variant with bound repressor domains and a gRNA from the description, at a concentration of 20 pM or less, is capable of binding to a double-stranded DNA target with an efficiency of at least 70%, at least 80%, at least 85%, at least 90%, or at least 95%. In one embodiment, an RNP of a long-term repressor fusion protein comprising a dCasX variant with bound repressor domains and a gRNA variant exhibits higher binding of a target sequence in the target nucleic acid compared to an RNP comprising a comparable long-term repressor fusion protein comprising a reference dCasX protein with bound repressor domains and a gRNA in a comparable assay system, wherein the target nucleic acid PAM sequence is TTC.In another embodiment, an RNP of a long-term repressor fusion protein comprising a dCasX variant with bound repressor domains and a gRNA variant exhibits higher binding affinity to a target sequence in the target nucleic acid compared to an RNP comprising a comparable long-term repressor fusion protein comprising a reference dCasX protein with bound repressor domains and a gRNA in a comparable assay system, wherein the target nucleic acid PAM sequence is ATC.In another embodiment, an RNP of a long-term repressor fusion protein comprising a dCasX variant with bound repressor domains and a gRNA variant exhibits higher binding affinity to a target sequence in the target nucleic acid compared to an RNP comprising a comparable long-term repressor fusion protein comprising a reference dCasX protein with bound repressor domains and a gRNA in a comparable assay system, wherein the nucleic acid PAM sequence. Petition 870250104944, dated 11 / 17 / 2025, page 59 / 689 55 / 579 target is CTC. In another embodiment, an RNP of a long-term repressor fusion protein comprising a dCasX variant with bound repressor domains and a gRNA variant exhibits higher binding affinity of a target sequence in the target nucleic acid compared to an RNP of a comparable long-term repressor fusion protein comprising a reference dCasX protein with bound repressor domains and a gRNA in a comparable assay system, wherein the PAM sequence of the target nucleic acid is GTC.In other embodiments, an RNP of a repressor fusion protein comprising the dCasX variant with bound repressor domains and a gRNA exhibits higher binding affinity for a target sequence in the target nucleic acid compared to an RNP comprising a comparable repressor fusion protein comprising the reference dCasX protein with bound repressor domains and the gRNA in a comparable assay system, wherein the target nucleic acid PAM sequence is GTC, TTC, ATC, or CTC. In the foregoing embodiments, the increased binding affinity for one or more PAM sequences is at least 1.5 times greater or more compared to the binding affinity of an RNP of any of the reference dCasX proteins (modified from SEQ ID NOS: 1-3) with bound repressor domains and the gRNA from SEQ ID NOS: 1731-1743 of Table 8 for the PAM sequences. c. dCasX Variant Proteins with Multi-Source Protein Domains

[00164] In certain embodiments, the description provides a chimeric variant dCasX protein for use in long-term repressor fusion proteins.

[00165] As used herein, a chimeric dCasX protein refers to a catalytically dead dCasX protein with Petition 870250104944, dated 11 / 17 / 2025, page 60 / 689 56 / 579 having at least two domains from different sources, as well as a catalytically killed dCasX protein containing at least one domain that is itself chimeric. Therefore, in some embodiments, a chimeric dCasX protein is one that includes at least two isolated or derived domains from different sources, such as from two different naturally occurring CasX proteins (e.g., from two different CasX reference proteins). In other embodiments, the chimeric dCasX protein is one that contains at least one domain that is a chimeric domain, for example, in some embodiments, part of a domain comprises a substitution from a different CasX protein (from a reference CasX protein or another CasX protein).

[00166] In some embodiments, at least one chimeric domain may be any of the NTSB, TSL, helical I, helical II, OBD, or RuvC domains, as described in this document. In the case of split or non-contiguous domains, such as helical I, RuvC, and OBD, a portion of the non-contiguous domain may be replaced by the corresponding portion from any other source. In some embodiments, the helical I-II domain (sometimes referred to as helical Ia) of the dCasX derived from SEQ ID NO: 2 is replaced by the corresponding helical I-II sequence from SEQ ID NO: 1, resulting in a chimeric dCasX protein.

[00167] In some embodiments, the helical I-II domain and the NTSB domain of dCasX derived from SEQ ID NO: 2 are replaced by the corresponding helical I-II and NTSB sequences from SEQ ID NO: 1, resulting in a chimeric dCasX protein.

[00168] A chimeric dCasX variant protein may comprise NTSB, TSL, helical II, helical I-II, helical II, OBD-I, and OBD-II domains of a CasX protein with SEQ ID NO: 2 and a RuvC-I and / or RuvC-II domain of a CasX protein with SEQ ID NO: 1, or vice versa. Petition 870250104944, dated 11 / 17 / 2025, p. 61 / 689 57 / 579 versa, where mutations or other sequence alterations are introduced to create the catalytically killed variant with improved variant properties, relative to the reference dCasX protein. As an example of the above, the chimeric RuvC domain comprises amino acids 660 to 823 of SEQ ID NO: 1 and amino acids 921 to 978 of SEQ ID NO: 2. As an alternative example of the above, a chimeric RuvC domain comprises amino acids 647 to 810 of SEQ ID NO: 2 and amino acids 934 to 986 of SEQ ID NO: 1.In a particular embodiment, a dCasX for use in long-term repressor fusion protein comprises an NTSB domain and a helical I-II domain of SEQ ID NO: 1 and a helical II domain of SEQ ID NO: 2; the latter being a chimeric domain, it being understood that dCasX variants have additional amino acid changes at selected locations (relative to the reference sequence) and the resulting chimeric dCasX proteins have improved characteristics compared to the reference dCasX proteins. The sequences in Table 2 having the NTSB domain and the helical I-II domain of SEQ ID NO: 1 and a helical II domain of SEQ ID NO: 2 include dCasX 491 (SEQ ID NO: 4), 515 (SEQ ID NO: 6), 516 (SEQ ID NO: 7), 518-520 (SEQ ID NO: 9-11), 522-527 (SEQ ID NO: 12-17), 532 (SEQ ID NO: 22), 593 (SEQ ID NO: 25), 676 (SEQ ID NO: 28), with an L169K substitution in the NTSB domain) and 812 (SEQ ID NO: 29).The coordinates of the CasX domains in the reference CasX proteins of SEQ ID NO: 1 and SEQ ID NO: 2 are provided in Table 1 below. Those skilled in the art will understand that the domain boundaries indicated in Table 1 below are approximate and that protein fragments whose boundaries differ from those given in the table below by 1, 2, or 3 amino acids may have the same activity as the domains described below. Table 1: Domain Coordinates in CasX Proteins from Repetition 870250104944, dated 11 / 17 / 2025, page 62 / 689 58 / 579 reference Domain Name Coordinates in SEQ ID NO: 1* Coordinates in SEQ ID NO: 2* OBD-I 1-55 1-57 II helical 56-99 58-101 NTSB 100-190 102-191 I-II helical 191-331 192-332 II helical 332-508 333-500 OBD-II 509-659 501-646 RuvC-I 660-823 647-810 TSL 824-933 811-920 RuvC-II 934-986 921-978 * amino acid position

[00169] In some embodiments, a dCasX variant protein used in the long-term repressor fusion proteins of the description comprises a sequence selected from the group consisting of SEQ IDs NOS: 4-29, as set forth in Table 2, wherein the sequence comprises a RuvC domain comprising one or more mutations that inactivate the cleavage activity of the RuvC domain. In other embodiments, a dCasX variant protein used in the long-term repressor fusion proteins of the description comprises a sequence that is at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 81% identical, at least about 82% identical, at least about 83% identical, at least about 84% identical, at least about 85% identical, at least about 86% identical, at least about 86% identical, at least about 87% identical, at least about 88% identical, at least about 89% identical,at least approximately 89% identical, at least approximately 90% identical, at least approximately 91% identical, at least approximately 92% identical, at least approximately 93% identical, at least approximately 94% identical, by, Petition 870250104944, dated 11 / 17 / 2025, page 63 / 689 59 / 579 less about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or at least about 99.5% identical to a sequence of SEQ IDs NOs: 4-29, as set out in Table 2. In some embodiments, the dCasX variant protein comprises a RuvC domain comprising one or more mutations that inactivate the cleavage activity of the RuvC domain. In some embodiments, the long-term repressor fusion protein comprising dCasX retains the ability to form an RNP with a gRNA. In a particular embodiment, the dCasX variant protein used in the long-term repressor fusion protein of the gene repressor systems described comprises a sequence of SEQ ID NO: 4 (dCasX 491).In another particular embodiment, the dCasX variant protein used in the long-term repressor fusion protein of the gene repressor systems described comprises a sequence of SEQ ID NO: 6 (dCasX 515). In another particular embodiment, the dCasX variant protein used in the long-term repressor fusion protein of the gene repressor systems described comprises a sequence of SEQ ID NO: 29 (dCasX 812). Table 2: Variant Sequences of dCasX SEQ ID NO dCasX Amino acid sequence 4 dCasX491 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVM TPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQP LFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDAC- MGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAG KENLEYPSVTLPPQPHTKEGVDAYNEVIARVRM- Petition 870250104944, dated 11 / 17 / 2025, pp. 64 / 689 60 / 579 SEQ ID NO dCasX Amino acid sequence WVNLNLWQKLKLSRDDAKPLLRLLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKKKFARYQLGDLLLLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVP- MEVNFNFNDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAK- LAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEAQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 5 dCasX514 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVM TPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDAC-MGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAG KENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAEN- Petition 870250104944, de 17 / 11 / 2025, pág. 65 / 689 61 / 579 SEQ ID NO dCasX Amino acid sequence SILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTIHTSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 6 dCasX515 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVM TPDLRERLENLRKKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDAC- MGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVP- MEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQR- Petition 870250104944, de 17 / 11 / 2025, pág. 66 / 689 62 / 579 SEQ ID NO dCasX Amino acid sequence TIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRF- SHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 7 dCasX516 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVM TPDLRERLENLRKKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDAC- MGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAG KENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNHNLWQKLKLSRDDAKPLLRLLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVP-MEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPL SRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAK- LAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRF- Petition 870250104944, dated 11 / 17 / 2025, page 67 / 689 63 / 579 SEQ ID NO dCasX Sequência de aminoacidos SHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 8 dCasX517 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVM TPDLRERLENLRKKPENIPQPISNTSRANLNKLLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAITNYFGRCVNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGAPVGKALSDAC- MGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAG KENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKKKKFARYQLGDLLLLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVP- MEVNFNFNDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAK-LAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 9 dCasX518 RQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRV MTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLK- Petition 870250104944, dated 11 / 17 / 2025, pp. 68 / 689 64 / 579 SEQ ID NO dCasX Sequência de aminoacidos PEMDEKGNLTTAG- FACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCVNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALS DACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAG KENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLA- GYKRQEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAK- LAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEAQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 10 dCasX519 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNLTTAGFACQCGQP LFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDPHYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDAC- MGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAG KENLEYPSVTLPPQPHTCEGVDAYNEVIARVRM- Petition 870250104944, of 17 / 11 / 2025, p. 69 / 689 65 / 579 SEQ ID NO dCasX Amino acid sequence WVNLNLWQKLKLSRDDAKPLLRLLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVP- MEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHIQLRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRF- SHRPVQEKFVCLNCGFETHAAEAQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 11 dCasX520 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVM TPDLRERLENLRKKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDAC-MGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAG KENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAEN- Petition 870250104944, de 17 / 11 / 2025, pág. 70 / 689 66 / 579 SEQ ID NO dCasX Amino acid sequence SILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTTQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 12 dCasX522 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVM TPDLRERLENLRKKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDAC- MGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKKKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVP- MEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKRSLGNPTHILRIGESYKEKQR- Petition 870250104944, de 17 / 11 / 2025, pág. 71 / 689 67 / 579 SEQ ID NO dCasX Amino acid sequence TIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEAQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 13 dCasX523 QEIKRINKIRRRLVKDSNTKKAGKTYPMKTLLVRVMT PDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVY- WEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDAC- MGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAG KENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVP-MEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMV RNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRF- Petition 870250104944, dated 11 / 17 / 2025, page 72 / 689 68 / 579 SEQ ID NO dCasX Sequência de aminoacidos SHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 14 dCasX524 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVM TPDLRERLENLRKKPENIPQPISNTSRANLNKLLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAITNYFGRCVNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALS DACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAG KENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKKKKFARYQLGDLLLLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVP- MEVNFNFNDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAK-LAYEGLSKTYLSKTLAQYTSKTCSNCGFTIHSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDI SSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 15 dCasX525 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVM TPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVY- Petition 870250104944, dated 11 / 17 / 2025, page 73 / 689 69 / 579 SEQ ID NO dCasX Sequência de aminoacidos WEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAITNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALS DACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAG KENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDAKPLLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAEN- SILDISGFSKQYNCAFIWQKDGVKKLNLLYLIINIFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKAKNLADDMVRNTA RDLLYYAATQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 16 dCasX526 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQP LFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDAC- MGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAG Petition 870250104944, dated 11 / 17 / 2025, page 74 / 689 70 / 579 SEQ ID NO dCasX Amino acid sequence KENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVP- MEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAAKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAK- LAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 17 dCasX527 QEIKRINKIRRRLVKDSNTKKAGKTRGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNLTTAGFACQCGQ PLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDPHYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDAC- MGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAG KENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDAKPLLRLKGFPSFPLVERQANEVDWDMVCCNVKLINEKEDGKVFWQNL AGYKRQEALRPAYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEERRSEDAQSKAALTDWLRAKASFVIEG- Petition 870250104944, of 17 / 11 / 2025, p. 75 / 689 71 / 579 SEQ ID NO dCasX Sequence of amino acids LKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 18 dCasX528 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVM TPDLRERLENLRKKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASYPVGKALSDAC- MGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVP- MEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVI- Petition 870250104944, de 17 / 11 / 2025, pág. 76 / 689 72 / 579 SEQ ID NO dCasX Sequence of amino acids ALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYD RVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 19 dCasX529 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVM TPDLRERLENLRKKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASNPVGKALSDAC- MGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAG KENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVP-MEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPL SRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAK- LAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNN- Petition 870250104944, dated 11 / 17 / 2025, page 77 / 689 73 / 579 SEQ ID NO dCasX Sequência de aminoacidos DISSWTKGRSGEALSLLKKRF- SHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 20 dCasX530 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVM TPDLRERLENLRKKPENIPQPISNTSRANLNKLLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAITNYFGRCVNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALS DACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAG KENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKKKKFARYQLGDLLLLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGWGKLRFKKIKPEAFEANRFYTVINKKSGEIVP- MEVNFNFNDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAK-LAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRF- SHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 21 dCasX531 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVM TPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVY- Petition 870250104944, dated 11 / 17 / 2025, page 78 / 689 74 / 579 SEQ ID NO dCasX Sequência de aminoacidos WEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAITNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALS DACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAG KENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDAKPLLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAEN- SILDISGFSKQYNCAFIWQKDGVKKLNLYLIINIFKGYGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKNLADDMVRNTA RDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSPYRKKLKEVWKPAV 22 dCasX532 QEIKRINKIRRRLVKDSNTKKAGKTRGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQ PLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDAC- MGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAG Petition 870250104944, dated 11 / 17 / 2025, page 79 / 689 75 / 579 SEQ ID NO dCasX Amino acid sequence KENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVP- MEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAK- LAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRF- SHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 23 dCasX533 QEIKRINKIRRRLVKDSNTKKAGKTRGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNLTTAGFACQCGQ PLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDPHYSIHVTKESTHPVKPLAQIAGNRYASYPVGKALSDAC- MGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAG KENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDAKPLLRLKGFPSFPLVERQANEVDWDMVCCNVKLINEKEDGKVFWQNL AGYKRQEALRPAYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEERRSEDAQSKAALTDWLRAKASFVIEG- Petition 870250104944, of 17 / 11 / 2025, p. 80 / 689 76 / 579 SEQ ID NO dCasX Sequence of amino acids LKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 24 dCasX535 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVM TPDLRERLENLRKKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASSPVGKALSDAC- MGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVP- MEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVI- Petition 870250104944, de 17 / 11 / 2025, pág. 81 / 689 77 / 579 SEQ ID NO dCasX Amino acid sequence ALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 25 dCasX593 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVM TPDLRERLENLRKKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDAC- MGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAG KENLEYPSVTLPPQPHTKEGVDAYNEVIARRWWVNLNLWQKLKLSRDDAKPLLRLLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVP-MEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPL SRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAK- LAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNN- Petition 870250104944, dated 11 / 17 / 2025, page 82 / 689 78 / 579 SEQ ID NO dCasX Sequência de aminoacidos DISSWTKGRSGEALSLLKKRF- SHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 26 dCasX668 QEIKRINKIRRRLVKDSNTKKAGKTRGPMKTLLVRV MTPDLRERLENLRKKPENIPQPISNTSRANLNKLLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAITNYFGRCVNAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASSPVGKALSDAC- MGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAG KENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKKKKFARYQLGDLLLLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVP- MEVNFNFNDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAK-LAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 27 dCasX672 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVM TPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVY- Petition 870250104944, dated 11 / 17 / 2025, page 83 / 689 79 / 579 SEQ ID NO dCasX Sequência de aminoacidos WEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAITNYFGRCNVAEHEKLIKLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASSPVGKALS DACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAG KENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDAKPLLLRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAEN- SILDISGFSKQYNCAFIWQKDGVKKLNLLYLIINIFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKAKNLADDMVRNTA RDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSPYRKKLKEVWKPAV 28 dCasX676 QEIKRINKIRRRLVKDSNTKKAGKTRGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQ PLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLIKLAQLKPEKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASSPVGKALSDAC- MGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAG Petition 870250104944, dated 11 / 17 / 2025, page 84 / 689 80 / 579 SEQ ID NO dCasX Amino acid sequence KENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRLLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYLSSEEDRKKKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVP- MEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAK- LAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRF- SHRPVQEKFVCLNCGFETHAAEAQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV 29 dCasX812 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPASKKIDQNKLKPEMDEKGNLTTAGFACQCGQP LFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQRALDPHYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDAC- MGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAG KENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDAKPLLRLCKFPSFPLVERQANEVDWDMVCCNVKLINEKEDGKVFWQNL AGYKRQEALRPAYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEERRSEDAQSKAALTDWLRAKASFVIEG- Petition 870250104944, of 17 / 11 / 2025, p. 85 / 689 81 / 579 SEQ ID NO dCasX Sequence of amino acids LKEADKDEFCRCELKLQKWYGDLRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVALTFERREVLDSSNIKPMNLIGVARGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFANLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHAAEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV d. Affinity for gRNA

[00170] In some embodiments, a long-term repressor fusion protein comprising a dCasX with bound repressor domains has improved affinity for gRNA relative to a comparable long-term repressor fusion protein comprising a reference dCasX protein with corresponding bound repressor domains, leading to the formation of the ribonucleoprotein (RNP) complex. The increased affinity of the long-term repressor fusion protein for gRNA may, for example, result in a lower Kd for the generation of an RNP complex, which may, in some cases, result in a more stable formation of the ribonucleoprotein complex. In some embodiments, the Kd of a long-term repressor fusion protein for gRNA is increased relative to a reference dCasX protein and bound repressor domains by a factor of at least about 1.1, at least about 1.2, at least about 1.3, at least about Petition 870250104944, dated 11 / 17 / 2025, p. 86 / 689 82 / 579 1.4, at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100. In some embodiments, the long-term repressor fusion protein comprising the dCasX variant has an affinity of increased binding of approximately 1.1 to approximately 10 times with gRNA compared to the long-term repressor fusion protein comprising the catalytically killed variant of the reference CasX protein SEQ ID NO: 2.

[00171] In some embodiments, increasing the affinity of the long-term repressor fusion protein for gRNA results in increased stability of the ribonucleoprotein complex when delivered to mammalian cells, including in vivo delivery to an individual. This increased stability can affect the function and utility of the complex in an individual's cells, as well as result in improved pharmacokinetic properties in the blood when delivered to an individual. In some embodiments, the increased affinity of the long-term repressor fusion protein and the resulting increased stability of the ribonucleoprotein complex allows a lower dose of the long-term repressor fusion protein to be delivered to the individual or cells while still having the desired activity; for example, gene repression and / or epigenetic modification in vivo or in vitro. The increased ability to form RNP and maintain Petition 870250104944, dated 11 / 17 / 2025, page 87 / 689 83 / 579 las in stable form can be evaluated using in vitro assays known in the art.

[00172] In some embodiments, a higher affinity (stronger binding) of a dCasX variant long-term repressor fusion protein to a gRNA allows for a greater amount of transcriptional repression events and / or epigenetic modification when both the long-term repressor fusion protein and the gRNA remain in an RNP complex. Increased transcriptional repression events can be assessed using assays described in this document.

[00173] Methods for measuring the binding affinity of long-term repressor fusion protein with a gRNA include in vitro methods using purified long-term repressor fusion protein and a gRNA. The binding affinity with the long-term repressor fusion protein can be measured by fluorescence polarization if the gRNA or the long-term repressor fusion protein is labeled with a fluorophore. Alternatively or additionally, the binding affinity can be measured by biolayer interferometry, electrophoretic mobility change assays (EMSAs), or filter binding. Additional standard techniques for quantifying absolute affinities of RNA-binding proteins, such as dCasX proteins described, for specific gRNAs, such as reference gRNAs and variants thereof, include, among others, isothermal calorimetry (ITC) and surface plasmon resonance (SPR). e. Improved specificity for a target nucleic acid sequence

[00174] In some embodiments, a long-term repressor fusion protein comprising a dCasX variant protein with linked repressor domains has improved specificity for a target nucleic acid sequence that is complementary to the se Petition 870250104944, dated 11 / 17 / 2025, page 88 / 689 84 / 579 gRNA targeting sequence relative to a reference dCasX protein with bound repressor domains. As used in this document, specificity, sometimes referred to as target specificity, refers to the degree to which a CRISPR / Cas system ribonucleoprotein complex binds to off-target sequences that are similar to, but not identical to, the target nucleic acid sequence; for example, a long-term repressor fusion protein containing RNPs with a higher degree of specificity would exhibit reduced off-target methylation of sequences relative to an RNP of a reference dCasX with bound repressor domains. The specificity and reduction of potentially deleterious off-target effects of long-term repressor fusion proteins may contribute to achieving an acceptable therapeutic index for use in mammalian subjects.Without wishing to be bound by theory, it is possible that amino acid changes in helical domains I and II that increase the specificity of the long-term repressor fusion protein for the target nucleic acid strand may thereby increase the specificity of the long-term repressor fusion protein for the target nucleic acid overall. In some embodiments, amino acid changes that increase the specificity of long-term repressor fusion proteins for target nucleic acid may also result in decreased affinity of long-term repressor fusion proteins for DNA, but the overall benefit and safety of the composition are enhanced. f. Repressor fusion proteins with heterologous proteins

[00175] Also contemplated within the scope of the description are long-term repressor fusion proteins comprising a heterologous protein fused to the long-term repressor fusion protein for use in the systems of the description. This includes long-term repressor fusion proteins comprising N fusions Petition 870250104944, dated 11 / 17 / 2025, page 89 / 689 85 / 579 terminals or C-terminals to a heterologous protein or domain thereof. In some embodiments, the long-term repressor fusion protein is fused to one or more proteins or domains thereof that have a different activity of interest.

[00176] In some cases, a heterologous polypeptide (a fusion partner) for use with a long-term repressor fusion protein provides subcellular localization, i.e., the heterologous polypeptide contains a subcellular localization sequence (e.g., a nuclear localization signal (NLS) to target the nucleus, a sequence to keep the fusion protein out of the nucleus, a nuclear export sequence (NES), a sequence to keep the fusion protein retained in the cytoplasm, a mitochondrial localization signal for targeting mitochondria, a chloroplast localization signal for targeting a chloroplast, an ER retention signal, and the like).

[00177] In some cases, a long-term repressor fusion protein includes (is fused to) a nuclear localization signal (NLS). In some cases, a long-term repressor fusion protein is fused to 2 or more, 3 or more, 4 or more, or 5 or more, 6 or more, 7 or more, or 8 or more NLSs. In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 20 amino acids) the N-terminus and / or C-terminus of the long-term repressor fusion protein. In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 20 amino acids) the N-terminus of the long-term repressor fusion protein. In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 20 amino acids) the C-terminus of the long-term repressor fusion protein. In some cases, one or more NLSs (3 or Petition 870250104944, dated 11 / 17 / 2025, p. 90 / 689 86 / 579 plus, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 20 amino acids) either the N-terminus or the C-terminus of the long-term repressor fusion protein. In some cases, a single NLS is positioned at the N-terminus and a single NLS is positioned at the C-terminus of the long-term repressor fusion protein. One skilled in the art will understand that an NLS at or near the N- to C-terminus of a protein may be within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids from the N- to C-terminus. In some embodiments, the N-terminal bound NLS of dCasX or the long-term repressor fusion protein is identical to the C-terminal bound NLS. In other embodiments, the N-terminal bound NLS of dCasX or the long-term repressor fusion protein is different from the C-terminal bound NLS. Representative configurations of long-term repressor fusion proteins with NLS are shown in FIG. 61.In some embodiments, NLSs suitable for use with a long-term repressor fusion protein in the systems described comprise sequences with at least about 85%, at least about 90%, or at least about 95% identity to, or are identical to, sequences derived from: the SV40 virus large T antigen NLS having the amino acid sequence PKKKRKV (SEQ ID NO: 30); The nucleoplasmin NLS (e.g., the split nucleoplasmin NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 31); the c-MYC NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 32) or RQRRNELKRSP (SEQ ID NO: 33). In some embodiments, the NLS and a short peptide linker attached to the N-terminal of the long-term repressor fusion protein is the sequence PKKKRKVSR (SEQ ID NO: 34). In some embodiments, the NLS and a short peptide linker attached to the N-terminal of the long-term repressor fusion protein is the sequence PKKKRKVSRVNGSGSGGG (SEQ ID NO: 3298). Petition 870250104944, dated 11 / 17 / 2025, p. 91 / 689 In some embodiments, the NLS and a short peptide linker attached to the C-terminal of the long-term repressor fusion protein is the sequence TSPKKKRKV (SEQ ID NO: 3273). In some embodiments, the NLS attached to the N-terminal of the long-term repressor fusion protein is selected from the group consisting of N-terminal sequences as set forth in Table 3. In some embodiments, the NLS comprises a sequence selected from the group consisting of SEQ ID NOS: 30-97, 3273, and 3298. In some embodiments, the NLS comprises a sequence selected from the group consisting of SEQ ID NOS: 34-97. In some embodiments, the NLS attached to the C-terminal of the long-term repressor fusion protein is selected from the group consisting of C-terminal sequences as set forth in Table 4.In some embodiments, NLSs suitable for use with a long-term repressor fusion protein in the systems described include sequences with at least about 80%, at least about 90%, or at least about 95% identity or identical to one or more sequences from Table 3 or Table 4. Those skilled in the art will appreciate that any of the NLS sequences set forth in Table 3 and Table 4 can be fused to or proximal to the N- to C-terminus of the long-term repressor fusion proteins described herein. Table 3: N-terminal NLS Amino Acid Sequences NLS Amino Acid Sequence* SEQ ID NO PKKKRKVSR 34 PKKKRKVGGSPKKKRKVGGSPKKKRKVGGSPKKKRKVSR 35 PKKKRKVGGSPKKKRKVGGSPKKKRKVGGSPKKKRKVGGSPKKKRKVGGSPKKKRKVSR 36 PAAKRVKLDSR 37 PAAKRVKLDGGSPAAKRVKLDSR 38 Petition 870250104944, dated 11 / 17 / 2025, p. 92 / 689 88 / 579 Sequência de Aminoacidos de NLS* SEQ ID NO PAAKRVKLDGGSPAAKRVKLDGGSPAAKRVKLDGGSPAAKRVKLDSR 39 PAAKRVKLDGGSPAAKRVKLDGGSPAAKRVKLDGGSPAAKRVKLDGGSPAAKRVKLDGGSPAAKRVKLDSR 40 KRPAATKKAGQAKKKKSR 41 KRPAATKKAGQAKKKKGGSKRPAATKKAGQAKKKKSR 42 PAAKRVKLDGGSPKKKRKVSR 43 PAAKKKLDGGSPKKKRKVSR 44 PAAKKKLDSR 45 PAAKKKKLDGGSPAAKKKKLDGGSPAAKKKLDSR 46 PAAKKKLDGGSPAAKKKLDGGSPAAKKKLDGSPAAKKKLDSR 47 PAKRARRGYKCSR 48 PAKRARRGYKCGSPAKRARRGYKCSR 49 PRRKREESR 50 PYRGRKESR 51 PLRKRPRRSR 52 PLRKRPRRGSPLRKRPRRSR 53 PAAKRVKLDGGKRTADGSEFESPKKKRKVGGS 54 PAAKRVKLDGGKRTADGSEFESPKKKRKVPPPPG 55 PAAKRVKLDGGKRTADGSEFESPKKKRKVGIHGVPAAPG 56 PAAKRVKLDGGKRTADGSEFESPKKKRKVGGGSGGGSPG 57 PAAKRVKLDGGKRTADGSEFESPKKKRKVVPGGGSGGG- SPG 58 PAAKRVKLDGGKRTADGSEFESPKKKRKVAEAAAKE- AAAKEAAAKAPG 59 PAAKRVKLDGGSPKKKRKVGGS 60 Petition 870250104944, de 17 / 11 / 2025, pág. 93 / 689 89 / 579 NLS Amino Acid Sequence* SEQ ID NO PAAKRVKLDPPPPKKKRKVPG 61 PAAKRVKLDPG 62 PAAKRVKLDGGGSGGGSGGGS 63 PAAKRVKLDPPP 64 PAAKRVKLDGGGSGGGSGGGSPPP 65 PKKKRKVPPP 66 PKKKRKVGGS 67 *The residues in bold are NLS residues, while the residues not in bold are linkers. Table 4: Amino Acid Sequences of C-terminal NLS NLS Amino Acid Sequence SEQ ID NO GSPKKKRKVGGSPKKKRKVGGSPKKKRKVGGSPKKKRKV 68 GSPKKKRKVGGSPKKKRKVGGSPKKKRKVGGSPKKKRKVGGSPKKKRKVGGSPKKKRKV 69 GSPAAKRVKLDGGSPAAKRVKLD 70 GSPAAKRVKLDGGSPAAKRVKLDGGSPAAKRVKLDGGSPAAKRVKLD 71 GSKRPAATKKAGQAKKKK 72 KRPAATKKAGQAKKKKGGSKRPAATKKAGQAKKKK 73 GSKLGPRKATGRWGS 74 GSKRKGSPERGERKRHWGS 75 GSPKKKRKVGSGSKRPAATKKAGQAKKKKLE 76 GPKRTADSQHSTPPKTKRKVEFEPKKKRKV 77 GGGSGGGSKRTADSQHSTPPKTKRKVEFEPKKKRKV 78 Petition 870250104944, dated 11 / 17 / 2025, page 94 / 689 90 / 579 NLS Amino Acid Sequence SEQ ID NO AEAAAKEAAAKEAAAKAKR- TADSQHSTPPKTKRKVEFEPKKKRKV 79 GPPKKKRKVGGSKRTADSQHSTPPKTKRKVEFEPKKKRKV 80 GPAAEAAKEAAAKEAAAKAPAAKRVKLD 81 GPGGGSGGGSGGGSPAAKRVKLD 82 GPPAAKRVKLD 91 GGSPKKKRKVGGSPKKKRKV 92 GGSGGSGGSPKKKRKVGGSPKKKRKV 93 VGGGSGGGSGGGSPAAKRVKLD 94 VPPPPAAKRVKLD 95 VPPPGGGSGGGSGGGSPAAKRVKLD 96 VGSPAAKRVKLD 97

[00178] In some embodiments, one or more NLSs are linked to the long-term repressor fusion protein or to an NLS adjacent to an optional linker peptide. In some embodiments, the linker peptide is selected from the group consisting of SR, GS, GP, TS, VGS, GGS, (G)n (SEQ ID NO: 98), (GS)n (SEQ ID NO: 99), (GSGGS)n (SEQ ID NO: 100), (GGSGGS)n (SEQ ID NO: 101), Petition 870250104944, dated 11 / 17 / 2025, page 95 / 689 91 / 579 (GGGS)n (SEQ ID NO: 102), GGSG (SEQ ID NO: 103), GGSGG (SEQ ID NO: 104), GSGSG (SEQ ID NO: 105), GSGGG (SEQ ID NO: 106), GGGSG (SEQ ID NO: 107), GSSSG (SEQ ID NO: 108), GPGP (SEQ ID NO: 109), GGP, PPP, VPPP, PPAPPA (SEQ ID NO: 110), PPPG (SEQ ID NO: 111), PPPGPPP (SEQ ID NO: 112), PPP(GGGS)n (SEQ ID NO: 113), (GGGS)nPPP (SEQ ID NO: 114), AEAAAKEAAAKEAAAKA (SEQ ID NO: 115), VPPPGGGSGGGSGGGS (SEQ ID NO: 116), TGGGPGGGAAAGSGS (SEQ ID NO: 117), GGGSGGGSGGGSPPP (SEQ ID NO: 118), TPPKTKRKVEFE (SEQ ID NO: 119), GGSGGGS (SEQ ID NO: 120), GSGSGGG (SEQ ID NO: 121), SSGNSNANSRGPSFSSGLVPLSLRGSH (SEQ ID NO: 122), GGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE (SEQ ID NO: 123) e GGSGGG (SEQ ID NO: 124), onde n é 1 a 5.

[00179] In general, NLSs (or multiple NLSs) are strong enough to drive the accumulation of a long-term repressor fusion protein in the nucleus of a eukaryotic cell. Detection of accumulation in the nucleus can be performed by any suitable technique. For example, a detectable marker can be fused to a long-term repressor fusion protein so that its location within a cell can be visualized. Cell nuclei can also be isolated from cells, the contents of which can then be analyzed by any suitable process for detecting protein, such as immunohistochemistry, Western blot, or enzyme activity assay. Accumulation in the nucleus can also be determined indirectly. IV. Long-Term Repressor Domain Fusion Proteins

[00180] The description provides long-term fusion protein systems comprising a DNA-binding protein linked to multiple repressor domains, wherein the system is capable of binding to a PCSK9 target nucleic acid and repressing gene transcription. Petition 870250104944, dated 11 / 17 / 2025, page 96 / 689 92 / 579 PCSK9, including through epigenetic modification of the target nucleic acid. Exemplary DNA-binding proteins for use in fusion proteins include zinc finger (ZF) proteins, TALE (transcription activator-like effector), and catalytically killed CRISPR proteins.

[00181] In some embodiments, the description provides long-term repressor fusion protein systems comprising a catalytically killed CRISPR protein, such as a dCasX, linked to multiple repressor domains that, when complexed with a guide ribonucleic acid (gRNA) comprising a targeting sequence complementary to a PCSK9 target nucleic acid sequence, the system is able to bind to a PCSK9 target nucleic acid and repress or silence the transcription of a PCSK9 gene. Examples of gene repression processes that decrease transcription include, among others, those that inhibit the formation of a transcription initiation complex, those that decrease the rate of transcription initiation, those that decrease the rate of transcription elongation, those that decrease transcription processivity, and those that antagonize transcriptional activation (e.g., by blocking the binding of a transcriptional activator).Gene repression can constitute, for example, the prevention of activation, as well as the inhibition of expression below an existing level. Transcriptional repression includes both reversible and irreversible inactivation of gene transcription; the latter can result from epigenetic modification of the target nucleic acid.

[00182] Among the repressor domains that have the ability to repress or silence genes, the Krüppel-associated box repressor domain (KRAB) is among the most powerful in human genome systems (Alerasool, N., et al. An efficient KRAB domain for CRISPRi applications. Nat. Methods 17:1093 (2020)). The domains Petition 870250104944, dated 11 / 17 / 2025, page 97 / 689 93 / 579 KRAB domains are present in approximately 400 transcription factors based on human zinc finger protein which, through the binding of dCasX linked to the target nucleic acid, are able to recruit additional repressor domains, such as, among others, Trim28 (also known as Kap1 or Tif1-beta) which, in turn, assembles a protein complex with chromatin regulators, such as CBX5 / HP1a and SETDB1, that induce repression of gene transcription, but in a temporary and limited way. Representative and non-limiting examples of KRAB domains suitable for use in the systems described include ZIM3 (SEQ ID NO: 129) and ZNF10 (SEQ ID NO: 128).The description provides additional repressor domains that are of human origin, as well as repressor domains that are of non-human origin with markedly distinct sequences (referred to in this document as RD1) that resulted in enhanced transcriptional repression compared to ZIM3 and ZNF10 when incorporated into long-term repressor fusion protein construct modalities, as described in this document.

[00183] In some embodiments, the description provides systems in which the modification transmitted by the use of the LTRP:gRNA system is epigenetic and therefore the silencing of the PCSK9 gene can be inherited by mechanisms other than the replication of edited DNA. As used in this document, epigenetic modification means a modification in DNA or DNA-associated histones, apart from a change in the DNA sequence itself (e.g., a substitution, deletion, or rearrangement), wherein the modification is either a direct modification by a component of the system or is indirect by the recruitment of one or more additional cellular components, but wherein the target nucleic acid sequence of the DNA itself is not edited to change the sequence. For example, DNA methyltransferase 3A (DNMT3A) (or its catalytic domain) directly modifies DNA by methylation. Petition 870250104944, dated 11 / 17 / 2025, page 98 / 689 94 / 579 of the same, while KRAB recruits KAP1 / TIFie corepressor complexes that act as potent transcriptional repressors and can additionally recruit factors associated with DNA methylation and repressive chromatin formation, such as heterochromatin protein 1 (HP1), histone deacetylases, and histone methyltransferases (Ying, Y., et al. The Krüppel-associated box repressor domain induces reversible and irreversible regulation of endogenous mouse genes by mediating different chromatin states. Nucleic Acids Res. 43(3): 1549 (2015)). Furthermore, the catalytically inactive cofactor DNMT3L helps establish a heritable methylation pattern after DNA replication, along with endogenous DNMT1 in the cell. The ATRXDNMT3-DNMT3L (ADD) domain of DNMT3A is known to have two main functions: 1) allosterically regulating the catalytic activity of DNMT3A by serving as an autoinhibitory domain of methyltransferase and 2) specifically interacting with histone H3 tails that are unmethylated at lysine (K)4, leading to preferential methylation of DNA bound to chromatin H3 tails that are unmethylated at K4 (Zhang, Y., et al. Chromatin methylation activity of Dnmt3a and Dnmt3a / 3L is guided by interaction of the ADD domain with the histone H3 tail. Nucleic Acids Research 38:4246 (2010)). In some modalities, the inclusion of the ADD domain enhances the transcriptional repression of targeted genes when incorporated into LTRP, compared to otherwise equivalent LTRP without the ADD domain.In other embodiments, the inclusion of the ADD domain enhances the specificity of transcriptional repression of targeted genes when compared to otherwise equivalent LTRPs without the ADD domain. Supporting data for the above are provided in the Examples, as well as in WO2023049742A2, incorporated by reference in this document.

[00184] In some embodiments, the repressor fusion protein Petition 870250104944, dated 11 / 17 / 2025, page 99 / 689 The long-term 95 / 579 fusion protein comprises a DNA-binding protein linked to a first, second, and third repressor domain, wherein each of the repressor domains is different, and the fusion protein is capable of binding to the target nucleic acid of PCKS9. In some embodiments, the long-term repressor fusion protein comprises a DNA-binding protein linked to a first, second, third, and fourth repressor domain, wherein each of the repressor domains is different. In any of the foregoing embodiments, the fusion protein is capable of forming an RNP with a gRNA of the system that binds to the target nucleic acid when the DNA-binding domain comprises a catalytically killed CRISPR protein.

[00185] In some embodiments, the DNA-binding protein comprises a TALE that can bind to, but not cleave, the target nucleic acid. In some embodiments, the DNA-binding protein comprises a modified zinc finger protein that binds to, but does not cleave, the target nucleic acid. In some embodiments, the DNA-binding protein comprises a catalytically killed CRISPR protein that can become a complex with a gRNA to form an RNP that can bind to, but not cleave, the target nucleic acid. In some embodiments, the long-term repressor fusion protein comprises a catalytically killed CRISPR protein sequence, a first repressor domain (hereinafter referred to as RD1), a DNMT3A catalytic domain (hereinafter referred to as DNMT3A) of the DNMT3A protein as the second domain, and a DNMT3L interaction domain (hereinafter referred to as DNMT3L) of the DNMT3L protein as the third domain.In some embodiments, the long-term repressor fusion protein comprises a catalytically killed CRISPR protein sequence, an RD1, a DNMT3A as the second domain, a DNMT3L as the third domain, and an ATRX-DNMT3-DNMT3L domain (of the... Petition 870250104944, dated 11 / 17 / 2025, page 100 / 689 96 / 579 (referred to as ADD) of the DNMT3A protein as the fourth domain. In some embodiments, the long-term repressor fusion protein comprises a dCasX and further comprises a first and second NLS and one or more linker peptides described in this document. In some embodiments, the long-term repressor fusion protein is capable of forming an RNP with a gRNA that binds to the target nucleic acid. It has been found that the use of the aforementioned domains, when configured in selected orientations relative to a dCasX in a long-term repressor fusion protein, can result in pronounced epigenetic modification of a PCSK9 target nucleic acid when complexed with a gRNA with a targeting sequence complementary to defined regions of the PCSK9 gene, and that the combination of repressor domains works in synchrony, resulting in an additive or synergistic effect on transcriptional silencing of the target gene, depending on the configuration.

[00186] Representative amino acid sequences of the components used in long-term repressor fusion protein constructs are provided in this document.

[00187] In some embodiments, the dCasX of the long-term repressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NOS: 4-29, or a sequence with at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity with it. In some embodiments, the dCasX of the long-term repressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NOS: 4-29. In some embodiments, the dCasX of the long-term repressor fusion protein comprises Petition 870250104944, dated 11 / 17 / 2025, p. 101 / 689 97 / 579 the sequence with SEQ ID NO: 4, or a sequence with at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity with it. In some embodiments, the dCasX of the long-term repressor fusion protein comprises the sequence with SEQ ID NO: 4.

[00188] In some embodiments, the RD1 of the long-term repressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NOS: 128-1726 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with it. In some embodiments, the RD1 of the long-term repressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NOS: 128-1726.In some embodiments, the RD1 of the long-term repressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NO:128 or SEQ ID NO:129, or a sequence with at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with it. In some embodiments, the RD1 of the long-term repressor fusion protein comprises a sequence selected from the group consisting of SEQ ID NO:128 or SEQ ID NO:129. In another embodiment, the first repressor domain (RD1) of the repressor fusion protein... Petition 870250104944, dated 11 / 17 / 2025, p. 102 / 689 98 / 579 long-term comprises a sequence selected from the group consisting of SEQ IDs NOS: 130-1726, or a sequence with at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with it. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence selected from the group consisting of SEQ IDs NOS: 130-1726.In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence selected from the group consisting of SEQ IDs NOS: 130-224, or a sequence with at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with it. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence selected from the group consisting of SEQ IDs NOS: 130-224.In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence selected from the group consisting of SEQ IDs NOS: 130-138, or a sequence with at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with it. In another embodiment, the first repressor domain (RD1). Petition 870250104944, dated 11 / 17 / 2025, p. 103 / 689 99 / 579 of the long-term repressor fusion protein comprises a sequence selected from the group consisting of SEQ IDs NOS: 130138. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence of SEQ ID NO: 135 or a sequence with at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with it.In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence of SEQ ID NO: 131 or a sequence with at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with it. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence of SEQ ID NO: 135.In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence of SEQ ID NO: 130 or a sequence with at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with it. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence of SEQ ID. Petition 870250104944, dated 11 / 17 / 2025, p. 104 / 689 100 / 579 NO: 130. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence of SEQ ID NO: 131. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence of SEQ ID NO: 132. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence of SEQ ID NO: 133. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence of SEQ ID NO: 134. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence of SEQ ID NO: 135. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence of SEQ ID NO: 136.In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence with SEQ ID NO: 137. In another embodiment, the first repressor domain (RD1) of the long-term repressor fusion protein comprises a sequence with SEQ ID NO: 138.

[00189] In some embodiments, a second repressor domain of the long-term repressor fusion protein is a DNMT3A, comprising a sequence of SEQ ID NO: 126, or a sequence with at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity therewith. In some embodiments, the second repressor domain of the long-term repressor fusion protein comprises a sequence of SEQ ID NO: 126. Petition 870250104944, dated 11 / 17 / 2025, p. 105 / 689 101 / 579

[00190] In some embodiments, the third repressor domain of the long-term repressor fusion protein is a DNMT3L, comprising a sequence of SEQ ID NO: 127, or a sequence with at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with it.

[00191] In some embodiments, the optional fourth repressor domain of the long-term repressor fusion protein is an ADD, comprising a sequence with SEQ ID NO: 125, or a sequence with at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity therewith. In some embodiments, the optional fourth repressor domain of the long-term repressor fusion protein comprises a sequence with SEQ ID NO: 125. In some embodiments, the C-terminal of ADD is linked to the N-terminal of DNMT3A.The addition of ADD to long-term repressor fusion proteins comprising RD1, DNMT3A, and DNMT3L has been found to significantly enhance or increase long-term repression and / or epigenetic modification of the target nucleic acid, as well as repression specificity, compared to long-term repressor fusion proteins without ADD. Exemplary data for the enhanced repression and specificity of long-term repressor fusion proteins comprising ADD are presented in the Examples and are described in WO2023049742A2, incorporated by reference. Petition 870250104944, dated 11 / 17 / 2025, page 106 / 689 102 / 579 in this document. In some embodiments, the fusion protein comprises one or more linker peptides selected from the group consisting of SEQ ID NOs: 98-124, 3278-3289 (exemplary sequences are shown in Tables 5 and 58) and one or more NLS comprising a sequence selected from the group consisting of SEQ ID NOs: 30-97, 32998 and 3299. Exemplary configurations of long-term repressor fusion proteins comprising the ADD are presented in FIG. 61.

[00192] In some embodiments, the long-term repressor fusion protein comprises, from the N-terminus to the C-terminus, a DNMT3A, a DNMT3L, a DNA-binding protein, and an RD1. In some embodiments, the long-term repressor fusion protein comprises, from the N-terminus to the C-terminus, an ADD, a DNMT3A, a DNMT3L, a DNA-binding protein, and an RD1. In some embodiments, the DNA-binding protein may be a zinc finger, a TALE, or a catalytically killed CRISPR protein. In some embodiments, the long-term repressor fusion protein comprises an NLS at the N-terminus, at the C-terminus, or both. In some embodiments, the long-term repressor fusion protein comprises one or more linkers between DNMT3A and DNMT3L, between DNMT3L and the DNA-binding protein, and / or between the DNA-binding protein and RD1.

[00193] In some embodiments, the long-term repressor fusion protein comprises, from the N-terminus to the C-terminus, a DNA-binding protein, an RD1, a DNMT3A, and a DNMT3L. In some embodiments, the long-term repressor fusion protein comprises, from the N-terminus to the C-terminus, a DNA-binding protein, an RD1, an ADD, a DNMT3A, and a DNMT3L. In some embodiments, the DNA-binding protein may be a zinc finger, a TALE, or a catalytically killed CRISPR protein. In some embodiments, the long-term repressor fusion protein with Petition 870250104944, dated 11 / 17 / 2025, p. 107 / 689 103 / 579 involves an NLS at the N-terminus. In some embodiments, the long-term repressor fusion protein comprises an NLS between RD1 and DNMT3A. In some embodiments, the long-term repressor fusion protein comprises an NLS at the N-terminus, the C-terminus, or both. In some embodiments, the long-term repressor fusion protein comprises one or more linkers between an N-terminal NLS and the DNA-binding protein, between the DNA-binding protein and RD1, between RD1 and DNMT3A, or optionally, ADD and / or between DNMT3A and DNMT3A.

[00194] In some embodiments, the long-term repressor fusion protein comprises, from the N-terminus to the C-terminus, a DNA-binding protein, a DNMT3A, a DNMT3L, and an RD1. In some embodiments, the long-term repressor fusion protein comprises, from the N-terminus to the C-terminus, a DNA-binding protein, an ADD, a DNMT3A, a DNMT3L, and an RD1. In some embodiments, the DNA-binding protein may be a zinc finger, a TALE, or a catalytically killed CRISPR protein. In some embodiments, the long-term repressor fusion protein comprises an NLS at the N-terminus, at the C-terminus, or both. In some embodiments, the long-term repressor fusion protein comprises one or more linkers between an N-terminal NLS and the DNA-binding protein, between the DNA-binding protein and DNMT3A, or optionally ADD, between DNMT3A and DNMT3L, and / or between DNMT3L and RD1.

[00195] In some embodiments, the long-term repressor fusion protein comprises, from the N-terminus to the C-terminus, an RD1, a DNMT3A, a DNMT3L, and a DNA-binding protein. In some embodiments, the long-term repressor fusion protein comprises, from the N-terminus to the C-terminus, an RD1, an ADD, a DNMT3A, a DNMT3L, and a DNA-binding protein. In some embodiments, Petition 870250104944, dated 11 / 17 / 2025, p. 108 / 689 104 / 579 The DNA-binding protein can be a zinc finger, a TALE, or a catalytically killed CRISPR protein. In some embodiments, the long-term repressor fusion protein comprises an N-terminal, C-terminal, or both NLS. In some embodiments, the long-term repressor fusion protein comprises one or more linkers between RD1 and DNMT3A, or optionally ADD, between DNMT3A and DNMT3L, between DNMT3L and the DNA-binding protein, and / or between the DNA-binding protein and a C-terminal NLS.

[00196] In some embodiments, the long-term repressor fusion protein comprises, from the N-terminus to the C-terminus, a DNMT3A, a DNMT3L, an RD1, and a DNA-binding protein. In some embodiments, the long-term repressor fusion protein comprises, from the N-terminus to the C-terminus, an ADD, a DNMT3A, a DNMT3L, an RD1, and a DNA-binding protein. In some embodiments, the DNA-binding protein may be a zinc finger, a TALE, or a catalytically killed CRISPR protein. In some embodiments, the long-term repressor fusion protein comprises an NLS at the N-terminus, at the C-terminus, or both. In some embodiments, the long-term repressor fusion protein comprises one or more linkers between DNMT3A and DNMT3L, between DNMT3L and RD1, between RD1 and the DNA-binding protein, and / or between the DNA-binding protein and a C-terminal NLS.

[00197] In some embodiments, the long-term repressor fusion protein comprises, from the N-terminus to the C-terminus, a DNMT3A, a DNMT3L, an RD1, a DNA-binding protein, and a second RD1. In some embodiments, the long-term repressor fusion protein comprises, from the N-terminus to the C-terminus, an ADD, a DNMT3A, a DNMT3L, an RD1, a DNA-binding protein, and a second RD1. In one embodiment of the above, the second RD1 Petition 870250104944, dated 11 / 17 / 2025, p. 109 / 689 105 / 579 may be identical in sequence to the first RD1. In another embodiment of the foregoing, the second RD1 may be different in sequence from the first RD1. In some embodiments, the DNA-binding protein may be a zinc finger, a TALE, or a catalytically killed CRISPR protein. In some embodiments, the long-term repressor fusion protein comprises an NLS at the N-terminal, the C-terminal, or both. In some embodiments, the long-term repressor fusion protein comprises one or more linkers between DNMT3A and DNMT3L, between DNMT3L and RD1, between RD1 and the DNA-binding protein, between the DNA-binding protein and the second RD1, and / or between the second RD1 and a C-terminal NLS.

[00198] In some cases, the long-term repressor fusion protein also comprises one or more NLS. In some embodiments, the long-term repressor fusion protein comprises an N-terminal to C-terminal configuration of NLS-ADD-DNMT3A-DNMT3L-DNA-binding protein-RD1-NLS, NLS-DNA-binding protein-RD1-NLS-ADD-DNMT3A-DNMT3L, NLS-DNA-binding protein-ADD-DNMT3A-DNMT3L-RD1-NLS), NLS-RD1-ADDDNMT3A-DNMT3L-DNA-binding protein-NLS, NLS-ADDDNMT3A-DNMT3L-RD1-DNA-binding protein-NLS, or NLSADD-DNMT3A-DNMT3L-RD1-DNA-binding protein-RD1-NLS. In some embodiments, the DNA-binding protein may be a zinc finger, a TALE, or a catalytically killed CRISPR protein. In some embodiments, the long-term repressor fusion protein is able to bind to and repress or silence transcription of the PCSK9 gene.

[00199] In some embodiments of the long-term repressor fusion protein, one or more linker peptides can be inserted between any two adjacent domains of the repressor fusion protein. Petition 870250104944, dated 11 / 17 / 2025, page 110 / 689 106 / 579 long-term repressor. In some embodiments, the long-term repressor fusion protein comprises an N-terminal to C-terminal configuration of NLS-ADD-DNMT3A-Ligand2-DNMT3L-Ligand1-Ligand3A-DNA-binding protein-Ligand3B-RD1-NLS (configuration 1). In some embodiments, the long-term repressor fusion protein comprises an N-terminal to C-terminal configuration of NLS-Ligand3A-DNA-binding protein-Ligand3BR-RD1-NLS-Ligand1-ADD-DNMT3A-Ligand2-DNMT3L (configuration 2). In some embodiments, the long-term repressor fusion protein comprises an N-terminal to C-terminal configuration of NLS-Ligand3A-DNA-binding protein-Ligand1-ADD-DNMT3A-Ligand2-DNMT3L-Ligand3B-RD1-NLS (configuration 3). In some embodiments, the long-term repressor fusion protein comprises an N-terminal to C-terminal configuration of NLS-RD1Ligand3A-ADD-DNMT3A-Ligand2-DNMT3L-Ligand1-DNA-binding protein-Ligand3B-NLS (configuration 4).In some embodiments, the long-term repressor fusion protein comprises an N-terminal to C-terminal configuration of NLS-ADD-DNMT3ALigand2-DNMT3L-Ligand3A-RD1-Ligand1-DNA-binding protein-Ligand3B-NLS (configuration 5). In some embodiments, the DNA-binding protein of the preceding configurations may be a zinc finger, a TALE, or a catalytically killed CRISPR protein. Schematics of the configurations are depicted in FIG. 61. In some embodiments of the LTRP configurations 1-5, the NLS may comprise a sequence selected from the group consisting of SEQ ID NOs: 30-97, 3298, and 3299 (Tables 3 and 4), the ligand sequences may comprise independently selected sequences from the group consisting of SEQ ID NOs: 98-124, 3278-3289 (representative ligands shown in Tables 5 and 58). In some embodiments, the NLS may comprise a sequence of SEQ ID NO: 30, e. Petition 870250104944, dated 11 / 17 / 2025, p. 111 / 689 107 / 579 The binding sequences may comprise independently selected sequences from the group consisting of SEQ ID NOS: 120 and 122-124. In some embodiments, the DNA-binding protein may be a dCasX sequence selected from the group consisting of SEQ ID NOS: 4-29, or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with it.In some embodiments, the second suppressor domain is a DNMT3A domain comprising the sequence SEQ ID NO: 126, or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with it. In some embodiments, the third repressor is a DNMT3L domain comprising the sequence with SEQ ID NO: 127, or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with it.In some embodiments, the fourth repressor is an ADD domain comprising the sequence SEQ ID NO: 125, or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about . Petition 870250104944, dated 11 / 17 / 2025, p. 112 / 689 108 / 579 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with it. In some embodiments, long-term repressor fusion proteins are able to bind to and repress or silence the target nucleic acid of the PCSK9 gene.

[00200] In some embodiments, the description provides a system comprising a long-term repressor fusion protein comprising two RD1 domains, a second repressor domain, a third repressor domain, and a fourth repressor domain operationally linked to a DNA-binding protein; for example, a zinc finger, TALE, or catalytically killed CRISPR protein. In some embodiments, the DNA-binding protein comprises a catalytically killed CasX comprising a sequence selected from the group consisting of SEQ IDs NOS: 4-29, or a sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the same.In some embodiments, the sequences of the two RD1s are identical. In other embodiments, the two RD1s comprise different sequences. In some embodiments, the two RD1s are located N-terminally to the DNA-binding protein. In some embodiments, the two RD1s are located C-terminally to the DNA-binding protein. In some embodiments, one RD1 is located N-terminally to the DNA-binding protein and one RD1 is located C-terminally to the DNA-binding protein.

[00201] In some embodiments, the repressor fusion protein Petition 870250104944, dated 11 / 17 / 2025, p. 113 / 689 109 / 579 long-term comprises two RD1s. In some embodiments, the long-term repressor fusion protein comprises an N-terminal to C-terminal configuration of NLS-ADD-DNMT3A-Ligand2DNMT3L-Ligand3A-a RD1a-Ligand1-DNA-Ligand3BRD1a-Ligand4-NLS (configuration 6a), wherein the RD1a sequences are identical (see FIG. 61 as a schematic of the long-term repressor fusion protein). In some embodiments, the long-term repressor fusion protein comprises an N-terminal to C-terminal configuration of NLS-ADD-DNMT3A-Ligand2DNMT3L-Ligand3A-a RD1a-Ligand1-DNA protein-Ligand3BRD1b-Ligand4-NLS (configuration 6b), wherein the RD1a and RD1b sequences are different (see FIG. 61 as a schematic of the long-term repressor fusion protein). In some embodiments, the DNA-binding protein may be a zinc finger, a TALE, or a catalytically killed CRISPR protein.In some embodiments, a long-term repressor protein modality of the paragraph is able to bind to and repress or silence the expression of the PCSK9 gene.

[00202] In some embodiments, the description provides a long-term repressor fusion protein of configuration 6a, in which the two RD1 sequences are identical. In some embodiments of the long-term repressor fusion protein of configuration 6a, the DNA-binding protein comprises a dCasX of the sequence of SEQ ID NO: 4, or a sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with it, the first and the second. Petition 870250104944, dated 11 / 17 / 2025, p. 114 / 689 110 / 579 second copies of RD1a comprise a sequence selected from the group consisting of SEQ IDs 130-1726, or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identity with it; the second repressor domain is a DNMT3A comprising the sequence with SEQ ID 126, or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with it, the third repressor is a DNMT3L comprising the sequence SEQ ID NOs: 127, or a sequence variant with at least about 70%,at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% identity with it, the fourth suppressor is an ADD comprising the sequence with SEQ ID NO: 125, or a sequence with at least approximately 70%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately with 99% identity to it, the NLSs comprise independently selected sequences from the group consisting of SEQ ID NOS: 30-97 (Tables 3 and 4), the L1 ligand comprises a sequence, Petition 870250104944, dated 11 / 17 / 2025, p. 115 / 689111 / 579 cia of SEQ ID NO: 123, the L2 linker comprises a sequence of SEQ ID NO: 122, the L3A linker comprises a sequence of SEQ ID NO: 124, the L3B linker comprises a sequence of SEQ ID NO: 120, and the L4 linker comprises a sequence of SEQ ID NO: 3288 or SEQ ID NO: 3289. In some embodiments of the long-term repressor fusion protein of configuration 6a, the linker sequences are independently selected from the group consisting of SEQ ID NOS: 98-124, 3278-3289 (exemplary sequences shown in Tables 5 and 58).In some embodiments of the configuration 6a long-term repressor fusion protein, where two copies of RD1 are identical, each RD1 comprises a sequence selected from the group consisting of SEQ ID NOS: 130, 131, and 135, or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95% identity with it. A diagram of configuration 6a is shown in FIG. 61. In some embodiments, the configuration 6a long-term repressor fusion protein is able to form an RNP with a gRNA of the description and is able to bind to and repress or silence the target nucleic acid of the gene.

[00203] In some embodiments of the 6b configuration long-term repressor fusion protein, wherein the two RD1 sequences are different, the DNA-binding protein comprises a dCasX of the sequence of SEQ ID NO: 4, or a sequence with at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, Petition 870250104944, dated 11 / 17 / 2025, p. 116 / 689 112 / 579 at least about 98%, or at least about 99% identity with it, the first copy of the first repressor domain (RD1a) comprises a sequence selected from the group consisting of SEQ ID NOS: 130-1726, or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identity with it, the second copy of the first repressor domain (RD1b) comprises a different sequence selected from the group consisting of SEQ ID NOS: 1301726, or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identity with it, the second suppressor domain is a DNMT3A comprising the sequence SEQ ID NO: 126,or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with it, the third repressor is a DNMT3L comprising the sequence with SEQ ID NOs: 127, or a sequence variant with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about, 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identification with it, the fourth repressor is an ADD comprising the sequence Petition 870250104944, dated 11 / 17 / 2025, p. 117 / 689 113 / 579 of SEQ ID NO: 125, or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with it, the NLSs comprise sequences independently selected from the group consisting of SEQ ID NOS: 30-97 from Tables 3 and 4, the L1 ligand comprises a sequence of SEQ ID NO: 123, the L2 ligand comprises a sequence of SEQ ID NO: 122, the L3A ligand comprises a sequence of SEQ ID NO: 124, the L3B ligand comprises a sequence of SEQ ID NO: 120, and the L4 ligand comprises a sequence of SEQ ID NO: 3288 or SEQ ID NO: 3289.In some embodiments of the long-term repressor fusion protein of the 6b configuration, the linker sequences are selected from the group consisting of SEQ ID NOS: 98-124, 32783289 (exemplary sequences shown in Tables 5 and 58).In some embodiments of the 6b configuration long-term repressor fusion protein, where the two copies of RD1 are different, RD1a comprises a sequence with SEQ ID NO: 130, or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identity with it, and RD1b comprises a sequence with SEQ ID NO: 131, or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identity with it. She. In some forms of the fusion protein. Petition 870250104944, dated 11 / 17 / 2025, p. 118 / 689 114 / 579 long-term repressor of configuration 6b, wherein the two copies of RD1 are different, RD1a comprises a sequence of SEQ ID NO: 130, or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identity with it, and RD1b comprises a sequence of SEQ ID NO: 135, or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identity with it. I identify with her.In some embodiments of the 6b configuration long-term repressor fusion protein, where the two copies of RD1 are different, RD1a comprises a sequence with SEQ ID NO: 131, or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identity with it, and RD1b comprises a sequence with SEQ ID NO: 130, or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identity with it. she.In some embodiments of the 6b configuration long-term repressor fusion protein, where the two copies of RD1 are different, RD1a comprises a sequence with SEQ ID NO: 131, or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least cer. Petition 870250104944, dated 11 / 17 / 2025, p. 119 / 689 115 / 579 approximately 93%, at least about 94%, or at least about 95% identity with it, and RD1b comprises a sequence with SEQ ID NO: 135, or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least less about 93%, at least about 94%, or at least about 95% identity with it.In some embodiments of the 6b configuration long-term repressor fusion protein, where the two copies of RD1 are different, RD1a comprises a sequence with SEQ ID NO: 135, or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identity with it, and RD1b comprises a sequence with SEQ ID NO: 130, or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identity with it. she.In some embodiments of the 6b configuration long-term repressor fusion protein, where the two copies of RD1 are different, RD1a comprises a sequence with SEQ ID NO: 135, or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identity with it, and RD1b comprises a sequence with SEQ ID NO: 131, or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 91%. Petition 870250104944, dated 11 / 17 / 2025, p. 120 / 689 116 / 579 92%, at least about 93%, at least about 94%, or at least about 95% identity with it. In some embodiments of the 6b configuration long-term repressor fusion protein, where the two copies of RD1 are different, RD1a and RD1b are independently selected from the group consisting of the SEQ ID NOS sequences: 132-134 and 136-138. In some embodiments of the 6b configuration long-term repressor fusion protein, where the two copies of RD1 are different, RD1a and RD1b are independently selected from the group consisting of the SEQ ID NOS sequences: 130, 131, and 135. A schematic of the 6b configuration is shown in FIG. 61. In some embodiments, the long-term repressor fusion protein is able to form an RNP with a gRNA of the description and is able to bind to and repress or silence the target nucleic acid of the gene. Table 5: Amino acid sequences of exemplary ligands for long-term repressor fusion proteins Ligand Position Ligand Set Amino Acid Sequence SEQ ID NO L1 Initial / Original Assessment GGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE 123 Set 85 ASPAAPAPASPAAPAPSAPAA 3278 Set 43 GSGGSGGSGGSPVPSTPPTPSPSTPPTP 3279 Set 36 PPTPSPSPVPSTPPTNSSSTPPTPS 3280 Set 1 ASAAAPAAASAAASAPSAAAA 3281 Set 66 AASPAAPSAPPAAASP 3282 L2 Initial / Original Assessment SSGNSNANSRGPSFSSGLVPLSLRGS H 122 Petition 870250104944, dated 11 / 17 / 2025, p. 121 / 689 117 / 579 Ligand Position Ligand Set Amino Acid Sequence SEQ ID NO L3A Initial Assessment / Original GGSGGG 124 Set 85 IRAHGD 3283 Set 43 AFPAAPAPA 3284 Set 36 GSGNSSGSGGS 3285 Set 1 ASAAAPAAA 3286 Set 66 PPTP 3287 L3B Initial Assessment / Original GGSGGGS 120 L4v1 Initial Assessment / Original GSGS 3288 L4v2 Initial Assessment / Original GSGSGSG 3289

[00204] In some embodiments of the long-term repressor fusion protein comprising a dCasX and optionally an ADD, and which is configured as configuration 1, the long-term repressor protein comprises a sequence selected from the group consisting of SEQ ID NOS: 22836-22855 or a sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity therewith. In some embodiments of the long-term repressor fusion protein comprising a dCasX and optionally an ADD, and which is configured as configuration 1, the long-term repressor protein comprises a sequence selected from the group consisting of SEQ ID NOS: 22836-22855. In some embodiments of the Petition 870250104944, dated 11 / 17 / 2025, p. 122 / 689 118 / 579 long-term repressor fusion protein comprising a dCasX and optionally an ADD, and which is configured as configuration 1, the long-term repressor protein comprises a sequence selected from the group consisting of SEQ ID NOS: 22838 and 22847. In some embodiments of the long-term repressor fusion protein comprising a dCasX and optionally an ADD, and which is configured as configuration 1, the long-term repressor protein comprises a sequence selected from the group consisting of SEQ ID NOS: 22839 and 22848. In some embodiments of the long-term repressor fusion protein comprising a dCasX and optionally an ADD, and which is configured as configuration 1, the long-term repressor protein comprises a sequence selected from the group consisting of SEQ ID NOS: 22840 and 22849.

[00205] In some embodiments of the system comprising a DNA-binding protein, and is configured as a selected configuration from the group consisting of configuration 1, configuration 2, configuration 3, configuration 4, configuration 5, configuration 6a and configuration 6b, described above, the long-term repressor fusion protein comprises a dCasX and is capable of complexing with a gRNA with a targeting sequence complementary to the PCSK9 target nucleic acid in a cell to form an RNP and, upon binding of an RNP to the PCSK9 target nucleic acid in a cell, the target nucleic acid is epigenetically modified and transcription of the PCSK9 gene is repressed.In some embodiments, transcription of the PCSK9 gene is repressed by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about. Petition 870250104944, dated 11 / 17 / 2025, p. 123 / 689 119 / 579 95% or at least 99%. In some embodiments, the transcription of the PCSK9 gene in at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80% or more of the cells in a cell population is repressed. In some embodiments, transcriptional repression is analyzed in an in vitro assay, including cell-based assays, when compared to untreated cells or cells treated with a comparable system comprising a non-targeted spacer.In some modalities, transcriptional repression is analyzed in vivo, in cells obtained from an individual in whom the long-term repressor fusion protein and gRNA with a targeting sequence complementary to the PCSK9 target nucleic acid of a gene in a cell are administered; either as a protein and gRNA, or as nucleic acids (e.g., a gRNA and an mRNA encoding the long-term repressor fusion protein), where the individual is selected from a group consisting of mouse, rat, pig, non-human primate, and human.

[00206] Above all, preferably, repression of the PCSK9 gene results in complete inhibition of gene expression, so that no gene product is detectable. However, those skilled in the art will appreciate that incomplete inhibition can still be useful and desirable for various applications. In some embodiments, repression of PCSK9 gene transcription by a system embodiment is sustained for at least about 8 hours, at least about 1 day, at least about 7 days, at least 2 weeks, or by Petition 870250104944, dated 11 / 17 / 2025, page 124 / 689 120 / 579 less about 3 weeks, at least about 1 month, or at least about 2 months, when tested in an in vitro assay, including cell-based assays. In some embodiments, the repression of PCSK9 gene transcription by the system compositions is sustained for at least about 7 days, at least 2 weeks, at least about 3 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months in target cells from an individual when the composition comprising the system of description is administered as a therapeutically effective dose. In some embodiments, the individual is selected from the group consisting of mouse, rat, pig, non-human primate, and human.In some embodiments, the use of an LTRP of configurations 1, 4, 5, and 6, when used in an LTRP:gRNA system of the embodiments, results in off-target methylation or off-target activity that is less than about 10%. 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, or less than about 0.1% in cells.

[00207] In some embodiments, transcriptional repression of the PCSK9 gene in cells treated with an LTRP:gRNA system of the embodiments may be inherited and is stable across one or more cell divisions. In some embodiments, transcriptional repression is stable across 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 cell divisions, or more. V. mRNA compositions encoding long-term repressor fusion proteins

[00208] In another aspect, the description refers to messenger RNA (mRNA) compositions comprising sequences of the components Petition 870250104944, dated 11 / 17 / 2025, p. 125 / 689 121 / 579 individual tes, as well as the complete mRNA sequences of the long-term repressor domain fusion protein constructs described. In some embodiments, mRNA compositions are useful in transcriptional repression and epigenetic modification of a gene, for example, a PCSK9 gene, when used to express the long-term repressor fusion protein. In some cases, the mRNA is designed for use in certain delivery formulations; for example, nanoparticles, such as synthetic nanoparticles or lipid nanoparticles (LNPs). The description also provides methods used to design the mRNA sequences of the mRNAs used in the compositions. In some cases, an mRNA encoding the long-term repressor fusion protein can be co-formulated in a nanoparticle (such as an LNP) with a gRNA comprising a targeting sequence complementary to a sequence of a PCSK9 gene.Through delivery to the target cell via LNP, the long-term repressor domain fusion protein is expressed from mRNA and can complex with gRNA as an RNP capable of binding to the target nucleic acid of PCSK9. In other cases, the mRNA encoding the long-term repressor domain fusion protein and gRNA can be formulated into separate nanoparticles and delivered separately or as a mixture.

[00209] In some embodiments, the mRNA compositions described have been modified to result in one or more enhanced characteristics compared to an unmodified mRNA encoding the same long-term repressor protein and therefore may have a positive impact on the effectiveness of mRNA-based delivery. Exemplary enhanced characteristics of the mRNA described in this document include, among others, improved expression upon delivery to a cell, reduced immunogenicity, increased stability, and enhanced manufacturability compared Petition 870250104944, dated 11 / 17 / 2025, page 126 / 689 122 / 579 with unmodified mRNA. In some cases, modifications to the mRNA result in an improved characteristic of at least about 1.1 to about 100,000 times improved compared to unmodified mRNA. In some embodiments, an improved feature of the modified mRNA is at least about 1.1 to about 10,000 times better, at least about 1.1 to about 1,000 times better, at least about 1.1 to about 500 times better, at least about 1.1 to about 400 times better, at least about 1.1 to about 300 times better, at least about 1.1 to about 200 times better, at least about 1.1 to about 100 times better, at least about 1.1 to about 50 times better, at least about 1.1 to about 40 times better, at least about 1.1 to about 30 times better, at least about 1.1 to about 20 times better, at least about 1.1 to about 10 times better, at least about 1.1 to about 9 times better, by minus about 1,1 to approximately 8 times better, at least approximately 1.1 to approximately 7 times better, at least approximately 1.1 to approximately 6 times better, at least approximately 1.1 to approximately 5 times better, at least approximately 1.1 to approximately 4 times better, at least approximately 1.1 to approximately 3 times better, at least approximately 1.1 to approximately 2 times better, at least approximately 1.1 to approximately 1.5 times better, at least approximately 1.5 to approximately 3 times better, at least approximately 1.5 to approximately 4 times better, at least approximately 1.5 to approximately 5 times better, at least approximately 1.5 to approximately 10 times better, at least approximately 5 to approximately 10 times better, at least approximately 10 to approximately 20 times better, at least 10 to approximately 30 times better, at least less than 10 to approximately 50 times better, or at least 10 to about 100 times better, compared to unmodified mRNA. In some embodiments, an unimproved feature of the modified mRNA is at least about 10 to about 1,000 times better. Petition 870250104944, dated 11 / 17 / 2025, page 127 / 689 123 / 579 times improved compared to unmodified mRNA.

[00210] Optimization of coding sequences and untranslated regions (UTRs) can be useful when distributing an mRNA that encodes a protein of interest, as opposed to a DNA template that would be transcribed into an mRNA. DNA templates are long-lived, can replicate, and can produce many RNA transcripts over their lifetimes. For DNA templates, transcription efficiency and pre-mRNA processing are the main determinants of protein expression levels. In contrast, mRNAs generally have a much shorter half-life, on the order of hours, as they are vulnerable to degradation in the cytoplasm and cannot produce more copies of themselves.As such, mRNA stability and translation efficiency can be determinants of protein expression levels for mRNA-based delivery, and specific UTR sequences and coding sequences that dictate mRNA stability and translation efficiency can therefore be leveraged to improve the effectiveness of mRNA-based delivery. a. 5' cap

[00211] In some embodiments of the mRNA described, the mRNA comprises a 5' cap attached 5' to the 5' UTR of the mRNA sequence of any of the embodiments described in this document. In some embodiments, the 5' cap is a 7-methylguanylate cap. In some embodiments, the 5' cap comprises m7G(5')ppp(5')mAG. In other embodiments, the 5' cap comprises m7G(5')ppp(5'(A,G(5')ppp(5')A or G(5')ppp(5')G. Exemplary caps are known in the art and described, for example, in WO 2017 / 053297, the contents of which are incorporated by reference herein. b. Untranslated region (UTR) 5'

[00212] The 5' UTR of an mRNA molecule can be a key determinant of both mRNA stability and efficiency. Petition 870250104944, dated 11 / 17 / 2025, page 128 / 689 124 / 579 with which it is translated into protein. Specifically, the 5' UTR, together with the 5' cap structure, serves as a binding site and recruitment platform for the translation pre-initiation complex, as well as additional regulatory proteins that can positively or negatively affect translation. Structures within the 5' UTR can enhance translation by recruiting initiation factors or other protein or RNA factors, reduce translation by physically blocking ribosome binding and scanning, and contribute to mRNA stability by affecting both hydrolysis and nuclease digestion.

[00213] An exemplary 5' UTR sequence for use in the mRNA description is provided in Table 6. Table 6 lists the RNA sequence and the RNA sequence with N1-methyl-pseudouridine substituted in place of uridine. Table 6: UTR 5' sequences SEQ ID NO Nucleic acid sequence* Description 22787 AAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA RNA sequence of UTR 5' 3300 AAAmΨAAGAGAGAAAAGAA- GAGmΨAAGAAGAAAmΨAmΨAAGA N1-methylpseudouridine substituted RNA sequence of UTR 5' *'mψ' = N1-methyl-pseudouridine

[00214] In some embodiments, the UTR 5' comprises the sequence with SEQ ID NO: 22787, or a sequence with at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% identity with it. In some embodiments, the UTR 5' comprises the sequence with SEQ ID NO: 22787. In some embodiments, the UTR 5' consists of the sequence with SEQ ID NO: Petition 870250104944, dated 11 / 17 / 2025, p. 129 / 689 125 / 579 22787. In some embodiments, the UTR 5' comprises the sequence with SEQ ID NO: 3300, or a sequence with at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% identity with it. In some embodiments, the UTR 5' comprises the sequence with SEQ ID NO: 3300. In some embodiments, the UTR 5' consists of the sequence with SEQ ID NO: 3300. c. UTR 3'

[00215] 3' UTR sequences can impact mRNA stability and translation efficiency, and thus can determine both subcellular localization and tissue-specific expression. Factors influencing these properties include microRNA binding sites, AU-rich elements that recruit an array of RNA-binding proteins, Pumilio-binding elements, and other binding sites for RNA-binding proteins. While many of these interactions with the 3' UTR are known to negatively affect stability or expression, some can enhance translation. The effects of a 3' UTR sequence can be highly cell-type specific due to differential expression of microRNAs and RNA-binding proteins, providing opportunities to manipulate tissue-specific expression in a therapeutic mRNA. In some embodiments, the 3' UTR for use in the mRNA described is a mouse 3' UTR.In some embodiments, UTR 3' is a UTR 3' of the mouse HBA gene from Table 7.

[00216] UTR 3' sequences exemplifying the description are provided in Table 7. Table 7 lists the RNA sequence and the RNA sequence with N1-methyl-pseudouridine substituted in place of uridine. Table 7: UTR 3' sequences Petition 870250104944, dated 11 / 17 / 2025, p. 130 / 689 126 / 579 SEQ ID NO Nucleic acid sequence* Description 3125 GCUGCCUUCUGCGGGGCUUGCCUUCUG GCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAG 3' UTR RNA sequence 3310 GCmΨGCCmΨmΨCmΨGCGGGGCmΨmΨG CCmΨmΨCmΨGGCCAmΨGCCCmΨmΨCm ΨmΨCmΨCmΨCCCmΨmΨGCACCmΨGmΨAC- CmΨCmΨmΨGGmΨCmΨmΨmΨGAAmΨAA AGCCmΨGAGmΨAGGAAG RNA sequence replaced by N1-methylpseudouridine from the 3' UTR *'mψ' = Nl-methyl-pseudouridine

[00217] In some embodiments, UTR 3' comprises the sequence with SEQ ID NO: 3125, or a sequence with at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% identity with it. In some embodiments, UTR 3' comprises the sequence with SEQ ID NO: 3125. In some embodiments, UTR 3' consists of the sequence with SEQ ID NO: 3125. In some embodiments, UTR 3' comprises the sequence with SEQ ID NO: 3310, or a sequence with at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% identity with it. In some modes, UTR 3' comprises the sequence of SEQ ID NO: 3310. In some modes, UTR 3' consists of the sequence of SEQ ID NO: 3310. d. Poly(A) sequence

[00218] The inclusion of a 3' poly(A) tail in an mRNA sequence can contribute to the stability and efficiency of mRNA translation. Generally, longer poly(A) tails are associated with increased mRNA stability, thus allowing its Petition 870250104944, dated 11 / 17 / 2025, p. 131 / 689 127 / 579 translation and promoting high protein expression.

[00219] In some embodiments, an mRNA described comprises a poly(A) sequence with at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 185, or at least about 190 adenine nucleotides. In some embodiments, the poly(A) sequence of an mRNA described comprises 80 adenine nucleotides. In some embodiments, the poly(A) sequence comprises the nucleic acid sequence of AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 3057). In some embodiments, the poly(A) sequence comprises the nucleic acid sequence of AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 3307). e. Sequence Modifications to mRNA

[00220] In some embodiments, an mRNA sequence from the description has been modified by codon optimization of the sequence encoding the long-term repressor protein using one or more parameters to enhance expression in the target cell. Non-limiting examples of such parameters include codon usage in human host cells (e.g., using the codon adaptation index (CAI)), minimizing rare codons, using codon usage tables derived from biological products intended for use as Petition 870250104944, dated 11 / 17 / 2025, p. 132 / 689 128 / 579 therapeutic, using an mRNA stability index or reducing the GC content. Codon optimization methods and codon use in various organisms are known in the art. See, for example, www.genscript.com / tools / codon-frequency-table.

[00221] In some embodiments, mRNA sequences for long-term repressor protein constructs with codon optimized for expression in a human cell are provided in this document. In other cases, various naturally occurring or modified nucleosides may be used to produce the modified mRNA according to the present invention.In some embodiments, an mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); Nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, pseudouridine (e.g., N-1-methylpseudouridine), 2-thiouridine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); Intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages).In some embodiments, the mRNA comprises one or more non-standard nucleotide residues. The non-standard nucleotide residues may include, for example, 5-methylcytidine (“5mC”), pseudouridine (“ψυ”), and / or 2-thiouridine (“2sU”). In a particular embodiment, one or more or all of the uridine residues in the mRNA described are replaced by 1-methylpseudouridine. In some embodiments, all of the residues... Petition 870250104944, dated 11 / 17 / 2025, p. 133 / 689 129 / 579 uridine residues in the mRNA description are replaced by 1-methylpseudouridine. See, for example, U.S. Pat. No. 8,278,036 or WO2011012316, incorporated by reference herein, for a discussion of such residues and their incorporation into mRNA. In some embodiments, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or 100% of the uridine nucleosides in the mRNA sequence are replaced by N1-methylpseudouridine.

[00222] In some embodiments, modifications to mRNA by codon optimization and / or substitution of modified nucleotides in the sequence result in an improved characteristic of at least about 1.1 to about 100,000 times improved compared to unmodified mRNA. In some embodiments, an improved feature of the modified mRNA is at least about 1.1 to about 10,000 times better, at least about 1.1 to about 1,000 times better, at least about 1.1 to about 500 times better, at least about 1.1 to about 400 times better, at least about 1.1 to about 300 times better, at least about 1.1 to about 200 times better, at least about 1.1 to about 100 times better, at least about 1.1 to about 50 times better, at least about 1.1 to about 40 times better, at least about 1.1 to about 30 times better, at least about 1.1 to about 20 times better, at least about 1.1 to about 10 times better.at least approximately 1.1 to approximately 9 times better, at least approximately 1.1 to approximately 8 times better, at least approximately 1.1 to approximately 7 times better, at least approximately 1.1 to approximately 6 times better, at least approximately 1.1 to approximately 5 times better, at least approximately 1.1 to approximately 4 times better, at least approximately 1.1 to approximately 3 times better, at least approximately 1.1 to approximately 2 times better, at least approximately 1.1 to approximately 1.5 times better, Petition 870250104944, dated 11 / 17 / 2025, page 134 / 689 130 / 579 at least about 1.5 to about 3 times better, at least about 1.5 to about 4 times better, at least about 1.5 to about 5 times better, at least about 1.5 to about 10 times better, at least about 5 to about 10 times better, at least about 10 to about 20 times better, at least 10 to about 30 times better, at least 10 to about 50 times better, or at least 10 to about 100 times better compared to unmodified mRNA. In some embodiments, an unimproved feature of the modified mRNA is at least about 10 to about 1,000 times improved compared to unmodified mRNA. f. LTRP mRNA component sequences

[00223] The present invention provides mRNA comprising sequences encoding the components used in the long-term repressor fusion proteins of the description. In some embodiments, the mRNA comprises sequences encoding DNA-binding proteins, including TALE, ZF, and catalytically killed CRISPR proteins. In some embodiments, the mRNA comprises the sequence with SEQ ID NO: 3274 encoding dCasX 515 (SEQ ID NO: 6), or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with it.In some embodiments, the mRNA comprises the sequence SEQ ID NO: 3276 encoding dCasX 812 (SEQ ID NO: 29), or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with it. In some embodiments, the mRNA comprises the sequence SEQ ID. Petition 870250104944, dated 11 / 17 / 2025, p. 135 / 689 131 / 579 NO: 3275 encoding dCasX 491 (SEQ ID NO:4), or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the same. In some embodiments, the mRNA comprises the sequence with SEQ ID NO: 22736 that encodes dCasX 676 (SEQ ID NO: 28), or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with it.

[00224] In some embodiments, the mRNA sequence encoding dCasX 491 (SEQ ID NO: 4) has pseudouridine nucleosides that replace one or more, or all, of the uridines in the sequence (SEQ ID NO: 22729). In some embodiments, the mRNA sequence encoding dCasX 515 (SEQ ID NO: 6) has pseudouridine nucleosides that replace one or more, or all, of the uridines in the sequence (SEQ ID NO: 22728). In some embodiments, the mRNA sequence encoding dCasX 676 (SEQ ID NO: 28) has pseudouridine nucleosides that replace one or more, or all, of the uridines in the sequence. In some embodiments, the mRNA sequence encoding dCasX 812 (SEQ ID NO: 29) has pseudouridine nucleosides that replace one or more, or all, of the uridines in the sequence (SEQ ID NOS: 22730 or 22731).

[00225] In some embodiments, the description provides mRNA sequences that encode the RD1 domains. In some embodiments, the mRNA sequence encoding RD1 comprises a sequence selected from the group consisting of SEQ ID NOS: 3120, 22804, and 3334-6527, or a sequence with at least approximately Petition 870250104944, dated 11 / 17 / 2025, p. 136 / 689 132 / 579 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95% identity with it. In some embodiments, the mRNA sequence encoding RD1 comprises a sequence selected from the group consisting of SEQ ID NOs: 3120, 22804, and 3334-6527. In another embodiment, the mRNA sequence encoding RD1 comprises a sequence selected from the group consisting of SEQ ID NOS: 3334-3342 and 4931-4939, or a sequence with at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with it.In another embodiment, the mRNA sequence encoding RD1 comprises a sequence selected from the group consisting of SEQ ID NOs: 3334-3342 and 4931-4939. In another embodiment, the mRNA sequence encoding RD1 comprises a sequence of SEQ ID NO: 3334 or SEQ ID NO: 4931, or a sequence with at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with it. In another embodiment, the mRNA sequence encoding RD1 comprises a sequence with SEQ ID NO: 3334 or SEQ ID NO: 4931. In another embodiment, the mRNA sequence encoding RD1 comprises a sequence with SEQ ID NO: 3335 or SEQ ID NO: 4932, or a sequence with at least about 70%, at least about 80%, at least about 90%, of the RD1 sequence. Petition 870250104944, dated 11 / 17 / 2025, p. 137 / 689 133 / 579 minus approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% identity with it. In another embodiment, the mRNA sequence encoding RD1 comprises a sequence with SEQ ID NO: 3335 or SEQ ID NO: 4932. In another embodiment, the mRNA sequence encoding RD1 comprises a sequence with SEQ ID NO: 3336 or SEQ ID NO: 4933, or a sequence with at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with it. In another embodiment, the mRNA sequence encoding RD1 comprises a sequence with SEQ ID NO: 3336 or SEQ ID NO: 4933.In another embodiment, the mRNA sequence encoding RD1 comprises a sequence of SEQ ID NO: 3337 or SEQ ID NO: 4934, or a sequence with at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity therewith. In another embodiment, the mRNA sequence encoding RD1 comprises a sequence with SEQ ID NO: 3337 or SEQ ID NO: 4934. In another embodiment, the mRNA sequence encoding RD1 comprises a sequence with SEQ ID NO: 3338 or SEQ ID NO: 4935, or a sequence with at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about... Petition 870250104944, dated 11 / 17 / 2025, p. 138 / 689 134 / 57994%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identification with her. In another embodiment, the mRNA sequence encoding RD1 comprises a sequence with SEQ ID NO: 3338 or SEQ ID NO: 4935. In another embodiment, the mRNA sequence encoding RD1 comprises a sequence with SEQ ID NO: 3339 or SEQ ID NO: 4936, or a sequence with at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with it. In another embodiment, the mRNA sequence encoding RD1 comprises a sequence with SEQ ID NO: 3339 or SEQ ID NO: 4936.In another embodiment, the mRNA sequence encoding RD1 comprises a sequence with SEQ ID NO: 3340 or SEQ ID NO: 4937, or a sequence with at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with it. In another embodiment, the mRNA sequence encoding RD1 comprises a sequence with SEQ ID NO: 3340 or SEQ ID NO: 4937.In another embodiment, the mRNA sequence encoding RD1 comprises a sequence of SEQ ID NO: 3341 or SEQ ID NO: 4938, or a sequence with at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about... Petition 870250104944, dated 11 / 17 / 2025, p. 139 / 689 135 / 579 98%, or at least about 99% identity with it. In another embodiment, the mRNA sequence encoding RD1 comprises a sequence with SEQ ID NO: 3341 or SEQ ID NO: 4938.

[00226] In some embodiments, the mRNA comprises a sequence encoding the second repressor domain. In some embodiments, the second repressor domain comprises DNMT3A. In some embodiments, the mRNA sequence encoding DNMT3A comprises a sequence with SEQ ID NO: 3128 or SEQ ID NO: 3331, or a sequence with at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with it. In some embodiments, the mRNA sequence encoding DNMT3A comprises a sequence with SEQ ID NO: 3128 or SEQ ID NO: 3331.

[00227] In some embodiments, the mRNA comprises a sequence encoding the third repressor domain. In some embodiments, the third repressor domain comprises DNMT3L. In some embodiments, the mRNA sequence encoding DNMT3L comprises a sequence with SEQ ID NO: 3119 or SEQ ID NO: 3332, or a sequence with at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity with it. In some embodiments, the mRNA sequence encoding DNMT3L comprises a sequence with SEQ ID NO: 3119 or SEQ ID NO: 3332.

[00228] In some embodiments, mRNA comprises a se Petition 870250104944, dated 11 / 17 / 2025, p. 140 / 689 136 / 579 sequence encoding the fourth repressor domain. In some embodiments, the fourth repressor domain comprises ADD. In some embodiments, the mRNA sequence encoding ADD comprises a sequence with SEQ ID NO: 3296 or SEQ ID NO: 22726, or a sequence with at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity with it. In some embodiments, the mRNA sequence encoding ADD comprises a sequence with SEQ ID NO: 3296 or SEQ ID NO: 22726.

[00229] In some embodiments, the mRNA described comprises a Kozak sequence. In some embodiments, the Kozak sequence comprises a GCCACCAUGG (SEQ ID NO: 22805). In some embodiments, an mRNA described comprises a Kozak sequence and two bases upstream of the NLS (to produce methionine and alanine upstream of the NLS). In some embodiments, the mRNA comprises the sequence GCCACCAUGGCC (SEQ ID NO: 22806) between UTR 5' and the sequence encoding NLS.

[00230] In another embodiment, the mRNA comprises an NLS. In some embodiments, the sequence encoding the NLS comprises a sequence selected from the group consisting of SEQ ID NO: 3291 and SEQ ID NO: 22807, or a sequence with at least about 70%, at least about 80%, or at least about 90% identity with it. In another embodiment, the mRNA comprises a sequence selected from the group consisting of SEQ ID NO: 3291 and SEQ ID NO: 22807. In another embodiment, for examples of these embodiments in which the long-term repressor protein comprises more than one NLS, the mRNA comprises two or more NLSs. Petition 870250104944, dated 11 / 17 / 2025, p. 141 / 689 137 / 579 sequences selected independently from the group consisting of SEQ ID NO: 3291 and SEQ ID NO: 22807.

[00231] In some embodiments, the mRNA comprises sequences that encode the ligands of the long-term repressor fusion protein. In some embodiments, the sequence encoding the ligand comprises a sequence selected from the group consisting of SEQ ID NOS: 3312-3327 and 22808-22828, or a sequence with at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95% identity therewith. In some embodiments, the sequence encoding the ligand comprises a sequence selected from the group consisting of SEQ ID NOS: 3312-3327 and 22808-22828.In some embodiments, for example those embodiments where the long-term repressor fusion protein comprises more than one ligand, the sequences encoding the ligands are independently selected from the group consisting of SEQ ID NOS: 3312-3327 and 22808-22828, or a sequence with at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95% identity with it.

[00232] In some embodiments, the mRNA comprises a sequence encoding the long-term repressor fusion protein of the configurations provided in this document. In some embodiments, the mRNA encodes the configuration 1 long-term repressor fusion protein and the mRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 6529-8134, 974111347, 14628-16233 and 17841-19446, or a sequence with at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least Petition 870250104944, dated 11 / 17 / 2025, p. 142 / 689 138 / 579 approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99% identity with it. In some embodiments, the mRNA comprises a sequence that is selected from the group consisting of SEQ ID NOS: 6529-8134, 9741-11347, 14628-16233 and 17841-19446.

[00233] In some embodiments, the mRNA comprises a sequence encoding the configuration-1 long-term repressor fusion protein and comprises a sequence encoding RD1 comprising a sequence of SEQ ID NO: 130. In some embodiments, the mRNA comprises a sequence of SEQ ID NOS: 6531, 9744, 14630, or 17843, or a sequence with at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with it. In some embodiments, the mRNA comprises a sequence with SEQ ID NOS: 6531, 9744, 14630, or 17843. In some embodiments, the mRNA comprises a sequence encoding the configuration 1 long-term repressor fusion protein and comprises a sequence encoding RD1 with SEQ ID NO: 131.In some forms, the mRNA comprises a SEQ ID sequence NOS: 6529, 9742, 14628 or . 17841, or a sequence with at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity therewith. In some embodiments, the mRNA comprises a SEQ ID sequence NOS: 6529, 9742, 14628, or 17841. In some Petition 870250104944, dated 11 / 17 / 2025, p. 143 / 689 In embodiments 139 / 579, the mRNA comprises a sequence encoding the configuration-1 long-term repressor fusion protein, and the mRNA comprises a sequence encoding RD1 of SEQ ID NO: 132. In some embodiments, the mRNA comprises a sequence of SEQ ID NOS: 6535, 9748, 14634, or 17847, or a sequence with at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity therewith. In some embodiments, the mRNA comprises a sequence with SEQ ID NOS: 6535, 9748, 14634, or 17847. In some embodiments, the mRNA comprises a sequence encoding the configuration 1 long-term repressor fusion protein and comprises a sequence encoding RD1 with SEQ ID NO: 133.In some embodiments, the mRNA comprises a sequence with SEQ ID NOS: 6536, 9749, 14635, or 17848, or a sequence with at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity therewith. In some embodiments, the mRNA comprises a sequence with SEQ ID NOS: 6536, 9749, 14635, or 17848. In some embodiments, the mRNA comprises a sequence encoding the configuration 1 long-term repressor fusion protein, and the mRNA comprises a sequence encoding RD1 with SEQ ID NO: 134. In some embodiments, the mRNA comprises a sequence with SEQ ID NOS: 6534, 9747, 14633, or 17846, or a sequence with at least about 80%, at least about 90%, at least about 91%, at least cerPetição 870250104944, dated 11 / 17 / 2025, p. 144 / 689. 140 / 579 approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least approximately 99% of identity with it. In some embodiments, the mRNA comprises a sequence with SEQ ID NOS: 6534, 9747, 14633, or 17846. In some embodiments, the mRNA comprises a sequence encoding the configuration 1 long-term repressor fusion protein and comprises a sequence encoding RD1 with SEQ ID NO: 135. In some embodiments, the mRNA comprises a sequence with SEQ ID NOS: 6530, 9743, 14629, or 17842, or a sequence with at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity therewith.In some embodiments, the mRNA comprises a sequence with SEQ ID NOS: 6530, 9743, 14629, or 17842. In some embodiments, the mRNA comprises a sequence encoding the configuration 1 long-term repressor fusion protein and comprises a sequence encoding RD1 with SEQ ID NO: 136. In some embodiments, the mRNA comprises a sequence with SEQ ID NOS: 6537, 9750, 14636, or 17849, or a sequence with at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity therewith. In some embodiments, the mRNA comprises a sequence with SEQ ID NOS: 6537, 9750, 14636, or 17849. In some embodiments, the mRNA comprises a sequence encoding the long repressor fusion protein. Petition 870250104944, dated 11 / 17 / 2025, p. 145 / 689 141 / 579 configuration timeframe 1 and comprises a sequence encoding RD1 of SEQ ID NO: 137. In some embodiments, the mRNA comprises a sequence of SEQ ID NOS: 6533, 9746, 14632 or 17845, or a sequence with at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with it. In some embodiments, the mRNA comprises a sequence with SEQ ID NOS: 6533, 9746, 14632, or 17845. In some embodiments, the mRNA comprises a sequence encoding the configuration 1 long-term repressor fusion protein and comprises a sequence encoding RD1 with SEQ ID NO: 138.In some embodiments, the mRNA comprises a sequence with SEQ ID NOS: 6532, 9745, 14631, or 17844, or a sequence with at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity therewith. In some embodiments, the mRNA comprises a sequence with SEQ ID NOS: 6532, 9745, 14631, or 17844.

[00234] The description provides an mRNA encoding a configuration 5 long-term repressor fusion protein. In some embodiments, the mRNA comprises a sequence that is selected from the group consisting of SEQ ID NOS: 8135-9740, 11348-12953, 16234-17839, and 19447-21052, or a sequence with at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, by Petition 870250104944, dated 11 / 17 / 2025, p. 146 / 689 142 / 579 less about 97%, at least about 98%, or at least about 99% identity with it. In some embodiments, the mRNA comprises a sequence that is selected from the group consisting of SEQ ID NOS: 8135-9740, 11348-12953, 16234-17839 and 19447-21052.

[00235] The description provides an mRNA encoding a long-term repressor fusion protein of the 6a configuration, where the encoded RD1 is identical. In some embodiments, the mRNA comprises a sequence that is selected from the group consisting of SEQ ID NOS: 12954-14553 and 21053-22652, or a sequence with at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with it. In some embodiments, the mRNA comprises a sequence that is selected from the group consisting of SEQ ID NOS: 12954-14553 and 21053-22652.

[00236] The description provides an mRNA encoding a long-term repressor fusion protein of configuration 6b, where the encoded RD1 is different. In some embodiments, the mRNA comprises a sequence that is selected from the group consisting of SEQ ID NOS: 14554-14626 and 22653-22725, or a sequence with at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with it. In some embodiments, the mRNA comprises a sequence that is selected from the group consisting of SEQ ID NOS: 14554-14626 and 22653-22725.

[00237] In some embodiments of the mRNA that encodes a Petition 870250104944, dated 11 / 17 / 2025, p. 147 / 689 143 / 579 long-term repressor fusion protein of LTRP:gRNA systems from the description, upon distribution of the system to a target cell, the mRNA is expressed and the long-term repressor fusion protein is able to complex with a gRNA and bind to the target DNA, resulting in repression or silencing of PCSK9 gene transcription in the cell. VI. Nucleic Acids: Guide Systems

[00238] In another aspect, the description refers to specifically designed guide ribonucleic acids (gRNAs) comprising a scaffold and a complementary linked targeting sequence (and therefore capable of hybridizing with) a target nucleic acid sequence of a PCSK9 gene. The gRNA described in this document can be used with long-term repressor proteins and systems comprising them to repress transcription of the target nucleic acid PCSK9 in a eukaryotic cell. As used in this document, the term gRNA covers naturally occurring molecules and gRNA variants, including chimeric gRNA variants comprising different gRNA domains. The gRNAs in the description comprise a scaffold and a targeting sequence complementary to a target nucleic acid of a cell linked to the 3' end of the scaffold.

[00239] In some embodiments, systems comprising an mRNA encoding a long-term repressor fusion protein comprise a dCasX protein and one or more gRNAs, upon expression of the dCasX protein in a transfected cell, form a ribonucleoprotein (RNP) complex with the gRNA, which can target and bind to specific sites in the target nucleic acid sequence of the cell. The gRNA provides target specificity to the complex by including a targeting sequence (or spacer) with a nucleotide sequence that is complementary to a sequence. Petition 870250104944, dated 11 / 17 / 2025, page 148 / 689 144 / 579 cia of the target nucleic acid sequence, while the long-term repressor fusion protein of the system provides site-specific activity, such as binding and repression of the target sequence that is guided to a target site (e.g., stabilized at a target site) within a target nucleic acid sequence by virtue of its association with gRNA.

[00240] Modalities of gRNAs and mRNA and gRNA formulations for use in the repression and / or epigenetic modification of PCSK9 target nucleic acids are described below in this document. a. gRNA variants and reference gRNA

[00241] As used in this document, a reference gRNA refers to a CRISPR guide ribonucleic acid comprising a wild-type sequence of a naturally occurring gRNA. In some embodiments, a gRNA scaffold from the description may be subjected to one or more mutagenesis methods, such as the mutagenesis methods described in WO2023235818A2, WO2022120095A1, and WO2020247882A1, incorporated by reference in this document, which may include Deep Mutational Evolution (DME), deep mutational scanning (DMS), error-prone PCR, cassette mutagenesis, random mutagenesis, scaled extension PCR, gene shuffling, domain switching, or chemical modification in order to generate one or more gRNA variants with enhanced or varied properties relative to the modified gRNA scaffold.The activity of the gRNA scaffold from which a gRNA variant was derived can be used as a benchmark against which the activity of the gRNA variant is compared, thereby measuring improvements in the function or other characteristics of the gRNA scaffold.

[00242] Table 8 provides the sequences of the tracr and reference gRNA scaffold sequences. In some embodiments, the of Petition 870250104944, dated 11 / 17 / 2025, p. 149 / 689 Description 145 / 579 provides gRNA variants in which the gRNA has a framework comprising a sequence with one or more nucleotide modifications relative to a reference gRNA sequence from any of the SEQ IDs NOS: 1731-1743 in Table 8. Table 8: tracr sequences and reference gRNA scaffold SEQ ID NO. Nucleotide sequence 1731 ACAUCUGGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGACUAUGUCGUAUGGACGAAGCGCUUAUUUAUCGGAGAGAAACCGAUAAGUAAAACGCAUCAAAG 1732 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG 1733 ACAUCUGGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGACUAUGUCGUAUGGACGAAGCGCUUAUUUAUCGGAGA 1734 ACAUCUGGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGACUAUGUCGUAUGGACGAAGCGCUUAUUUAUCGG 1735 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGAGA 1736 GUUUACACACUCCCUCUCAUAGGGU 1738 GUUUACACACUCCCUCUCAUGAGGU 1739 UUUUACAUACCCCCUCUCAUGGGAU 1740 GUUUACACACUCCCUCUCAUGGGGG 1741 CCAGCGACUAUGUCGUAUGG 1742 GCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGC Petition 870250104944, dated 11 / 17 / 2025, page 150 / 689 146 / 579 SEQ ID NO. Nucleotide sequence 1743 GGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGA b. gRNA domains and their functions

[00243] The gRNAs of the systems described comprise two segments: a targeting sequence and a protein-binding segment. The targeting segment of a gRNA includes a nucleotide sequence (referred to interchangeably as a spacer, a target, or a targeting sequence) that is complementary to (and therefore hybridizes with) a specific sequence (a target site) within the target nucleic acid sequence (e.g., a strand of a double-stranded target DNA, a target ssRNA, a target ssDNA, etc.), described in more detail below. The targeting sequence of a gRNA is capable of binding to a target nucleic acid sequence, including, in the context of the present invention, a coding sequence, a complement to a coding sequence, a non-coding sequence, and accessory elements.The protein-binding segment (or activator or protein-binding sequence) of the gRNA interacts with (e.g., binds to) a dCasX protein as a complex, forming an RNP (described in more detail below). As used in this document, framework refers to all parts of the guide except for the targeting sequence, which is composed of several regions, described in more detail below. The properties and characteristics of CasX gRNA, both wild-type and variants, are described in WO2020247882A1, US20220220508A1 and. WO2022120095A1, incorporated by reference in this document.

[00244] In the case of a reference gRNA, the gRNA occurs naturally Petition 870250104944, dated 11 / 17 / 2025, p. 151 / 689 147 / 579 Generally as a double-stranded guide RNA (dgRNA), in which the director and activator portions each have a duplex-forming segment that are complementary to each other and hybridize to form a double-stranded duplex (dsRNA to gRNA duplex). The term director or director RNA is used in this document to refer to a crRNA-like molecule (crRNA: CRISPR RNA) of a CasX double-stranded guide RNA (and therefore of a CasX single-stranded guide RNA when the activator and director are linked to each other; for example, by intervening nucleotides). The crRNA has a 5' region that anneals to the tracrRNA followed by the nucleotides of the targeting sequence.In the case of gRNA for use in the systems described, the scaffolds are designed so that the activating and targeting portions are covalently linked to each other (rather than hybridizing to each other) and comprise a single molecule and can be referred to as a single-molecule gRNA, single guide RNA, single-molecule guide RNA, single-molecule guide RNA, or sgRNA. The gRNA variants described for use in the systems are all single-molecule versions.

[00245] Collectively, the assembled gRNAs described comprise distinct structured regions or domains: the RNA triplex, the scaffold stem loop, the extended stem loop, the pseudo-knot, and the targeting sequence which, in the embodiments described, is complementary to a target nucleic acid sequence and is located at the 3' end of the gRNA. The RNA triplex, the scaffold stem loop, the pseudo-knot, and the extended stem loop, together with the unstructured triplex loop that joins portions of the triplex, are referred to as the gRNA scaffold. In some cases, the scaffold stem further comprises a bubble. In other cases, the scaffold further comprises a triplex loop region. Petition 870250104944, dated 11 / 17 / 2025, p. 152 / 689 148 / 579 In still other cases, the framework also includes an unstructured 5' region. In some embodiments, the gRNA frameworks described for use in LTRP:gRNA systems comprise a framework stem loop with the sequence CCAGCGACUAUGUCGUAGUGG (SEQ ID NO: 1822), or a sequence with at least 1, 2, 3, 4, or 5 mismatches with it.

[00246] Each of the structured domains contributes to establishing the overall RNA fold of the guide and retaining the guide's functionality; particularly the ability to properly complex with the dCasX protein. For example, the guide scaffold stem interacts with the helical domain I of the dCasX protein, while residues within the triplex, triplex loop, and pseudo-knot stem interact with the OBD of the dCasX protein. Together, these interactions confer the guide's ability to bind and form an RNP with the LTRP that retains stability, while the spacer (or targeting sequence) directs and defines the specificity of the RNP to bind to a specific DNA sequence.

[00247] Site-specific binding of a target nucleic acid sequence (e.g., genomic DNA) by the LTRP dCasX protein can occur at one or more locations (e.g., a sequence of a PCSK9 target nucleic acid) determined by the base-pairing complementarity between the gRNA targeting sequence and the target nucleic acid sequence. Thus, for example, the gRNA described has sequences complementary to, and therefore can hybridize with, the target nucleic acid, which is adjacent to a sequence complementary to a TC spacer adjacent motif (PAM) or a PAM sequence, such as ATC, CTC, GTC, or TTC. Because the targeting sequence of a guide sequence hybridizes with a sequence of a target nucleic acid sequence, a targeting sequence can be modified by a user to Petition 870250104944, dated 11 / 17 / 2025, p. 153 / 689 149 / 579 hybridize with a specific target nucleic acid sequence, provided the location of the PAM sequence is considered. In some embodiments, for the design of a targeting sequence, the target nucleic acid comprises a PAM sequence located 5' from the targeting sequence with at least a single nucleotide separating the PAM from the first nucleotide of the target nucleic acid complementary to that of the targeting sequence. This feature distinguishes the systems described in this document from Cas9 systems and results in the ability of the systems described to modify different locations in the DNA sequence when compared to Cas9 systems. In some embodiments, the PAM is located on the non-targeted strand of the target region, i.e., the strand that is complementary to the target nucleic acid. In some embodiments, the gRNA targeting sequence is complementary to a PCSK9 target nucleic acid sequence one nucleotide from an ATC PAM sequence.In some embodiments, the gRNA targeting sequence is complementary to a PCSK9 target nucleic acid sequence one nucleotide from a CTC PAM sequence. In some embodiments, the gRNA targeting sequence is complementary to a PCSK9 target nucleic acid sequence one nucleotide from a GTC PAM sequence. In some embodiments, the gRNA targeting sequence is complementary to a PCSK9 target nucleic acid sequence one nucleotide from a TTC PAM sequence. By selecting the gRNA targeting sequences, defined regions of the sequence or target nucleic acid sequences delimiting a particular location within the target nucleic acid can be repressed using the LTRP:gRNA systems described in this document. In some embodiments, the gRNA targeting sequence has between 15 and 22 consecutive nucleotides. In some embodiments, the targeting sequence... Petition 870250104944, dated 11 / 17 / 2025, p. 154 / 689 150 / 579 The targeting sequence has 15, 16, 17, 18, 19, 20, 21, or 22 consecutive nucleotides. In some embodiments, the targeting sequence consists of 22 consecutive nucleotides. In some embodiments, the targeting sequence consists of 21 consecutive nucleotides. In some embodiments, the targeting sequence consists of 20 consecutive nucleotides. In some embodiments, the targeting sequence consists of 19 consecutive nucleotides. In some embodiments, the targeting sequence consists of 18 consecutive nucleotides. In some embodiments, the targeting sequence consists of 17 consecutive nucleotides. In some embodiments, the targeting sequence consists of 16 consecutive nucleotides. In some embodiments, the targeting sequence consists of 15 consecutive nucleotides.By selecting gRNA targeting sequences, defined regions of the PCSK9 target nucleic acid sequence can be repressed and / or epigenetically modified using the LTRP:gRNA systems described in this document.

[00248] The gene repressor systems of the present invention can be designed to target any region of, or proximal to, a PCSK9 gene or region of a PCSK9 gene for which transcription repression is sought. When the entire gene is to be repressed, designing a guide with a targeting sequence complementary to a sequence that encompasses or is proximal to the transcription start site (TSS) is contemplated by the description. TSS targeting occurs at different positions within the promoter region, depending on the promoter sequence and the concentration of initiation substrate. The core promoter serves as a binding platform for the transcription machinery, which comprises Pol II and its associated general transcription factors (GTFs) (Haberle, V. et al. Eukaryotic core promoters and the functional Petition 870250104944, dated 11 / 17 / 2025, page 155 / 689 151 / 579 transcription initiation base (Nat Rev Mol Cell Biol. 19(10):621 (2018)). It has been proposed that variability in TSS selection involves DNA scrunching (shrinkage) and anti-scrunching, whose characteristics are: (i) forward and backward movement of the leading edge of RNA polymerase, but not the trailing edge, relative to the DNA, and (ii) expansion and contraction of the transcription bubble. In some embodiments, the target nucleic acid sequence bound by an RNP of the LTRP:gRNA system is within 1.5 kilobases (kb) of a transcription initiation site (TSS) in the PCSK9 gene. In some embodiments, the target nucleic acid sequence linked by an RNP of the system is within 20 base pairs (bp), 50 bp, 100 bp, 150 bp, 200 bp, 250 bp, 500 bp, 1 kb, or 1.5 kb upstream of a TSS of the PCSK9 gene.In some embodiments, the target nucleic acid sequence linked by an RNP of the system is within 20 bp, 50 bp, 100 bp, 150 bp, 200 bp, 250 bp, 500 bp, 1 kb, or 1.5 kb downstream of a PCSK9 gene TSS. In some embodiments, the target nucleic acid sequence linked by an RNP of the system is within 1.5 kb upstream to 1.5 kb downstream, 1 kb upstream to 1 kb downstream, 500 bp upstream to 500 bp downstream, 300 bp upstream to 300 bp downstream, or 100 bp upstream to 100 bp downstream of a PCSK9 gene TSS. In some embodiments, the target nucleic acid sequence linked by an RNP of the system is within 20 bp, 50 bp, 100 bp, 150 bp, 200 bp, 250 bp, 500 bp, 1 kb, or 1.5 kb of a PCSK9 gene enhancer. In some embodiments, the target nucleic acid sequence linked by an RNP of the description system is within 1 kb 3' to an untranslated 5' region of the PCSK9 gene.In other embodiments, the target nucleic acid sequence bound by an RNP of the system is within the open reading frame of the PCSK9 gene, including introns (if present). In some embodiments, the targeting sequence of a gRNA of the system description is complementary to... Petition 870250104944, dated 11 / 17 / 2025, p. 156 / 689 152 / 579 a sequence of an exon of the PCSK9 gene. In one particular embodiment, the gRNA targeting sequence of the description system is complementary to an exon 1 sequence of the PCSK9 gene. In other embodiments, the gRNA targeting sequence of the description system is complementary to an intron sequence of the PCSK9 gene. In other embodiments, the gRNA targeting sequence of the description system is complementary to an intron-exon junction sequence of the PCSK9 gene. In other embodiments, the gRNA targeting sequence of the description system is complementary to a regulatory element sequence of the PCSK9 gene. In other embodiments, the gRNA targeting sequence of the description system is complementary to an intergenic region sequence of the PCSK9 gene.In other embodiments, the targeting sequence of a gRNA from the description system is complementary to a sequence of an exon junction, an intron, and / or a regulatory element of the PCSK9 gene. In cases where the targeting sequence is complementary to a regulatory element sequence, such regulatory elements include, among others, promoter regions, enhancer regions, intergenic regions, 5' untranslated regions (UTR 5'), 3' untranslated regions (UTR 3'), conserved elements, and regions comprising cis regulatory elements. The promoter region is intended to enclose nucleotides within 5 kb of the initiation point of the coding sequence or, in the case of gene enhancer elements or conserved elements, it may be thousands of bp, hundreds of thousands of bp, or even millions of bp away from the coding sequence of the PCSK9 gene.In the foregoing, the targets are those in which the PCSK9 gene encoding the target is intended to be repressed so that the PCSK9 gene product is not expressed or is expressed at a lower level in a cell. In some embodiments, through... Petition 870250104944, dated 11 / 17 / 2025, p. 157 / 689 153 / 579 By binding the RNP of the system to the target nucleic acid binding site, the system is able to repress transcription of the 5' PCSK9 gene at the RNP binding site. In other embodiments, by binding the RNP of the system to the target nucleic acid binding site, the system is able to repress transcription of the 3' PCSK9 gene at the RNP binding site.

[00249] In some embodiments, the target nucleic acid comprises a PAM sequence located 5' from the targeting sequence with at least one nucleotide separating the PAM from the first nucleotide of the targeting sequence. In some embodiments, the PAM is located on the non-targeted strand of the target region, i.e., the strand that is complementary to the target nucleic acid and is shown below as Table 9, representing targeting sequences for PCSK9 target nucleic acid for binding to the gRNA scaffolds described; for example, gRNA 174 (SEQ ID NO: 1744), 235 (SEQ ID NO: 1745), 316 (SEQ ID NO: 1746) or chemically modified versions thereof. In some embodiments, the PAM sequence is TTC.

[00250] In some embodiments, the gRNA targeting sequence comprises a sequence selected from the group consisting of SEQ ID NOS: 1824-2544, 2672, 2675, 2694, and 2714, as set forth in Table 9. In some embodiments, the gRNA targeting sequence comprises a sequence selected from the group consisting of SEQ ID NOS: 1824-1880, 1883, 1884, 1888, 1889, 2672, 2675, 2694, and 2714. In some embodiments, the gRNA targeting sequence comprises a sequence selected from the group consisting of SEQ ID NOS: 1834, 1849, 1853, 1855-1858, 1860, 1862, 1863, 1867, 1869, 1870, 1872, 1874, and 1875. In some embodiments, the gRNA targeting sequence comprises a sequence selected from the group consisting of Petition 870250104944, dated 11 / 17 / 2025, p. 158 / 689 154 / 579 in SEQ IDs 1855, 1867, and 1869. In one particular embodiment, the gRNA targeting sequence consists of a sequence with SEQ ID 1855. In another particular embodiment, the gRNA targeting sequence consists of a sequence with SEQ ID 1867. In yet another particular embodiment, the gRNA targeting sequence consists of a sequence with SEQ ID 1869. In some embodiments, the preceding targeting sequences are linked to the gRNA scaffolds of gRNA 174 (SEQ ID 1744), 235 (SEQ ID 1745), or 316 (SEQ ID 1746), or chemically modified versions thereof. Table 9: Specific Targeting Sequences for PCSK9 using long-term repressor fusion proteins Targeting Sequence RNA Sequence SEQ ID PAM Sequence TG-06-342 AAUUACAGGCAACAGGAAGG 1824 TTC TG-06-343 CCCCAUGUAAGAGAGGAAG U 1825 TTC TG-06-344 CAGUUUCUGCCUCGCCGCGG 1826 TTC TG-06-345 GCCUCGCCGCGGCACAGGUG 1827 TTC TG-06-346 CCCACCUGUGCCGCGGCGAG 1828 TTC TG-06-347 CUCCUUCACCCACCUGUGCC 1829 TTC TG-06-348 AGGCAUUCACUCCUUCACCC 1830 TTC TG-06-349 CUGUGCCUGGUGCAGUUCCC 1831 TTC TG-06-350 GUGUCAUAAAGAAAUUGCCU 1832 TTC TG-06-351 UUAUGACACAGAACUCAUGC 1833 TTC TG-06-001 GAGGAGGACGGCCUGGCCGA 1834 TTC TG-06-002 ACCGCUGCGCCAAGGUGGCGG 1835 TTC TG-06-004 GCCAGGCCGUCCUCCUCGGA 1836 TTC TG-06-005 GUGCUCGGGUGCUUCGGCCA 1837 TTC TG-06-117 ACUUUGUUUGCAAAGACCUC 1838 TTC TG-06-118 GAGUGAAAUGGCCUGCUCUG 1839 TTC TG-06-119 GAGCAGGCCAUUUCACUCGG 1840 TTC Petition 870250104944, dated 11 / 17 / 2025, p. 159 / 689 155 / 579 Targeting Sequence RNA Sequence SEQ ID PAM Sequence TG-06-120 CUCGGAAUCUGCUGUGCAUC 1841 TTC TG-06-121 GGAAGGGCUGUCGAUACUGG 1842 TTC TG-06-123 UCCCAGUAUCGACAGCCCUU 1843 TTC TG-06-124 CAGUAUCGACAGCCCUUCCA 1844 TTC TG-06-125 AGAAAGAGCAAGCCUCAUGU 1845 TTC TG-06-128 AGAAAUCAACUGGACAAGCA 1846 TTC TG-06-131 UGAACAUGGUGUGUAAAAGG 1847 TTC TG-06-132 AGAAGAUUCAAU UUGCAAAG 1848 TTC TG-06-133 AUGGUAGGCACAAGCUCAGC 1849 TTC TG-06-134 GAAUUCUAUGGUAGGCACAA 1850 TTC TG-06-135 GGAAAGCUGAGCUUGUGCCU 1851 TTC TG-06-138 AGGGAUUUAUACUACAAAGA 1852 TTC TG-06-139 AGGAGCAGCUAGUUGGUAAG 1853 TTC TG-06-140 AAACUUAGCCUGGACCCCCU 1854 TTC TG-06-141 ACUGGCCUUAACCUGGCAGC 1855 TTC TG-06-142 UUCCACUGGCCUUAACCUGG 1856 TTC TG-06-143 GAAUCAAUCCUACUGUGGAC 1857 TTC TG-06-144 GUGGGCAGCGAGGAGUCCAC 1858 TTC TG-06-145 UGGGUCCACCUUGUCUCCUG 1859 TTC TG-06-146 GAAGUCUCACUGGUCAGCAG 1860 TTC TG-06-147 GUGUUUCCUGGGUCCACCUU 1861 TTC TG-06-149 AGCCCAGUUAGGAUUUGGGA 1862 TTC TG-06-150 UCCCUCUGCGCGUAAUCUGA 1863 TTC TG-06-151CUCUGCGCGUAAUCUGACGC 1864 TTC TG-06-152 GCCUCGCCCUCCCCAAACAG 1865 TTC TG-06-153 GUUAAUGUUUAAUCAGAUAG 1866 TTC TG-06-154 AGGGUGUGGGUGCUUGACGC 1867 TTC TG-06-155 GCAGCGACGUCGAGGCGCUC 1868 TTC TG-06-157 GGGUCUGAGCCUGGAGGAGU 1869 TTC TG-06-158 GGAGCAGGGCGCGUGAAGGG 1870 TTC Petition 870250104944, dated 11 / 17 / 2025, page 160 / 689 156 / 579 Targeting Sequence RNA Sequence SEQ ID PAM Sequence TG-06-159 GCGCGCCCCUUCACGCGCCC 1871 TTC TG-06-160 CGCGCCCUGCUCCUGAACUU 1872 TTC TG-06-161 GCUCCUGCACAGUCCUCCCC 1873 TTC TG-06-167 CACUGAAUAGCGCAGCCGCA 1874 TTC TG-06-168 GUGGGAAGGUUCGCGGGGUU 1875 TTC TG-06-169 CGGGGUUGGGAGACCCGGAG 1876 TTC TG-06-170 UCGGCCUCCGGGUCUCCCAA 1877 TTC TG-06-171 CAGUACGUUCCAGGCAUUCA 1878 TTC TG-06-449 GCUGAAACAGAUGGAAUACU 1879 TTC TG-06-249 AAACCAAAUCGGAACCCACU 1880 TTC TG-06-386 UGUUGCCUGUAAUUGGAAUU 1881 TTC TG-06-122 CCUUUGUUUCUUCCCAGUAU 1883 TTC TG-06-127 UCCUCCUGCCUGGUACACAA 1884 TTC TG-06-137 GAAUGUACCUAUAUGACGUC 1885 TTC TG-06-431 CCCCGGCCUCCCAUCCCUAC 1886 TTC TG-06-433 CUUGGCACGAUCUUGGGGAC 1887 TTC TG-06-445 GAUUUGGUUUGGAAAACAUG 1888 TTC TG-06-446 CUCCAGGCCCUCCACCCUCC 1889 TTC TG-06-447 CACCCCGCCCCUGUCUCGGG 1890 TTC TG-06-355 CCAACCUCAGAAACUUGGAG 1891 TTC TG-06-356 GAGGUUGGAGAAGGUAGCCA 1892 TTC TG-06-359 AUCUACUCAUUCAGUCUAUG 1893 TTC TG-06-360 CCUCAUAGACUGAAUGAGUA 1894 TTC TG-06-361GUCUAUGAGGGGAAGGCAAU 1895 TTC TG-06-362 AGCCAUUGCCUUCCCCUCAU 1896 TTC TG-06-363 UCCCUCUAAAAAUGCCACUU 1897 TTC TG-06-364 CUUCACCCCACAAGCAUCCU 1899 TTC TG-06-365 UGAGUUAUUCUUUCCCUUCA 1900 TTC TG-06-366 GGGCAGAGGGGACCAUCCUC 1901 TTC TG-06-367 UCCUUCAGGGCAGAGGGGAC 1902 TTC Petition 870250104944, dated 11 / 17 / 2025, page 161 / 689 157 / 579 Targeting Sequence RNA Sequence SEQ ID PAM Sequence TG-06-368 CCUCCUGCCUUCCUCCUUCA 1903 TTC TG-06-369 GGGGCCUCCCCUCCACCACA 1904 TTC TG-06-370 GACUUACCCUGAUUGCUGGU 1905 TTC TG-06-371 CCCACACCCUGGCCAGCAAA 1906 TTC TG-06-372 CAAAUGGGUUCCCCCACACC 1907 TTC TG-06-373 CCCUCUUCUCAAAUGGGUUC 1908 TTC TG-06-374 UUUGGGCAACUUAUGGGAGG 1909 TTC TG-06-375 UCCCUCACCAAUUACCCCUC 1910 TTC TG-06-376 CAUCCCAUAUCCUCCACUGC 1911 TTC TG-06-377 AUCUGUCGCAGAGCCCAGGG 1912 TTC TG-06-378 GAGGGAGGUGGGGGAGGAAG 1913 TTC TG-06-379 UCCCCCACCUCCCUCAGAAC 1914 TTC TG-06-380 UGGGCCUGGGGACAUCCAUG 1915 TTC TG-06-381 UCAUUCUCUGGGUGCACGGU 1916 TTC TG-06-382 CUGGGUGCACGGUAACGACC 1917 TTC TG-06-383 CUGCUGCUCCCCUUUGGGAC 1918 TTC TG-06-384 UGCUCCAGGGGAGGCCUUUG 1919 TTC TG-06-385 1920 1924 TTC TG-06-454 CCCCUGCCCCUUCAGCGUGGU 1925 TTC TG-06-455CGAGGCCCAAUUGGCGUCCUU 1926 TTC TG-06-456 CUGCCUGAGUCCUAAAGGAC 1927 TTC TG-06-457 CACAGCCUGAGGGCCAGAAG 1928 TTC TG-06-458 GGCCCUCAGGCUGUGGGAAG 1929 TTC TG-06-459 UCCCGGGGCGAGACCACUAG 1930 TTC TG-06-460 CGGGGCGAGACCACUAGCUU 1931 TTC TG-06-461 AAGUAUUACCAGCCCAGGAC 1932 TTC Petition 870250104944, dated 11 / 17 / 2025, page 162 / 689 158 / 579 Targeting Sequence RNA Sequence SEQ ID PAM Sequence TG-06-462 GUGUCCCCAGCUUGGAGUC 1933 TTC TG-06-463 UCUCACUAGCUGUGGUGGUGCUU 1934 TTC TG-097-TCUCUCCAC C TG-06-465 GUUGACCAUGAG UGAACUUA 1938 TTC TG-06-466 CCUCUGCCCCAGGAGCUGUG 1939 TTC TG-06-467 GCCACUCACAGCUGCCUGCC 1940 TTC TG-06-4GUG66TCGCCUGUG TG-06-469 UUGUUCCCUGAGCCUUGAC 1942 TTC TG-06-470 CUGAGCCUUGACUUUCUCG 1943 TTC TG-06-471 CGAGGGAUGUUGUGGGGUUG 1944 TTCGCGTC52 TG-06UCCU4-4 TG-06-473 AGUUACCACUGCUCCAAAAC 1946 TTC TG-06-474 GGAAAUAGUGAG UACCCCAU 1947 TTC TG-06-475 GGUGGUCAUACAGCCUCUGC 1948 TTC TG-06-476 ACAUCCGA TG-06-477 AAACUUCCACCAGAGAGAGAAA 1950 TTC TG-06-478 AACUCCACCAGAGAGAGAAGAA 1951 TTC TG-06-479 CUSTOMIZATION 1952 TTC TG-056AA-TCAUCCAA1952 TG-06-481 AUUCCCACAAAAUAACCAUU 1954 TTC TG-06-482 AAAUUCGAUUCCCACAAAAU 1955 TTC TG-06-483 UUCAAAUUCGAUUCCCACA 1956 TTC TG-06-47 TAACUAAGA1 CAUGAAGA TG-06-485UUUAGUCAUGAGAAACUGAG 1958 TTC TG-06-486 AAUGCACCUGGGGUGACCUC 1962 TTC TG-06-490 GAUGGGUCUUUGAAGCAUUA 1963 TTC TG-06-491 CAUGACACCAUCAUCGCUGA 1964 TTC Petition 870250104944, dated 11 / 17 / 2025, page 163 / 689 159 / 579 Targeting Sequence RNA Sequence SEQ ID PAM Sequence TG-06-492 GCGAUGAUGGUGUCAUGAGA 1965 TTC TG-06-493 UGGUACCUCCUAAUCCUAGA 1966 TTC TG-06-494 AGGAUUAGGAGGUACCAUGA 1967 TTC TG-06-495 CAGCUCUGUAUCUUUGUUCA 1968 TTC TG-06-496 GACGGUGGAUAAAGAACUGU 1969 TTC TG-06-497 UUAUCCACCGUCUGAAAUGU 1970 TTC TG-06-498 CGUCAGGUGCUACCAUCAAA 1971 TTC TG-06-499 UCCAAGCAGACUCUGAUUGC 1972 TTC TG-06-500 1973 TTC TG-06-501 1977 TTC TG-06-505 UAAGCAGGUAUGUCUGCCUG TG-06-509 CUUGAAGGCUGAUUAUUAAA 1982 TTC TG-06-510 CUUGCCAUGUGAUUGAUCUC 1983 TTC TG-06-511 GCCAUCAGUGAAAGCAGCUU 1984 TTC TG-06-512 UGGAGAGCUUGCAGAAUCAU 1985 TTC TG-06-513 GCUCAUGAUUCUGCAAGCUC 1986 TTC TG-06-514 UUUAUAUAGCAACACAAAAG 1987 TTC TG-06-5151988 TTC TG-06-516 GGUCCACGGUUUCCUGGCUC 1993 TTC TG-06-520 UGGCUCCAAAGCCCAUGAUU 1994 TTC TG-06-521 CUCAAUUUAUUCUGACUGGG 1995 TTC Petition 870250104944, dated 11 / 17 / 2025, page 164 / 689 160 / 579 Targeting Sequence RNA Sequence SEQ ID PAM Sequence TG-06-522 GACUGGGGCAUGGGGGAGGG 1996 TTC TG-06-523 UCUGUACCUGCACCCAGCUC 1997 TTC TG-06-524 GGCAAAAAUGGGAACUACGG 1998 TTC TG-06-525 AAUGUGCCAUUCAGGCAAAA 1999 TTC TG-06-526 UUAUCAUCUACUAUACUCUG 2001 TTC TG-06-527 UCCAUGUCAUCAUGUUCCUC 2002 TTC TG-06-528 AUGUCAUCAUGUUCCUCCUU 2003 TTC TG-06-529 UCCUUGCAUGGGGCCAGGAU 2004 TTC TG-06-530 GCACCACCACGUAGGUGCCA 2005 TTC TG-06-531 AUGGCCUUCUUCCUGGCUUC 2009 TTC TG-06-535 UGGUGAAGAUGAGUGGCGAC 2010 TTC TG-06-536 CAAAAAGGGUGGCUCACCAG TG-06-539 2014 TTC TG-06-540 GUCUCCUCGUCUUUCAAAUG 2018 TTC TG-06-544 UUUGAAAGACGAGGAGACUG 2019 TTC TG-06-5452020 TTC TG-06-546 UGUGCCAAUGCAUCUGCUGC 2021 CUCUGGAUAGCAACAGCUUC 2024 TTC TG-06-550 UUAGGACAGGGUGGCCCUCC 2025 TTC TG-06-551 GCCCCUCCACCGAAGAUGUG 2026 TTC Petition 870250104944, dated 11 / 17 / 2025, page 165 / 689 161 / 579 Targeting Sequence RNA Sequence SEQ ID PAM Sequence TG-06-552 GCACCACCUUCUAGCCCACC 2029 TTC TG-06-553 AGCCCACCUCGUUUCCUGGC 2030 TTC TG-06-5CUGAAATCUUUCU4 UGGCU0 TG-06-555 CACUGAUUCUCCACAUGGCA 2032 TTC TG-06-556 CCACAUGGCAGGCGGUGCUU 2033 TTC TG-06-557 UAGCCUCCUGCAGACAGUGA 2034 TTC TG-06-5GUGATCCG TG03CUGAUCCG CU03 TG-06-559 AAGGCAGGGACCCUGUCAAU 2036 TTC TG-06-560 AUCAGUCUGCAACACACACG 2037 TTC TG-06-561 CCUCUGCAGCACACAGUAGG 2038 TTC TG-06-562 TG-06-562 TB0CAUGAUCGCUU TG-06-563 GGAGGAGACUGACCAGUG 2040 TTC TG-06-564 UCUCCCCAAGGCUGAGCA 2041 TTC TG-06-565 UCGUCUCCCCAAGGCUGA 2042 TTC TG-06-56GTC6 TG06-56GTCAC20GUCCUUGGC TG-06-567 GUUUGCCUUCUGCUUGGCCU 2044 TTC TG-06-568 AACUGGGCAUCAUACCAGCC 2045 TTC TG-06-569 AGUCCUGCCCAGCCCCUUAU 2046 TTC TG-046-TCAUGUAA270 TG-06-571 UUUAAGGACUAAAUGAGGUC 2048 TTC TG-06-572 CGACCAUUUAGUCCUUAA 2049 TTC TG-06-573 GUACCUAGCAUCUGCUGAAU 2050 TTC TG-06-5CGAUGAACU74 GCGUACU4 TG-06-575GACCUAAGUGGUAACAGCCU 2055 TTC TG-06-579 CUCUCCUUACCCUUCAGACC 2056 TTC TG-06-580 CUCUGCCCAGUUGGAGCCGG 2057 TTC TG-06-581 CCGCUGCCAUGCGCAGGGAG 2059 TTC Petition 870250104944, dated 11 / 17 / 2025, page 166 / 689 162 / 579 Targeting Sequence RNA Sequence SEQ ID PAM Sequence TG-06-582 CCAGCCUGCUCAGUGCAAAC 2060 TTC TG-06-583 GUUUGUCCCCUAGUCUAUUC 2061 TTC TG-06-584 CUCCAUGUCCAUCCUGCCCA 2062 TTC TG-06-585 GAACUUGACAUCUUGUCUGG 2063 TTC TG-06-586 GAAUCCUUCCUAAAAGGAAU 2064 TTC TG-06-587 UAAAAGGAAUCCUCUAUGCC 2065 TTC TG-06-588 GCAGGCCUUGAAGUUGCCCC 2066 TTC TG-06-589 ACGGGAUGCUCUGGGCAAAG 2067 TTC TG-06-590 UCCGCCCGGUACCGUGGAGG 2068 TTC TG-06-591 GCUCAGAUGGGGUGGGGCAG 2069 TTC TG-06-592 UGUUGCCCCACAAAUGCAAA 2070 TTC TG-06-593 AAAUGGUGAGAAAAGGAAAA 2071 TTC TG-06-594 CACCAUUUGGAAUGGCCCAG 2072 TTC TG-06-591 TG-06-599 UUCCACUGUUCCCUCUGCCU 2077 TTC TG-06-600 CUCUGCCUGGUGUGGCCUGG 2078 TTC TG-06-601 AUAGCCAGGCCACACCAGGC 2079 TTC TG-06-602 AUGGCCGCUCCUCCUCCCUC 2080 TTC TG-06-603 AGCAGGGACUUGCAGGUGGC 2081 TTC TG-06-604 CCGACUGAAUUGCCACCUGC 2082 TTC TG-06-605GUCGGGGAGACUCUAGAUG 2083 TTC TG-06-606 CUCCUCAGGCCAGGUGCAUC 2084 TTC TG-06-607 UUCCUCCUCAGGCCAGGUG 2085 TTC TG-06-608 AGUUGGA6-TCGUCGUUAAGU6 TG-06-8 CCCCAAGCUCUGCGUCCUGG 2087 TTC TG-06-610 CCCCUUACCGGUUCUCGUGG 2088 TTC TG-06-611 CAAGAUCCCACGAGAACCGG 2089 TTC Petition 870250104944, dated 11 / 17 / 2025, p. 167 / 689 163 / 579 Targeting Sequence RNA Sequence SEQ ID PAM Sequence TG-06-612 CGUGGGAUCUUGGGAAGCCA 2090 TTC TG-06-613 UCUAUGAGCUUUGUCGUUCU 2091 TTC TG-06-614 AUGAGCUUUGUCGUUCUUGU 2092 TTC TG-06-615 CACAGGUUGCUGUGAGACUC 2093 TTC TG-06-616 ACUCAUUGGAGUCUCACAGC 2094 TTC TG-06-617 AAAAGCAUCUGCACAUCCUU 2095 TTC TG-06-618 CUUCCAAAAGCAUCUGCACA 2096 TTC TG-06-619 CCACACCCAGCCCACCAGCU 2097 TTC TG-06-620 GCUGCUGCCACCCUUGCCAG 2098 TTC TG-06-621 UACAGAGGAGCUGUCCUCUC 2099 TTC TG-06-622 UUUCCAACCAGGUGAGACCU 2100 TTC TG-06-623 AACCAGGUGAGACCUCUUCA 2101 TTC TG-06-624 GUGGAGCCCUGGAGCCCCCU 2102 TTC TG-06-625 CAGUCCCUGCCUGGACUGGG 2103 TTC TG-06-626 AACCGAGGGGUGGUAAUUU 2104 TTC TG-06-627 CUUGCCUCUUCCGUGUCCCA 2105 TTC TG-06-628 GUGUCCCAACAGCGGCAGCA 2106 TTC TG-06-629 UUGUCUCAUAAGGACACUUG 2108 TTC TG-06-630 GGCCCAAGGGAGAGGAGGGA 2109 TTC TG-06-631 CUCCUCUCCCUUGGGCCAGA 2110 TTC TG-06-632 UCCCCAUAGGAUGCCCAUGG 2111 TTC TG-06-633 CCAUAGGAUGCCCAUGGAUU 2112 TTC TG-06-634 CGCUUCCCCCGAGUGUCUGU 2113 TTC TG-06-635CCCGAGUGUCUGUGGGAGGU 2114 TTC TG-06-636 ACACCCCUGCCUGCCAUUCC 2116 TTC TG-06-637 GGAGUCAUAUCAAGGGCUUG 2117 TTC TG-06-638 UCCAGCCAAGCCCUUGAUAU 2118 TTC TG-06-639 UCAUUCAUGGGGAGGCCACA 2119 TTC TG-06-640 UUCAUGGGGAGGCCACAAGC 2120 TTC TG-06-641 UGGGGAGGCCACAAGCAUGG 2121 TTC Petition 870250104944, dated 11 / 17 / 2025, pp. 168 / 689 164 / 579 Targeting Sequence RNA Sequence SEQ ID PAM Sequence TG-06-642 CUGUCAGGUAUUGAGUGAGG 2122 TTC TG-06-643 UUCACAUUUGUAUCCCCAGC 2124 TTC TG-06-644 UUUCAUUCACAUUUGUAUCC 2125 TTC TG-06-645 GGAUACACUGUUGGGGGAAG 2126 TTC TG-06-646 CCCAACAGUGUAUCCAGAAG 2127 TTC TG-06-647 UUAAACAAAUGCUACUUGAC 2128 TTC TG-06-648 GAGCUCUCAGCCUAUCCAGA 2129 TTC TG-06-649 GGAUAGGCUGAGAGCUCUGA 2130 TTC TG-06-650 GCUGGAUACACAGGCUCGCA 2131 TTC TG-06-651 UGAUCCUUCUGCUGGAUACA 2132 TTC TG-06-652 UGGAGGAAGCGCUGUUCUAG 2133 TTC TG-06-653 AGCCAAGACCUACGGGGGCA 2134 TTC TG-06-654 ACUGCUUGGACACCGUCCCC 2135 TTC TG-06-655 UUCCACUGCUUGGACACCGU 2136 TTC TG-06-656 AUCCACGUUCAUUCCACUGC 2137 TTC TG-06-657 GCACAGGCCACACUCCCUCC 2138 TTC TG-06-658 CAUCUGACCUGGAGGCCACC 2139 TTC TG-06-659 CAGGCGUAUGGCAGGGCUGC 2141 TTC TG-06-660 CUCUCACCAGGCAGCCCUGC 2142 TTC TG-06-661 AGCCUCUGGAAAGCUCUGUG 2143 TTC TG-06-662 UGAGGCCACCUUGCCACGCU 2144 TTC TG-06-663 CUGUGACAGCGUGGCAAGGU 2145 TTC TG-06-664 AAACCCCCGGGUGAGGCAGG 2146 TTC TG-06-6652147 TTC TG-06-666 2151 TTC TG-06-670 AAGUGCAGGCGGUUCCUGCC 2152 TTC TG-06-671 AUCUGCCGCUGGGGCCACAC 2153 TTC Petition 870250104944, dated 11 / 17 / 2025, pp. 169 / 689 165 / 579 Targeting Sequence RNA Sequence SEQ ID PAM Sequence TG-06-672 GCUGCAAGAUGAUGUGACCA 2154 TTC TG-06-673 GGUCCCACCCGUGCUUGGGA 2155 TTC TG-06-674 UCGUCGUGUGUGAGGAGGGA 2156 TTC TG-06-675 CUCCUCACACACGACGAGGA 2157 TTC TG-06-676 AGUCCAUGUUCCUCGUCGUG 2158 TTC TG-06-677 GCUCGAUGUGCGUUGUGUGG 2159 TTC TG-06-678 CUCCCUGGACGUAAGCCACA 2160 TTC TG-06-679 AGAUAAGCCUGUAUUCCCCG 2161 TTC TG-06-680 CCGGGCUGUCUGCCAAUGUA 2162 TTC TG-06-681 AGGCUGACGGGACAACUGGA 2163 TTC TG-06-682 CUCAGAGCUUCCAGGCUGAC 2164 TTC TG-06-683 UGAGCACCUGUAUCCCCUGC 2165 TTC TG-06-684 ACAUUCCAUAGCCCAGCUCA 2166 TTC TG-06-685 AUAGCCCAGCUCAAUAUCUA 2167 TTC TG-06-686 UCUAGGGUGGCGAGCACUGU 2168 TTC TG-06-687 GGUCGGAGCUCACAGCGCUC 2169 TTC TG-06-688 CAGUGUAGCACCGGGCACAU 2170 TTC TG-06-689 CUCAUCAAUAGGCAUCUACC 2171 TTC TG-06-690 GUAGCAUGCUGAGCAGGUCC 2172 TTC TG-06-691 UCUGUAGCAUGCUGAGCAGG 2173 TTC TG-06-692 UGCCGAGACUGGAGGCCUUA 2174 TTC TG-06-693 GACUGUGCCCAGACCUACUA 2175 TTC TG-06-694 GGCAAGGUCGGCCCCCAGUC 2176 TTC TG-06-695UGUGGGAUCUCAGAGCAGCC 2177 TTC TG-06-696 GUGGCUGCUCUGAGAUCCCA 2178 TTC TG-06-697 CCAAAUAAGGUCUGGCUCAC 2179 TTC TG-06-698 CUCAUAAAGACACAUCACAG 2180 TTC TG-06-699 CCAAUCUCCACCUCUGUCUCUC 2181 TTC TG-06-700 UCCCAGAGCCUUCGCUGCUG 2182 TTC TG-06-701 CAGAGCCUUCGCUGCUGAGU 2183 TTC Petition 870250104944, dated 11 / 17 / 2025, page 170 / 689 166 / 579 Targeting Sequence RNA Sequence SEQ ID PAM Sequence TG-06-702 CAGACUCAGCAGCGAAGGCU 2184 TTC TG-06-703 CUGCUGAGUCUGGGAAUUUG 2185 TTC TG-06-704 GUCACAAAUUCCCAGACUCA 2186 TTC TG-06-705 UAUGGCUUCGGUCACAAAUU 2187 TTC TG-06-706 CAUCUCAGGACAGGUGAGCA 2188 TTC TG-06-707 GAAGUGCCAUCUGAACAGCA 2189 TTC TG-06-708 GAUGGCACUUCAGAAGACUC 2190 TTC TG-06-709 GAAGACUCAGGAGACCCUGG 2191 TTC TG-06-710 UGGCUGCAGGGGUUCCAGGG 2192 TTC TG-06-711 GCUGGCGCAGGACUCUGCUA 2193 TTC TG-06-712 GGCAGGUUGCUGCUAGCAGA 2194 TTC TG-06-713 UUUGGCCUGGCUGAGAGUUU 2195 TTC TG-06-714 GAGACCUGCGCUGGAGCGGA 2196 TTC TG-06-715 UCUCUCCCUUCUCCAUCCAG 2197 TTC TG-06-716 CCAUCCAGCAGGCUGGACCU 2198 TTC TG-06-717 CCUAUACCCUAACCUUUGUC 2199 TTC TG-06-718 CCAAGAAGCAGGCACUGGGG 2200 TTC TG-06-719 CUGAGGUUGUGACUCGUGUG 2201 TTC TG-06-720 GCCUCACACGAGUCACAACC 2202 TTC TG-06-721 CCACUCCACAAGAUACCAGG 2203 TTC TG-06-722 UGUCUCUAAAAUGAGCCGGC 2204 TTC TG-06-723 CCUUUGAGUCUUUAAAUCUU 2205 TTC TG-06-724 CAGCUCACCGAGUCUGCAAG 2206 TTC TG-06-725UGCAGACUCGGUGAGCUGUG 2207 TTC TG-06-726 UAUGAAGGAGCAGGAUGACU 2208 TTC TG-06-727 UGGCAGUAGCAUUGCCAGCU 2209 TTC TG-06-728 CCAGUGCCUGGGAUGUGGC 2216 TG-06-729 GUGUGUUAACUAUAAGGUUG 2212 TTC TG-06-730 AAGGCGACAUUUGUGGGAGA 2214 TTC TG-06-731 CGGUGGUCACUCUGUAUGCU 2215 TTC Petition 870250104944, of 17 / 11 / 2025, p. 171 / 689 167 / 579 Targeting Sequence RNA Sequence SEQ ID PAM Sequence TG-06-732 CGAAGUCGGUGACCAUGACC 2216 TTC TG-06-733 AGAAUGUGCCCGAGGAGGAC 2217 TTC TG-06-734 ACAGACAGGUAAGCACGGCC 2218 TTC TG-06-735 CACAACUGAUGGCAGGGAGG 2219 TTC TG-06-736 UCCAUCCAGCCACCUGCUGA 2220 TTC TG-06-737 CAUGUGGUCCUUGUGUUCGU 2221 TTC TG-06-738 UCGAGCAGGCCAGCAAGUGU 2222 TTC TG-06-739 CUUGGCAGUUGAGCACGCGC 2223 TTC TG-06-740 UGGUUAGGAGACAUUAGCUC 2224 TTC TG-06-741 CUGGCUCUGCGGCAGAGGCU 2225 TTC TG-06-742 UCUGCACUCGUGGCCACUGG 2226 TTC TG-06-743 CAGUGGCCACGAGUGCAGAU 2227 TTC TG-06-744 UCAUCUGCACUCGUGGCCAC 2228 TTC TG-06-745 GCCUUGACCCUGGGUCAAUG 2229 TTC TG-06-746 ACAGUUCCACACUGCUCCCC 2230 TTC TG-06-747 AUGUGGAGGGACUCGAGGUG 2231 TTC TG-06-748 AUCUUCUGCUUGCAUACCUC 2232 TTC TG-06-749 GCUUGCAUACCUCUGAGACC 2233 TTC TG-06-750 AGACUGUGACUACAUUUAGU 2235 TTC TG-06-751 CAACUGGACUUCAUAGGGGG 2236 TTC TG-06-752 CUGUCCACCGGCCCCCAGAG 2237 TTC TG-06-753 UUCUGUGGCCAGACAUUGAG 2238 TTC TG-06-754 GUGGCCAGACAUUGAGCAAG 2239 TTC TG-06-755UCAACUAUUUAGCAGCUACG 2240 TTC TG-06-756 CCAGCUUGAGCUGUGCGACC 2241 TTC TG-06-757 CAGCGAGUUCCCCAGCUUGA 2242 TTC TG-06-758 ACUGUAAGCCCCAGCCCCCA 2243 TTC TG-06-759 CAGCCUUUGAAACAGGCUAG 2244 TTC TG-06-760 AAGGCUGAGAAGGCCCAGGA 2245 TTC TG-06-761 GCCCUGAGACUUUCCUACAG 2246 TTC Petition 870250104944, dated 11 / 17 / 2025, page 172 / 689 168 / 579 Targeting Sequence RNA Sequence SEQ ID PAM Sequence TG-06-762 UGCCUUUAAAACUGAACAUC 2247 TTC TG-06-763 GCCCCAUCAGGUGACCCCUU 2248 TTC TG-06-764 GGAACUGACCUGACUGAGCC 2249 TTC TG-06-765 UGAAGGUCAGUGUGUGGAGG 2250 TTC TG-06-766 GGGAAAGAACAGCCUCCCCU 2251 TTC TG-06-767 UUCCCAGAAAGGCCUUCCCC 2252 TTC TG-06-768 CCAGGCCUGGUGCUCUGGCC 2254 TTC TG-06-769 CCAGGAAGUCCUCCAGAGGC 2255 TTC TG-06-770 UGGAGAAGUCCCUUCUUUGG 2256 TTC TG-06-771 UUGGGGUCCCAGUCAGUGUA 2257 TTC TG-06-772 CAUACACUGACUGGGACCCC 2258 TTC TG-06-773 UCCUCUGUAAGCAGGUGAUU 2259 TTC TG-06-774 UCACCUCCACACUCAGUGCC 2260 TTC TG-06-775 CCUCCACACUCAGUGCCACU 2261 TTC TG-06-776 AGCUCCAUCCUUGGCCACGG 2262 TTC TG-06-777 GGAUCCUGGCCUCUAAGAUG 2263 TTC TG-06-778 CCCCUGUCAAAUGAGGUGUG 2264 TTC TG-06-779 AGGUCCCCUUGCACAUCAGG 2265 TTC TG-06-780 AACCCUCUGCCAGCCUGUGA 2266 TTC TG-06-781 GGCCUCAGUGGCACUCCACU 2267 TTC TG-06-782 UUCUGGCCUCAGUGGCACUC 2268 TTC TG-06-783 UUAGACAAUCCUGGGAGCAA 2269 TTC TG-06-784 AGUUUGUGUGGGUCCAACAC 2271 TTC TG-06-785CCCGCCACUCUCUGAGGCCU 2272 TTC TG-06-786 CCACCCAACUUCCCCCGCCA 2273 TTC TG-06-787 CAUUCCUCUGAGGCUCAUCC 2274 TTC TG-06-788 UCUGAGGCUCAUCCUCUUGG 2275 TTC TG-06-789 AUGAAAGAGAGG CACCAAGA 2276 TTC TG-06-790 UGGAAAGGGAUAAUAAGGUU 2277 TTC TG-06-791 UAUAUUCAGACGCUUAGACA 2278 TTC Petition 870250104944, dated 11 / 17 / 2025, page 173 / 689 169 / 579 Targeting Sequence RNA Sequence SEQ ID PAM Sequence TG-06-792 GACGCUUAGACAGAGCCAGG 2279 TTC TG-06-793 CUGUGCCUGGCUCUGUCUAA 2280 TTC TG-06-794 AGCUCAUGUGACCGUGCGGG 2281 TTC TG-06-795 GCAGGAAUUGCCAGCCUGCC 2283 TTC TG-06-796 UGCAGAAUUUGAUGUUGCAA 2284 TTC TG-06-797 UUCUUCCCUGGGAGGCCUCA 2286 TTC TG-06-798 UCCCUGGGAGGCCUCAGUCU 2287 TTC TG-06-799 CUGGGAGGCCUCAGUCUGUU 2288 TTC TG-06-800 CAGCAGCAUUUCCACUGGCU 2289 TTC TG-06-801 CAGCCCCUCCCUUACAGGCA 2290 TTC TG-06-802 UCCCAGGCCUGGAGUUUAUU 2291 TTC TG-06-803 GAAUAAACUCCAGGCCUGGG 2292 TTC TG-06-804 GAAAAGCCAGCUGGUCCAGC 2293 TTC TG-06-805 GGGACGAUGCCUGCCUCUAC 2294 TTC TG-06-806 UGUGGCACGUGGGCUUCUUG 2295 TTC TG-06-807 UGUGGCACGUUCCUGGAGGC 2296 TTC TG-06-808 GCCUCCAGGAACGUGCCACA 2297 TTC TG-06-809 UGGAGGCCGAACCCUUCUGG 2298 TTC TG-06-810 AAAGCCAGAAGGGUUCGGCC 2299 TTC TG-06-811 GGCUUUGGAAGGAGUCGUCA 2300 TTC TG-06-812 UCCCCAGCCUCCCGCAUGGC 2301 TTC TG-06-813 AGCCCCUUCCUCCCCAGCCU 2302 TTC TG-06-814 GACUCUGCGAUGAUGGAGGU 2303 TTC TG-06-815GGAGGGAGAGUGGCCAGGAC 2304 TTC TG-06-816 GGUACCCGCCCCCUCCCCAU 2305 TTC TG-06-817 CAUUAGCCUUGCUCAAGUUG 2306 TTC TG-06-818 UCGGGUGAGAGGUCUGAGCC 2307 TTC TG-06-819 CCUAGAUACCCGCAGUGUCC 2308 TTC TG-06-820 UUCCCCUAGAUACCCGCAGU 2309 TTC TG-06-821 AGACUUGAAGUG GAACAAGA 2310 TTC Petition 870250104944, dated 11 / 17 / 2025, page 174 / 689 170 / 579 Targeting Sequence RNA Sequence SEQ ID PAM Sequence TG-06-822 PAM 2311 TTC TG-06-823 TTC 2312 TTC TG-06-824 TG-06-824 TGGGC TG-06-825 GCCGCCAGGGAAGCCUUGAU 2314 TTC TG-06-826 CUGGCGCCGAAUUUAAAGG 2315 TTC TG-06-827 CCCCGUGGCACUUAGAACCA 2316 TTC TAGGU7GU7GU7GUUCUCA828 TG-06-829 UAUCUCCCCCACUAGGAUGU 2318 TTC TG-06-830 CAAUGCCCCUGUGGAGUUUA 2319 TTC TG-06-831 UUAGGAGGGACAUUUGAGU 2320 TTC TG-06-3GGGUCGU32 TCAGGU32 TG-06-833 UCUCUGGUCCCCCUGCAG 2323 TTC TG-06-834 AGGACACUCAGUCUGAGUG 2324 TTC TG-06-835 UCUGUGCACUCGCCUCCCUC 2325 TTC TG-06-836 TTC TG-06-836 TG 2GCUGCCCUGC TG-06-837 UCCAUCAAGCUCCCGAUCAA 2327 TTC TG-06-838 CCUGGCCAUAAGACAGCAA 2328 TTC TG-06-839 CGAAGGUAAGCCGCCUGCAG 2329 TTC TG-06-840 TGAGGUGCUAGTCU84 CCCCUGCACCAGGCAUUGCA 2331 TTC TG-06-842 CUGCCAAAGAUGUCAUCAU 2332 TTC TG-06-843 CUGAGGACCAGCGGGUACUG 2333 TTC TG-06-844 CCCUUGU6-TGTCGUGUAAGGA4GAGCCCCAUUCUCAUUAAU 2336 TTC TG-06-846 UUAAGCCCUCCCUCUCUCCUA 2337 TTC TG-06-847 UGGUAGGAGAGAGGAGGGCU 2338 TTC TG-06-848 GUGGAGGUCCCCACUCUC39 TG-06-849 CACUCCUGGAGAAACUGGAG 2340 TTC TG-06-850 CCAGGAGUGGGAAGCGGCGG 2341 TTC TG-06-851 GUAAGCUUACAGGGCUGGCC 2342 TTC Petition 870250104944, of 17 / 11 / 2025, p. 175 / 689 171 / 579 Targeting Sequence RNA Sequence SEQ ID PAM Sequence TG-06-852 CGCGGCUGGGGGCUGCUGGA 2343 TTC TG-06-853 CGCUGUGGCUCUCUCCAGCA 2344 TTC TG-06-854 UAGAGGCUGCAGGACAGUGC 2345 TTC TG-06-855 AUGCUCACCUGGGUGUGAGC 2346 TTC TG-06-856 ACCAAAAUGCUG CAACCAGA 2348 TTC TG-06-857 GGCUUGAGGAGAUGGGGUCU 2349 TTC TG-06-858 AAAUGGAGAUAGAGAGAAAU 2350 TTC TG-06-859 AAACACAGAGUGGUUUCAAA 2351 TTC TG-06-860 UCCAAACACAGAGUGGUUUC 2352 TTC TG-06-861 GCCAGUGGGCAGCUCUGAAU 2353 TTC TG-06-862 GAGCUGCCCACUGGCAGAAC 2354 TTC TG-06-863 UUGGGCAGGAGUGAGCUCCU 2355 TTC TG-06-864 GGGGACAAGGAGCUCACUCC 2356 TTC TG-06-865 AAGCCCAUUCUAAAGCAGAU 2357 TTC TG-06-866 AAAGCAGAUUCCCAUUUCCG 2358 TTC TG-06-867 CAUUUCCGUCUUUGACUCUA 2359 TTC TG-06-868 GUCUUUGACUCUAAGGCCCA 2360 TTC TG-06-869 CCUUAUGCACCCACUGCCCG 2361 TTC TG-06-870 AUACUCUGGUUCUGCCACUU 2362 TTC TG-06-871 AUGCCCUUUGAGCCUGGGCA 2364 TTC TG-06-872 CCUCAUCUCAGCUUCCUCCU 2365 TTC TG-06-873 UCCUCCAUAAGAGGGAAAAA 2366 TTC TG-06-874 GCCCCAGCCGGGGGCCUCUCUC 2367 TTC TG-06-875AACCGAGGUCUGCCCUCCUU 2368 TTC TG-06-876 CCCUGCCGCUCUAUAGCUGU 2369 TTC TG-06-877 UCAUACAGAGAUGUUAAGUA 2370 TTC TG-06-878 AGCUAUCGUUACUAUUGUUU 2371 TTC TG-06-879 UCACCCGUUGCUUUAAAAUC 2372 TTC TG-06-880 ACACAGGGUAGCCCGUCAAU 2373 TTC TG-06-881 UGCUACCUGCCUGGGCAUGU 2374 TTC Petition 870250104944, dated 11 / 17 / 2025, page 176 / 689 172 / 579 Targeting Sequence RNA Sequence SEQ ID PAM Sequence TG-06-882 GGUUGGGUGGAGCAUGAGAG 2375 TTC TG-06-883 AGGCUGGAGUCCCACUGUCU 2377 TTC TG-06-884 CCUGGACGUGCUCAGACGAG 2378 TTC TG-06-885 CGUCUGAGCACGUCCAGGGG 2379 TTC TG-06-886 CAAACCCUCAGUGGUUCCCC 2380 TTC TG-06-887 GGGCACAGCAGGCUGGCAUG 2381 TTC TG-06-888 UGAACAUCCAUCCUACAUGC 2382 TTC TG-06-889 GGAAAGAUGAGCAUAGUCAG 2383 TTC TG-06-890 CUCCAAGCCAGGACCCCUGA 2384 TTC TG-06-891 GGGGUCCUGGCUUGGAGGGA 2385 TTC TG-06-892 AAAGGGGAGCGUGAUUAGAG 2386 TTC TG-06-893 AACUGACAUGUGCCUGUGAG 2387 TTC TG-06-894 AUGACUUCAAACUGACAUGU 2388 TTC TG-06-895 UGGAGUCAGAUUCCAUGACU 2389 TTC TG-06-896 CUUAGCUGCAAAAUGGUGCU 2390 TTC TG-06-897 CUAUGUCCUAGCUGCAAGGG 2391 TTC TG-06-898 GAGCACAUGCCAGGCUCCAU 2392 TTC TG-06-899 CACGUGUCAUCUUCACAGUG 2393 TTC TG-06-900 CAGUGUCCCUGUGAGUAGGU 2394 TTC TG-06-901 CUUUCCAUCUUACAAAUGAG 2395 TTC TG-06-902 UAGCCAAUGGGUGUGUCUGA 2397 TTC TG-06-903 GAGGUGGUUCAGACAGACCC 2398 TTC TG-06-904 GACAGACCCCGGCUUACCCU 2399 TTC TG-06-905AGGGUAAGCCGGGGUCUGUC 2400 TTC TG-06-906 GCCUGCUGGCUGCAUAGGGA 2401 TTC TG-06-907 ACCCAGAGAUGGAGACAGUG 2402 TTC TG-06-908 CAGGGAAAACCAUCUUCU-2006 TG-06-9 CUGAGAAACUAAGGCUCAGA 2404 TTC TG-06-910 UUGAGUUGUUUCUAGGUUUC 2405 TTC TG-06-911 AGGUUUCCUAGCUCUUGCCU 2406 TTC Petition 870250104944, of 17 / 11 / 2025, p. 177 / 689 173 / 579 Targeting sequence RNA sequence SEQ ID PAM sequence TG-06-912 UAGCUCUUGCCUCAGACCUU 2407 TTC TG-06-913 CAUCUGAGCUGGCUUUCCCUC 2408 TTC TG406GCCC-3TCTCG UCU TG-06-915 UGCUGCCAUGCCCCAGGUCU 2410 TTC TG-06-916 AGACCUGGGGCAUGGCAGCA 2411 TTC TG-06-917 AUGCUCCUUGACUUUGCAUU 2412 TTC TG-06UGCCGGTCGG TCUG32 TG-06-919 GUGUCAGUUUGUGCCACCAC 2414 TTC TG-06-920 GCCAUUGUGGACAGCAUG 2415 TTC TG-06-921 ACUCCAAGUGGAGUGGGGUA 2416 TTC TG-06-9AUGCCAUTCAU UUAUU TG-06-923 GCAUGCAGCUCAGGCACCCC 2418 TTC TG-06-924 UGCCUUGCUGGUCAUGCUAA 2419 TTC TG-06-925 AGGGCUUGGCCAUCCUUG 2420 TTC 2420 TG-06AGCCTCAGCUUG TG-06-927 AGAAACCUGAGU GUAUAACU 2422 TTC TG-06-928 UUUAAGAGUGGACUCCUAG 2423 TTC TG-06-929 GGUCUGGAUGGUGCAUUCCU 2424 TTC TG-06GAGU-930 TCUGCAAUCCAU TG-06-931 AGGUCUCUUCCUGGCACUG 2426 TTC TG-06-932 CCCUCCUGGAGCAUCUUUU 2427 TTC TG-06-933 UCCACUAAAAAUAGAUGCUCC 2428 TTC TG-06-93AUTCAUGUAGUAGUA TG-06-935UCCACCUUGUCAGCGAGACC 2431 TTC TG-06-936 CUGCCUCAUGGCCUGUUUCG 2432 TTC TG-06-937 AGGCAAAGCAAAGGCCCCCA 2433 TTC TG-06-938 UGGUACUCCCUGCACAUCCC 2434 TTC TG-06-939 CACCUCUCUGGUUUCCUGGU 2435 TTC TG-06-940 GAUCCAGGCCAGACCACACU 2436 TTC TG-06-941 CAGCUCAGCAGCUAGGCCAC 2437 TTC Petition 870250104944, dated 11 / 17 / 2025, page 178 / 689 174 / 579 Targeting Sequence RNA Sequence SEQ ID PAM Sequence TG-06-942 GCCCAAGGUCCUUGUCAUCU 2438 TTC TG-06-943 GAGGGAUGAGUUUGGCACUG 2440 TTC TG-06-944 UGCCUGGUUUGAGGGCCGUG 2441 TTC TG-06-945 GCACCAUGGCUGACCAAACA 2442 TTC TG-06-946 GUGAGGCCAAAGUGCAGACG 2443 TTC TG-06-947 CCCUCCGACAUCCUUCUGUG 2444 TTC TG-06-948 UUUCUCCCUCCGACAUCCUU 2445 TTC TG-06-949 UUUCCUCGGGCUCUGGCAGG 2446 TTC TG-06-950 UCGGGCUCUGGCAGGUGACC 2447 TTC TG-06-951 CUGGUGCUGCCUGUAGUGCU 2448 TTC TG-06-952 GACUUGUCCCUCUCUCAGCC 2449 TTC TG-06-953 GCCUUUCCGGGGCUGCUGGC 2450 TTC TG-06-333 GGGGCUGCUGGCCUGGCCCU TTC TG-06-959 GCCCACGGACAUCGGCACAU 2456 TTC TG-06-960 GUGCCAGGCAUUCAAUCCUC 2457 TTC TG-06-961 AUCCUCAGGUCUCCACCAAG 2458 TTC TG-06-962 UCCCAUGGAUAGGGGAGGGG 2459 TTC TG-06-963 CAUGGAUAGGGGAGGGGCG 2460 TTC TG-06-964 CCACAGUUAGCUGGAGAUGA 2461 TTC TG-06-965GGAUGGCAUCUAGCCAGAGG 2462 TTC TG-06-966 UGAGCCACCUUUACUCUGCU 2463 TTC TG-06-967 UCCUCUGUGAAGUAGGGGUG 2465 TTC TG-06-968 CAGAGGAAGAAACCUGGAAC 2466 TTC TG-06-335 UGCUGUGUGAGCUUGGCAGG 2467 TTC TG-06-970 GGCUCAGUUCCUGCUGUGUG 2468 TTC TG-06-971 GAGCCAGCCCAAUCUGCGUU 2469 TTC Petition 870250104944, dated 11 / 17 / 2025, page 179 / 689 175 / 579 Targeting Sequence RNA Sequence SEQ ID PAM Sequence TG-06-972 UACUUCACCCGGCUGGGCUC 2470 TTC TG-06-973 CCCGGCUGGGCUCCUCAUUU 2471 TTC TG-06-974 CAGCCUCACUGUUACCCGUA 2472 TTC TG-06-336 CCUUCCCAGCCUCACUGUUA 2473 TTC TG-06-976 UGGUCUGUGUUCCCCUUCCC 2474 TTC TG-06-977 GCCAUCACUCACCGAGCUUC 2475 TTC TG-06-978 AUGCCUGCAGGCAUCGUUCU 2476 TTC TG-06-337 GUUAUCACCCAGGCCUGAUU 2477 TTC TG-06-338 CUGGCCUGGCGGAGAUGCUU 2478 TTC TG-06-339 AAGGCAUGGUCGGGGGAGAG 2479 TTC TG-06-341 AGUUACAAAAGCAAAACAGG 2480 TTC TG-06-983 UAGUAUAAGCAAGGCAUCAC 2481 TTC TG-06-984 ACAAACUUACCCAGUUCCUA 2482 TTC TG-06-985 CAUCACCAGUACUUGCCCAA 2483 TTC TG-06-986 UAGGAUAGGCACAAGGAGUA 2484 TTC TG-06-987 UUCCUAGGAUAGGCACAAGG 2485 TTC TG-06-988 UGCAGCUCCCCUGACCACCC 2486 TTC TG-06-989 UGAUUUAAAAUU CAGAACAG 2491 TTC TG-06-994 GAACAGUCUCAGAUUGGGUG 2492 TTC TG-06-995UAAACACAAGCAAUCCUCCU 2493 TTC TG-06-996 AGACCCUGGGCAACAGUGAG 2494 TTC TG-06-997 AUGUGGGGACCUCCAGGCCCU 2495 TTC TG-06-998 CCAGCCUUAGAGUGGAGGGC 2496 TTC TG-06-999 CUCUCUCUCUAGACAGCCCA 2497 TTC TG-06-1000 UUAUGACAUGGGCUGUCUAG 2498 TTC TG-06-1001 GACAAUUCAGUGUCAUAUAU 2499 TTC Petition 870250104944, dated 11 / 17 / 2025, page 180 / 689 176 / 579 Targeting Sequence RNA Sequence SEQ ID PAM Sequence TG-06-1002 AUAUAUGACACUGAAUUGUC 2500 TTC TG-06-1003 GUGUCAUAUUG GAAAGCAU 2501 TTC TG-0425GAUGUCAU TTC TG-06-1005 AGCCAUUGCCAUCUGUCAAC 2503 TTC TG-06-1006 AACGGGCAGAGAUAACACAG 2504 TTC TG-06-1007 GUGGAGGGGAAGGAGAGAGA 2505 TTCCA6 TCAGAUC0-1AUCC06-1006 TTC TG-06-1009 AACAUCACAGAGCUGUCAGG 2507 TTC TG-06-1010 UGCAGAGGGUUAGCAGGUGG 2508 TTC TG-06-1011 UCCCGAUCCCUGGUGACCA 2509AG TTCCA0 TGU1CUCUUCAUGGUGACCA 2509 2511 TTC TG-06-1013 UAGUGGCCAGCGUUGCUCAC 2512 TTC TG-06-1014 UGCUAGAUCCCACAGGUCUC 2513 TTC TG-06-1015 GCAUCCUCCUAAGCUUUAG 25-CUAG06 TTC-14 TTC 2515 TTC TG-06-1017 GGAUUCAUCUGCAAAGCUUA 2516 TTC TG-06-1018 CUGGAGGGGCUGACCAUUUC 2517 TTC TG-06-1019 UUUCUGGAGGGGCUGACC T0G206 T0G218 UCUUAGUACUUUACACUAAU 2519 TTC TG-06-1021 CCCGCCCACUACGGCUGUGU 2520 TTC TG-06-1022 UAUAUCAGAACAGCCGUA 2521 TTC TG-06-1023 UCAUCCAUCAGAAC25GAUAUAUGAAGAAUGGCGGC 2523 TTC TG-06-1025 AAAACAGAGUUGGAGUCUUG 2524 TTC TG-06-1026 UAGCUUCUAAGAGUAUAAAA 2525 TTC TG-06-1027 UGUGUGGCAAAGUUCUUAGC 2526 TTC TG-06-1028 AAAUUUGGUUCAUGUGUGGC 2527 TTC TG-06-1029 UCCCUAGUGUAUCUACUUCC 2528 TTC TG-06-1030 CUAGUGUAUCUACUUCCUUU 2529 TTC TG-06-1031 AAAGGAAGUAGAUACACUAG 2530 TTC Petition 870250104944, dated 11 / 17 / 2025, page 181 / 689 177 / 579 Targeting Sequence RNA Sequence SEQ ID PAM Sequence TG-06-1032 UCUGCUACCUCUUGUGUAUA 2531 TTC TG-06-1033 AGUCUGCUUGUUCCAAGCUC 2532 TTC TG-06-1034 GAGCUUGGAACAAGCAGACU 2533 TTC TG-06-1035 AAGCUCAGAAGGUAACUGCA 2534 TTC TG-06-1036 CUGCUAACAUCGAGGUGUGU 2535 TTC TG-06-1037 UAAAACUGACUGUAGUUGUC 2536 TTC TG-06-1038 UUCCCAGGAUGCCUUCAACG 2537 TTC TG-06-1039 GCCCGUUGAAGGCAUCCUGG 2538 TTC TTC TTC TG-06-148 GCCGGGCCCACCUUUUCAGU 2694 TTC TG-06-1046 UCUCUUACAUGGGGGGAAAC 2714 TTC c. gRNA modifications

[00251] In another aspect, the description refers to gRNAs (sometimes referred to as gRNA variants in this document) comprising modifications relative to a reference gRNA from which the gRNAs were derived. The gRNAs can be used in the long-term gene repressor fusion protein systems described in this document. In some embodiments, a gRNA variant for use in the systems of the description comprises one or more nucleotide substitutions, insertions, deletions, or domains exchanged or substituted relative to a gRNA sequence of the description that enhances a feature. Exemplary regions for Petition 870250104944, dated 11 / 17 / 2025, p. 182 / 689 178 / 579 modifications and exchanged regions or domains include the RNA triplex, pseudo-knot, scaffold stem loop, and extended stem loop. In some embodiments, the gRNA variant described comprises at least one first exchanged region from a different gRNA, resulting in a chimeric gRNA. A representative example of such a chimeric gRNA is guide 316 (SEQ ID NO: 1746), in which the extended stem loop of scaffold gRNA 235 (SEQ ID NO: 1745) is replaced by the extended stem loop of scaffold gRNA 174 (SEQ ID NO: 1744), wherein the resulting 316 variant retains the ability to form an RNP with a long-term repressor fusion protein and exhibits an improved characteristic compared to precursor 235 when evaluated in an in vitro or in vivo assay under comparable conditions.

[00252] All gRNAs that have one or more enhanced functions, features, or add one or more new functions when the gRNA scaffold variant is compared to a gRNA scaffold from which it was derived, while retaining the functional properties of being able to complex with the long-term repressor fusion protein and guide the holo RNP ribonucleoprotein complex to the target nucleic acid of PCSK9 are predicted to be within the scope of the description. In some embodiments, the gRNA has a selected enhanced feature from the group consisting of increased pseudo-knot stability, increased triplex region stability, increased scaffold stem stability, extended stem stability, reduced off-target folding intermediates, increased binding affinity to a long-term repressor fusion protein, and increased repression activity when complexed with a long-term repressor fusion protein or any combination thereof.In some cases, the improved characteristic is evaluated in an in vitro assay, including the assays in the Examples. In other cases, a. Petition 870250104944, dated 11 / 17 / 2025, p. 183 / 689 179 / 579 enhanced feature is evaluated in vivo.

[00253] Table 10 provides exemplary gRNA variant scaffold sequences from the description that are used as gRNA scaffolds or for the generation of gRNAs for use in the LTRP:gRNA systems described. In some embodiments, the gRNA variant scaffold for use in the systems comprises a sequence selected from the group consisting of SEQ ID NOS: 17441746, or a sequence with at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least less about 97%, at least about 98%, at least about 99% sequence identity with it, wherein the gRNA variant retains the ability to form an RNP with a long-term repressor fusion protein from the description. In some embodiments, the variant gRNA framework for use in the systems comprises a sequence selected from the group consisting of SEQ IDs NOS: 1744-1746.In some embodiments, the variant gRNA scaffold for use in the systems comprises SEQ ID NO: 1744. In some embodiments, the variant gRNA scaffold for use in the systems comprises SEQ ID NO: 1745. In some embodiments, the variant gRNA scaffold for use in the systems comprises SEQ ID NO: 1746. It will be understood that in those embodiments where a vector comprises a DNA coding sequence for a gRNA, the thymine (T) bases may be replaced by uracil (U) bases of any of the gRNA sequence embodiments described in this document. Similarly, any RNA sequence disclosed in this document may be encoded by DNA in which the uracil bases are replaced by thymine. In some embodiments, the description provides gRNA variants from Table 10 that are chemically modified. Petition 870250104944, dated 11 / 17 / 2025, p. 184 / 689 180 / 579 fixed, described below. In some embodiments, the gRNA comprises a scaffold comprising a sequence with SEQ ID NOS: 1744-1746 and is chemically modified. Table 10: gRNA scaffold sequences SEQ ID NO: Framework variant ID Nucleotide sequence 1744 174 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 1745 235 ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCCGCUUACGGACUUCGGUCCGUAAGAGGCAUCAGAG 1746 316 ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAGAG

[00254] The additional gRNA variants contemplated for use in the systems described are selected from the group consisting of SEQ ID NOS: 1747-1821. In some embodiments, the gRNA comprises a scaffold comprising a sequence of SEQ ID NOS: 1747-1821 and is chemically modified.

[00255] Guide scaffolds can be made by various methods, including recombinant or solid-phase RNA synthesis. However, scaffold length can affect fabricability when using solid-phase RNA synthesis, with longer lengths resulting in increased manufacturing costs, decreased purity and yield, and higher synthesis failure rates. For use in particle formulations, such as lipid nanoparticle (LNP) formulations, solid-phase RNA synthesis Petition 870250104944, dated 11 / 17 / 2025, page 185 / 689 Solid 181 / 579 scaffold is preferred for generating the quantities needed for commercial development. Although previous experiments identified gRNA scaffold 235 (SEQ ID NO: 1745) as having enhanced properties compared to gRNA scaffold 174 (SEQ ID NO: 1744), its increased length (in nucleotides) made its use for LNP formulations problematic due to restrictions on synthetic fabrication. Consequently, alternative sequences were sought. In some embodiments, the description provides variant gRNA scaffolds with improved fabrication capabilities compared to the gRNA scaffold from which it was derived. In some embodiments, the description provides gRNA in which the gRNA scaffold and the attached targeting sequence have a sequence length of less than about 115 nucleotides, less than about 110 nucleotides, or less than about 100 nucleotides.In one particular embodiment, the gRNA 316 scaffold (SEQ ID NO: 1746) has a shorter sequence compared to the 235 scaffold from which it was derived. The gRNA 316 scaffold was designed in which the 235 scaffold sequence was modified by a domain exchange where the extended stem loop of scaffold 174 replaced the extended stem loop of scaffold 235, resulting in the chimeric gRNA scaffold 316, having the sequence ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAGAG (SEQ ID NO: 1746), with 89 nucleotides, compared to the 99 nucleotides of the gRNA 235 scaffold. The resulting 316 scaffold had the added advantage that the extended stem loop does not contain CpG motifs; a potentiated property that confers reduced potential to elicit an immune response. In some modes, the shorter sequence length of the 316 framework provides improvements in fidelity. Petition 870250104944, dated 11 / 17 / 2025, page 186 / 689 182 / 579 high in the ability to synthetically create the guide with the correct and complete sequence, as well as an enhanced ability to be successfully incorporated into an LNP. In some embodiments, the description provides chemically modified gRNA 316 variants, described below. d. chemically modified gRNAs

[00256] In some embodiments, gRNAs have one or more chemical modifications. In some embodiments, the chemical modification is the addition of a 2'O-methyl group to one or more nucleotides in the sequence. In some embodiments, the chemical modification is the substitution of a phosphorothioate bond between two or more nucleotides in the sequence. In some embodiments, the chemical modification is a substitution of phosphorothioate bonds between two or more nucleotides at each terminal end of the gRNA. In some embodiments, the gRNA comprises a substitution of phosphorothioate bonds between two or more nucleotides located at 1, 2, 3, or 4 nucleotides from the 5' terminal end, the 3' terminal end, or both terminal ends of the gRNA. In some embodiments, the gRNA comprises an addition of a 2'O-methyl group to one or more nucleotides of the gRNA.In some embodiments, one or more nucleotides located 1, 2, 3, or 4 nucleotides from the 5' end, the 3' end, or both ends of the gRNA are modified by the addition of a 2'O-methyl group. In some embodiments, the first 1, 2, or 3 nucleosides from the 5' end of the backbone (i.e., A, C, and U in the case of gRNA 174, 235, and 316) are modified by the addition of a 2'O-methyl group, and each of the modified nucleosides is linked to the adjacent nucleoside by a phosphorothioate linkage. Similarly, the last 1, 2, or 3 nucleotides from the 3' end of the targeting sequence linked to the 3' end of the backbone are modified in a similar manner. Petition 870250104944, dated 11 / 17 / 2025, p. 187 / 689 183 / 579 In some embodiments, the description provides gRNA with chemical modifications selected from the group consisting of the SEQ ID NOS sequences: 2948-2956, 2958-2966, and 2968-2976, as set out in Table 22, or a sequence with at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least less about 97%, at least about 98%, or at least about 99% sequence identity with it. In any of the above items, the 20 nucleotides at the 3' end of the sequence are replaced by a targeting sequence complementary to a PCSK9 target nucleic acid sequence; optionally, the targeting sequence is selected from the group consisting of SEQ ID NOS: 1824-1880, 1883, 1884, 1888, 1889, 2672, 2675, 2694, and 2714, but includes the chemical modifications at the 3' end of the gRNA.In some embodiments, the 20 nucleotides at the 3' end of the sequence are replaced by a targeting sequence complementary to a PCSK9 target nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1834, 1849, 1853, 1855, 1858, 1860, 1862, 1863, 1867, 1869, 1870, 1872, 1874, and 1875, but includes the chemical modifications at the 3' end of the gRNA. In some embodiments, the 20 nucleotides at the 3' end of the sequence are replaced by a targeting sequence complementary to a PCSK9 target nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1855, 1867, and 1869, but includes the chemical modifications at the 3' end of the gRNA.In some embodiments, the chemically modified gRNA comprises a sequence selected from the group consisting of the sequences with SEQ IDs: 2948-2956, 2958-2966, and 2968-2976, with the 20 nucleotides at the 3' end replaced by a targeting sequence selected from the group consisting of the sequences with SEQ IDs:. Petition 870250104944, dated 11 / 17 / 2025, pp. 188 / 689 184 / 579 1824-1880, 1883, 1884, 1888, 1889, 2672, 2675, 2694 and 2714, but with chemical modifications at the 3' end. In some embodiments, the chemically modified gRNA comprises the sequence SEQ ID NO: 2968 with the 20 nucleotides at the 3' end replaced by a targeting sequence selected from the group consisting of SEQ ID NOs: 1824-1880, 1883, 1884, 1888, 1889, 2672, 2675, 2694 and 2714, but with chemical modifications at the 3' end. In some embodiments, the chemically modified gRNA comprises the sequence SEQ ID NO: 2968 with the 20 nucleotides at the 3' end replaced by a targeting sequence selected from the group consisting of SEQ ID NOs: 1855, 1867, and 1869, but with the chemical modifications at the 3' end. In some embodiments, the modified gRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 22780-22803.In some embodiments, the modified gRNA comprises a selected sequence from the group consisting of SEQ ID NOS: 22788-22790. A schematic of the structure of gRNA variants 174, 235, and 316 is shown in FIGS. 7-9, respectively, and the chemically modified versions of gRNA variants 235 and 316 are shown in FIGS. 10-14. In some embodiments, the chemically modified gRNA exhibits enhanced stability compared to a gRNA without chemical modifications. e. Complex Formation with Repressor Fusion Proteins

[00257] Upon distribution or expression of the system components in a target cell, the gRNA variant is able to complex with a long-term repressor fusion protein such as an RNP, and bind to the target nucleic acid of the PCSK9 gene. In some embodiments, a gRNA variant has an enhanced ability to form an RNP complex with a long-term repressor fusion protein when compared to a reference gRNA. Improve Petition 870250104944, dated 11 / 17 / 2025, page 189 / 689 185 / 579 The formation of the ribonucleoprotein complex can, in some modes, improve the efficiency with which functional RNPs are assembled. In some modes, more than 90%, more than 93%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% of RNPs comprising a gRNA variant and its targeting sequence and a long-term repressor fusion protein of the description are competent for gene repression of a target nucleic acid. VII. Polynucleotides and Vectors

[00258] The description refers to polynucleotides that encode long-term repressor fusion proteins and / or gRNA, which are useful in the repression and epigenetic modification of the PCSK9 gene. The description provides polynucleotides that encode mRNA that encodes the long-term repressor fusion protein, for example, DNA polynucleotides that encode the corresponding mRNA.

[00259] A long-term repressor fusion protein or an mRNA encoding the long-term repressor fusion protein of the description can be prepared by in vitro synthesis using conventional methods as known in the art. Several commercial synthetic apparatuses are available, for example, automated synthesizers by Applied Biosystems, Inc., Beckman, etc. When using synthesizers, naturally occurring amino acids or nucleotides (as applicable) can be substituted for non-natural amino acids or nucleotides. The particular sequence and manner of preparation will be determined by convenience, economy, required purity, and the like. A gRNA can also be produced synthetically; for example, by using a T7 RNA polymerase system known in the art.

[00260] The long-term repressor fusion protein and / or gRNA can also be prepared by recombinant production of a se Petition 870250104944, dated 11 / 17 / 2025, p. 190 / 689 186 / 579 polynucleotide sequence encoding the long-term repressor fusion protein or gRNA of any of the embodiments described in this document using standard recombinant techniques known in the art and incorporating the coding gene into an appropriate expression vector for a host cell.For the production of the encoded long-term repressor fusion protein and / or gRNA of any of the embodiments described in this document, the methods include transforming an appropriate host cell with an expression vector comprising the coding polynucleotide and culturing the host cell under conditions that cause or permit the resulting long-term repressor fusion protein or gRNA of any of the embodiments described in this document to be expressed or transcribed in the transformed host cell, which are recovered by methods described in this document or by standard purification methods known in the art or as described in the Examples. Standard recombinant techniques in molecular biology are used to produce the polynucleotides and expression vectors of the present invention.

[00261] A long-term repressor fusion protein and / or gRNA of the description may also be isolated and purified according to conventional recombinant synthesis methods. A lysate may be prepared from the expression host and the lysate purified using high-performance liquid chromatography (HPLC), exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification techniques. Most often, the compositions used will comprise 50% or more by weight of the desired product, more generally 75% or more by weight, preferably 95% or more by weight, and for therapeutic purposes, generally 99.5% or more by weight, relative to contaminants related to the method of product preparation and its purification. Typically, the percentages Petition 870250104944, dated 11 / 17 / 2025, page 191 / 689 187 / 579 ens will be based on the total protein. Thus, in some cases, a long-term repressor fusion protein or gRNA of the present invention is at least 80% pure, at least 85% pure, at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure (e.g., free from contaminants or other macromolecules, etc.).

[00262] Additionally, the description provides vectors comprising polynucleotides encoding the long-term repressor fusion proteins and gRNAs described in this document. In some cases, the vectors are used for the expression and retrieval of the CasX and gRNA components of the LTRP:gRNA system, where the syste...

Claims

CLAIMS 1. A system for transcriptional repression of a subtilisin / kexin Type 9 proprotein convertase gene (PCSK9), characterized in that it comprises: (a) a guide ribonucleic acid (gRNA) comprising a targeting sequence complementary to a target nucleic acid sequence of the PCSK9 gene, and (b) an mRNA encoding a long-term repressor fusion protein (LTRP), wherein the LTRP comprises: a. a catalytically killed CasX (dCasX); b. a catalytic domain 3A of DNA methyltransferase (DNMT) (DNMT3A); c. a DNMT3 interaction domain (DNMT3L); and d. a first repressor domain (RD1), wherein the LTRP is capable of forming a ribonucleoprotein (RNP) with the gRNA.

2. System according to claim 1, characterized in that the LTRP comprises, from the N- to the C-terminal: a. the DNMT3A; b. the DNMT3L; c. the dCasX; and d. the RD1.

3. System according to claim 1, characterized in that the LTRP comprises, from the N- to the C-terminal: a. the DNMT3A; b. the DNMT3L; c. the RD1; and d. the dCasX.

4. System, according to claim 3, characterized in that the LTRP further comprises an additional RD1 C Petition 870250104944, dated 11 / 17 / 2025, page 585 / 689 2 / 36 terminal to dCasX.

5. System according to claim 4, characterized in that the RD1 sequences are identical.

6. System according to claim 4, characterized in that the RD1 sequences are different.

7. System, according to any one of claims 1 to 6, characterized in that the LTRP comprises an ATRX-DNMT3-DNMT3L domain of DNMT3A DNA (ADD) linked to the N-terminal of DNMT3A.

8. System according to any one of claims 1 to 7, characterized in that the LTRP comprises one or more binding peptides.

9. System according to claim 8, characterized in that at least one of the one or more linking peptides comprises a sequence independently selected from the group consisting of SEQ IDs NOS: 98-124 and 3278-3289.

10. System according to any one of claims 1 to 9, characterized in that the LTRP comprises one or more nuclear locator signals (NLS).

11. System according to claim 10, characterized in that at least one of one or more NLS comprises a sequence selected from the group consisting of SEQ ID NOS: 30-97.

12. System according to claim 10 or 11, characterized in that at least one of one or more NLS is a simian virus 40 (SV40) NLS.

13. System according to claim 12, characterized in that the NLS of SV40 comprises the sequence SEQ ID NO:

30.

14. System, according to claim 10 or 11, as per Petition 870250104944, dated 11 / 17 / 2025, page 586 / 689 3 / 36, characterized by the fact that at least one of one or more NLS is a c-MYC NLS.

15. System according to claim 14, characterized in that the NLS of c-MYC comprises the sequence with SEQ ID NO:

32.

16. System according to any one of claims 1 or 2 or 8 to 15, characterized in that the LTRP comprises, from the N- to the C-terminal, a. a first NLS; b. DNMT3A; c. a first linker peptide; d. DNMT3L; e. a second linker peptide; f. a third linker peptide; g. dCasX; h. a fourth linker peptide; i. RD1; and j. a second NLS.

17. System according to claim 16, characterized in that the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 22836 and SEQ ID NOS: 22838-22846.

18. System according to any one of claims 7 to 15, characterized in that the LTRP comprises, from the N- to the C-terminal, a. a first NLS; b. the ADD; c. the DNMT3A; d. a first linker peptide; e. the DNMT3L; f. a second linker peptide; g. a third linker peptide; h. the dCasX; i. a fourth linker peptide; j. the RD1; and k. a second NLS.

19. System according to claim 18, characterized in that the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 22837 and SEQ ID NOS: 22847-22855.

20. System according to any one of claims 1, 3 or 8 to 15, characterized in that the LTRP comprises, from the N- to the C-terminal, a. a first NLS; b. DNMT3A; c. a first linker peptide; d. DNMT3L; e. a third linker peptide; f. RD1; g. a second linker peptide; h. dCasX; i. a fourth linker peptide; and j. a second NLS.

21. System according to any one of claims 7 to 15, characterized in that the LTRP comprises, from the N- to the C-terminal, a. a first NLS; b. the ADD; c. the DNMT3A; d. a first linker peptide; e. the DNMT3L; f. a second linker peptide; g. the RD1; h. a third linker peptide; i. the dCasX; j. a fourth linker peptide; and k. a second NLS.

22. System according to any one of claims 7 to 15, characterized in that the LTRP comprises, from the N- to the C-terminal, a. a first NLS; b. the ADD; c. the DNMT3A; d. a first linker peptide; e. the DNMT3L; f. a second linker peptide; g. the RD1; h. a third linker peptide; i. the dCasX; j. a fourth linker peptide; k. an additional RD1; l. a fifth linker; and m. a second NLS.

23. System according to claim 22, characterized in that the sequences of RD1 and the additional RD1 are identical.

24. System according to claim 22, characterized in that the sequences of RD1 and the additional RD1 are different.

25. System, according to any of the claims Petition 870250104944, of 11 / 17 / 2025, p. 589 / 689 6 / 36 tions 1 to 24, characterized in that dCasX comprises a sequence selected from the group consisting of SEQ ID NOS: 4-29 or a sequence with at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

26. System according to claim 25, characterized in that dCasX comprises a sequence with SEQ ID NO: 4 or a sequence with at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with it.

27. System according to claim 26, characterized in that dCasX consists of a sequence of SEQ ID NO:

4.

28. System, according to any one of claims 1 to 27, characterized in that RD1 comprises a sequence selected from the group consisting of SEQ ID NOS: 1281726 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

29. System, according to any one of claims 1 to 27, characterized in that RD1 comprises a sequence selected from the group consisting of SEQ ID NOS: 130 Petition 870250104944, dated 11 / 17 / 2025, page 590 / 689 7 / 36 138 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

30. System, according to any one of claims 1 to 27, characterized in that RD1 comprises the sequence with SEQ ID NO: 130 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with it.

31. System, according to any one of claims 1 to 27, characterized in that RD1 comprises the sequence with SEQ ID NO: 131 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with it.

32. System, according to any one of claims 1 to 27, characterized in that RD1 comprises the sequence with SEQ ID NO: 132 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with it.

33. System, according to any one of claims 1 to 27, characterized in that RD1 comprises the sequence with SEQ ID NO: 133 or a sequence with at least about 70%, at least about 80%, at least about 85%, or at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with it.

34. System, according to any one of claims 1 to 27, characterized in that RD1 comprises the sequence with SEQ ID NO: 134 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with it.

35. System, according to any one of claims 1 to 27, characterized in that RD1 comprises the sequence with SEQ ID NO: 135 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with it.

36. System, according to any one of claims 1 to 27, characterized in that RD1 comprises the sequence with SEQ ID NO: 136 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with it.

37. System, according to any one of claims 1 to 27, characterized in that RD1 comprises the sequence with SEQ ID NO: 137 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or, as per Petition 870250104944, dated 11 / 17 / 2025, p. 592 / 689 9 / 36, less than about 99% sequence identity with it.

38. System, according to any one of claims 1 to 27, characterized in that RD1 comprises the sequence with SEQ ID NO: 138 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with it.

39. System according to any one of claims 7 to 15, 18, 19 or 19 to 38, characterized in that the ADD comprises a sequence with SEQ ID NO: 125 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

40. System, according to any one of claims 1 to 39, characterized in that DNMT3A comprises a sequence with SEQ ID NO: 126 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with it.

41. System, according to any one of claims 1 to 40, characterized in that DNMT3L comprises a sequence with SEQ ID NO: 127 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

42. System, according to any one of claims 1 to 41, characterized in that dCasX comprises a sequence selected from the group consisting of SEQ ID NOS: 3122 and 22727 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

43. System, according to any one of claims 1 to 41, characterized in that RD1 comprises a sequence selected from the group consisting of SEQ ID NOS: 33346527 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

44. System, according to any one of claims 1 to 41, characterized in that RD1 comprises a sequence selected from the group consisting of SEQ ID NOS: 33343342 and 4931 to 4939 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

45. System, according to any one of claims 1 to 41, characterized in that RD1 comprises a sequence selected from the group consisting of SEQ ID NOS: 3335 and SEQ ID NO: 4932 or a sequence with at least about 70%, Petition 870250104944, 11 / 17 / 2025, p. 594 / 689 11 / 36 at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

46. ​​A system, according to any one of claims 1 to 41, characterized in that RD1 comprises a sequence selected from the group consisting of SEQ ID NOS: 3339 and SEQ ID NO: 4936 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with it.

47. System, according to any one of claims 1 to 41, characterized in that RD1 comprises a sequence selected from the group consisting of SEQ ID NOS: 3334 and SEQ ID NO: 4931 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

48. System, according to any one of claims 1 to 41, characterized in that RD1 comprises a sequence selected from the group consisting of SEQ ID NOS: 3336 and SEQ ID NO: 4933 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

49. System, according to any one of claims 1 to 41, characterized in that RD1 comprises a selected sequence from the group consisting of SEQ ID NOS: 3337 and SEQ ID NO: 4934 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

50. System, according to any one of claims 1 to 41, characterized in that RD1 comprises a sequence selected from the group consisting of SEQ ID NOS: 3338 and SEQ ID NO: 4935 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

51. System, according to any one of claims 1 to 41, characterized in that RD1 comprises a sequence selected from the group consisting of SEQ ID NOS: 3340 and SEQ ID NO: 4937 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

52. A system according to any one of claims 1 to 41, characterized in that RD1 comprises a sequence selected from the group consisting of SEQ ID NOS: 3341 and SEQ ID NO: 4938 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with it. Petition 870250104944, dated 11 / 17 / 2025, p. 596 / 689 13 / 36 53. System, according to any one of claims 1 to 41, characterized in that RD1 comprises a sequence selected from the group consisting of SEQ ID NOS: 3342 and SEQ ID NO: 4939 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

54. System, according to any one of claims 1 to 53, characterized in that DNMT3A comprises a sequence selected from the group consisting of SEQ ID NOS: 3128 and SEQ ID NO: 3331 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

55. System, according to any one of claims 1 to 54, characterized in that the sequence encoding DNMT3L comprises a sequence selected from the group consisting of SEQ ID NOS: 3119 and SEQ ID NO: 3332 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

56. System, according to any one of claims 7 to 15, 18, 19 or 21 to 55, characterized in that the ADD comprises a sequence selected from the group consisting of SEQ ID NOS: 3127, 3296 and 22726 or a sequence with at least about 70%, at least about 80%, at least about 85%, Petition 870250104944, 11 / 17 / 2025, p. 597 / 689 14 / 36 at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

57. System according to any one of claims 1, 2, 8 to 17 or 25 to 55, characterized in that the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NOS: 6529-8134 and 14628-16233 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

58. System, according to any one of claims 1, 2, 8 to 17 or 25 to 55, characterized in that the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NOS: 6529-6537 and 14628-14636 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

59. A system according to any one of claims 1, 2, 8 to 17 or 25 to 55, characterized in that the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 6529 and SEQ ID NO: 14628 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it. Petition 870250104944, dated 11 / 17 / 2025, p. 598 / 689 15 / 36 60. System according to any one of claims 1, 2, 8 to 17 or 25 to 55, characterized in that the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 6530 and SEQ ID NO: 14629 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

61. System according to any one of claims 1, 2, 8 to 17 or 25 to 55, characterized in that the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 6531 and SEQ ID NO: 14630 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

62. System, according to any one of claims 1, 2, 8 to 17 or 25 to 55, characterized in that the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 6532 and SEQ ID NO: 14631 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

63. System, according to any one of claims 1, 2, 8 to 17 or 25 to 55, characterized in that the mRNA encoding the LTRP comprises a sequence selected from the group Petition 870250104944, dated 11 / 17 / 2025, page 599 / 689 16 / 36 po consisting of SEQ ID NO: 6533 and SEQ ID NO: 14632 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

64. System according to any one of claims 1, 2, 8 to 17 or 25 to 55, characterized in that the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 6535 and SEQ ID NO: 14632 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

65. System, according to any one of claims 1, 2, 8 to 17 or 25 to 55, characterized in that the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 6534 and SEQ ID NO: 14633 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

66. System, according to any one of claims 1, 2, 8 to 17 or 25 to 55, characterized in that the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 6536 and SEQ ID NO: 14635 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

67. System, according to any one of claims 1, 2, 8 to 17 or 25 to 55, characterized in that the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 6537 and SEQ ID NO: 4636 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

68. System according to any one of claims 1, 2, 7 to 15, 18, 19 or 25 to 56, characterized in that the mRNA encoding LTRP comprises a sequence selected from the group consisting of SEQ ID NOS: 9742-11347 and 17840-19446 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

69. A system according to any one of claims 1, 2, 7 to 15, 18, 19 or 25 to 56, characterized in that the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NOS: 9742-9750 and 17841-17849 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it. Petition 870250104944, dated 11 / 17 / 2025, p. 601 / 689 18 / 36 70. System according to any one of claims 1, 2, 7 to 15, 18, 19 or 25 to 56, characterized in that the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 9742 and SEQ ID NO: 17841 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

71. System according to any one of claims 1, 2, 7 to 15, 18, 19 or 25 to 56, characterized in that the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 9743 and SEQ ID NO: 17842 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

72. System according to any one of claims 1, 2, 7 to 15, 18, 19 or 25 to 56, characterized in that the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 9744 and SEQ ID NO: 17843 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

73. System, according to any one of claims 1, 2, 7 to 15, 18, 19 or 25 to 56, characterized in that the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 9745 and SEQ ID NO: 17844 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

74. System according to any one of claims 1, 2, 7 to 15, 18, 19 or 25 to 56, characterized in that the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 9746 and SEQ ID NO: 17845 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

75. System according to any one of claims 1, 2, 7 to 15, 18, 19 or 25 to 56, characterized in that the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 9747 and SEQ ID NO: 17846 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

76. System, according to any one of claims 1, 2, 7 to 15, 18, 19 or 25 to 56, characterized in that the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 9748 and SEQ ID NO: 17847 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

77. System according to any one of claims 1, 2, 7 to 15, 18, 19 or 25 to 56, characterized in that the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NO: 9749 and SEQ ID NO: 17848 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

78. System according to any one of claims 1, 2, 7 to 15, 18, 19 or 25 to 56, characterized in that the mRNA encoding the LTRP comprises a sequence selected from SEQ ID NO: 9750 and SEQ ID NO: 17849 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

79. A system according to any one of claims 1, 3, 8 to 15, 20 or 25 to 55, characterized in that the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NOS: 8135-9740 and 16234-17839 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it. Petition 870250104944, dated 11 / 17 / 2025, p. 604 / 689 21 / 36 80. System according to any one of claims 1, 3, 8 to 15, 20 or 25 to 55, characterized in that the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NOS: 8135-8143 and 16234-16242 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

81. System according to any one of claims 1, 3, 7 to 15, 21 or 25 to 56, characterized in that the mRNA encoding LTRP comprises a sequence selected from the group consisting of SEQ ID NOS: 11348-12953 and 19447-21052 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

82. System according to any one of claims 1, 3, 7 to 15, 21 or 25 to 56, characterized in that the mRNA encoding LTRP comprises a sequence selected from the group consisting of SEQ ID NOS: 11348-11356 and 19447-19455 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

83. System, according to any one of claims 1, 3 to 5, 7 to 15, 22, 23 or 25 to 56, characterized in that the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NOS: 12954-14553 and 2105322652 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

84. System according to any one of claims 1, 3 or 4, 6 to 15, 22 or 24 to 56, characterized in that the mRNA encoding the LTRP comprises a sequence selected from the group consisting of SEQ ID NOS: 14554-14626 and 22653-22725 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

85. System according to any one of claims 1 to 84, characterized in that the mRNA is codon-optimized.

86. System according to claim 85, characterized in that the mRNA is codon-optimized for expression in a human cell.

87. System according to any one of claims 1 to 86, characterized in that the mRNA is chemically modified, optionally in which one or more or all of the uridine residues of the mRNA are replaced by 1-methyl-pseudouridine.

88. System according to any one of claims 1 to 87, characterized in that the mRNA comprises a 5' untranslated region (UTR), a 3' UTR, a poly(A) sequence and / or a 5' cap.

89. System, according to claim 88, characterized by the fact that the 5'UTR comprises a sequence of SEQ ID NO: 3300. Petition 870250104944, dated 11 / 17 / 2025, page 606 / 689 23 / 36 90. System according to claim 88 or 89, characterized in that the 3'UTR comprises a sequence of SEQ ID NO: 3310.

91. System, according to any one of claims 88 to 90, characterized in that poly(A) comprises a sequence of SEQ ID NO: 3057.

92. System according to any one of claims 88 to 91, characterized in that the 5' cap is attached 5' to the mRNA.

93. A system according to any one of claims 87 to 92, characterized in that the mRNA comprises the selected sequence from the group consisting of SEQ ID NOS: 14628-16233, 16234-17839, 17841-19446, 19447-21052, 21053-22652 and 22653-22725 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

94. System, according to any one of claims 87 to 92, characterized in that the mRNA comprises a sequence with SEQ ID NO: 14628-14635 or 14636 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

95. System, according to any one of claims 87 to 92, characterized in that the mRNA comprises Petition 870250104944, dated 11 / 17 / 2025, p. 607 / 689 24 / 36 a sequence with SEQ ID NO: 14628 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the same.

96. System, according to any one of claims 87 to 92, characterized in that the mRNA comprises a sequence with SEQ ID NO: 14629 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with it.

97. System, according to any one of claims 87 to 92, characterized in that the mRNA comprises a sequence with SEQ ID NO: 14630 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with it.

98. System according to any one of claims 87 to 92, characterized in that the mRNA comprises a sequence with SEQ ID NO: 14631 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity therewith. Petition 870250104944, dated 11 / 17 / 2025, pp. 608 / 689 25 / 36 99. System, according to any one of claims 87 to 92, characterized in that the mRNA comprises a sequence with SEQ ID NO: 14632 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with it.

100. System, according to any one of claims 87 to 92, characterized in that the mRNA comprises a sequence with SEQ ID NO: 14633 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with it.

101. System, according to any one of claims 87 to 92, characterized in that the mRNA comprises a sequence with SEQ ID NO: 14634 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with it.

102. System, according to any one of claims 87 to 92, characterized in that the mRNA comprises a sequence with SEQ ID NO: 14635 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with it.

103. System, according to any one of claims 87 to 92, characterized in that the mRNA comprises a sequence with SEQ ID NO: 14636 or a sequence with at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with it.

104. System according to any one of claims 1 to 103, characterized in that the target nucleic acid sequence of the PCSK9 gene is within 1.5 kb of a transcription start site (TSS) in the gene.

105. System according to any one of claims 1 to 103, characterized in that the target nucleic acid sequence of the PCSK9 gene is within 500 bps upstream to 500 bps downstream of a TSS of the gene.

106. System according to any one of claims 1 to 103, characterized in that the target nucleic acid sequence of the PCSK9 gene is within 300 bps upstream to 300 bps downstream or is within 100 bps upstream to 100 bps downstream of a TSS of the gene.

107. System, according to any one of claims 1 to 103, characterized in that the target nucleic acid sequence of the PCSK9 gene is within 100 bps upstream to 100 bps downstream of a TSS of the gene.

108. System, according to any one of claims 1 to 107, characterized in that the target nucleic acid sequence of the PCSK9 gene is within 1 kb of a gene enhancer. Petition 870250104944, dated 11 / 17 / 2025, page 610 / 689 27 / 36 109. A system according to any one of claims 1 to 107, characterized in that the target nucleic acid sequence of the PCSK9 gene is within the 3' untranslated region of the PCSK9 gene.

110. System according to any one of claims 1 to 109, characterized in that the gRNA targeting sequence comprises a sequence selected from the group consisting of the following SEQ IDs: 1824-1881, 1883-1897, 1899-1934, 1937-1991, 1993-1999, 2001-2026, 2029-2057, 2059-2106, 2108-2114, 2116-2122, 2124-2139, 2141-2209, 2211-2212, 2214-2233, 2235-2252, 2254-2269, 2271-2281, 2283-2284, 2286-2320, 2322-2334, 2236-2346, 2348-2362, 2364-2375, 2377-2395, 2397-2428, 2430-2438, 2440-2463, 2465-2509, 2511-2544, 2672, 2675, 2694 and 2714.

111. System according to any one of claims 1 to 109, characterized in that the gRNA targeting sequence comprises a sequence selected from the group consisting of the SEQ ID NOS: 1824-1880, 1883, 1884, 1888, 1889, 2672, 2675, 2694 and 2714.

112. System according to any one of claims 1 to 109, characterized in that the gRNA targeting sequence comprises a sequence selected from the group consisting of the following SEQ IDs: 1834, 1849, 1853, 1855-1858, 1860, 1862, 1863, 1867, 1869, 1870, 1872, 1874 and 1875.

113. System according to any one of claims 1 to 109, characterized in that the gRNA targeting sequence comprises a sequence selected from the group consisting of SEQ IDs NOS: 1855, 1867 and 1869.

114. System, according to any of the claims 110 to 113, characterized in that the gRNA targeting sequence comprises a sequence in which 1, 2, 3, 4 or 5 nucleotides are removed from the 3' end of the sequence.

115. System according to any one of claims 1 to 114, characterized in that the gRNA is a single-molecule gRNA (sgRNA).

116. System according to any one of claims 1 to 115, characterized in that the gRNA comprises a scaffold loop stem comprising the sequence CCAGCGACUAUGUCGUAGUGG (SEQ ID NO: 1822) or a sequence with 1, 2, 3, 4 or 5 mismatches therewith.

117. System, according to any one of claims 1 to 116, characterized in that the gRNA comprises a scaffold comprising a sequence selected from the group consisting of SEQ ID NOS: 1744-1746 or a sequence with at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity with it.

118. System, according to any one of claims 1 to 116, characterized in that the gRNA comprises a scaffold comprising a sequence with SEQ ID NOS: 1746 or a sequence with at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity with it.

119. System, according to any one of claims 1 to 118, characterized in that the gRNA comprises Petition 870250104944, dated 11 / 17 / 2025, p. 612 / 689 29 / 36 a sequence selected from the group consisting of SEQ ID NOS: 3074-3081, 3145-3147, 3150-3153 and 3158-3176 or a sequence with at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity with it.

120. System according to any one of claims 1 to 119, characterized in that the gRNA is chemically modified.

121. System according to claim 120, characterized in that the chemical modification to the gRNA comprises an addition of a 2'O-methyl group to one or more nucleotides of the gRNA.

122. System according to claim 121, characterized in that one or more nucleotides located 1, 2, 3 or 4 nucleotides from the 5' terminal end, the 3' terminal end or both terminal ends of the gRNA are modified by the addition of a 2'O-methyl group.

123. System according to any one of claims 120 to 122, characterized in that the chemical modification of the gRNA comprises a substitution of a phosphorothioate bond between two or more nucleotides of the gRNA.

124. System according to claim 123, characterized in that the chemical modification comprises a substitution of phosphorothioate linkages between two or more nucleotides located 1, 2, 3 or 4 nucleotides from the 5' terminal end, 3' terminal end or both terminal ends of the gRNA.

125. System, according to any one of claims 104 to 124, characterized in that the gRNA comprises Petition 870250104944, dated 11 / 17 / 2025, p. 613 / 689 30 / 36 a sequence selected from the group consisting of SEQ ID NOS: 3074-3081, 3147, 3151, 3153, 3159-3164, 3166-3171, 3173-3176 and 22788-22803 or a sequence with at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity with it.

126. System according to any one of claims 104 to 124, characterized in that the gRNA comprises a sequence selected from the group consisting of SEQ IDs NOS: 22788-22803.

127. System, according to any one of claims 104 to 124, characterized in that the gRNA comprises a sequence selected from the group consisting of SEQ IDs NOS: 22788-22790.

128. Lipid nanoparticle, characterized in that it comprises the system as defined in any one of claims 1 to 127.

129. Lipid nanoparticle (LNP), characterized in that it comprises mRNA, as defined in any one of claims 87 to 103, and gRNA, as defined in any one of claims 120 to 127.

130. LNP, according to claim 128 or 129, characterized in that the LNP comprises one or more components selected from the group consisting of ionizable lipids, one or more auxiliary phospholipids, one or more polyethylene glycol (PEG) modified lipids, and cholesterol or a derivative thereof.

131. LNP, according to any of claims 128 to 130, characterized in that the LNP comprises an ionizable lipid, an auxiliary phospholipid, a lipid modified by polyethylene glycol (PEG) and cholesterol or a derivative thereof.

132. LNP, according to any one of claims 128 to 131, characterized in that it comprises a cationic lipid comprising a pKa of 5 to 8.

133. Pharmaceutical composition, characterized in that it comprises: a. the system, as defined in any one of claims 1 to 127; or b. the LNP, as defined in any one of claims 128 to 132, and one or more pharmaceutically suitable excipients.

134. Pharmaceutical composition, characterized in that it comprises a plurality of lipid nanoparticles, as defined in any one of claims 128 to 132, and a pharmaceutically acceptable carrier or diluent.

135. Pharmaceutical composition, according to claim 134, characterized in that the average diameter of lipid nanoparticles in plurality is between about 20 nm and about 200 nm.

136. Pharmaceutical composition, according to claim 134 or 135, characterized in that the pharmaceutical composition is formulated for a route of administration selected from the group consisting of intravenous, intra-arterial, intraportal vein injection, intraperitoneal, intramuscular, intracerebroventricular, intracisternal, intrathecal, intracranial, intralumbar, intraocular, subcutaneous and oral routes.

137. Method for repressing the transcription of a PCSK9 gene in a cell population, characterized in that it comprises introducing into the cells of the population: Petition 870250104944, dated 11 / 17 / 2025, pp. 615 / 689 32 / 36 a. the system, as defined in any one of claims 1 to 127; b. the LNP, as defined in any one of claims 128 to 132; c. the pharmaceutical composition, as defined in any one of claims 133 to 136; or d. combinations of two or more of (a)-(c), thereby repressing the transcription of the PCSK9 gene in the cell population.

138. Method, according to claim 137, characterized in that the transcription of the PCSK9 gene is repressed in at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or more of the cells in the population.

139. Method, according to claim 137 or 138, characterized in that the transcription of the PCSK9 gene in the cells of the population decreases by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90% compared with an untreated cell.

140. A method according to any one of claims 137 to 139, characterized in that the cells of the population are eukaryotic cells.

141. Method, according to claim 140, characterized in that the eukaryotic cells are selected from the group consisting of rodent cells, mouse cells, rat cells and non-human primate cells.

142. Method according to claim 140, characterized in that the eukaryotic cells are human cells.

143. A method according to any one of claims 140 to 142, characterized in that the eukaryotic cells are selected from the group consisting of liver cells, intestinal cells, kidney cells, central nervous system cells, smooth muscle cells, macrophages, retinal cells, and arterial wall cells.

144. Method, according to any one of claims 137 to 143, characterized in that the repression of the PCSK9 gene in the cell population occurs in vitro or ex vivo.

145. Method, according to any one of claims 137 to 143, characterized in that the repression of the PCSK9 gene of the cell population occurs in vivo in an individual.

146. Method according to claim 145, characterized in that the individual is selected from the group consisting of a rodent, a mouse, a rat, and a non-human primate.

147. Method according to claim 145, characterized in that the individual is a human.

148. A method according to any one of claims 137 to 147, characterized in that the repression of transcription is stable during one or more cell divisions.

149. Method, according to any one of claims 137 to 148, characterized in that the suppression of transcription is hereditary.

150. Method, according to any of claims 137 to 149, characterized in that the repression is reversible. Petition 870250104944, dated 11 / 17 / 2025, pp. 617 / 689 34 / 36 151. Method according to claim 150, characterized in that the suppression is reversible by the use of a DNMT inhibitor.

152. Method according to claim 151, characterized in that the DNMT inhibitor is a cytidine analogue.

153. Method according to claim 151 or 152, characterized in that the DNMT inhibitor is selected from the group consisting of azacitidine, decitabine, clofarabine and zebularine.

154. A method for treating a PCSK9-related disease in an individual in need thereof, characterized in that it comprises administering a therapeutically effective dose of: a. the system, as defined in any one of claims 1 to 127; b. the LNP, as defined in any one of claims 128 to 132; or c. the pharmaceutical composition, as defined in any one of claims 133 to 136, thereby treating the PCSK9-related disease.

155. Method according to claim 154, characterized in that the individual is treated with a therapeutically effective dose of LNP.

156. Method according to claim 154 or 155, characterized in that the LNP is administered by a route of administration selected from the group consisting of intravenous, intra-arterial, intraportal vein injection, intraperitoneal, intramuscular, intracerebroventricular, intracisternal, intrathecal, intracranial, intralobar, intraocular, subcutaneous and oral routes.

157. Method, according to any one of claims 154 to 156, characterized in that the disease related to PCSK9 is selected from the group consisting of autosomal dominant hypercholesterolemia (ADH), hypercholesterolemia, elevated total cholesterol levels, hyperlipidemia, elevated low-density lipoprotein (LDL) levels, elevated LDL cholesterol levels, reduced high-density lipoprotein levels, hepatic steatosis, coronary artery disease, ischemia, stroke, peripheral vascular disease, thrombosis, type 2 diabetes, high blood pressure, atherosclerosis, obesity, aortic stenosis, elevated PCSK9 levels, or a combination thereof.

158. A method, according to any one of claims 154 to 157, characterized in that the method results in improvement in at least one clinically relevant outcome selected from the group consisting of a change from baseline in LDL cholesterol, a decrease in atheromatous plaque volume, a reduction in coronary plaque, a reduction in atherosclerotic cardiovascular disease (ASCVD), cardiovascular death, non-fatal myocardial infarction, ischemic stroke, non-fatal stroke, coronary revascularization, unstable angina, and visual acuity.

159. A method, according to any one of claims 154 to 158, characterized in that the method results in improvement in at least two clinically relevant outcomes selected from the group consisting of a change from baseline in LDL cholesterol, a decrease in atheromatous plaque volume, a reduction in coronary plaque, a reduction in atherosclerotic cardiovascular disease (ASCVD), cardiovascular death, non-fatal myocardial infarction, ischemic stroke, non-fatal stroke, coronary revascularization, unstable angina, and visual acuity.

160. Kit, characterized in that it comprises the system, as defined in any of claims 1 to 127, the LNP, as defined in any of claims 128 to 132, the pharmaceutical composition, as defined in any of claims 133 to 136, or combinations thereof, and a suitable container.

161. Kit according to claim 160, characterized in that it comprises a buffer, an excipient, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a marker, a marker visualization reagent, instructions for use, or any combination thereof.

162. Composition, characterized in that it comprises: a. the system, as defined in any one of claims 1 to 127; b. the LNP, as defined in any one of claims 128 to 132; or c. the pharmaceutical composition, as defined in any one of claims 133 to 136.

163. Composition according to claim 162, characterized in that it is for use in the manufacture of a medicament for the treatment of a PCSK9-related disease in an individual in need thereof.

164. Composition according to claim 162, characterized in that it is for use in the treatment of a PCSK9-related disease in an individual in need thereof.

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