Mitochondrial targeting sequences for delivery of RNA to the mitochondria

Abstract

The present disclosure relates to mitochondrial delivery systems and methods for targeting RNA into the mitochondria using mitochondrial targeting sequences.

Description

[0001] MITOCHONDRIAL TARGETING SEQUENCES FOR DELIVERY OF RNA TO THE MITOCHONDRIA

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims priority to, and the benefit of, U.S. Provisional Patent Application No. 63 / 625,361, filed January 26, 2024, which is incorporated by reference herein in its entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under Grant Nos. EY017141 and EY027414 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] REFERENCE TO SEQUENCE LISTING

[0007] The sequence listing submitted on December 6, 2024, as an .XML file entitled “11348- 025WOl_ST26” created on December 5, 2024, and having a file size of 182,730 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

[0008] FIELD

[0009] The present disclosure relates to mitochondrial delivery systems and methods for targeting RNA into the mitochondria using mitochondrial targeting sequences.

[0010] BACKGROUND

[0011] Human mitochondrial DNA (mtDNA) encodes 37 genes (2 for ribosomal RNAs, 22 for tRNAs and 13 for proteins) that are essential for cell viability. These genes are buffered against the effect of mutations because somatic cells typically contain around 1 ,000 copies of mtDNA; however, the mitochondrial genome is prone to oxidative damage induced by both exogenous and endogenous reactive oxygen species (ROS), as well as potential replication errors. With advancing age, deletions and point mutations accumulate in mtDNA resulting in detrimental phenotypic manifestations. Furthermore, inborn errors of mtDNA form the basis of multiple pediatric disorders, including Leber’s hereditary optic neuropathy (LHON; mutation in ND4), Leigh syndrome (LS; mutation in ATP6), Neuropathy, Ataxia, Retinitis Pigmentosa (NARP; mutation in ATP6), and Mitochondrial encephalopathy lactic acidosis and stroke-like episodes syndrome (MELAS; mutation in tRNAL“1 : JR) / TL1). Significant obstacles remain to correcting any mtDNA mutations in living animals since there is no delivery system for nucleic acids into mitochondria. Thus, there is a need to address the aforementioned problems by generating mitochondrial delivery systems for delivery of RNA into the mitochondria.

[0012] SUMMARY

[0013] Disclosed herein are chimeric nucleic acids for targeting RNA molecules into the mitochondria using a novel mitochondrial targeting sequence. The present disclosure also provides a mitochondrial genome editing system comprising a chimeric nucleic acid for targeting RNA molecules into the mitochondria. The present disclosure also provides a method of treating mitochondrial diseases using the mitochondrial genome editing system comprising a chimeric nucleic acid for targeting RNA molecules into the mitochondria.

[0014] In one aspect, disclosed herein is a chimeric nucleic acid comprising a first nucleic acid sequence operably fused to a second nucleic acid sequence; wherein the second nucleic acid sequence comprises a mitochondrial targeting sequence (MTS) comprising a mitochondrial ribosomal protein S12 (MRPS 12) gene, or a fragment thereof.

[0015] In some aspects, disclosed herein is a chimeric nucleic acid of any preceding aspect, wherein the MTS comprises at least 50% identity with SEQ ID NO: 1. In some embodiments, the MTS comprises at least 60% identity with SEQ ID NO: 1. In some embodiments, the MTS comprises at least 70% identity with SEQ ID NO: 1. In some embodiments, the MTS comprises at least 80% identity with SEQ ID NO: 1. In some embodiments, the MTS comprises at least 90% identity with SEQ ID NO: 1. In some embodiments, the MTS comprises 100% identity with SEQ ID NO: 1.

[0016] In some aspects, disclosed herein is a chimeric nucleic acid of any preceding aspect, wherein the chimeric nucleic acid comprises a CRISPR nuclease gene, or a fragment thereof. In some aspects, disclosed herein is a chimeric nucleic acid of any preceding aspect, wherein the chimeric nucleic acid comprises a ten-eleven translocation (TET) gene. In some aspects, disclosed herein is a chimeric nucleic acid of any preceding aspect, wherein the chimeric nucleic acid comprises a reporter gene. In some embodiments, the reporter gene is either a monomeric red fluorescent protein mCherry (mCherry) gene or a green fluorescent protein (GFP) gene.

[0017] In some embodiments, the second nucleic acid sequence comprises a CRISPR nuclease gene. In some embodiments, the first nucleic acid sequence comprises a ten-eleven translocation (TET) gene. In some embodiments, the first nucleic acid sequence comprises a reporter gene. In some embodiments, the first nucleic acid sequence comprises a guide RNA sequence.

[0018] In one aspect, disclosed herein is a mitochondrial genome editing system comprising: a chimeric nucleic acid comprising a first nucleic acid sequence operably fused to a second nucleic acid sequence; wherein the second nucleic acid sequence comprises a mitochondrial targeting sequence (MTS) comprising a mitochondrial ribosomal protein S12 (MRPS12) gene, or a fragment thereof; and an RNA-guided nuclease.

[0019] In some embodiments, the RNA-guided nuclease is selected from Cas9, a mutant Cas9 with nickase activity (Cas9H840A or Cas9D10A), or a mutant Cas9 with no nuclease activity (dCas9).

[0020] In one aspect, disclosed herein is a mitochondrial genome editing system, wherein the MTS directs the chimeric nucleic into a mitochondrion. In one aspect, disclosed herein is a mitochondrial genome editing system, wherein the first nucleic acid sequence is integrated into a mitochondrial genome in the presence of the RNA-guided nuclease (or CRISPR nuclease). In one aspect, disclosed herein is a mitochondrial genome editing system, wherein the first nucleic acid sequence comprises a targeted gene modification. In some embodiments, the targeted gene modification is selected from the group consisting of insertions, deletions, base pair conversions, methylation, and any variations thereof.

[0021] In one aspect, disclosed herein is a method of treating a mitochondrial disease in a subject in need thereof, wherein the method comprises administering to the subject the mitochondrial genome editing system of any preceding aspects. In some aspects, the mitochondrial genome editing system is imported into a cell and further imported into a mitochondrion. In some aspects, disclosed herein is the method of any preceding aspect, wherein the mitochondrial disease comprises a mitochondrial DNA (mtDNA) mutation. In some aspects, the mitochondrial disease is selected from the group consisting of oxidative phosphorylation defects, Barth’s syndrome, Complex I deficiency, Complex II deficiency, Complex III deficiency, Complex IV deficiency Complex V deficiency, cytochrome c oxidase (COX) deficiency, Leigh Disease, Leber’s Hereditary Optic Neuropathy, and any related diseases thereof. In some aspects, the MTS is selected from the group consisting of cytochrome c oxidase (COX), ATP synthase, subunit c of human ATP synthase, hexokinase I, hexokinase IV, voltage-dependent anion channel 2 (Porin or VDAC), and any variations thereof. BRIEF DESCRIPTION OF FIGURES

[0022] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.

[0023] FIGS. 1A-1D show the correction of m.8993 T>G in NARP cells using prime editing. The schematic diagram illustrates (A) a Prime editor with pegRNA structure in dark grey. (B) the constructs of pSP-A6pegR (top) and MTS-Cas9RT (bottom), and (C) the Msp I site in mutated (top) and wildtype (bottom) hATP6 PCR products. (D) An agarose gel displays PCR products subjected to Msp 1 digestion. MW: Ikb DNA ladder, with DNA sizes labeled in bp on the left. The lanes in the gel represent: lanes 1,3: modified NARP cells, lane 2: unmodified NARP cells. PBS: primer-binding site, EF-1P: human elongation factor-1 alpha promoter, A6pegR: guide RNA for prime editor targeting to ATP6T8993G, U6: U6 promoter, Cas9H840A: a nickase version of Cas9, MMLV: Moloney Murine Leukemia Virus, RT: reverse transcriptase, MTSRNA: RNA targeting sequence. MTS: mitochondrial targeting sequence of the cytochrome oxidase subunit VIII.

[0024] FIGS. 2A-2E show the sanger sequencing of the 261bp amplicons and their alignment with a plasmid carrying the wild type (WT) HATP6 (A). This alignment verifies the correction of the m.8993 T>G mutation (arrows) in the modified NARP cells (B-D) but not in unmodified NARP cells (E). The edited nucleotide base is highlighted in gray, while the 20-nt protospacer is marked by a long rectangular box. Sequences in FIGS. 2A-2E are SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24.

[0025] FIG. 3 shows the AmpliconEZ next generation sequencing and their alignment with a plasmid carrying mutated(mut) hATP6. This alignment verifies the correction of m.8993 T>G mutation in the modified NARP cells (NGS_S1-S3) but not in unmodified NARP cells (NGS- NARP). Sequences marked by a long rectangular box represent the 20-nt protospacer, while sequences highlighted in grey indicate corrected nucleotide bases. Sequences in FIG. 3 are SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27.

[0026] FIGS. 4A-4E show the correction of the m.l 1778 G>A mutation in a LHON cybrid cell line (HFF3) using prime editing. The schematic diagram illustrates (A) pSP-N4pegR, (B) MTS-Cas9RT, and (C) the site of SfaN I in wildtype (top) and mutated (bottom) hND4 PCR products. (D) An agarose gel displays PCR products subjected to SfaN I digestion. MW: Ikb DNA ladder, with DNA sizes labeled in bp on the left. The lanes in the gel represent: lane 1 : unedited HFF3 cells, lane 2: edited-unsorted HFF3 cells, and lane 3: edited- sorted HFF3 cells. (E) Sanger sequencing of the 301 bp amplicons and their alignment with a plasmid carrying the mutated WD4(muthND4) confirm the correction of the m.H778G>A mutation (arrows) in both edited-unsorted and edited-sorted cells, but not in the unedited control cells. The sequence highlighted in grey corresponds to the editing site, while the region marked by a long rectangular box represents the sequence replaced by the RT-template. EF-1P: human elongation factor- 1 alpha promoter, N4pegR: guide RNA for prime editor targeting to ND4G11778A, U6: U6 promoter, Cas9H840A: a nickase version of Cas9, RT: Moloney Murine Leukemia virus reverse transcriptase, MTSRNA: RNA targeting sequence. MTS: mitochondrial targeting sequence of the cytochrome oxidase subunit VIII. Sequences in FIGS. 4E are SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30.

[0027] FIGS. 5 A-5D show the base editor modification of mtDNA in a LHON cybrid cell line (HFF3). The schematic diagram illustrates the constructs of (A) pSP-mutND4gRNA3, (B) MTS-Cas9-ABE, and (C) MTS-Cas9-CBE. (D) Amplicon EZ next generation sequencing, aligned to the mutant hND4Gl 1778A (Original Sequence), confirmed A-to-G conversions mediated by ABE (HFF-ABE) and C-to-T conversions mediated by CBE (HFF-BE3) in HFF3 cells. No nucleotide base conversions were detected in the control (HFF). The sequence highlighted in rectangular grey box on top represents the 20-nt protospacer, while the sequences highlighted in small boxes indicate the conversed-nucleotide bases. EF-1P: human elongation factor- 1 alpha promoter, mutN4gR: guide RNA for base editors targeting to MT- ND4G11778A, MTSRNA: RNA targeting sequence, ABE: Adenine deaminase, CBE: Cytidine deaminase, Cas9D10A: a nickase version of Cas9, UGI: Uracil DNA glycosylase inhibitor, MTS : mitochondrial targeting sequence of the cytochrome oxidase subunit VIII. Sequences in FIGS. 5D are SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38.

[0028] FIGS. 6A-6E show the base editor modification of MT-ND4 in 293T cells. The schematic diagram illustrates the constructs of (A) pSP-wtND4gRNA and (B) MTS-Cas9- CBE. (C-E) Sanger sequencing of the 301 bp amplicons aligned to wild type human MT- ND4(C) has verified the CBE-mediated C-to-T conversion (arrow) in modified cells (D), while no such conversion was observed in control cells (E). Sequences highlighted in grey represent the 20-nt protospacer. U6: U6 promoter, EF-1P: human elongation factor- 1 alpha promoter, wtN4gR: guide RNA for base editors targeting to human mitochondrial gene ND4, MTSRNA: RNA targeting sequence, CBE: Cytidine deaminase, Cas9D10A: a nickase version of Cas9, UGI: Uracil DNA glycosylase inhibitor, MTS: mitochondrial targeting sequence of the cytochrome oxidase subunit VIII. Sequence in FIGS. 6A-6E is SEQ ID NO:39.

[0029] FIGS. 7A-7E show the base editor modification of MT-ND1 in 293T cells. The schematic diagram illustrates the constructs of (A) pSP-NDlgRNA and (B) MTS-Cas9-CBE. (C-E) Sanger sequencing of the 313 bp amplicons aligned to wildtype human MT -ND 1(C) has verified the CBE-mediated C-to-T conversion (arrow) in modified cells (D), while no such conversion was observed in control cells (E). Sequences highlighted in grey represent the 20- nt protospacer. U6: U6 promoter, EF-1P: human elongation factor-1 alpha promoter, NlgR: guide RNA for base editors targeting to human mitochondrial gene ND1, MTSRNA: RNA targeting sequence, CBE: Cytidine deaminase, Cas9D10A: a nickase version of Cas9, UGI: Uracil DNA glycosylase inhibitor, MTS: mitochondrial targeting sequence of the cytochrome oxidase subunit VIII. Sequences in FIGS. 7A-7E are SEQ ID NO:40 and SEQ ID NO:41.

[0030] FIGS. 8A-8E show the base editor modification of MT-ND2 in 293T cells. The schematic diagram illustrates the constructs of (A) pSP-ND2gRNA and (B) MTS-Cas9-CBE. (C-E) Sanger sequencing of the 324 bp amplicons aligned to wildtype human MT-ND2(C) has verified the CBE-mediated C-to-T conversion (arrow) in modified cells (D), while no such conversion was observed in control cells (E). Sequences highlighted in grey represent the 20- nt protospacer. U6: U6 promoter, EF-1P: human elongation factor-1 alpha promoter, N2gR: guide RNA for base editors targeting to human mitochondrial gene ND2, MTSRNA: RNA targeting sequence, CBE: Cytidine deaminase, Cas9D10A: a nickase version of Cas9, UGI: Uracil DNA glycosylase inhibitor, MTS: mitochondrial targeting sequence of the cytochrome oxidase subunit VIII. Sequence in FIGS. 8A-8E is SEQ ID NO:42.

[0031] FIGS. 9A-9E show the base editor modification of MT-ND3 in 293T cells. The schematic diagram illustrates the constructs of (A) pSP-ND3gRNA and (B) MTS-Cas9-CBE. (C-E) Sanger sequencing of the 339 bp amplicons aligned to wild type human MT-ND3 (C) has verified the CBE-mediated C-to-T conversion (arrow) in modified cells (D), while no such conversion was observed in control cells (E). Sequences highlighted in grey represent the 20- nt protospacer. U6: U6 promoter, EF-1P: human elongation factor-1 alpha promoter, N3gR: guide RNA for base editors targeting to human mitochondrial gene ND3, MTSRNA: RNA targeting sequence, CBE: Cytidine deaminase, Cas9D10A: a nickase version of Cas9, UGI: Uracil DNA glycosylase inhibitor, MTS: mitochondrial targeting sequence of the cytochrome oxidase subunit VIII. Sequences in FIGS. 9A-9E are SEQ ID NO:43 and SEQ ID NO:44.

[0032] FIGS. 10A-10E show the base editor modification of MT-ND5 in 293T cells. The schematic diagram illustrates the constructs of (A) pSP-ND5gRNA and (B) MTS-Cas9-CBE. (C-E) Sanger sequencing of the 305 bp amplicons aligned to wild type human MT-ND5(C) has verified the CBE-mediated C-to-T conversion (arrow) in modified cells (D), while no such conversion was observed in control cells (E). Sequences highlighted in grey represent the 20- nt protospacer. U6: U6 promoter, EF- 1P: human elongation factor-1 alpha promoter, N5gR: guide RNA for base editors targeting to human mitochondrial gene ND5, MTSRNA: RNA targeting sequence, CBE: Cytidine deaminase, Cas9D10A: a nickase version of Cas9, UGI: Uracil DNA glycosylase inhibitor, MTS: mitochondrial targeting sequence of the cytochrome oxidase subunit VIII. Sequence in FIGS. 10A-10E is SEQ ID NO:45.

[0033] FIGS. 11A-11E show the base editor modification of MT-ATP8 in 293T cells. The schematic diagram illustrates the constructs of (A) pSP-ATP8gRNA and (B) MTS-Cas9-CBE. (C-E) Sanger sequencing of the 329 bp amplicons aligned to wild type human MT-ATP8 (C) has verified the CBE-mediated C-to-T conversion (arrow) in modified cells (D), while no such conversion was observed in control cells (E). Sequences highlighted in grey represent the 20- nt protospacer. U6: U6 promoter, EF-1P: human elongation factor-1 alpha promoter, A8gR: guide RNA for base editors targeting to human mitochondrial gene ATP8, MTSRNA: RNA targeting sequence, CBE: Cytidine deaminase, Cas9D10A: a nickase version of Cas9, UGI: Uracil DNA glycosylase inhibitor, MTS: mitochondrial targeting sequence of the cytochrome oxidase subunit VIII. Sequence in FIGS. 11A-1 IE is SEQ ID NO:46.

[0034] FIGS 12A-12E show the base editor modification of MT-COX2 in 293T cells. The schematic diagram illustrates the constructs of (A) pSP-COX2gRNA and (B) MTS-Cas9-CBE. (C-E) Sanger sequencing of the 325 bp amplicons aligned to wild type human MT-COX2 (C) has verified the CBE-mediated C-to-T conversion (arrow) in modified cells (D), while no such conversion was observed in control cells (E). Sequences highlighted in grey represent the 20- nt protospacer. U6: U6 promoter, EF-1P: human elongation factor- 1 alpha promoter, C2gR: guide RNA for base editors targeting to human mitochondrial gene COX2. MTSRNA: RNA targeting sequence, CBE: Cytidine deaminase, Cas9D10A: a nickase version of Cas9, UGI: Uracil DNA glycosylase inhibitor, MTS: mitochondrial targeting sequence of the cytochrome oxidase subunit VIII. Sequence in FIGS. 12A-12E is SEQ ID NO:47.

[0035] FIGS. 13A-13E show the base editor modification of MT-COX3 in 293T cells. The schematic diagram illustrates the constructs of (A) pSP-COX3gRNA and (B) MTS-Cas9-CBE. (C-E) Sanger sequencing of the 338 bp amplicons aligned to wild type human MT-COX3 (C) has verified the CBE-mediated C-to-T conversion (arrow) in modified cells (D), while no such conversion was observed in control cells (E). Sequences highlighted in grey represent the 20- nt protospacer. U6: U6 promoter, EF-1P: human elongation factor- 1 alpha promoter, C3gR: guide RNA for base editors targeting to human mitochondrial gene COX3, MTSRNA: RNA targeting sequence, CBE: Cytidine deaminase, Cas9D10A: a nickase version of Cas9, UGI: Uracil DNA glycosylase inhibitor, MTS: mitochondrial targeting sequence of the cytochrome oxidase subunit VIII. Sequence in FIGS. 13A-13E is SEQ ID NO:48. FIGS. 14A-14C show the Cas9 nuclease mediated mtDNA deletion. (A) The schematic diagram illustrates the construct of pSP-A6gR-MTS2-Cas9. (B). qPCR reveals a significant reduction in mtDNA content in Cas9 nuclease-modified cells compared to unmodified controls, as determined by the ratio of mtDNA to nuclear DNA. (C) ATP -based cell viability assay of the modified 293T cells (A6MTS2C) demonstrates a significant decrease over time in culture compared to normal controls. U6: U6 promoter, A6gR: guide RNA targeting to MT-ATP6, MTSRNA: RNA targeting sequence, EF-1P: human elongation factor-1 alpha promoter, Cas9: Cas9 nuclease, MTS: mitochondrial targeting sequence of the cytochrome oxidase subunit VIII.

[0036] FIGS. 15A-15F show direct delivery of RNA to mitochondria in Hela cells. (A-B) The schematic diagram illustrates the constructs of (A) sc-U6-muthTLl-mCherry (A) and (B) sc- U6-wthTLl-GFP. (C) The scatterplot shows the load of the delivered gene muthTL-mCherry relative to endogenous mitochondrial genes ND1, ND4, and ATP6, as well as nuclear gene GAPDH. (D) Mutant hTLl (muthTL) significantly reduces ATP -based cell viability in glucose-free galactose media compared to naive cells (Control). However, the introduction of wildtype hTLl (Rescued) reverses this effect, leading to increased cell viability. (E) Mutant hTLl (muthTL) decreases mitochondrial ATP production rates compared to naive controls (Control), but wildtype hTLl(Rescued) reverses these effects, leading to an increased ratio of ATP production rate in mitochondria (mito) to glycolysis (glyco). Specifically, 1.73 for control, 1.03 for muthTL, and 1.84 for rescued cells. (F) Western blot electrophoresis patterns of mitochondrial protein in control cells and cells transfected with mutant hTLl (muthTL), wildtype hTLl (wthTL), and a combination of wild type and mutant hTLl (Rescued). ITR: AAV inverted terminal repeats, U6: U6 promoter, EF-1P: human elongation factor- 1 alpha promoter, muthTLl: human MT-tRNAleuA3243G, wthTLl: wildtype human MT-tRNAleu, MTSRNA: RNA targeting sequence.

[0037] FIGS. 16A-16G shows the delivery of RNA directly to mitochondria in MEL AS fibroblasts, rescuing their mitochondria function. (A) Mitochondrial-stress analysis indicates that the delivery of wildtype hTLl significantly improved mitochondria function in MELAS cells. This improvement is evident in heightened ATP production (B), maximal respiration (C), improved coupling efficiency (D), increased spare respiratory capacity (E), and reduced proton leak (F). Furthermore, (G) Western blot results demonstrate elevated expression levels of five ETC complexes in MELAS cells after the delivery of wildtype hTLl, compared to controls. ITR: AAV inverted terminal repeats, HSP: human mitochondrial heavy strand promoter, EF- 1P: human elongation factor- 1 alpha promoter, wthTLl : wildtype human MT-tRNAleu, MTSRNA: RNA targeting sequence, Oligo: oligomycin, Rot / AA: rotenone-antimycin A, FCCP: carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone, Crtl: Control, Trt: Rescued, CI, CII, CIII, CIV and CV: electron transport chain (ETC) complexes I-V, OCR: oxygen consumption rate.

[0038] FIGS. 17A-17H shows the delivery of RNA directly to mitochondria in normal C57BL / 6 mice, inducing the MELAS phenotype. H&E staining reveals neuronal cell loss in various brain regions, including the Context (A), Hippocampus (B), Thalamus-Midbrain (C, D), Caudate Putamen (E), corpus callosum-fornix (F), corpus callosum (G), and Hippocampus (H) in mice injected with mutant hTLl. TH: thalamus, M: midbrain, C: cortex, CC: corpus callosum, CP: caudate putamen, F: fomix, HC: hippocampus

[0039] FIGS. 18A- 18C shows the demethylation of mtDNA. The schematic diagram illustrates the constructs of (A) MTS-dCas9-TETl and (B) pSP-CSB-gRNA. (C) AmpliconEZ next generation sequencing on bisulfite converted mtDNA and their alignment with human mtDNA validate the demethylation of the targeted loci (arrows). Highlighted sequences represent bisulfite-converted demethylated Cs converted to Us (corresponding to Ts in DNA). EF-1P: human elongation factor- 1 alpha promoter, dCas9: catalytically inactive Cas9; MTSRNA: RNA targeting sequence; Tetl : Ten-Eleven Translocation enzyme: CSBgR: guide RNA targeting to D-loop conserved sequence blocks, MTS : mitochondrial targeting sequence of the cytochrome oxidase subunit VIII. Sequences in FIGS. 18A-18C are SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53.

[0040] DETAILED DESCRIPTION

[0041] Disclosed herein are mitochondrial targeting sequences that can target RNA into mitochondria. Using this sequence, guide RNA of CRISPR / Cas derived genomic editing tools, including Cas9 nucleases, Base editors, and Prime editors, are targeted into mitochondria in living eukaryotic cells to install a locus-specific modification. Other target RNAs of interest, such as MT-tRNA, are targeted into mitochondria to induce or rescue disease phenotypes. Using this sequence, guide RNA of CRISPR / Cas9, and enzymes for methylation / demethylation to install a locus-specific epigenetic modification in mitochondrial DNA.

[0042] The following description of the disclosure is provided as an enabling teaching of the disclosure. To this end, those skilled in the relevant art will recognize and appreciate that many changes can be made to the various embodiments of the invention described herein, while still obtaining the beneficial results of the present disclosure. It will also be apparent that some of the desired benefits of the present disclosure can be obtained by selecting some of the features of the present disclosure without utilizing other features. Accordingly, those who work in the art will recognize that many modifications and adaptations to the present disclosure are possible and can even be desirable in certain circumstances and are a part of the present disclosure. Thus, the following description is provided as illustrative of the principles of the present disclosure and not in limitation thereof.

[0043] Reference will now be made in detail to the embodiments of the invention, examples of which are illustrated in the drawings and the examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[0044] Terminology

[0045] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of’ and “consisting of’ can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed.

[0046] As used in this disclosure and in the appended claims, the singular forms “a”, “an”, “the”, include plural referents unless the context clearly dictates otherwise.

[0047] The following definitions are provided for the full understanding of terms used in this specification.

[0048] The terms "about" and "approximately" are defined as being “close to” as understood by one of ordinary skill in the art. In one non- limiting embodiment the terms are defined to be within 10%. In another non-limiting embodiment, the terms are defined to be within 5%. In still another non-limiting embodiment, the terms are defined to be within 1%.

[0049] As used herein, the terms "may," "optionally," and "may optionally" are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation "may include an excipient" is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.

[0050] “Composition” refers to any agent that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, a vector, polynucleotide, cells, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the term “composition” is used, then, or when a particular composition is specifically identified, it is to be understood that the term includes the composition per se as well as pharmaceutically acceptable, pharmacologically active vector, polynucleotide, salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.

[0051] A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also, for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount.

[0052] An "increase" can refer to any change that results in larger amount of a symptom, disease, composition, condition, or activity. A substance is also understood to increase the genetic output of a gene when the genetic output of the gene product with the substance is more relative to the output of the gene product without the substance. Also, for example, an increase can be a change in the symptoms of a disorder such that the symptoms are more than previously observed. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount.

[0053] By “reduce” or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic. It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “reduces bacterial growth” means reducing the rate of growth of a bacterium relative to a standard or a control.

[0054] As used herein, the terms “genetically modified” or “gene modification” refers to a living cell, tissue, or organism whose genetic material has been altered using genetic engineering techniques. The genetic modification results in an alteration that may or may not occur naturally by mating and / or natural recombination. Modified genes can be transferred within the same species, across species (creating transgenic organisms), and across kingdoms. New, exogenous genes can be introduced, or endogenous genes can be enhanced, altered, or knocked out. Examples of a gene modification is a gene insertion, a gene deletion, a base pair conversion, a methylation, an acetylation, a sumolyation, or any variation thereof.

[0055] The terms "cell," "cell line" and "cell culture" include progeny. It is also understood that all progenies may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that has the same function or biological property, as screened for in the originally transformed cell, are included.

[0056] A "control" is an alternative subject or sample used in an experiment for comparison purpose. A control can be "positive" or "negative."

[0057] As used herein, by a “subject” is meant an individual. Thus, the “subject” can include domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, chickens, ducks, geese, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), and birds. “Subject” can also include a mammal, such as a primate or a human. Thus, the subject can be a human or veterinary patient.

[0058] A "gene" refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular polypeptide or protein after being transcribed and translated. Any of the polynucleotides sequences described herein may be used to identify larger fragments or full-length coding sequences of the gene with which they are associated.

[0059] The terms “treat,” “treating,” “treatment,” and grammatical variations thereof as used herein, include partially or completely delaying, alleviating, mitigating or reducing the intensity of one or more attendant symptoms of a disorder or condition and / or alleviating, mitigating or impeding one or more causes of a disorder or condition. Treatments may be applied preventively, prophy tactically, palliatively or remedially.

[0060] As used herein, “operably fused” refers to two or more compositions, compounds, or molecules being bound or linked together in such a way the optimizes the intended function. When bound or linked, these compositions, compounds, or molecules can be linked covalently, electrostatic interaction, through hydrogen bonding, or any combinations thereof.

[0061] A “mitochondrial targeting sequence” (MTS) is a sequence that directs the transport of another sequence into to mitochondria. These sequences can be either an RNA, a DNA, or a peptide sequence that transport a molecule of the same family into the mitochondria. These sequences usually either encode a native mitochondrial protein to ensure proper direction into the mitochondria or untranslated regions of mRNAs to facilitate their transport to the mitochondria.

[0062] A “nucleotide” is a compound consisting of a nucleoside, which consists of a nitrogenous base and a 5 -carbon sugar, linked to a phosphate group forming the basic structural unit of nucleic acids, such as DNA or RNA. The four types of nucleotides are adenine (A), cytosine (C), guanine (G), and thymine (T), each of which are bound together by a phosphodiester bond to form a nucleic acid molecule.

[0063] A “nucleic acid” is a chemical compound that serves as the primary informationcarrying molecules in cells and make up the cellular genetic material. Nucleic acids are comprised nucleotides, which are the monomers made of a 5-carbon sugar (usually ribose or deoxyribose), a phosphate group, and a nitrogenous base. A nucleic acid can also be a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). A chimeric nucleic acid comprises two or more of the same kind of nucleic acid fused together to form one compound comprising genetic material.

[0064] A “3’ untranslated region” (3’-UTR) refers to a section of a nucleic acid, usually messenger RNA (mRNA), that immediately follows the translation terminal codon, or the final nucleotide triplet recognized during protein synthesis. The 3’-UTR often contains regulatory regions that influence gene expression and can range from 60-4000 nucleotides.

[0065] A “nuclease” is an enzyme capable of cleaving the phosphodiester bonds between nucleotides of nucleic acids. Nuclease can possess properties to cause double or single stranded breaks to target nucleic acids. Nucleases are commonly used in CRISPR technology to modify a host genome to express or inhibit a target gene.

[0066] As used herein, a “mitochondrial genome editing system” refers to a technology that gives the ability to change the mitochondrial DNA (mtDNA) of an organism. This system comprises components, compounds, and molecules require to modify the mtDNA to induce a specific modification to a target mtDNA.

[0067] A “mitochondrion” is a cellular membrane-bound compartment, or organelle found in most eukaryotic cells, which are essential for cellular respiration and cellular energy production. These cellular structures comprise their own genome consisting of 37 genes important for energy production, respiration, calcium regulation, heat generation, and mediating cell growth and death.

[0068] As used herein, the term “integrated” refers to the act or process of fusing one composition into another composition, cell, or organism to form a new entity.

[0069] A mitochondrial disease refers to any chronic (long- lasting), genetic, inherited disease that arises when mitochondria fail to properly function giving rise to secondary symptoms including, but not limited to heart disease, physical disabilities, seizures, muscle weakness, neurodegenerative disabilities, vomiting, blindness, deafness, and limited eye mobility. Improper mitochondria functions, or mitochondrial dysfunctions include, but are not limited to decreased energy production, increased, or decreased heat production, dysregulation of calcium signaling pathways, dysregulation of cell death and cell growth pathways, and altered respiration.

[0070] A “genome” refers to a complete set of genes or genetic material present within a cell, tissue, or organism. A genome can be nuclear (found within a cell’s nucleus) or mitochondrial (found within a cell’s mitochondria).

[0071] As used herein, a “mutation” refers to changing the structure of a gene, resulting in a variant form that may be transmitted to later generations. A mutation is caused by the alteration of single nucleotides in DNA, or the deletion, insertion, or rearrangement of larger sections of genes. A mutation can lead to the expression of a protein that has been changed physically or functionally leading to lethality, non-lethal dysfunction effects, or no effects.

[0072] “mtDNA” or “mitochondrial DNA” refers to the circular group of genes, or chromosome, found inside mitochondria. mtDNA has features such as maternal inheritance (can only be inherited from the mother of an offspring) and mutation rate.

[0073] As used herein, “import”, “importing” or “imported” refers to the process of bringing a foreign or external composition into a cell, tissue, or organism. Compositions can be imported passively (moved through without assistance), actively (moved through with enzymatic regulation), or osmotically (moved through based on concentration relative to water or another fluid).

[0074] As used herein, “catalytically impaired” refers to the state in which any composition, enzyme, or combination thereof is not capable of performing any intended functions. For example, a natural or engineered nuclease intended to convert an adenine nucleotide into a guanine nucleotide would be catalytically impaired if no conversion or an improper conversion to a thymine or cytosine was performed in the presence of said nuclease.

[0075] Compositions and Systems

[0076] The mitochondria are double membrane bound compartments, or organelles, found within many cell types of most eukaryotic organisms. These organelles are responsible for performing aerobic respiration, the process of chemically consuming oxygen (O2), to generate most of the cell’ s energy supply in the form of adenosine triphosphate (ATP). The mitochondria also comprise its own genome, commonly referred to as a “mitogenome”. The mitogenome comprises 37 genes, also referred to as mitochondrial DNA (mtDNA): 13 for subunits of the respiratory protein complexes I, III, IV, and V, 22 for mitochondrial tRNA, and 2 for rRNA. The mitogenome is susceptible to oxidative damage from both exogenous and endogenous reactive oxygen species (ROS) due to its proximity to the electron transport chain (ETC) and the absence of protective histones. Thus, mtDNA damage tends to be more extensive and persistent compared to nuclear DNA. These accumulated mtDNA damages, such as deletions, insertions, base pair substitutions, etc., lead to defective mitochondrial RNA and protein expression. Therefore, there is a need to provide a chimeric molecule or compound that can effectively repair mtDNA mutations.

[0077] The present disclosure provides a chimeric nucleic acid for targeting RNA molecules into the mitochondria. The present disclosure also provides a mitochondrial genome editing system comprising the chimeric nucleic acid for targeting RNA molecules into the mitochondria. The present disclosure also provides a method of treating mitochondrial diseases using the mitochondrial genome editing system comprising the chimeric nucleic acid for targeting RNA molecules into the mitochondria.

[0078] In one aspect, disclosed herein is a chimeric nucleic acid comprising a first nucleic acid sequence operably fused to a second nucleic acid sequence; wherein the second nucleic acid sequence comprises a mitochondrial targeting sequence (MTS) comprising a mitochondrial ribosomal protein S 12 (MRPS12) gene, or a fragment thereof. In some embodiments, the MTS is located in a non-coding region of the MRPS12 gene, or a fragment thereof. In some embodiments, the MTS is located at the 3’ untranslated region (3’-UTR) of the MRPS12 gene, or a fragment thereof.

[0079] In some embodiments, the MTS comprises at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1. In some embodiments, the MTS encodes at least 25 nucleotides. In some embodiments, the MTS encodes 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, or more nucleotides. In some embodiments, the MTS encodes 163 nucleotides.

[0080] In some aspects, disclosed herein is a chimeric nucleic acid of any preceding aspect, wherein the chimeric nucleic acid further comprises a CRISPR nuclease gene. In some embodiments, the CRISPR nuclease gene is a Cas9 gene, or any variant thereof. In some embodiments, the CRISPR nuclease gene is a catalytically impaired Cas9, dCas9, gene. In some embodiments, the CRISPR nuclease gene is a Cpfl gene, or any variant thereof. In some aspects, disclosed herein is a chimeric nucleic acid of any preceding aspect, wherein the chimeric nucleic acid comprises a ten-eleven translocation (TET) gene. In some embodiments, the chimeric nucleic acid comprises an adenine deaminase (ABE) gene. In some embodiments, the chimeric nucleic acid comprises a cytidine deaminase (CBE) gene. In some embodiments, the chimeric nucleic acid comprises a Moloney murine leukemia virus reverse transcriptase (MMLV-RT) gene.

[0081] In some aspects, disclosed herein is a chimeric nucleic acid of any preceding aspect, wherein the chimeric nucleic acid further comprises a reporter gene. In some embodiments, the reporter gene is either a monomeric red fluorescent protein Cherry (mCherry) gene or a green fluorescent protein (GFP) gene. In some embodiments, the reporter gene includes, but is not limited to a luciferase, yellow fluorescent protein (YFP), blue fluorescent protein (BFP), cyane fluorescent protein (CFP), monomeric red fluorescent protein (mRFP), Discosoma striata (DsRed), mCherry, mOrange, tdTomato, mSTrawberry, mPlum, photoactivatable GFP (PA- GFP), Venus, Kaede, monomeric kusabira orange (mKO), Dronpa, enhanced CFP (ECFP), Emerald, Cyan fluorescent protein for energy transfer (CyPet), super CFP (SCFP), Cerulean, photoswitchable CFP (PS-CFP2), photoactivatable RFP1 (PA-RFP1), photoactivatable mCherry (PA-mCherry), monomeric teal fluorescent protein (mTFPl), Eos fluorescent protein (EosFP), Dendra, TagBFP, TagRFP, enhanced YFP (EYFP), Topaz, Citrine, yellow fluorescent protein for energy transfer (YPet), super YFP (SYFP), enhanced GFP (EGFP), Superfolder GFP, T-Sapphire, Fucci, mK02, m0range2, mApple, Sirius, Azurite, EBFP, and / or EBFP2.

[0082] In some embodiments, the chimeric nucleic acid further comprises a CRISPR nuclease gene. In some embodiments, the chimeric nucleic acid further comprises a ten-eleven translocation (TET) gene. In some embodiments, the chimeric nucleic acid further comprises an adenine deaminase (ABE) gene. In some embodiments, the chimeric nucleic acid further comprises a cytidine deaminase (CBE) gene. In some embodiments, the chimeric nucleic acid further comprises a Moloney murine leukemia virus reverse transcriptase (MMEV-RT) gene. In some embodiments, the CRISPR nuclease gene is a Cas9 gene, or any variant thereof. In some embodiments, the CRISPR nuclease gene is a catalytically impaired Cas9, dCas9, gene. In some embodiments, the CRISPR nuclease gene is a Cpfl gene, or any variant thereof. In some embodiments, the chimeric nucleic acid sequence further comprises a reporter gene. In some embodiments, the first nucleic acid sequence comprises a guide RNA sequence.

[0083] Genome editing systems are a type of genetic engineering in which genetic material, such as nucleic acids, are inserted, deleted, modified, or replaced in a genome of a living organism. Unlike early genetic engineering techniques that randomly insert genetic material into a host genome, current genome editing techniques target the modifications to site specific gene locations. These editing systems commonly utilize engineered nuclease to regulate said modifications to induce changes to the genome. Genome editing techniques are used for many research purposes, including, but not limited to gene therapy and genetic engineering. For the purposes of gene therapy, inserting a functional gene into a cell or cellular compartments, such as the mitochondria, can target and remove defective genes to cure certain genetic diseases.

[0084] In one aspect, disclosed herein is a mitochondrial genome editing system comprising the chimeric nucleic acid of any preceding aspect. In one aspect, disclosed herein is a mitochondrial genome editing system comprising: a chimeric nucleic acid comprising a first nucleic acid sequence operably fused to a second nucleic acid sequence; wherein the second nucleic acid sequence comprises a mitochondrial targeting sequence (MTS) comprising a mitochondrial ribosomal protein S12 (MRPS12) gene, or a fragment thereof; and an RNA- guided nuclease.

[0085] In some embodiments, the RNA-guided nuclease is selected from Cas9, a mutant Cas9 with nickase activity (Cas9H840A or Cas9D10A), or a mutant Cas9 with no nuclease activity (dCas9). In some embodiments, the RNA-guided nuclease is a mutant Cas9 with nickase activity (Cas9H840A or Cas9D10A). In some embodiments, the RNA-guided nuclease is a mutant Cas9 with no nuclease activity (dCas9).

[0086] In one aspect, disclosed herein is a mitochondrial genome editing system, wherein the MTS directs the chimeric nucleic into a mitochondrion. In one aspect, disclosed herein is a mitochondrial genome editing system, wherein the first nucleic acid sequence is integrated into a mitochondrial genome in the presence of the RNA-guided nuclease (or CRISPR nuclease). In some embodiments, the RNA-guided nuclease gene is a Cas9 gene, or any variant thereof. In some embodiments, the RNA-guided nuclease gene is a catalytically impaired Cas9 or a dCas9 gene. In some embodiments, the RNA-guided nuclease gene is a Cpfl gene, or any variant thereof. In one aspect, disclosed herein is a mitochondrial genome editing system, wherein the first nucleic acid sequence comprises a targeted gene modification. In some embodiments, the targeted gene modification is selected from the group consisting of insertions, deletions, base pair conversions, methylation, and any variations thereof. In some embodiments, the RNA-guided nuclease is fused to a second mitochondrial targeting sequence. In some embodiments, the RNA-guided nuclease is fused to an N-terminus of a second mitochondrial targeting sequence. In some embodiments, the RNA-guided nuclease is fused to a C-terminus of a second mitochondrial targeting sequence. In some embodiments, the RNA-guided nuclease is fused to an N-terminus of a second mitochondrial targeting sequence and is fused to a C-terminus of a third mitochondrial targeting sequence.

[0087] In some embodiments, the RNA-guided nuclease is encoded by a third nucleic acid sequence.

[0088] Methods

[0089] The mitochondrial genome, or mitogenome, comprising the mtDNA is susceptible to mutations leading to mitochondrial diseases. These diseases are usually inherited at birth but can be acquired by accumulating with age. Diseases arising from mtDNA mutations can impair functions of various mitochondrial proteins, pathways, and process. Some symptoms of these diseases include, but are not limited to heart disease, physical disabilities, seizures, muscle weakness, neurodegenerative disabilities, vomiting, blindness, deafness, and limited eye mobility. Therefore, developing a treatment method for mitochondrial diseases using a mitochondrial genome editing system is needed.

[0090] In one aspect, disclosed herein is a method of treating a mitochondrial disease in a subject in need thereof, wherein the method comprises administering the mitochondrial genome editing system of any preceding aspects. In some aspects, the mitochondrial genome editing system is imported into a cell and further imported into a mitochondrion.

[0091] In one aspect, disclosed herein is a method for gene editing of a mitochondrial genome in a subject, wherein the method comprises administering the mitochondrial genome editing system of any preceding aspect to the subject. In some aspects, the mitochondrial genome editing system is imported into a cell and further imported into a mitochondrion. In one embodiment, the mitochondrial genome editing system comprises: a chimeric nucleic acid comprising a first nucleic acid sequence operably fused to a second nucleic acid sequence; wherein the second nucleic acid sequence comprises a mitochondrial targeting sequence (MTS) comprising a mitochondrial ribosomal protein S12 (MRPS12) gene, or a fragment thereof; and an RNA-guided nuclease. In some embodiments, the MTS comprises at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 1.

[0092] In one aspect, disclosed herein is a method for gene editing of a mitochondrial genome in a cell, wherein the method comprises administering the mitochondrial genome editing system of any preceding aspect to the cell.

[0093] In some aspects, disclosed herein is the method of any preceding aspect, wherein the mitochondrial disease comprises a mitochondrial DNA (mtDNA) mutation. In some aspects, disclosed herein is the method of any preceding aspect, wherein the mitochondrial disease is selected from the group consisting of oxidative phosphorylation defects, Barth’s syndrome, Complex I deficiency, Complex II deficiency, Complex III deficiency, Complex IV deficiency Complex V deficiency, cytochrome c oxidase (COX) deficiency, Leigh Disease, Leiber’s Hereditary Optic Neuropathy, and any related diseases thereof. In some embodiments, the mitochondrial disease is selected from, but is not limited to, Alper’s disease, camitine-acyl- carnitine deficiency, carnitine deficiency, co-enzyme Q10 deficiency, mitochondrial myopathy, Lethal Infantile Mitochondrial Myopathy, Maternal Myopathy and Cardiomyopathy, Neurogenic muscle weakness, Ataxia, and Retinitis Pigmentosa, Fatal Infantile Cardiomyopathy Plus (FICP), Mitochondrial Encephalomyopathy with Lactic Acidosis and Stroke like episodes (MELAS), MELAS-associated cardiomyopathy, Leiber’s hereditary optic neuropathy and Dystonia (LDYT), Myoclonic Epilepsy and Ragged Red Muscle Fibers (MERRF), Maternally inherited Hypertrophic Cardiomyopathy (MHCM), Chronic Progressive External Ophthalmoplegia (CPEO), Kearns Sayre Syndrome (KSS), Diabetes Mellitus, Diabetes Mellitus + Deafness, Chronic Intestinal Pseudoobstruction with myopathy and Ophthalmoplegia (CIPO), Maternally inherited DEAFness or aminoglycoside- induced DEAFness (DEAF), Progressive encephalopathy (PEM), SensoriNeural Hearing Loss, Encephalomyopathy, Mitochondrial cytopathy, Dilated Cardiomyopathy, Gastrointestinal reflux, Dementia and Chorea, Ataxia, Myoclonus, Exercise Intolerance, Epilepsy, Strokes, Optic atrophy & Cognitive Decline, Familial Bilateral Striatal Necrosis, Focal Segmental Glomerulosclerosis, Lethal Infantile Mitochondrial Myopathy, Myopathy and Diabetes Mellitus, Myoclonic Epilepsy and Psychomotor regression, MERME MERFF / MELAS overlap disease, Maternally inherited hypertrophic cardiomyopathy, maternally inherited cardiomyopathy, maternally inherited Leigh Syndrome (MILS), Mitochondrial Encephalocardiomyopathy, Multisystem Mitochondrial Disorder (myopathy, encephalopathy, blindness, hearing loss, peripheral neuropathy), Nonarteritic Anterior Ischemic Optic Neuropathy, Non-insulin Dependent Diabetes Mellitus, Progressive Encephalopathy, Progressive Myoclonus Epilepsy Rett Syndrome, Sudden Infant Death Syndrome, and Maturity-Onset Diabetes of the Young.

[0094] Methods for delivery of nucleic acids into cells is known in the art. Various delivery methods can use a viral vector (for example, adeno-associated virus (AAV) derived vectors), a nanoparticle, a lipid-like nanoparticle, or other methods known in the art.

[0095] In some embodiments, the second MTS or third MTS can further comprise a nucleic acid encoding cytochrome c oxidase (COX), C0X8, ATP synthase, subunit c of human ATP synthase, hexokinase I, hexokinase IV, voltage -dependent anion channel 2 (Porin or VDAC), or a variation or fragment thereof.

[0096] In some embodiments, the second MTS or third MTS can further comprise a nucleic acid encoding amine oxidase (flavin containing) A, pancreatic beta cell form, peripheral benzodiazepine receptor-related protein, metaxin 2, putative mitochondrial outer membrane protein import receptor (hTOM), glutathione transferase, cytochrome b5, peripheral benzodiazepine receptor, germ cell kinase anchor S-AKAP84, A kinase anchor protein, carnitine O-palmitoyltransferase I precursor, hexokinase II, amine oxidase (flavin containing) B, long chain fatty acid CoA ligase 2, long chain fatty acid CoA ligase 1 (palmitoyl-CoA ligase), voltage-dependent anion channel 1, metaxin 1, Human putative outer mitochondrial membrane 34kDa translocase (hTOM34), voltage-dependent anion channel 4 (outer mitochondrial membrane protein porin), cytochrome-b5 reductase, voltage-dependent anion channel 3 (outer mitochondrial membrane protein porin), Mitochondrial import receptor subunit TOM20 homolog (Mitochondrial 20kDa outer membrane protein), tumorous imaginal discs homolog precursor (HTID-1), or a variation or fragment thereof.

[0097] EXAMPLES

[0098] The following examples are set forth below to illustrate the compositions, devices, methods, and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.

[0099] Example 1. Introduction

[0100] Mitochondrial dysfunction can affect every organ in the body, especially those with high energy demands like the central nervous system, brain, heart, and eyes. To date, no FDA- approved treatment is available for any diseases caused by mtDNA mutation, mainly due to barriers in manipulating mtDNA. Four major approaches, including allotopic expression, mitochondrial replacement, ZFNs / TALENs, and MTSAAV, are currently being used, but each has a major drawback impeding its efficiency. A recently developed strategy uses mitochondrial-targeted TALE arrays to direct double- stranded DNA deaminases to enable base editing in mtDNA. However, this strategy induces high off-target editing. Besides, engineering TALE is time-consuming and challenging; it needs to re-design and re-construct the targeting domain for each novel target sequence. The resulting editing agents can vary substantially in activity and specificity for a targeted site.

[0101] CRISPR / Cas offers new techniques to overcome these difficulties. This system is powered by the principle of easily designed guide RNA(gRNA) that leads Cas nucleases to the locus of interest to initiate site-specific DNA modification. Two new strategies have been developed recently with enhanced editing efficiency and product purity, especially in slowly dividing or non-dividing cells: Base editors and Prime editors. The application of the CRISPR / Cas technologies to mitochondrial gene editing has been hampered because of the barrier in importing their nucleic acid components into the organelle. The technology disclosed herein can overcome these difficult challenges. In addition, this technology allows delivery of small RNAs, for example MT-tRNA, directly into mitochondria.

[0102] Example 2. Prime editor modification of mtDNA.

[0103] Prime editors offer the greatest versatility for generating targeted insertions, deletions, and base conversions to correct most disease-associated mtDNA mutations. This system requires two components: a reverse transcriptase (RT) fused with Cas9 nickase (Cas9RT), and a guide RNA containing the desired sequence to incorporate into the genome (pegRNA) (Fig 1A). To apply the prime editor to modify mtDNA, a cybrid cell line homoplasmic for ATP6T8993G was used. This mutation causes Leigh Syndrome (LS) and Neurogenic muscle weakness, Ataxia, and Retinitis Pigmentosa (NARP). As a first step, a pegRNA targeting the locus of T8993G in ATP6 was designed and linked to the RNA targeting sequence and followed by a fluorescence marker, mCherry, as shown in Fig IB (top, pSP-A6pegR). Then, a construct to deliver Cas9RT into mitochondria was made by fusing CAS9H840A nickase in frame with a 23 amino acid mitochondrial targeting sequence (MTS) from the cytochrome oxidase subunit VIII, a human codon-optimized Moloney murine leukemia virus reverse transcriptase (MMLV-RT), and GFP with a T2A sequence in between (MTS-Cas9RT, Fig IB bottom). After transfecting the cybrids with pSP-A6pegR and MTS-Cas9RT, PCR was performed on DNA extracted from FACS-sorted GFP and mCHERRY double-positive cells and digested with a restriction enzyme, Mspl. ATP6 harboring T8993G mutation gains a MspI site and generates two fragments of 130 and 131bp (Fig 1C top), while wildtype ATP6 (WTATP6) generates only one fragment of 261 bp (Fig 1C bottom). Since NARP cybrids are homoplasmic for ATP6T8993G, the presence of a 261bp fragment in Mspl digestion is evidence of successful modification. Fig ID shows the existence of this 261bp band in the modified cells (lanes 2, 3). Sanger DNA sequencing confirmed that this 261bp band was wildtype ATP6 (Fig 2), and AmpliconEZ next-generation sequencing confirmed that 6-12% of mtDNA were wildtype (Fig 3).

[0104] To apply the prime editor to modify mtDNA, another cybrid cell line (HFF3) homoplasmic for ND4G11778A was used. This mutation causes Leber’s Hereditary Optic Neuropathy (LHON). A pegRNA targeting the locus of G11778A in ND4 was designed and linked to the RNA targeting sequence and followed by a fluorescence marker, mCherry, as shown in Fig 4A (pSP-N4pegR). The MTS-Cas9RT in Fig 4B is the same as in Fig IB. After transfecting the HFF3 cybrids with pSP-N4pegR and MTS-Cas9RT, PCR was performed on DNA extracted from unsorted or FACS-sorted GFP and mCHERRY double-positive cells and digested with a restriction enzyme, SfaN I. Wildtype ND4 (WTND4) having a SfaN I site and generates two fragments of 168 and 133bp (Fig 4C top). In contrast, mutant ND4 (mutND4) generates only one fragment of 301bp (Fig 4C bottom). Since HFF3 cybrids are homoplasmic for ND4G11778A, the presence of 168 and 133 bp fragments in SfaN I digestion is evidence of successful modification. Fig 4D shows the existence of two small bands with expected size only in the modified cells (lanes 2, 3). Sanger DNA sequencing confirmed that modified HFF3 had both mutant and wildtype ND4 in the cell (heteroplasmy)(Fig 4E).

[0105] Example 3. Base editor modification of mtDNA.

[0106] Base editing is a new strategy to install a targeted base modification in an RNA- programmed manner. It uses a Cas9 nickase (Cas9D10A) to direct an adenine (ABE) or cytidine (CBE) deaminase to modify a targeted window of single-stranded DNA, resulting in A:T to G:C or C:G to T:A conversions, respectively. To apply the base editor to modify mtDNA, a gRNA targeting mutant MT-ND4 was designed and linked to the RNA targeting sequence, followed by a fluorescence marker, mCherry, as shown in Fig 5A (pSP- mutND4gRNA3-l). Then, constructs delivering Cas9-ABE or Cas9-CBE into mitochondria were made by fusing CAS9D10A in frame with a 23 amino acid mitochondrial targeting sequence (MTS) from the cytochrome oxidase subunit VIII and an ABE gene (Fig 5B) or a CBE gene and an Uracil DNA glycosylase inhibitor (UG1) gene (Fig 5C). After transfecting HFF3 with pSP-mutND4gRNA3-l and MTS-Cas9-ABE or MTS-Cas9-CBE, DNA was extracted, and AmpliconEZ next-generation sequencing shows that ABE induces successful conversions of A- to -G at A2, A4, and A7 (HFF-ABE), and CBE induces successful conversions of C- to -T at C3 and C6 (HFF-BE3), from the 5’-end of the protospacer (Fig 5D).

[0107] To apply base editing for mtDNA modification in another cell line (293T), a gRNA was designed targeting wildtype MT-ND4 and linked it to the RNA targeting sequence, followed by a fluorescence marker, mCherry, as illustrated in Fig 6A (pSP-wtND4gRNA). The MTS-Cas9-CBE in Fig 6B is the same as that in Fig 5C. After transfecting 293T cells with pSP-wtND4gRNA and MTS-Cas9-CBE, PCR was performed on DNA extracted from unsorted cells for Sanger sequencing. Sequence alignment with human mtDNA (Fig 6C) demonstrates that CBE induced a successful conversion of C- to -T at C6 from the 5 ’-end of the protospacer (Fig 6D). The abundance of targeted editing in the cells reached 11% on day 5 and increased to 16% on day 6 post-transfection. No nucleotide base conversion was detected in control cells (Fig 6E)

[0108] To employ the base editor for mtDNA modification in another locus in 293T cells, a gRNA was designed targeting wildtype MT-ND1 and linked it to the RNA targeting sequence, followed by a fluorescence marker, mCherry, as illustrated in Fig 7 A (pSP-NDlgRNA). The MTS-Cas9-CBE in Fig 7B is the same as that in Fig 5C. After transfecting 293T cells with pSP-NDlgRNA and MTS-Cas9-CBE, PCR was performed on DNA extracted from unsorted cells for Sanger sequencing. The sequence alignment with human mtDNA (Fig 7C) demonstrates that CBE induced a successful conversion of C- to -T at C5 from the 5 ’-end of the protospacer (Fig 7D), whereas no nucleotide base conversion was found in control cells (Fig 7E).

[0109] To employ the base editor for mtDNA modification in the ND2 gene in 293T cells, a gRNA was designed targeting wildtype MT-ND2 and linked it to the RNA targeting sequence, followed by a fluorescence marker, mCherry, as illustrated in Fig 8A (pSP-ND2gRNA). The MTS-Cas9-CBE in Fig 8B is the same as that in Fig 5C. After transfecting 293T cells with pSP-ND2gRNA and MTS-Cas9-CBE, PCR was performed on DNA extracted from unsorted cells for Sanger sequencing. The sequence alignment with human mtDNA (Fig 8C) demonstrates that CBE induced a successful conversion of C- to -T at C2, C3, and C6 from the 5 ’ -end of the protospacer (Fig 8D) , where as no nucleotide base conversion was found in control cells (Fig 8E).

[0110] To employ the base editor for mtDNA modification in the ND3 gene in 293T cells, a gRNA was designed targeting wildtype MT-ND3 and linked it to the RNA targeting sequence, followed by a fluorescence marker, mCherry, as illustrated in Fig 9A (pSP-ND3gRNA). The MTS-Cas9-CBE in Fig 9B is the same as that in Fig 5C. After transfecting 293T cells with pSP-ND3gRNA and MTS-Cas9-CBE, PCR was performed on DNA extracted from unsorted cells for Sanger sequencing. The sequence alignment with human mtDNA (Fig 9C) demonstrates that CBE induced a successful conversion of C- to -T at C5 and C6 from the 5’- end of the protospacer (Fig 9D), whereas no nucleotide base conversion was found in control cells (Fig 9E).

[0111] To employ the base editor for mtDNA modification in the ND5 gene in 293T cells, a gRNA was designed targeting wildtype MT-ND5 and linked it to the RNA targeting sequence, followed by a fluorescence marker, mCherry, as illustrated in Fig 10A (pSP-ND5gRNA). The MTS-Cas9-CBE in Fig 10B is the same as that in Fig 5C. After transfecting 293T cells with pSP-ND5gRNA and MTS-Cas9-CBE, PCR was performed on DNA extracted from unsorted cells for Sanger sequencing. The sequence alignment with human mtDNA (Fig IOC) demonstrates that CBE induced a successful conversion of C- to -T at C5 and C6 from the 5’- end of the protospacer (Fig 10D), whereas no nucleotide base conversion was found in control cells (Fig 10E).

[0112] To employ the base editor for mtDNA modification in the ATP8 gene in 293T cells, a gRNA was designed targeting wildtype MT-ATP8 and linked it to the RNA targeting sequence, followed by a fluorescence marker, mCherry, as illustrated in Fig 11A (pSP-ATP8gRNA). The MTS-Cas9-CBE in Fig 11B is the same as that in Fig 5C. After transfecting 293T cells with pSP-ATP8gRNA and MTS-Cas9-CBE, PCR was performed on DNA extracted from unsorted cells for Sanger sequencing. The sequence alignment with human mtDNA (Fig 11C) demonstrates that CBE induced a successful conversion of C- to -T at C6 from the 5 ’-end of the protospacer (Fig 11D), whereas no nucleotide base conversion was found in control cells (Fig HE).

[0113] To employ the base editor for mtDNA modification in the C0X2 gene in 293T cells, a gRNA was designed targeting wildtype MT-C0X2 and linked it to the RNA targeting sequence, followed by a fluorescence marker, mCherry, as illustrated in Fig 12A (pSP-COX2gRNA). The MTS-Cas9-CBE in Fig 12B is the same as that in Fig 5C. After transfecting 293T cells with pSP-COX2gRNA and MTS-Cas9-CBE, PCR was performed on DNA extracted from unsorted cells for Sanger sequencing. The sequence alignment with human mtDNA (Fig 12C) demonstrates that CBE induced a successful conversion of C- to -T at C6 from the 5 ’-end of the protospacer (Fig 12D), whereas no nucleotide base conversion was found in control cells (Fig 12E). To employ the base editor for mtDNA modification in the C0X3 gene in 293T cells, a gRNA was designed targeting wildtype MT-C0X3 and linked it to the RNA targeting sequence, followed by a fluorescence marker, mCherry, as illustrated in Fig 13A (pSP-COX3gRNA). The MTS-Cas9-CBE in Fig 13B is the same as that in Fig 5C. After transfecting 293T cells with pSP-COX3gRNA and MTS-Cas9-CBE, PCR was performed on DNA extracted from unsorted cells for Sanger sequencing. The sequence alignment with human mtDNA (Fig 13C) demonstrates that CBE induced a successful conversion of C- to -T at C5 and C6 from the 5’- end of the protospacer (Fig 13D), whereas no nucleotide base conversion was found in control cells (Fig 13E).

[0114] Example 4. Cas nuclease editing of mtDNA.

[0115] Most mitochondrial genetic diseases are heteroplasmic (having both mutant and wildtype mtDNA). The percentage of mutant mtDNA is key to the development and severity of clinical phenotypes. Therefore, reducing the level of mutant mtDNA provides a promising therapeutic strategy for mitochondrial diseases. To assess the potential of Cas nuclease in facilitating the deletion of targeted mtDNA, a gRNA targeting MT-ATP6 was designed and linked to the RNA targeting sequence, followed by MTS-Cas9 nuclease (Fig 14 A). The resultant constructs were transfected into 293T cells, leading to a highly significant decrease in mtDNA content (Fig 14B), and ATP-based cell viability (Fig 14C) compared to controls.

[0116] Example 5. Delivery of tRNA to mitochondria in vitro.

[0117] Mutations in mitochondrial tRNA represent a significant factor in human morbidity, being associated with a diverse range of pathologies. These range from isolated organ-specific diseases like myopathy or hearing loss, to multisystem disorders, including gastrointestinal dysmotility, encephalopathy, and life-threatening cardiomyopathy. Mitochondrial Encephalopathy, Lactic Acidosis, and Stroke-like episodes (MELAS) syndrome stand out as one of the most prevalent mitochondrial genetic disorders, leading to multi-organ failure in children and young adults. About 80% of MELAS cases arise from a point mutation in the mitochondrial tRNALeu (MT-7L / ) / m. 3243 A>G. Currently, no FDA-approved treatment exists for MELAS or any other mitochondrial disease. To determine whether MT-tRNA can be delivered into mitochondria and induce metabolic shifts in normal cells, mutant and wildtype human MT-TL1 were linked with the RNA targeting sequence and cloned under the control of the U6 promoter in an AAV2 self-complimentary backbone to generate constructs of sc-U6-muthTLl-mCherry (Fig 15A) and sc-U6-wthTLl-GFP (Fig 15B), with EF-la promoter-driven mCherry or GFP downstream, respectively.

[0118] Hela cells were transfected with sc-U6-muthTLl -mCherry and collected the cells on the 5thday post-infection for DNA extraction and qPCR. The efficiency of gene delivery was assessed as the ratio of mCherry DNA levels to those of endogenous mitochondrial genes, ND1, ND4 and ATP6, as well as the nuclear gene GAPDH. As presented in Fig 15C, the ratios in infected cells were 28%, 51%, 65%, and 2213% for ND1, ND4, ATP6, and GAPDH, respectively. An ATP-based cell viability assay was conducted on the transfected cells. As shown in Fig 15D, cells transfected with the mutated hTLl (muthTL) showed a significant reduction in cell viability compared to naive Hela cells (Control). However, introducing wildtype hTLl (Rescued) reversed this effect, leading to a marked improvement in cell viability. ATP production rates were measured using a Seahorse XF HS Mini Analyzer. The results indicated that cells transfected with the mutated hTLl (muthTL) exhibited a pronounced decrease in mitochondrial ATP production (MitoATP) and a slight reduction in glycolytic ATP production ATP production ratio of 1.03, compared to a ratio of 1.73 in naive Hela cells (control). Notably, the introduction of wildtype hTLl (Rescued) restored respiratory function, increasing the mitochondrial-to-glycolytic ATP production ratio to 1.84 (Fig 15E). Western blot confirmed that transfection with mutant hTLl (muthTL) led to a reduction in the expression of mitochondrial proteins ND4 and ATP6, compared to naive Hela cells (control). However, the introduction of the wild-type hTLl gene (Rescued) efficiently restored the expression of these mitochondrial proteins to levels comparable to those of control cells. Additionally, transfection with the wild-type hTLl gene alone (wthTL) did not induce any noticeable alterations in the expression of those proteins (Fig 15F).

[0119] To explore the therapeutic use of this strategy, MELAS fibroblasts homoplasmic for the m.3243A>G mutation were transfected with sc-U6-wthTLl-GFP and cells were collected on day 5 post transfection for mitochondrial-stress analysis. Specifically, the collected cells (rescued and naive control) were seeded in a seahorse plate containing glucose-free-galactose media and assessed 24 hours after seeding. These results demonstrated that the delivery of the wildtype hTLl gene led to significant improvements in mitochondria function (Fig 16A). This improvement is evident in heightened ATP production (Fig 16B), maximal respiration (Fig 16C), improved coupling efficiency (Fig 16D), increased spare respiratory capacity (Fig 16E), and reduced proton leak (Fig 16F). Western-blot using an antibody cocktail recognizing all five complexes (CLCV) of the mitochondrial electron transport chain (ETC) showed a noticeable increase in the expression of all complexes in the rescued cells from the two experimental replicates (Trt 1, and Trt 2) when compared to the naive control (Crtl, Fig 16G).

[0120] Example 6. Delivery of tRNA to mitochondria in vivo.

[0121] To detect the delivery of RNA directly into mitochondria in vivo, sc-U6-muthTLl- mCherry was packaged into AAV2 / 9 and injected it into the brains of 3-month-old C57BL / 6 mice via intracerebroventricular (ICV) injection. The mice were euthanized 6 months after injection, and their brains were dissected for histological analysis. This analysis showed distinct neuropathological changes, characterized by spongiform degeneration and notable loss of neuronal cells in various regions of the brain, including the hippocampus, cerebral cortex, thalamus, caudate putamen, fornix, midbrain, and corpus callosum (Fig 17A-17H).

[0122] Example 7. Epigenetic modification of mtDNA.

[0123] An increasing number of studies have shown that mtDNA replication and gene expression is also regulated by epigenetic mechanisms (mitoepigenetics), and their alteration has been suggested to underlie several pathological pathways, including Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, diabetic retinopathy, nonalcoholic fatty liver disease, and cancer. However, several technical challenges specific to interrogating the mitochondrial epigenome have hampered widespread studies to date. To alter mtDNA methylation at specific loci, MTS-dCas9 was fused in frame with the Ten-Eleven Translocation enzyme (TET1), an enzyme mediating DNA demethylation (MTS-dCas9-TETl, Fig 18A). Then, a guide RNA targeting mtDNA D-loop conserved sequence blocks was designed and linked to the RNA targeting sequence, followed by a fluorescence marker, mCherry, as shown in Fig 18B (pSP-CSB-gRNA). D-loop is a control region that contains the promoters driving transcription initiation of mtDNA heavy and light strands. After transfecting HFF3 cells with pSP-CSB-gRNA and MTS-dCas9-TETl, mtDNA was extracted for Bisulfite conversion and AmpliconEZ next-generation sequencing. Fig 18C shows that dCas9-TETl meditated demethylation at the targeted Cs in the modified cells (modified), which were converted to Us (Ts in DNA) by bisulfite (arrows).

[0124] It will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure without departing from the scope or spirit of the invention. Other embodiments of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the methods disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

[0125] SEQUENCES

[0126] 1. SEQ ID NO: 1 - MTSRNA cagaagaagtgacggctgggggcacagtgggctgggcgcccctgcagaacatgaaccttccgctcctggctgccacag ggtcctccgatgctggcctttgcgcctctagaggcagccactcatggattcaagtcctggctccgcctcttccatcaggacc act

[0127] 2. SEQ ID NO: 2 - MTS of the cytochrome oxidase subunit VIII atgtccgtcctgaegccgctgctgctgcggggcttgacaggctcggcccggcggctcecagtgcegcgcgcc

[0128] 3. SEQ ID NO: 3 - pSP-A6pegR cagaagaagtgacggctgggggcacagtgggctgggcgcccctgcagaacatgaaccttccgctcctggctgccacag ggtcctccgatgctggcctttgcgcctctagaggcagccactcatggattcaagtcctggctccgcctcttccatcaggacc actctagagggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgatccggtgccta gagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgt atataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggaccggtggatcccgccac catggtgagcaagggcgaggaggataacatggccatcatcaaggagttcatgcgcttcaaggtgcacatggagggctcc gtgaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacgagggcacccagaccgccaagctga aggtgaccaagggtggccccctgcccttcgcctgggacatcctgtcccctcagttcatgtacggctccaaggcctacgtga agcaccccgccgacatccccgactacttgaagctgtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgag gacggcggcgtggtgaccgtgacccaggactcctccctgcaggacggcgagttcatctacaaggtgaagctgcgcggc accaacttcccctccgacggccccgtaatgcagaagaagaccatgggctgggaggcctcctccgagcggatgtaccccg aggacggcgccctgaagggcgagatcaagcagcggctgaagctgaaggacggcggccactacgacgctgaggtcaa gaccacctacaaggccaagaagcccgtgcagctgcccggcgcctacaacgtcaacatcaagttggacatcacctcccac aacgaggactacaccatcgtggaacagtacgaacgcgccgagggccgccactccaccggcggcatggacgagctgta caagtaagaattctaactagagctcgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctccccc gtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtagg tgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagagaatagcaggcatgctggg gagcggccgcaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgac caaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggggcgc ctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcg gcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccttagcgcccgctcctttc gctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttag tgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcg ccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaactctatctcgggctattcttt tgatttataagggattttgccgatttcggtctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaat attaacgtttacaattttatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaac acccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcat gtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtca tgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacatt caaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacattt ccgtgtcgcccttattcccttttttgcggcattttgccttcctgUtttgctcacccagaaacgctggtgaaagtaaaagatgctg aagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaaga acgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcgg tcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaa gagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaagg agctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccatacc aaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactct agcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctgg ctggtttattgctgataaatctggagccggtgagcgtggaagccgcggtatcattgcagcactggggccagatggtaagcc ctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgc ctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaagga tctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtaga aaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggt ggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttct tctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagt ggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgg gctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagct atgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagag cgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatt tttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctgg ccttttgctcacatgtgagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaat taatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaatt atgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaac accgtactcattcaaccaatagccgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagt ggcaccgagtcggtgctacggccagggctattggttgaatgttttttgttttagagctagaaatagcaagttaaaataaggcta gtccgtttttagcgcgtgcgccaattctgcagacaaatggctctagaggtac SEQ ID NO: 4 - MTS-Cas9RT cctgagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggcccagtgagcaagggcgaggagctg ttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcg agggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccct cgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccg ccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaa gttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggca caagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttca agatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggc cccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcac atggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaaggaattctaactagagctc gctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtg ccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggt ggggcaggacagcaagggggaggattgggaagagaatagcaggcatgctggggagcggccgcaggaacccctagtg atggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctt tgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggggcgcctgatgcggtattttctccttacgcatc tgtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgtggt ggttacgcgcagcgtgaccgctacacttgccagcgccttagcgcccgctcctttcgctttcttcccttcctttctcgccacgttc gccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaa acttgatttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttcttta atagtggactcttgttccaaactggaacaacactcaactctatctcgggctattcttttgatttataagggattttgccgatttcgg tctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaattttatggtgcactct cagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggctt gtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcacc gaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcagg tggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaata accctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcgg cattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggtt acatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaa gttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatga cttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataacc atgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgg gggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatg cctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactg gatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagcc ggtgagcgtggaagccgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacg gggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtca gaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctc atgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcct ttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctacca actctttttccgaaggtaactggctlcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccac ttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgt cttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagc ccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagg gagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaac gcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagc ctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgtgagggcctatttccc atgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagt acaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttacc gtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccgggtcttcgagaagacctgttttaga gctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttttgttttagagct agaaatagcaagttaaaataaggctagtccgtttttagcgcgtgcgccaattctgcagacaaatggctctagaggtaccaga agaagtgacggctgggggcacagtgggctgggcgcccctgcagaacatgaaccttccgctcctggctgccacagggtc ctccgatgctggcctttgcgcctctagaggcagccactcatggattcaagtcctggctccgcctcttccatcaggaccactct agagggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgatccggtgcctagaga aggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatata agtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggaccggtatgtccgtcctgacgccg ctgctgctgcggggcttgacaggctcggcccggcggctcccagtgccgcgcgccgacaagaagtacagcatcggcctg gacatcggcaccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattcaaggtgctgg gcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggcc acccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatctgcaagagatcttcagca acgagatggccaaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagcacga gcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaag aaactggtggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgatcaagttccggggcc acttcctgatcgagggcgacctgaaccccgacaacagcgacgtggacaagctgttcatccagctggtgcagacctacaac cagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccatcctgtctgccagactgagcaagagc agacggctggaaaatctgatcgcccagctgcccggcgagaagaagaatggcctgttcggcaacctgattgccctgagcct gggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcagctgagcaaggacacctacgac gacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggccgccaagaacctgtccgacg ccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagcgcctctatgatcaagagatac gacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagattttctt cgaccagagcaagaacggctacgccggctacattgacggcggagccagccaggaagagttctacaagttcatcaagcc catcctggaaaagatggacggcaccgaggaactgctcgtgaagctgaacagagaggacctgctgcggaagcagcgga ccttcgacaacggcagcatcccccaccagatccacctgggagagctgcacgccattctgcggcggcaggaagatttttac ccaUcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctactacgtgggccctctggccag gggaaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtgga caagggcgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgccc aagcacagcctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaa gcccgccttcctgagcggcgagcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaag cagctgaaagaggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcc tccctgggcacataccacgatctgctgaaaattatcaaggacaaggacttcctggacaatgaggaaaacgaggacattctg gaagatatcgtgctgaccctgacactgtttgaggacagagagatgatcgaggaacggctgaaaacctatgcccacctgttc gacgacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctgagccggaagctgatcaacgg catccgggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaacttcatgcagct gatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacga gcacattgccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtg aaagtgatgggccggcacaagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacccagaagggaca gaagaacagccgcgagagaatgaagcggatcgaagagggcatcaaagagctgggcagccagatcctgaaagaacacc ccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctgcagaatgggcgggatatgtacgtggacca ggaactggacatcaaccggctgtccgactacgatgtggacgccatcgtgcctcagagctttctgaaggacgactccatcga caacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaagaggtcgtgaagaaga tgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgagag aggcggcctgagcgaactggataaggccggcttcatcaagagacagctggtggaaacccggcagatcacaaagcacgt ggcacagatcctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcacc ctgaagtccaagctggtgtccgatttccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgccc acgacgcctacctgaacgccgtcgtgggaaccgccctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggc gactacaaggtgtacgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaaggctaccgccaagtacttct tctacagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatccggaagcggcctctgatcgaga caaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctgagcatgcccc aagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcga taagctgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgc tggtggtggccaaagtggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatgga aagaagcagcttcgagaagaatcccatcgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatca agctgcctaagtactccctgttcgagctggaaaacggccggaagagaatgctggcctctgccggcgaactgcagaaggg aaacgaactggccctgccctccaaatatgtgaacttcctgtacctggccagccactatgagaagctgaagggctcccccga ggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcgagcagatcagcgagttct ccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccatcag agagcaggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccacc atcgaccggaagaggtacaccagcaccaaagagglgctggacgccaccctgatccaccagagcatcaccggcctgtac gagacacggatcgacctgtctcagctgggaggtgactctggaggatctagcggaggatcctctggcagcgagacaccag gaacaagcgagtcagcaacaccagagagcagtggcggcagcagcggcggcagcagcaccctaaatatagaagatgag tatcggctacatgagacctcaaaagagccagatgtttctctagggtccacatggctgtctgattttcctcaggcctgggcgga aaccgggggcatgggactggcagttcgccaagctcctctgatcatacctctgaaagcaacctctacccccgtgtccataaa acaataccccatgtcacaagaagccagactggggatcaagccccacatacagagactgttggaccagggaatactggtac cctgccagtccccctggaacacgcccctgctacccgttaagaaaccagggactaatgattataggcctgtccaggatctga gagaagtcaacaagcgggtggaagacatccaccccaccgtgcccaacccttacaacctcttgagcgggctcccaccgtc ccaccagtggtacactgtgcttgatttaaaggatgcctttttctgcctgagactccaccccaccagtcagcctctcttcgccttt gagtggagagatccagagatgggaatctcaggacaattgacctggaccagactcccacagggtttcaaaaacagtcccac cctgtttaatgaggcactgcacagagacctagcagacttccggatccagcacccagacttgatcctgctacagtacgtggat gacttactgctggccgccacttctgagctagactgccaacaaggtactcgggccctgttacaaaccctagggaacctcggg tatcgggcctcggccaagaaagcccaaatttgccagaaacaggtcaagtatctggggtatcttctaaaagagggtcagaga tggctgactgaggccagaaaagagactgtgatggggcagcctactccgaagacccctcgacaactaagggagttcctag ggaaggcaggcttctgtcgcctcttcatccctgggtttgcagaaatggcagcccccctgtaccctctcaccaaaccgggga ctctgtttaattggggcccagaccaacaaaaggcctatcaagaaatcaagcaagctcttctaactgccccagccctggggtt gccagatttgactaagccctttgaactctttgtcgacgagaagcagggctacgccaaaggtgtcctaacgcaaaaactggg accttggcgtcggccggtggcctacctgtccaaaaagctagacccagtagcagctgggtggcccccttgcctacggatgg tagcagccattgccgtactgacaaaggatgcaggcaagctaaccatgggacagccactagtcattctggccccccatgca gtagaggcactagtcaaacaaccccccgaccgctggctttccaacgcccggatgactcactatcaggccttgcttttggaca cggaccgggtccagttcggaccggtggtagccctgaacccggctacgctgctcccactgcctgaggaagggctgcaaca caactgccttgatatcctggccgaagcccacggaacccgacccgacctaacggaccagccgctcccagacgccgacca cacctggtacacggatggaagcagtctcttacaagagggacagcgtaaggcgggagctgcggtgaccaccgagaccga ggtaatctgggctaaagccctgccagccgggacatccgctcagcgggctgaactgatagcactcacccaggccctaaag atggcagaaggtaagaagctaaatgtttatactgatagccgttatgcttttgctactgcccatatccatggagaaatatacaga aggcgtgggtggctcacatcagaaggcaaagagatcaaaaataaagacgagatcttggccctactaaaagccctctttctg cccaaaagacttagcataatccattgtccaggacatcaaaagggacacagcgccgaggctagaggcaaccggatggctg accaagcggcccgaaaggcagccatcacagagactccagacacctctaccctcctcatagaaaattcatcaccctctggc ggctcaaaaagaaccgccgacggcagcgaattcgagtccgtcctgacgccgctgctgctgcggggcttgacaggctcgg cccggcggctcccagtgccgcgcgccggccgg SEQ ID NO: 5 - pSP-mutND4gRNA3-l aattctaactagagctcgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttcct tgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctat tctggggggtggggtggggcaggacagcaagggggaggattgggaagagaatagcaggcatgctggggagcggccg caggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgc ccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggggcgcctgatgcggt attttctccttacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgcattaag cgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccttagcgcccgctcctttcgctttcttccctt cctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggca cctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgtt ggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaactctatctcgggctattcttttgatttataagg gattttgccgatttcggtctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttac aattttatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgac gcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagagg ttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatg gtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatc cgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgccc ttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgg gtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatg atgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcataca ctattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgca gtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgctt ttltgcacaacatgggggatcalglaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagc gtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggca acaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctg ataaatctggagccggtgagcgtggaagccgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgta gttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaag cattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagat cctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaagg atcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccgg atcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgt agttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagt ggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggg gttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgc cacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggag cttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgt caggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgclggccttttgctcacat gtgagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgta aacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatg gactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccgcacatca taatcctctctcagttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtc ggtgcttttttgttttagagctagaaatagcaagttaaaataaggctagtccgtttttagcgcgtgcgccaattctgcagacaaa tggctctagaggtaccagaagaagtgacggctgggggcacagtgggctgggcgcccctgcagaacatgaaccttccgct cctggctgccacagggtcctccgatgctggcctttgcgcctctagaggcagccactcatggattcaagtcctggctccgcct cttccatcaggaccactctagagggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaatt gatccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtg ggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggaccggt ggatcccgccaccatggtgagcaagggcgaggaggataacatggccatcatcaaggagttcatgcgcttcaaggtgcac atggagggctccgtgaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacgagggcacccaga ccgccaagctgaaggtgaccaagggtggccccctgcccttcgcctgggacatcctgtcccctcagttcatgtacggctcca aggcctacgtgaagcaccccgccgacatccccgactacttgaagctgtccttccccgagggcttcaagtgggagcgcgtg atgaacttcgaggacggcggcgtggtgaccgtgacccaggactcctccctgcaggacggcgagttcatctacaaggtga agctgcgcggcaccaacttcccctccgacggccccgtaatgcagaagaagaccatgggctgggaggcctcctccgagc ggatgtaccccgaggacggcgccctgaagggcgagatcaagcagcggctgaagctgaaggacggcggccactacga cgctgaggtcaagaccacctacaaggccaagaagcccgtgcagctgcccggcgcctacaacgtcaacatcaagttggac atcacctcccacaacgaggactacaccatcgtggaacagtacgaacgcgccgagggccgccactccaccggcggcatg gacgagctgtacaagtaag SEQ ID NO: 6 - pSP-wtND4gRNA aattctaactagagctcgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttcct tgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctat tctggggggtggggtggggcaggacagcaagggggaggattgggaagagaatagcaggcatgctggggagcggccg caggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgc ccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggggcgcctgatgcggt attttctccttacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgcattaag cgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccttagcgcccgctcctttcgctttcttccctt cctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggca cctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgtt ggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaactctatctcgggctattcttttgatttataagg gattttgccgatttcggtctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttac aattttatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgac gcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagagg ttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatg gtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatc cgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgccc ttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgg gtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatg atgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcataca ctattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgca gtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgctt ttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagc gtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggca acaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctg ataaatctggagccggtgagcgtggaagccgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgta gttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaag cattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagat cctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaagg atcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccgg atcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgt agttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagt ggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggg gttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgc cacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggag cttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgt caggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacat gtgagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgta aacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatg gactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccgcgcatca taatcctctctcagttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtc ggtgcttttttgttttagagctagaaatagcaagttaaaataaggctagtccgtttttagcgcgtgcgccaattctgcagacaaa tggctctagaggtaccagaagaagtgacggctgggggcacagtgggctgggcgcccctgcagaacatgaaccttccgct cctggctgccacagggtcctccgatgctggcctttgcgcctctagaggcagccactcatggattcaagtcctggctccgcct cttccatcaggaccactctagagggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaatt gatccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtg ggggagaaccglatataaglgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggaccggt ggatcccgccaccatggtgagcaagggcgaggaggataacatggccatcatcaaggagttcatgcgcttcaaggtgcac atggagggctccgtgaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacgagggcacccaga ccgccaagctgaaggtgaccaagggtggccccctgcccttcgcctgggacatcctgtcccctcagttcatgtacggctcca aggcctacgtgaagcaccccgccgacatccccgactacttgaagctgtccttccccgagggcttcaagtgggagcgcgtg atgaacttcgaggacggcggcgtggtgaccgtgacccaggactcctccctgcaggacggcgagttcatctacaaggtga agctgcgcggcaccaacttcccctccgacggccccgtaatgcagaagaagaccatgggctgggaggcctcctccgagc ggatgtaccccgaggacggcgccctgaagggcgagatcaagcagcggctgaagctgaaggacggcggccactacga cgctgaggtcaagaccacctacaaggccaagaagcccgtgcagctgcccggcgcctacaacgtcaacatcaagttggac atcacctcccacaacgaggactacaccatcgtggaacagtacgaacgcgccgagggccgccactccaccggcggcatg gacgagctgtacaagtaag SEQ ID NO: 7 - MTS-Cas9-ABE tacacgtcgcaagaaccgaatatgttacttacaagaaatttttagcaatgagatggccaaagttgacgattctttctttcaccgtt tggaagagtccttccttgtcgaagaggacaagaaacatgaacggcaccccatctttggaaacatagtagatgaggtggcat atcatgaaaagtacccaacgatttatcacctcagaaaaaagctagttgactcaactgataaagcggacctgaggttaatctac ttggctcttgcccatatgataaagttccgtgggcactttctcattgagggtgatctaaatccggacaactcggatgtcgacaaa ctgttcatccagttagtacaaacctataatcagttgtttgaagagaaccctataaatgcaagtggcgtggatgcgaaggctatt cttagcgcccgcctctctaaatcccgacggctagaaaacctgatcgcacaattacccggagagaagaaaaatgggttgttc ggtaaccttatagcgctctcactaggcctgacaccaaattttaagtcgaacttcgacttagctgaagatgccaaattgcagctt agtaaggacacgtacgatgacgatctcgacaatctactggcacaaattggagatcagtatgcggacttatttttggctgccaa aaaccttagcgatgcaatcctcctatctgacatactgagagttaatactgagattaccaaggcgccgttatccgcttcaatgat caaaaggtacgatgaacatcaccaagacttgacacttctcaaggccctagtccgtcagcaactgcctgagaaatataagga aatattctttgatcagtcgaaaaacgggtacgcaggttatattgacggcggagcgagtcaagaggaattctacaagtttatca aacccatattagagaagatggatgggacggaagagttgcttgtaaaactcaatcgcgaagatctactgcgaaagcagcgg actttcgacaacggtagcattccacatcaaatccacttaggcgaattgcatgctatacttagaaggcaggaggatttttatccg ttcctcaaagacaatcgtgaaaagattgagaaaatcctaacctttcgcataccttactatgtgggacccctggcccgaggga actctcggttcgcatggatgacaagaaagtccgaagaaacgattactccatggaattttgaggaagttgtcgataaaggtgc gtcagctcaatcgttcatcgagaggatgaccaactttgacaagaatttaccgaacgaaaaagtattgcctaagcacagtttac tttacgagtatttcacagtgtacaatgaactcacgaaagttaagtatgtcactgagggcatgcgtaaacccgcctttctaagcg gagaacagaagaaagcaatagtagatctgttattcaagaccaaccgcaaagtgacagttaagcaattgaaagaggactact ttaagaaaattgaatgcttcgattctgtcgagatctccggggtagaagatcgatttaatgcgtcacttggtacgtatcatgacct cctaaagataattaaagataaggacttcctggataacgaagagaatgaagatatcttagaagatatagtgttgactcttaccct ctttgaagatcgggaaatgattgaggaaagactaaaaacatacgctcacctgttcgacgataaggttatgaaacagttaaag aggcgtcgctatacgggctggggacgattgtcgcggaaacttatcaacgggataagagacaagcaaagtggtaaaactat tctcgattttctaaagagcgacggcttcgccaataggaactttatgcagctgatccatgatgactctttaaccttcaaagaggat atacaaaaggcacaggtttccggacaaggggactcattgcacgaacatattgcgaatcttgctggttcgccagccatcaaa aagggcatactccagacagtcaaagtagtggatgagctagttaaggtcatgggacgtcacaaaccggaaaacattgtaatc gagatggcacgcgaaaatcaaacgactcagaaggggcaaaaaaacagtcgagagcggatgaagagaatagaagagg gtattaaagaactgggcagccagatcttaaaggagcatcctgtggaaaatacccaattgcagaacgagaaactttacctcta ttacctacaaaatggaagggacatgtatgttgatcaggaactggacataaaccgtttatctgattacgacgtcgatcacattgt accccaatcctttttgaaggacgattcaatcgacaataaagtgcttacacgctcggataagaaccgagggaaaagtgacaat gttccaagcgaggaagtcgtaaagaaaatgaagaactattggcggcagctcctaaatgcgaaactgataacgcaaagaaa gttcgataacttaactaaagctgagaggggtggcttgtctgaacttgacaaggccggatttattaaacgtcagctcgtggaaa cccgccaaatcacaaagcatgttgcacagatactagattcccgaatgaatacgaaatacgacgagaacgataagctgattc gggaagtcaaagtaatcactttaaagtcaaaattggtgtcggacttcagaaaggattttcaattctataaagttagggagataa ataactaccaccatgcgcacgacgcttatcttaatgccgtcgtagggaccgcactcattaagaaatacccgaagctagaaa gtgagtttgtgtatggtgattacaaagtttatgacgtccgtaagatgatcgcgaaaagcgaacaggagataggcaaggctac agccaaatacttcttttattctaacattatgaatttctttaagacggaaatcactctggcaaacggagagatacgcaaacgacct ttaattgaaaccaatggggagacaggtgaaatcgtatgggataagggccgggacttcgcgacggtgagaaaagttttgtcc atgccccaagtcaacatagtaaagaaaactgaggtgcagaccggagggttttcaaaggaatcgattcttccaaaaaggaat agtgataagctcatcgctcgtaaaaaggactgggacccgaaaaagtacggtggcttcgatagccctacagttgcctattctg tcctagtagtggcaaaagttgagaagggaaaatccaagaaactgaagtcagtcaaagaattattggggataacgattatgg agcgctcgtcttttgaaaagaaccccatcgacttccttgaggcgaaaggttacaaggaagtaaaaaaggatctcataattaa actaccaaagtatagtctgtttgagttagaaaatggccgaaaacggatgttggctagcgccggagagcttcaaaaggggaa cgaactcgcactaccgtctaaatacgtgaatttcctgtatttagcgtcccattacgagaagttgaaaggttcacctgaagataa cgaacagaagcaactttttgttgagcagcacaaacattatctcgacgaaatcatagagcaaatttcggaattcagtaagaga gtcatcctagctgatgccaatctggacaaagtattaagcgcatacaacaagcacagggataaacccatacgtgagcaggc ggaaaatattatccatttgtttactcttaccaacctcggcgctccagccgcattcaagtattttgacacaacgatagatcgcaaa cgatacacttctaccaaggaggtgctagacgcgacactgattcaccaatccatcacgggattatatgaaactcggatagattt gtcacagcttgggggtgactctggtggttcttccgtcctgacgccgctgctgctgcggggcttgacaggctcggcccggcg gctcccagtgccgcgcgccaccggtgtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgttgttt gcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcatt gtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcag gcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctcgataccgtcgacctctagct agagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaa gcataaagtgtaaagcctagggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcg ggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgct tcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggltat ccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggcc gcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaac ccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccg gatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgt tcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtcc aacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtg ctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagtt accttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagca gattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactca cgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaa agtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatc catagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccg cgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcct gcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaac gttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaagg cgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgc agtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagta ctcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgcca catagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatc cagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacagg aaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaa gcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacattt ccccgaaaagtgccacctgacgtcgacggatcgggagatcgatctcccgatcccctagggtcgactctcagtacaatctgc tctgatgccgcatagttaagccagtatctgctccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagct acaacaaggcaaggcttgaccgacaattgcatgaagaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacggg ccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagt tccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgt atgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtac atcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatg accttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaa tgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaa atcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtct atataagcagagctggtttagtgaaccgtcagatccgctagagatccgcggccgctaatacgactcactatagggagagcc gccaccatgtccgaagtcgagttttcccatgaglactggatgagacacgcattgactctcgcaaagagggcttgggatgaa cgcgaggtgcccgtgggggcagtactcgtgcataacaatcgcgtaatcggcgaaggttggaataggccgatcggacgcc acgaccccactgcacatgcggaaatcatggcccttcgacagggagggcttgtgatgcagaattatcgacttatcgatgcga cgctgtacgtcacgcttgaaccttgcgtaatgtgcgcgggagctatgattcactcccgcattggacgagttgtattcggtgcc cgcgacgccaagacgggtgccgcaggttcactgatggacgtgctgcatcacccaggcatgaaccaccgggtagaaatca cagaaggcatattggcggacgaatgtgcggcgctgttgtccgacttttttcgcatgcggaggcaggagatcaaggcccag aaaaaagcacaatcctctactgactctggtggttcttctggtggttctagcggcagcgagactcccgggacctcagagtccg ccacacccgaaagttctggtggttcttctggtggttcttccgaagtcgagttttcccatgagtactggatgagacacgcattga ctctcgcaaagagggctcgagatgaacgcgaggtgcccgtgggggcagtactcgtgctcaacaatcgcgtaatcggcga aggttggaatagggcaatcggactccacgaccccactgcacatgcggaaatcatggcccttcgacagggagggcttgtga tgcagaattatcgacttatcgatgcgacgctgtacgtcacgtttgaaccttgcgtaatgtgcgcgggagctatgattcactccc gcattggacgagttgtattcggtgttcgcaacgccaagacgggtgccgcaggttcactgatggacgtgctgcattacccag gcatgaaccaccgggtagaaatcacagaaggcatattggcggacgaatgtgcggcgctgttgtgttacttttttcgcatgcc caggcaggtctttaacgcccagaaaaaagcacaatcctctactgactctggtggttcttctggtggttctagcggcagcgag actcccgggacctcagagtccgccacacccgaaagttctggtggttcttctggtggttctgataaaaagtattctattggtttag ccatcggcactaattccgttggatgggctgtcataaccgatgaatacaaagtaccttcaaagaaatttaaggtgttggggaac acagaccgtcattcgattaaaaagaatcttatcggtgccctcctattcgatagtggcgaaacggcagaggcgactcgcctga aacgaaccgctcggagaaggta SEQ ID NO: 8 - MTS-Cas9-CBE atatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatggga ctttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggat agcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggac tttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcaga gctggtttagtgaaccgtcagatccgctagagatccgcggccgctaatacgactcactatagggagagccgccaccatga gctcagagactggcccagtggctgtggaccccacattgagacggcggatcgagccccatgagtttgaggtattcttcgatc cgagagagctccgcaaggagacctgcctgctttacgaaattaattgggggggccggcactccatttggcgacatacatcac agaacactaacaagcacgtcgaagtcaacttcatcgagaagttcacgacagaaagatatttctgtccgaacacaaggtgca gcattacctggtttctcagctggagcccatgcggcgaatgtagtagggccatcactgaattcctgtcaaggtatccccacgtc actctgtttatttacatcgcaaggctgtaccaccacgctgacccccgcaatcgacaaggcctgcgggatttgatctcttcagg tgtgactatccaaattatgactgagcaggagtcaggatactgctggagaaactttgtgaattatagcccgagtaatgaagccc actggcctaggtatccccatctgtgggtacgactgtacgttcttgaactgtactgcatcatactgggcctgcctccttgtctcaa cattctgagaaggaagcagccacagctgacattctttaccatcgctcttcagtcttgtcattaccagcgactgcccccacacat tctctgggccaccgggttgaaaagcggcagcgagactcccgggacctcagagtccgccacacccgaaagtgataaaaa gtattctattggtttagccatcggcactaattccgltggatgggctgtcataaccgatgaalacaaagtaccttcaaagaaattt aaggtgttggggaacacagaccgtcattcgattaaaaagaatcttatcggtgccctcctattcgatagtggcgaaacggcag aggcgactcgcctgaaacgaaccgctcggagaaggtatacacgtcgcaagaaccgaatatgttacttacaagaaattttta gcaatgagatggccaaagttgacgattctttctttcaccgtttggaagagtccttccttgtcgaagaggacaagaaacatgaa cggcaccccatctttggaaacatagtagatgaggtggcatatcatgaaaagtacccaacgatttatcacctcagaaaaaagc tagttgactcaactgataaagcggacctgaggttaatctacttggctcttgcccatatgataaagttccgtgggcactttctcatt gagggtgatctaaatccggacaactcggatgtcgacaaactgttcatccagttagtacaaacctataatcagttgtttgaaga gaaccctataaatgcaagtggcgtggatgcgaaggctattcttagcgcccgcctctctaaatcccgacggctagaaaacct gatcgcacaattacccggagagaagaaaaatgggttgttcggtaaccttatagcgctctcactaggcctgacaccaaatttta agtcgaacttcgacttagctgaagatgccaaattgcagcttagtaaggacacgtacgatgacgatctcgacaatctactggc acaaattggagatcagtatgcggacttatttttggctgccaaaaaccttagcgatgcaatcctcctatctgacatactgagagtt aatactgagattaccaaggcgccgttatccgcttcaatgatcaaaaggtacgatgaacatcaccaagacttgacacttctcaa ggccctagtccgtcagcaactgcctgagaaatataaggaaatattctttgatcagtcgaaaaacgggtacgcaggttatattg acggcggagcgagtcaagaggaattctacaagtttatcaaacccatattagagaagatggatgggacggaagagttgcttg taaaactcaatcgcgaagatctactgcgaaagcagcggactttcgacaacggtagcattccacatcaaatccacttaggcg aattgcatgctatacttagaaggcaggaggatttttatccgttcctcaaagacaatcgtgaaaagattgagaaaatcctaacct ttcgcataccttactatgtgggacccctggcccgagggaactctcggttcgcatggatgacaagaaagtccgaagaaacga ttactccatggaattttgaggaagttgtcgataaaggtgcgtcagctcaatcgttcatcgagaggatgaccaactttgacaag aatttaccgaacgaaaaagtattgcctaagcacagtttactttacgagtatttcacagtgtacaatgaactcacgaaagttaagt atgtcactgagggcatgcgtaaacccgcctttctaagcggagaacagaagaaagcaatagtagatctgttattcaagacca accgcaaagtgacagttaagcaattgaaagaggactactttaagaaaattgaatgcttcgattctgtcgagatctccggggta gaagatcgatttaatgcgtcacttggtacgtatcatgacctcctaaagataattaaagataaggacttcctggataacgaaga gaatgaagatatcttagaagatatagtgttgactcttaccctctttgaagatcgggaaatgattgaggaaagactaaaaacata cgctcacctgttcgacgataaggttatgaaacagttaaagaggcgtcgctatacgggctggggacgattgtcgcggaaact tatcaacgggataagagacaagcaaagtggtaaaactattctcgattttctaaagagcgacggcttcgccaataggaacttta tgcagctgatccatgatgactctttaaccttcaaagaggatatacaaaaggcacaggtttccggacaaggggactcattgca cgaacatattgcgaatcttgctggttcgccagccatcaaaaagggcatactccagacagtcaaagtagtggatgagctagtt aaggtcatgggacgtcacaaaccggaaaacattgtaatcgagatggcacgcgaaaatcaaacgactcagaaggggcaaa aaaacagtcgagagcggatgaagagaatagaagagggtattaaagaactgggcagccagatcttaaaggagcatcctgt ggaaaatacccaattgcagaacgagaaactttacctctattacctacaaaatggaagggacatgtatgttgatcaggaactg gacataaaccgtttatctgattacgacgtcgatcacattgtaccccaatcctttttgaaggacgattcaatcgacaataaagtgc ttacacgctcggataagaaccgagggaaaagtgacaatgttccaagcgaggaagtcgtaaagaaaatgaagaactattgg cggcagctcctaaatgcgaaactgataacgcaaagaaagttcgataacttaactaaagctgagaggggtggcttgtctgaa cttgacaaggccggatttattaaacgtcagctcgtggaaacccgccaaatcacaaagcatgttgcacagatactagattccc gaatgaatacgaaatacgacgagaacgataagctgattcgggaagtcaaagtaatcactttaaagtcaaaaltggtgtcgga cttcagaaaggattttcaattctataaagttagggagataaataactaccaccatgcgcacgacgcttatcttaatgccgtcgta gggaccgcactcattaagaaatacccgaagctagaaagtgagtttgtgtatggtgattacaaagtttatgacgtccgtaagat gatcgcgaaaagcgaacaggagataggcaaggctacagccaaatacttcttttattctaacattatgaatttctttaagacgga aatcactctggcaaacggagagatacgcaaacgacctttaattgaaaccaatggggagacaggtgaaatcgtatgggata agggccgggacttcgcgacggtgagaaaagttttgtccatgccccaagtcaacatagtaaagaaaactgaggtgcagacc ggagggttttcaaaggaatcgattcttccaaaaaggaatagtgataagctcatcgctcgtaaaaaggactgggacccgaaa aagtacggtggcttcgatagccctacagttgcctattctgtcctagtagtggcaaaagttgagaagggaaaatccaagaaac tgaagtcagtcaaagaattattggggataacgattatggagcgctcgtcttttgaaaagaaccccatcgacttccttgaggcg aaaggttacaaggaagtaaaaaaggatctcataattaaactaccaaagtatagtctgtttgagttagaaaatggccgaaaac ggatgttggctagcgccggagagcttcaaaaggggaacgaactcgcactaccgtctaaatacgtgaatttcctgtatttagc gtcccattacgagaagttgaaaggttcacctgaagataacgaacagaagcaactttttgttgagcagcacaaacattatctcg acgaaatcatagagcaaatttcggaattcagtaagagagtcatcctagctgatgccaatctggacaaagtattaagcgcata caacaagcacagggataaacccatacgtgagcaggcggaaaatattatccatttgtttactcttaccaacctcggcgctcca gccgcattcaagtattttgacacaacgatagatcgcaaacgatacacttctaccaaggaggtgctagacgcgacactgattc accaatccatcacgggattatatgaaactcggatagatttgtcacagcttgggggtgactctggtggttctactaatctgtcag atattattgaaaaggagaccggtaagcaactggttatccaggaatccatcctcatgctcccagaggaggtggaagaagtcat tgggaacaagccggaaagcgatatactcgtgcacaccgcctacgacgagagcaccgacgagaatgtcatgcttctgacta gcgacgcccctgaatacaagccttgggctctggtcatacaggatagcaacggtgagaacaagattaagatgctctctggtg gttcttccgtcctgacgccgctgctgctgcggggcttgacaggctcggcccggcggctcccagtgccgcgcgccgtttaa acccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaa ggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtg gggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggctt ctgaggcggaaagaaccagctggggctcgataccgtcgacctctagctagagcttggcgtaatcatggtcatagctgtttcc tgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctagggtgcctaatga gtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatc ggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgtt cggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaaga acatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccc cctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttcc ccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcg tggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccc cccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactgg cagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactac ggctacactagaagaacagtalttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatc cggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaaga agatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaa aggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttac caatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataac tacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatc agcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattg ttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcac gctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaag cggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcata attctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcgg cgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaa aacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactga tcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataaggg cgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacat atttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtcgacggatcg ggagatcgatctcccgatcccctagggtcgactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccc tgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatg aagaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagtt attaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcc tggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccatt gacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatc SEQ ID NO: 9 - pSP-A6gR-MTS2-Cas9 ccggtatgtccgtcctgacgccgctgctgctgcggggcttgacaggctcggcccggcggctcccagtgccgcgcgccga caagaagtacagcatcggcctggacatcggcaccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgccc agcaagaaattcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctgttcgaca gcggcgaaacagccgaggccacccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctg ctatctgcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttccacagactggaagagtccttcctggt ggaagaggataagaagcacgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccc caccatctaccacctgagaaagaaactggtggacagcaccgacaaggccgacctgcggctgatctatctggccctggcc cacatgatcaagttccggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggacaagctgttcat ccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccatcctg tctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaagaatggcctgttcg gcaacctgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcag ctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctgg ccgccaagaacctgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgag cgcctctatgatcaagagatacgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctg agaagtacaaagagattttcttcgaccagagcaagaacggctacgccggctacattgacggcggagccagccaggaaga gttctacaagttcatcaagcccatcctggaaaagatggacggcaccgaggaactgctcgtgaagctgaacagagaggacc tgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacctgggagagctgcacgccattctgcg gcggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctacta cgtgggccctctggccaggggaaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaa cttcgaggaagtggtggacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgccca acgagaaggtgctgcccaagcacagcctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtg accgagggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgtggacctgctgttcaagaccaac cggaaagtgaccgtgaagcagctgaaagaggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggcgt ggaagatcggttcaacgcctccctgggcacataccacgatctgctgaaaattatcaaggacaaggacttcctggacaatga ggaaaacgaggacattctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatcgaggaacggctga aaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctga gccggaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgc caacagaaacttcatgcagctgatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggcc agggcgatagcctgcacgagcacattgccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaa ggtggtggacgagctcgtgaaagtgatgggccggcacaagcccgagaacatcgtgatcgaaatggccagagagaacca gaccacccagaagggacagaagaacagccgcgagagaatgaagcggatcgaagagggcatcaaagagctgggcagc cagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctgcagaatgggc gggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggaccatatcgtgcctcagagctttct gaaggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccg aagaggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaa tctgaccaaggccgagagaggcggcctgagcgaactggataaggccggcttcatcaagagacagctggtggaaacccg gcagatcacaaagcacgtggcacagatcctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgg gaagtgaaagtgatcaccctgaagtccaagctggtgtccgatttccggaaggatttccagttttacaaagtgcgcgagatca acaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgccctgatcaaaaagtaccctaagctgga aagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaa ggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatccgg aagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcgg aaagtgctgagcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcct gcccaagaggaacagcgataagctgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgacagcc ccaccgtggcctattctgtgctggtggtggccaaagtggaaaagggcaagtccaagaaactgaagagtgtgaaagagctg ctggggatcaccatcatggaaagaagcagcttcgagaagaatcccatcgactttctggaagccaagggctacaaagaagt gaaaaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaacggccggaagagaatgctggcctctg ccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcctgtacctggccagccactatgag aagctgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcat cgagcagatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagc accgggataagcccatcagagagcaggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgcc ttcaagtactttgacaccaccatcgaccggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatccacca gagcatcaccggcctgtacgagacacggatcgacctgtctcagctgggaggcgacatgtccgtcctgacgccgctgctgc tgcggggcttgacaggctcggcccggcggctcccagtgccgcgcgccggccggcctgagggcagaggaagtctgcta acatgcggtgacgtcgaggagaatcctggcccagtgagcaagggcgaggagctgttcaccggggtggtgcccatcctg gtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaa gctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcg tgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccag gagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtga accgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaaca gccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgagga cggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaacca ctacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgacc gccgccgggatcactctcggcatggacgagctgtacaaggaattctaactagagctcgctgatcagcctcgactgtgcctt ctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaata aaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaaggggg aggattgggaagagaatagcaggcatgctggggagcggccgcaggaacccctagtgatggagttggccactccctctct gcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgag cgagcgagcgcgcagctgcctgcaggggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcata cgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgc tacacttgccagcgccttagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctc taaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcac gtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaact ggaacaacactcaactctatctcgggctattcttttgatttataagggattttgccgatttcggtctattggttaaaaaatgagctg atttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaattttatggtgcactctcagtacaatctgctctgatgcc gcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgctta cagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagg gcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtg cgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataa tattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctca cccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaaca gcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtatt atcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtc acagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggcc aacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttg atcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaac gttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttg caggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtggaagccgcg gtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatgg atgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatac tttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgt gagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctg cttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactg gcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccg cctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaag acgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgac ctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtat ccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctg tcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaa cgcggcctttttacggttcctggccttttgctggccttttgctcacatgtgagggcctatttcccatgattccttcatatttgcatat acgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaa agtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatt tcttggctttatatatcttgtggaaaggacgaaacaccgtactcattcaaccaatagccgttttagagctagaaatagcaagtta aaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttttgttttagagctagaaatagcaagttaaa ataaggctagtccgtttttagcgcgtgcgccaattctgcagacaaatggctctagaggtaccagaagaagtgacggctggg ggcacagtgggctgggcgcccctgcagaacatgaaccttccgctcctggctgccacagggtcctccgatgctggcctttg cgcctctagaggcagccactcatggattcaagtcctggctccgcctcttccatcaggaccactctagagggcagagcgcac atcgcccacagtccccgagaagttggggggaggggtcggcaattgatccggtgcctagagaaggtggcgcggggtaaa ctgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtg aacgttctttttcgcaacgggtttgccgccagaacacagga

[0129] 10. SEQ ID NO: 10 - MTS-dCas9-TETl cctgagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggcccagtgagcaagggcgaggagctg ttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcg agggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccct cgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccg ccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaa gttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggca caagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttca agatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggc cccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcac atggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaaggaattctaactagagctc gctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtg ccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggt ggggcaggacagcaagggggaggattgggaagagaatagcaggcatgctggggagcggccgcaggaacccctagtg atggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctt tgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggggcgcctgatgcggtattttctccttacgcatc tgtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgtggt ggttacgcgcagcgtgaccgctacacttgccagcgccttagcgcccgctcctttcgctttcttcccttcctttctcgccacgttc gccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaa acttgatttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttcttta atagtggactcttgttccaaactggaacaacactcaactctatctcgggctattcttttgatttataagggattttgccgatttcgg tctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaattttatggtgcactct cagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggctt gtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcacc gaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcagg tggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaata accctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcgg cattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggtt acatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaa gttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatga cttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataacc atgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgg gggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatg cctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactg gatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagcc ggtgagcglggaagccgcggtalcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacg gggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtca gaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctc atgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcct ttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctacca actctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccac ttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgt cttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagc ccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagg gagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaac gcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagc ctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgtgagggcctatttccc atgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagt acaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttacc gtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccgggtcttcgagaagacctgttttaga gctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttttgttttagagct agaaatagcaagttaaaataaggctagtccgtttttagcgcgtgcgccaattctgcagacaaatggctctagaggtaccaga agaagtgacggctgggggcacagtgggctgggcgcccctgcagaacatgaaccttccgctcctggctgccacagggtc ctccgatgctggcctttgcgcctctagaggcagccactcatggattcaagtcctggctccgcctcttccatcaggaccactct agagggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgatccggtgcctagaga aggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatata agtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggaccggtatgtccgtcctgacgccg ctgctgctgcggggcttgacaggctcggcccggcggctcccagtgccgcgcgccgacaagaagtacagcatcggcctg gacatcggcaccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattcaaggtgctgg gcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggcc acccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatctgcaagagatcttcagca acgagatggccaaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagcacga gcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaag aaactggtggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgatcaagttccggggcc acttcctgatcgagggcgacctgaaccccgacaacagcgacgtggacaagctgttcatccagctggtgcagacctacaac cagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccatcctgtctgccagactgagcaagagc agacggctggaaaatctgatcgcccagctgcccggcgagaagaagaatggcctgttcggcaacctgattgccctgagcct gggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcagctgagcaaggacacctacgac gacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggccgccaagaacctgtccgacg ccatcctgctgagcgacatcctgagagtgaacaccgagalcaccaaggcccccctgagcgcctctatgatcaagagatac gacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagattttctt cgaccagagcaagaacggctacgccggctacattgacggcggagccagccaggaagagttctacaagttcatcaagcc catcctggaaaagatggacggcaccgaggaactgctcgtgaagctgaacagagaggacctgctgcggaagcagcgga ccttcgacaacggcagcatcccccaccagatccacctgggagagctgcacgccattctgcggcggcaggaagatttttac ccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctactacgtgggccctctggccag gggaaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtgga caagggcgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgccc aagcacagcctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaa gcccgccttcctgagcggcgagcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaag cagctgaaagaggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcc tccctgggcacataccacgatctgctgaaaattatcaaggacaaggacttcctggacaatgaggaaaacgaggacattctg gaagatatcgtgctgaccctgacactgtttgaggacagagagatgatcgaggaacggctgaaaacctatgcccacctgttc gacgacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctgagccggaagctgatcaacgg catccgggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaacttcatgcagct gatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacga gcacattgccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtg aaagtgatgggccggcacaagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacccagaagggaca gaagaacagccgcgagagaatgaagcggatcgaagagggcatcaaagagctgggcagccagatcctgaaagaacacc ccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctgcagaatgggcgggatatgtacgtggacca ggaactggacatcaaccggctgtccgactacgatgtggacgccatcgtgcctcagagctttctgaaggacgactccatcga caacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaagaggtcgtgaagaaga tgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgagag aggcggcctgagcgaactggataaggccggcttcatcaagagacagctggtggaaacccggcagatcacaaagcacgt ggcacagatcctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcacc ctgaagtccaagctggtgtccgatttccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgccc acgacgcctacctgaacgccgtcgtgggaaccgccctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggc gactacaaggtgtacgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaaggctaccgccaagtacttct tctacagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatccggaagcggcctctgatcgaga caaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctgagcatgcccc aagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcga taagctgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgc tggtggtggccaaagtggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatgga aagaagcagcttcgagaagaatcccatcgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatca agctgcctaagtactccctgttcgagctggaaaacggccggaagagaatgctggcctctgccggcgaactgcagaaggg aaacgaactggccctgccctccaaatatgtgaacttcctgtacctggccagccactatgagaagctgaagggctcccccga ggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcgagcagatcagcgagttct ccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccatcag agagcaggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccacc atcgaccggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtac gagacacggatcgacctgtctcagctgggaggcgactccgtcctgacgccgctgctgctgcggggcttgacaggctcgg cccggcggctcccagtgccgcgcgccggccggcctctgcccacctgcagctgtcttgatcgagttatacaaaaagacaaa ggcccatattatacacaccttggggcaggaccaagtgttgctgctgtcagggaaatcatggagaataggtatggtcaaaaa ggaaacgcaataaggatagaaatagtagtgtacaccggtaaagaagggaaaagctctcatgggtgtccaattgctaagtg ggttttaagaagaagcagtgatgaagaaaaagttctttgtttggtccggcagcgtacaggccaccactgtccaactgctgtg atggtggtgctcatcatggtgtgggatggcatccctcttccaatggccgaccggctatacacagagctcacagagaatctaa agtcatacaatgggcaccctaccgacagaagatgcaccctcaatgaaaatcgtacctgtacatgtcaaggaattgatccag agacttgtggagcttcattctcttttggctgttcatggagtatgtactttaatggctgtaagtttggtagaagcccaagccccaga agatttagaattgatccaagctctcccttacatgaaaaaaaccttgaagataacttacagagtttggctacacgattagctcca atttataagcagtatgctccagtagcttaccaaaatcaggtggaatatgaaaatgttgcccgagaatgtcggcttggcagcaa ggaaggtcgacccttctctggggtcactgcttgcctggacttctgtgctcatccccacagggacattcacaacatgaataatg gaagcactgtggtttgtaccttaactcgagaagataaccgctctttgggtgttattcctcaagatgagcagctccatgtgctac ctctttataagctttcagacacagatgagtttggctccaaggaaggaatggaagccaagatcaaatctggggccatcgaggt cctggcaccccgccgcaaaaaaagaacgtgtttcactcagcctgttccccgttctggaaagaagagggctgcgatgatga cagaggttcttgcacataagataagggcagtggaaaagaaacctattccccgaatcaagcggaagaataactcaacaaca acaaacaacagtaagccttcgtcactgccaaccttagggagtaacactgagaccgtgcaacctgaagtaaaaagtgaaac cgaaccccattttatcttaaaaagttcagacaacactaaaacttattcgctgatgccatccgctcctcacccagtgaaagagg catctccaggcttctcctggtccccgaagactgcttcagccacaccagctccactgaagaatgacgcaacagcctcatgcg ggttttcagaaagaagcagcactccccactgtacgatgccttcgggaagactcagtggtgccaatgctgcagctgctgatg gccctggcatttcacagcttggcgaagtggctcctctccccaccctgtctgctcctgtgatggagcccctcattaattctgagc cttccactggtgtgactgagccgctaacgcctcatcagccaaaccaccagccctccttcctcacctctcctcaagaccttgcc tcttctccaatggaagaagatgagcagcattctgaagcagatgagcctccatcagacgaacccctatctgatgaccccctgt cacctgctgaggagaaattgccccacattgatgagtattggtcagacagtgagcacatctttttggatgcaaatattggtggg gtggccatcgcacctgctcacggctcggttttgattgagtgtgcccggcgagagctgcacgctaccactcctgttgagcacc ccaaccgtaatcatccaacccgcctctcccttgtcttttaccagcacaaaaacctaaataagccccaacatggttttgaactaa acaagattaagtttgaggctaaagaagctaagaataagaaaatgaaggcctcagagcaaaaagaccaggcagctaatgaa ggtccagaacagtcctctgaagtaaatgaattgaaccaaattccttctcataaagcattaacattaacccatgacaatgttgtca ccgtgtccccttatgctctcacacacgttgcggggccctataaccattgggtcggccgg SEQ ID NO: 11 - pSP-CSB-gRNA aattctaactagagctcgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttcct tgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctat tctggggggtggggtggggcaggacagcaagggggaggattgggaagagaatagcaggcatgctggggagcggccg caggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgc ccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggggcgcctgatgcggt attttctccttacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgcattaag cgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccttagcgcccgctcctttcgctttcttccctt cctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggca cctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgtt ggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaactctatctcgggctattcttttgatttataagg gattttgccgatttcggtctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttac aattttatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgac gcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagagg ttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatg gtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatc cgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgccc ttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgg gtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatg atgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcataca ctattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgca gtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgctt ttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagc gtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggca acaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctg ataaatctggagccggtgagcgtggaagccgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgta gttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaag cattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagat cctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaagg atcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccgg atcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgt agttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagt ggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggg gttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgc cacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggag cttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgt caggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacat gtgagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgta aacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatg gactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccgaGAG ATGTGTTTAAGTGCTGgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaa gtggcaccgagtcggtgcttttttgttttagagctagaaatagcaagttaaaataaggctagtccgtttttagcgcgtgcgcca attctgcagacaaatggctctagaggtaccagaagaagtgacggctgggggcacagtgggctgggcgcccctgcagaa catgaaccttccgctcctggctgccacagggtcctccgatgctggcctttgcgcctctagaggcagccactcatggattcaa gtcctggctccgcctcttccatcaggaccactctagagggcagagcgcacatcgcccacagtccccgagaagttggggg gaggggtcggcaattgatccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccg cctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgcca gaacacaggaccggtggatcccgccaccatggtgagcaagggcgaggaggataacatggccatcatcaaggagttcat gcgcttcaaggtgcacatggagggctccgtgaacggccacgagttcgagatcgagggcgagggcgagggccgcccct acgagggcacccagaccgccaagctgaaggtgaccaagggtggccccctgcccttcgcctgggacatcctgtcccctca gttcatgtacggctccaaggcctacgtgaagcaccccgccgacatccccgactacttgaagctgtccttccccgagggctt caagtgggagcgcgtgatgaacttcgaggacggcggcgtggtgaccgtgacccaggactcctccctgcaggacggcga gttcatctacaaggtgaagctgcgcggcaccaacttcccctccgacggccccgtaatgcagaagaagaccatgggctgg gaggcctcctccgagcggatgtaccccgaggacggcgccctgaagggcgagatcaagcagcggctgaagctgaagga cggcggccactacgacgctgaggtcaagaccacctacaaggccaagaagcccgtgcagctgcccggcgcctacaacgt caacatcaagttggacatcacctcccacaacgaggactacaccatcgtggaacagtacgaacgcgccgagggccgccac tccaccggcggcatggacgagctgtacaagtaag SEQ ID NO: 12 - pSP-N4pegR aattctaactagagctcgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttcct tgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctat tctggggggtggggtggggcaggacagcaagggggaggattgggaagagaatagcaggcatgctggggagcggccg caggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgc ccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggggcgcctgatgcggt attttctccttacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgcattaag cgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccttagcgcccgctcctttcgctttcttccctt cctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggca cctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgtt ggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaactctatctcgggctattcttttgatttataagg gattttgccgatttcggtctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttac aattttatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgac gcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagagg ttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatg gtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatc cgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgccc ttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgg gtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatg atgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcataca ctattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgca gtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgctt ttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagc gtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggca acaattaatagactggatggaggcggataaagltgcaggaccacttctgcgctcggcccttccggctggctggtttattgctg ataaatctggagccggtgagcgtggaagccgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgta gttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaag cattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagat cctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaagg atcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccgg atcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgt agttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagt ggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggg gttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgc cacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggag cttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgt caggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacat gtgagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgta aacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatg gactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccgctcgggc catgattatagtagttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtc ggtgcatccacagccatactataatcatggccctttttttagcgcgtgcgccaattctgcagacaaatggctctagaggtacc agaagaagtgacggctgggggcacagtgggctgggcgcccctgcagaacatgaaccttccgctcctggctgccacagg gtcctccgatgctggcctttgcgcctctagaggcagccactcatggattcaagtcctggctccgcctcttccatcaggaccac tctagagggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgatccggtgcctaga gaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtat ataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggaccggtggatcccgccacca tggtgagcaagggcgaggaggataacatggccatcatcaaggagttcatgcgcttcaaggtgcacatggagggctccgt gaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacgagggcacccagaccgccaagctgaag gtgaccaagggtggccccctgcccttcgcctgggacatcctgtcccctcagttcatgtacggctccaaggcctacgtgaag caccccgccgacatccccgactacttgaagctgtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgagga cggcggcgtggtgaccgtgacccaggactcctccctgcaggacggcgagttcatctacaaggtgaagctgcgcggcacc aacttcccctccgacggccccgtaatgcagaagaagaccatgggctgggaggcctcctccgagcggatgtaccccgagg acggcgccctgaagggcgagatcaagcagcggctgaagctgaaggacggcggccactacgacgctgaggtcaagacc acctacaaggccaagaagcccgtgcagctgcccggcgcctacaacgtcaacatcaagttggacatcacctcccacaacg aggactacaccatcgtggaacagtacgaacgcgccgagggccgccactccaccggcggcatggacgagctgtacaagt aag SEQ TD NO: 13 - pSP-NDlgRNA aattctaactagagctcgctgatcagcctcgactgtgccttctagttgccagccatctgtgttgcccctcccccgtgccttcct tgaccctggaaggtgccactcccactgicctttcciaataaaaigaggaaattgcatcgcattgtcigagtaggtgtcattctat tctggggggtggggtggggcaggacagcaagggggaggattgggaagagaatagcaggcatgctggggagcggccg caggaacccctagt.galggagttggccactocctctctgcgcgctcgctcgclcactgaggccgggcgaccaaaggtcgc ccgacgcccgggcttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggggcgcctgatgcggt atttciccttacgcatctgtgcggtatttcacaccgeatacgicaaagcaaccatagtacgcgcccigtagcggcgcaitaag cgcggcgggtgtggtggttacgcgcagcgigaccgctacacttgccagcgccttagcgcccgctcclttegctttcttccctt ccttctcgccacgttcgccggctitccccgtcaagctciaaatcgggggctccctttagggttccgatttagtgctUacggca cctcgaccccaaaaaactigattgggtgatggttcacgtagtgggccatcgcccigatagacggtltticgccctttgacgtt ggagtrxacgttcttaalagt:ggactcttgU£caaact:ggaacaacacteaactctatcicgggctaitettugait.tataagg gattttgccgaittcggtctattggttaaaaaatgagctgattaacaaaaatttaacgcgaattttaacaaaataitaacgtttac aatttatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgac gcgcccigacgggctigtctgctcccggeatccgcitacagacaagctgtgaccgtctccgggagcigcatglgtcagagg ttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctattttataggttaatgtcatgataataatg glttcttagacgtcaggtggcacttitcggggaaatgtgcgcggaacccctatiigtttattttctaaaiacaticaaatatgiatc cgctcatgagacaataaccctgataaatgcttcaataatatgaaaaaggaagagtatgagtatcaacattccgtgtcgccc ltattcccttitUgcggcaUtigccttcctgl:t.Utgctcacccagaaacgctggtgaaagtaaaagalgctgaagatcagtlgg gtgcacgagtgggttacatcgaactggatctcaacagcggtaagatcctgagagttttcgccccgaagaacgttttccaatg atgagcacttitaaagitctgciatgtggcgcggtattatcccgiaugacgcegggcaagagcaactcggtcgccgcataea ctattctcagaatgactggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaatatgca gtgctgcealaaccatgagigataacactgcggccaacttactictgacaacgatcggaggaccgaaggagciaaccgctt tttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagc glgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactat;iact.ggcgaactacttactctagcitcccggca acaaltaatagac£ggatggaggcggalaaagtlgcaggaccactlctgcgcE.cggcccttccggclggcE.ggttt.aUgci.g ataaatctggagccggtgagcgtggaagccgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgta gttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagaiaggigcctcactgattaag catggtaactgteagaccaagttactcatatatacttagattgatttaaaactcatttttaattaaaaggatctaggtgaagat cctittigataaictcaigaccaaaatccctiaacgtgagtutcgt.tccactgagcgtcagaccccgtagaaaagatcaaagg atctctgagatccttttttctgcgcgtaatctgctgctgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccgg atcaagagctaccaaclctitttccgaaggtaactggcticagcagagcgcagataccaaaiacigtictctagtgiagccgt agtaggccaccacttcaagaactctgtagcaccgcctacatacctcgctetgctaatcctgttaccagtggctgctgccagt ggcgataagicgtgtcitaccgggttggactcaagacgatagtaccggataaggcgcagcggicgggctgaacggggg gltcgigcacacagcccagcltggagcgaacgacciacaccgaactgagalacctacagcgtgagclatgagaaagcgc cacgcttcccgaagggagaaaggcggacaggt.atccggtaagcggcagggtcggaacaggagagcgcacgagggag cticcagggggaaacgcctggtatctttatagtcclgtcgggtttcgccacctctgactgagcgtcgatittgtgatgctcgi caggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggcctttgctcacat gtgagggcctatlicccatgaitccttcatatitgeatatacgaiacaaggctgtagagagataattggaattaatttgactgta aacacaaagatatagtacaaaatacgtgacgtagaaagtaataattctgggtagttgcagttttaaaattatgttttaaaatg gactaicaiatgctiaccgtaacitgaaagiaittcgaittctlggcttatatatet.tgiggaaaggacgaaacaccgciatcaa cattactaataaggttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtc ggtgcttttttgttiagagctagaaatagcaagtaaaataaggctagtccgtttitagcgcgigcgccaattctgcagacaaa tggctctagaggtaccagaagaagtgacggctgggggcacagtgggctgggcgcccctgcagaacatgaacctccgct ccLggcigccacagggtectccgaigctggcctttgcgcclctagaggcagccactcatggattcaagtcctggctccgcet cttccatcaggaccactctagagggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaat gatccggtgcctagagaaggiggcgcggggtaaactgggaaagtgaigt£gtgtactggctccgeclttltcccgagggt.g ggggagaaccgiaiataaglgcagiagtcgccgtgaacgttcutttcgcaacgggtugccgccagaacacaggaccggt ggatcccgccaccatggt.gagcaagggcgaggaggata^atggccatcat.caaggagtcalgcgctteaaggtgcac atggagggctccgtgaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacgagggcacccaga ccgccaagctgaaggtgaccaagggtggccccctgccctcgcctggg^atcctgicccctcagt.tcatgtacggctcca aggcctacgtgaagcaccccgccgacaiceccgaclacitgaagcigtecttecccgagggcitcaagigggagcgcgtg atgaacttcgaggacggcggcgtggtgaccgtgacccaggactcctccctgcaggacggcgagttcatctacaaggtga agcigcgcggcaccaactcccctccgacggccccgtaatgcagaagaagaccatgggcigggaggcclcciccgagc ggatgtaccccgaggacggcgccclgaagggcgagalcaagcagcggclgaagclgaaggacggcggccaciacga cgctgaggtcaagaccacctacaaggccaagaagccegtgcagcigcccggcgcciacaacgtcaaeatcaagtggac atcaccteccacaacgaggactacaccatcgtggaacagtacgaacgcgccgagggccgccactccaccggcggcatg gacgagagtacaagtaag SEQ ID NO: 14 - pSP-ND2gRNA aattetaactagagctcgctgatcagcctcgactgtgccttctagttgccagcx’atctgit.gtitgccccteccccgigccttcct tgaccctggaaggtgccactcccactgtcctitcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctat tctggggggtggggtggggcaggacagcaagggggaggattgggaagagaatagcaggcatgctggggagcggecg caggaacccciagigatggagttggceactccctctctgcgcgctcgctcgctcaclgaggccgggcgaccaaaggtcgc ccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggggcgcctgatgcggt atittctccttacgcaictgtgcggtaittcacaccgcatacgtcaaagcaaccat.agtacgcgccctgiagcggcgcattaag cgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccttagcgcccgctcctttcgctttcttccctt cctitctcgccacgticgccggcittccecgtcaagctctaaaicgggggctccctttagggttccgatttagtgcttacggca cctcgaccccaaaaaactgattgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgt ggaglccacgttcittaatagtggacicttgtccaaactggaacaacactcaactciatctcgggctatictittgatitataagg gatiUgccgatUcggictatiggtlaaaaaatgagclgatltaacaaaaaittaacgcgaatUtaacaaaatatlaacgtUac aatittat.ggtgcactctcagtacaatctgctctgatgccgcatagtaagccagccccgacacccgccaacacccgctgac gcgccctgacgggcttgtctgctcccggcaiccgctiacagacaagctgigaccgtctccgggagctgcatgtgtcagagg titcaccgt£atcaccgaaacgcgcgagacgaaagggcctegt.gatacgccta.ltUataggitaatgtcatgataaiaatg gtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctalttgttlatittclaaatacatcaaataigtatc cgctcatgagacaataaccctgataaatgcltcaataatattgaaaaaggaagaglatgagtattcaacatt.tccgtgtcgccc tuttcccutlttgcggcattttgccticctgtittgcicacccagaaacgclggtgaaagiaaaagatgctgaagatcagttgg gtgcacgagtgggtiacatcgaactggatctcaacagcggtaagatccttgagagtlttcgccccgaagaacgitttccaatg atgagcactt.t.taaagt.tctgetai:gtggcgcggtattatcccgtattgacgccgggcaagagcaact.cggtcgccgcataca ctattctcagaatgactggtigagiactcaccagtcacagaaaagcalcttacggatggcaigacagtaagagaaitatgca gtgctgccataaccatgagtgataacactgcggccaactactctgacaacgateggaggaccgaaggagctaaccgctt ttttgcacaacatgggggalcatgtaactcgccugatcgitgggaaccggagctgaatgaagccataccaaacgacgagc gtgacaccacgatgcctgtagcaatggcaacaacgtgcgcaaactataactggcgaactactactctagcttcccggca acaatiaatagaciggatggaggcggataaagttgcaggaccacitctgcgctcggccctlccggctggctggtitattgctg ataaatctggagccggtgagcgtggaagccgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgta gltaictacacgacggggagtcaggcaactatggatgaacgaaatagacagaicgctgagataggtgcctcactgaitaag cattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcattttaattaaaaggatctaggtgaagat ccttlttgataatctcatgaccaaaatcccttaacgtgagitticgitccactgagcgtcagaccccgtagaaaagatcaaagg atctcttgagatccttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccgg at.caagagctaccaactcttlttccgaaggtaactggcttcagcagagcgcagataccaaatactgtt.cttctagtgtagccgt agttaggccaccacttcaagaactcigtagcaccgcctacatacclcgciclgctaalccigttaccagtggctgctgecagt ggcgataagtcgtgtctacegggtiggaetcaa.gacgata.gttaccggataaggcgcagcggtcgggctgaacggggg gttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctaigagaaagcgc cacgct.tccegaagggagaaaggcggaeagglatccggtaagcggcagggtcggaacaggagagcgcacgagggag cttccagggggaaacgcctggtatclttatagtcctgtcgggtt.cgccacctcigacttgagcgicgait.Utgigalgctcgt. caggggggcggagcctatggaaaaacgccagcaacgcggccttttacggttcctggccttttgctggccttttgctcacat gtgagggcctaittcccatgat.lcctcatattlgcatalacgatacaaggcigttagagagataattggaatiaatttgactgta aacacaaagaiaUaglacaaaalacgtgacglagaaaglaataalllcllggglagLUgcagUllaaaaltalglLUaaaalg gactatcatatgcitaccgtaactigaaagtatttcgatttcitggcittatatatcitgtggaaaggacgaaacaccgtccatcat agcaggcagtggtttagagctagaaatagcaagtaaaataaggctagtccgtatcaactgaaaaagtggcaccgagt cgglgctitttgttagagctagaaatagcaagtaaaataaggctagtccgtttagcgcgigcgccaattctgcagacaa atggctctagaggtaccagaagaagtgacggctgggggcacagtgggctgggcgcccctgcagaacatgaaccttccg cl.cctggetgccacagggtcciccgatgctggectttgcgcctctagaggcagccactcatggattcaagtcctggctccgc ctcttccatcaggaccaciclagagggcagagcgcacatcgcccacagiccccgagaagitggggggagggglcggca atigatccggtgcct.agagaaggtggcgcggggtaaactgggaaagtgatgicgtgtactggctccgcctttttcccgagg gtgggggagaaccgtatataagtgcagtagicgccgtgaacgttcttttcgcaacgggtttgccgccagaacacaggacc ggtggatcccgccaccalggt.gagcaagggcgaggaggataacatggccaicatcaaggagttcalgcgcttcaaggt.g cacatggagggclccgtgaacggccacgagucgagalcgagggcgagggcgagggccgcccctacgagggcaccc agaccgccaagctgaaggtgaccaagggtggccccctgcccttcgcctgggacatcctgtcccctcagttcatgtacggct ccaaggcctacgigaagcaccccgccgacatccccgactactigaagctgtccttccccgagggcticaagtgggagcgc gtgatgaactcgaggacggcggcgtggtgaccgtgacccaggactcctccctgcaggacggcgagttcatctacaaggt gaagctgcgcggcaccaaettcccctecgacggccccgtaatgcagaagaagaccaigggcigggaggcclcciccga gcggatgtaccccgaggacggcgccctgaagggcgagatcaagcagcggctgaagctgaaggacggcggccactac gacgctgaggtcaag:Mjeacctacaaggccaagaagcccgtgcagcigcccggcgcctacaacgt.caacatcaagUgg acatcacctcccacaacgaggaclacaccaicglggaacagiacgaacgcgccgagggccgccactccaccggcggca lggacgagcigt.acaagt.aag SEQ ID NO: 15 - pSP-ND3gRNA aattctaactagagctcgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttcct tgaccctggaaggtgccacicccactgicctt.tcciaataaaatgaggaaatgcatcgcattgtcigagtaggtgtcattctat tetggggggtggggtggggcaggacagcaagggggaggattgggaagagaatagcaggcatgctggggagcggccg caggaaeccctagt.gatggagt.tggccactccctctctgcgcgetcgctcgctcactgaggccgggcgaccaaaggtcgc ccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggggcgcctgatgcggt atttctccttacgcatctgtgcggtattcacaccgcatacgteaaagcaaccatagtacgcgccctgtagcggcgcataag cgcggcgggtgtgglggttacgcgcagcgtgaccgctacacttgecagcgccttagcgccegctccttcgctttctccctt ccttctcgccacgtcgccggcttccccgtcaagctctaaatcgggggctccctttagggtccgattagtgcttacggca cctcgaccccaaaaaactigaLitgggigatggttcacgtagigggccaicgcccigatagaeggtUticgcccittgacgit ggagtccacgttetttaatagtggactcttgttccaaactggaacaacactcaactetatctcgggctattctttgatttataagg gattitgccgaitcggtctattggitaaaaaatgagctgattiaacaaaaatttaacgcgaattttaacaaaataitaacgittac aattttatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgac gcgcccigacgggctigtctgctcccggeatccgcitacagacaagclgtgaccgtctccgggagcigcatglgtcagagg tttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctattttataggttaatgtcatgataataatg glttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatu.gtt.tattttctaaaiacaticaaatatgiatc cgctcalgagacaataaccctgataaaigctcaataatatigaaaaaggaagaglatgagtateaacaltccgtgtcgccc ttattccctttttgcggcalttigccttcctgt.tttgctcacecagaaacgct.ggtgaaagtaaaagatgctgaagatcagtlgg gtgcacgagtgggttacatcgaactggatctcaacagcggtaagaiccttgagagttttcgccccgaagaacgtttccaatg atgagcacUitaaagiteLgctatgtggcgcggtatatcccgia.ltgacgccgggcaagagcaactcggtcgccgcaiaca ctaitctcagaatga£ttggttgagtactcaccagicacagaaaagcatcttacggaiggcatgacagiaagagaatatgca gtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgctt ttitgcacaacatgggggatcalgtaactcgccttgatcgtgggaaccggagctgaatgaagccataccaaacgacgagc glgacaccacgatgcclglageaatggcaacaacgUgcgcaaactaUaactggcgaactactlaclclagcLtcccggca acaattaatagactggatggaggcggataaagitgcaggaccactlctgcgcicggcccttccggctggciggtttattgctg ataaatctggagccggtgagcgtggaagccgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgta gttatctacacgacggggagtcaggcaactaiggaigaacgaaaiagacagatcgctgagaiaggigcctcactgattaag caltggtaactgtcagaccaagitlactcatalatactttagaltgaittaaaacitcatttttaatttaaaaggatctaggtgaagat cx'ttttigataatctcaigaccaaaatccctiaacgtgagt.lttcgttccactgagcgtcagaa'ccgtagaaaagatcaaagg atcttcitgagatccittitticlgcgcgiaatcigcigctgcaaacaaaaaaaccaccgctaceagcggiggtitgtitgccgg atcaagagctaccaaclctittccgaaggtaactggcttcagcagagcgcagataccaaaiactgticttctagtgiagccgt agttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgtaccagtggctgctgccagt ggcgataagicgtgtcitaccgggtt.ggactcaagacgatagt^eggataaggcgcagcggicgggctgaacggggg gucgigcacacagcccagcttggagcgaacgacciacaccgaactgagaiacctacagcgtgagclatgagaaagcgc cacgctcccgaagggagaaaggcggacaggtatccggtaagcggcagggteggaacaggagagcgcacgagggag cttccagggggaaacgcctggtatctttatagtcctgtcgggtticgccacctctgacttgagcgtcgatitttgtgatgctcgi caggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggcctttgctcacat gtgagggcctattcccaigaitccttcalatitgeatatacgaiacaaggctgltagagagataattggaaitaatttgactgta aacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatg gaciaicatatgctiaccgtaacttgaaag^itcgaittctggctttatatatcttgt.gga:iaggacgaaacaccgaaatcca ccccUacgagiggttttagagctagaaalagcaagltaaaataaggctaglccgttatcaacttgaaaaaglggcaccgagt cggtgcttUtgtttagagci.agaaatagcaagttaaaataaggetagicegttttagcgcgtgcgccaattctgcagacaa atggcictagaggtaccagaagaagtgacggctgggggcacagtgggctgggcgcccctgcagaacatgaaccttccg ctcctggctgccacagggtcctccgat.gclggccttlgcgcctctagaggcagccactcatggattcaagtcctggctccgc ctetlccateaggaccactetagagggcagagcgcacalcgcccacagtccccgagaagtggggggaggggtcggca at.tgatecggtgccuigagaf!ggf.ggcgcggggtaaactgggaaagtgaf.gtcgtgtacAggctocgccE.t.tu.cccgagg glgggggagaaccgtatataagtgcagtagtegccgigaacgUcititcgcaacgggittgccgccagaacacaggacc ggtggatcccgccaccatggtgagcaagggcgaggaggataacatggccaicatcaaggagttcatgcgcttcaagglg cacatggagggctccgtgaacggecacgagtcgagatcgagggcgagggcgagggcegcccc^egagggcaccc agaccgccaagctgaaggtgaccaagggtggccccctgcccitcgccigggacatcctgtcccctcagttcatgtacggct ccaaggcctacgtgaagcaccccgccgacatccccgactacttgaagctgtcctccccgagggcttcaagtgggagcgc gtgatgaacttcgaggacggcggcgtggtgaccgtgacccaggactcctecctgcaggacggcgagttcatctacaaggt gaagctgcgcggcaccaacttcccctccgacggccccgtaatgcagaagaagaccatgggctgggaggcctcctccga gcggatgtaccccgaggacggcgecctgaagggcgagatcaagcagcggctgaagclgaaggacggcggccactae gacgctgaggtcaagaccacctacaaggccaagaagcccgtgcagctgcccggcgcctacaacgtcaacatcaagttgg acaicacctcccacaacgaggactacaccatcgtggaacagt.acgaacgcgccgagggccgccactccaccggcggca tggacgagctgtacaagtaag SEQ ID NO: 16 - pSP-ND5gRNA aatictaactagagcicgctgaicagccicgactgigccitctagttgccagccatctgttgttigcccctcccccgtgccticct tgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaatgcatcgcattgtctgagtaggtgtcattctat iciggggggtggggtggggcaggacagcaagggggaggatigggaagagaatagcaggcatgciggggagcggccg caggaacccctagtgatggagtggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgc ccgacgcccgggcttigcccgggcggcctcagtgagcgagcgagegcgcagctgcet.gcaggggcgcctgatgcggt attUciccttacgcatctgtgcggtattcacaccgcatacgtcaaagcaaccatagtacgcgccclgtagcggcgcattaag cgcggcggglgt.ggtggt.tacgcgcagcgtgacegctacactigccagcgccti^gcgcccgctccttcgctticttcectt ccttctcgccacgtcgccggctitccccgtcaagctctaaatcgggggctccctitagggttccgattiagtgctttacggca cctcgaccccaaaaaacttgattgggtgat.ggitcacgtagtgggccatcgccctgatagacggitlitcgcccttgacgtl ggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaactctatcicgggctattcttttgattlataagg gattttgccgatttcggtctattggtaaaaaatgagctgattaacaaaaatttaacgcgaattttaacaaaatattaacgtttac aattltaiggtgcaciclcagiaeaaictgctctgatgccgcatagitaagccagccccgacacccgccaacacccgctgac gcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagagg ttttcaccgtcatcaccgaaacgcgcgagacgaaagggccicgtgatacgcctatttt.tataggtlaaigteatgataataatg gtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgiatc cgctcatgagacaataacccLgaiaaatgcttcaaiaataitgaaaaaggaagagtatgaglaticaacatttccgtgtcgccc ttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgg glgcacgagigggtacatcgaactggatctcaacagcggiaagatcctigagagtttcgccccgaagaacgituccaatg atgagcacttttaaagtctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcataca ctattctcagaatgacttggtigagiactcaccagtcacagaaaagcatctlacggatggcatgacagtaagagaattatgca gtgctgccataaccatgagtgataacactgcggccaacttactctgacaacgatcggaggaccgaaggagciaaccgctt ttttgcacaacat.gggggatcatglaa.ctcgccltgatcgugggaaceggagctgaatgaagccaia.ccaaacgacgagc glgacaccacgatgcctgtagcaatggcaacaacgtlgcgcaaactattaactggcgaactactiactclagcttcccggca acaataatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctg ataaatciggagccggtgagcgtggaagccgcggtatcattgcagcactggggccagalggtaagccctcccgtaicgia gttatetacacgacggggagtcaggcaactatggatgaacgaaatagacagategctgagataggtgcctcactgattaag catiggt.aacigteagaccaagtitacteatatatactttagattgattaaaacticattttaattt.aaaaggatctaggtgaagat ccttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaagg atctlctigagatccttttlttctgcgcgtaalctgctgcttgcaaacaaaaaaaccaecgctaccagcggtggit.tgitigccgg atcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgt agttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatccigttaccagtggctgctgccagt ggcgataagtcglgtcitaccgggttggactcaagacgatagtaccggataaggcgcagcggicgggctgaacggggg gtcgtgeacacagcccagetggagcgaaegacctacaccgaactgagatacctacagcgigagctatgagaaagcgc cacgcitcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggag cttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgEgatgctegt caggggggcggagcctatggaaaaacgceagcaacgcggcctitiaeggttcctggccttltgciggccttitgctcacat gtgagggcctatttcccatgatccttcatattgcatatacgatacaaggctgtagagagataattggaattaatttgactgta aacacaaagaiatiaglacaaaatacgtgacgLagaaagtaaiaattictgggtagtUgcagtutaaaatiatgttiaaaaig gactatcatatgcttaccgtaacttgaaagtattcgatttcttggctttatatatcttgtggaaaggacgaaacaccgcagccg gaagcctattcgcgittiagagctagaaatagcaagttaaaaiaaggctagtecgtiatcaactigaaaaagtggcaccgagt cggtgcttttttgttttagagctagaaatagcaagttaaaataaggctagtccgttttagcgcgtgcgccaatctgcagacaa atggctctagaggtaccagaagaagtgacggctgggggcacagtgggctgggcgccccigcagaacatgaaccttccg ctcctggctgccacagggtcctccgatgctggcctttgcgcctctagaggcagccactcatggattcaagtcctggctccgc ctcitccatcaggaccactctagagggcagagcgcacatcgcccacaglccccgagaagt.tggggggaggggtcggca augatccggtgcciagagaaggiggcgcggggtaaactgggaaagtgaigtegtgtaaggclccgcctimcccgagg gtgggggagaaccgta.tataagtgcagtagicgccgtgaa.cgtctlttcgcaaegggttgccgccagaacacaggace ggtggatcccgccaccatggtgagcaagggcgaggaggaiaacatggccatcatcaaggagtcatgcgcttcaaggtg cacat.ggagggctccgtgaacggccacgagttogagatcgagggcgagggcgagggccgcccctacgagggcaccc agaccgccaagctgaaggtgaccaagggtggccccclgccctlcgcctgggacatcctgtcccctcagltcatglacggct. ccaaggcctacgtgaagcaccccgccgacatccccgactacttgaagctgtccttccccgagggcttcaagtgggagcgc gtgatgaactcgaggacggcggcgtggtgaccgtgacccaggaclcciccctgcaggacggcgagttcaiciacaaggi gaagctgcgcggcaccaactlcccctccgacggccccglaatgcagaagaagaccatgggclgggaggcctcclccga geggatgtaccccgaggacggcgccctgaagggcgagatcaagcagcggctgaagctgaaggacggcggccactac gacgctgaggtcaagaccacctacaaggccaagaagcccgtgcagctgcccggcgcctacaacgtcaacatcaagttgg acatcacctcccacaacgaggactacaccaicglggaacagiacgaacgcgccgagggccgccactccaccggcggca tggacgagctgiacaagiaag SEQ ID NO: 17 - pSP-ATP8gRNA aattctaactagagctegctgatcagcctcgactgtgccttctagttgccagccatctgttgtitgcccctcccccgtgccttcct tgaccctggaaggtgccactcccactgicctttcciaataaaaigaggaaattgcatcgcatgtctgagtaggtgtcattctat tctggggggtggggtggggcaggacagcaagggggaggattgggaagagaaiagcaggcatgctggggagcggccg caggaacccctagtgatggagttggccactccctetctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgc ccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggggcgcctgatgcggt atttctcctacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgcataag cgcggcgggtgtggtggttacgcgcagcgigaccgctacacitgccagcgccttagcgcccgctcclttcgcttteitccctt. cctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggca cctcgaccccaaaaaacttgalttgggt.gatggtcacgtagtgggccaicgcccigatagaeggtltticgcccittgacgit. ggaglccacgUcittaalagtggacictgtlccaaactggaacaacaclcaactciatctcgggclatictittgatitaiaagg gatttt.gccgattcggtctattggitaaaaaat,gagct.gatttaacaaaaattaacgcgaatttaacaaaataitaacgtttac aattttatggtgcactctcagtacaatctgciclgatgccgcatagttaagccagccccgacacccgccaacacccgctgac gcgcccigacgggctigtct.gctcccggeatccgcttacagacaagctgtgaccgtx:tccgggagctgcatgtgtcagagg ttitcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttitataggttaatgtcaigataaiaatg gRtettagacgtcaggtggcactttcggggaaatgtgcgcggaacccctattt.gtt.tattt.tctaaatacaticaaatatgtatc cgctcatgagacaataaccctgataaaigcticaataatatigaaaaaggaagaglatgagtattcaacatttccgtgtcgccc ttaUccclttlttgcggcattttgccttcctgtttttgcicacccagaaacgclggtgaaaglaaaagatgctgaagalcagttgg gl.gcacgagtgggtt.acatcgaaciggatcteaacagcggtaagatccttgaga.gitticgccccgaagaacgtitccaatg atgagcacttlaaagtlctgctatgtggcgcggiaitatcccgtattgacgccgggcaagagcaactcggicgccgeataca ctattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgca gtgctgccataaccatgagtgataacactgcggccaactiacitelgacaacgatcggaggaccgaaggagctaaccgctl tttgcacaacatgggggatcatgtaactcgcctgatcgtgggaaccggagctgaatgaagccataccaaacgacgagc gtgacaccacgaigcctgtagcaatggcaacaacgiigcgcaaaciataaclggcgaaciaciiactctagctlcccggca acaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctg ataaatctggagccggtgagcgtggaagccgeggtatcattgcagcactggggccagatggtaagccetcccgiatcgta gttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaag cattggiaactgicagaceaagU.lacLcataiataclitagaltgaittaaaacttcatittiaaitiaaaaggatctaggtgaagat cctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaagg atcitcit.gagatccutittctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggt.ggtitgtitgccgg atcaagagctaccaactcttlttccgaaggtaaclggcttcagcagagcgcagataccaaatactgitcttctagigtagccgt agtta.ggecaccacttcaagaacictgiagcaccgcetacatacetcgctctgctaatcctgitaccagt.ggctgcigccagt ggcgataagtcgtgtcttaccgggtggactcaagacgatagitaccggataaggcgcagcggtcgggctgaacggggg gltcgigcacacagcccagcitggagcgaacgaccta.caccgaactgagatacctacagcgtgagclatgagaaagcgc cacgcttcccgaagggagaaaggcggacaggiatccggiaagcggcagggtcggaacaggagagcgcacgagggag cttccagggggaaacgcetggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgt caggggggcggagcctaiggaaaaacgccagcaacgcggccttttacggticctggcctittgctggccUtlgctcacat glgagggcclaUtcccalgatlcctcatattlgcatalacgaiacaaggctgtlagagagataaUggaaltaaiagactgta aacacaaagataitagtacaaaatacgtgacgtagaaagtaataatitcttgggiagtttgcagitttaaaattaigltttaaaatg gactatcatatgctaccgtaactgaaagtattcgatttctggcttatatatcttgtggaaaggacgaaacaccgcccaact aaatactaccgtagitttagagclagaaatagcaagtaaaataaggclagtccgitaicaacitgaaaaagtggcaccgagt cgglgctittitgttttagagctagaaatagcaagttaaaaiaaggctagtccgtttltagcgcgtgcgccaattctgcagacaa al.ggctctagaggtaccagaagaa.gtgacggctgggggcacagtgggctgggcgcccct.gcagaacatgaaccl.tccg ctcctggctgccacagggtcciccgatgctggccttgcgcciclagaggcagccactcatggattcaagtcctggctccgc ctcttccatcaggaccactctagagggcagagcgcacatcgcccacagtcx'ccgagaagtiggggggaggggtcggca attgatccggtgcctagagaaggtggcgcgggglaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagg glgggggagaaccgtatataagtgcagtagtcgccgtgaacgttcittttcgcaacgggittgccgccagaacacaggacc gglggatcccgccaccalggtgagcaagggcgaggaggataacatggccaicatcaaggagttcalgcgcitcaaggtg cacatggagggctccgtgaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacgagggcaccc agaccgccaagcigaaggtgaccaagggiggccccctgcccitcgccigggaeatcctgtcccctcagttcatgtacggci ccaaggcctacgtgaagcaccccgccgacatccccgactacttgaagctgtccttccccgagggctcaagtgggagcgc gtgatgaacttcgaggacggcggcgtggtgaccgigacccaggactccteccigcaggacggcgagitcatctacaaggt. gaagctgcgcggcaccaacttcccctccgacggccccgtaatgcagaagaagaccatgggctgggaggcctcctccga gcggatgtaccccgaggacggcgecctgaagggcgagalx'aagcagcggctgaagclgaaggacggcggccactac gacgctgaggtcaagaccacctacaaggccaagaagcccgtgcagctgcccggcgcclacaaegtcaacatcaagtgg acatcacctcccacaacgaggactacaccatcgtggaacagtacgaacgcgccgagggccgccactccaccggcggca tggacgagctgtacaagtaag SEQ ID NO: 18 - pSP-COX2gRNA aatctaactagagcicgctgaicagccicgactgigccttctagttgccagccatclgtlgttlgcccctcccccgtgcctcct t.gaccct.ggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcat.cgcat.gtctgagtaggtgtcattotat tctggggggtggggtggggcaggacagcaagggggaggatigggaagagaatagcaggcatgciggggagcggccg caggaacccctagtgatggagttggccactocctctctgcgcgctcgctogctcactgaggccgggcgaccaaaggtcgc ccgacgcccgggcttigcccgggcggcctcaglgagcgagcgagcgcgcagctgcctgcaggggcgcctgatgcggt atttctccttacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgcattaag cgcggcggglgtggtggttacgcgcagcgtgaccgctacactigccagcgccttogcgcccgctccttogcttottoccti cctttctcgccacgttcgccggctttecccgtcaagctctaaatcgggggctccctttagggtccgatttagtgctttacggca cctcgaccccaaaaaactgattigggtgatggitcacgtagtgggccatcgccctgatogacggittttcgccct.ttgacgt ggagtccacgttcttaatagtggactctgtccaaactggaacaacactcaactctatctcgggctattcttttgatttataagg gatt.tgccgatttoggtotatiggt.laaaaaatgagctgattaacaaaaaittaacgcgaatltaacaaaatat.taacgt.ttoc aattttatggtgcactctcagtacaatctgctctgatgccgcatagitaagccagccccgacacccgccaacacccgctgac gcgccctgacgggctgtotgctcccggcatocgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagagg ttttcaccglcaicaccgaaacgcgcgagacgaaagggcctcglgatacgcctattittataggtlaatgtcatgataataatg gttottogacgtcaggt.ggcacttt.tcggggaaatgtgcgcggaacccctat.tgttatt.ttotaaatacat.caaatatgtato cgcicatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgccc tto.ttoccltttgcggcattgccttoctgttttgctoacccagaaacgclggtgaaagtoaaagat.gctgaagalcagt.gg gtgcacgagigggtiacatcgaact.ggatctoaacagcggiaagatcctgagagt.ttcgccccgaagaacgt.ltccaatg atgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcataca ctattctcagaatgactggtigagiactoaccagtcacagaaaagcatcttacggatggcaigacagtaagagaatatgca glgctgccataaccatgagtgataacactgcggccaacttacltctgacaacgatoggaggaccgaaggagclaaccgclt tttgcacaacatgggggatoatgtaactcgcctgatogtgggaaccggagctgaatgaagccataccaaacgacgagc gtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggca acaatiaatagaciggatggaggcggataaagttgcaggaccactctgcgctcggccctlccggctggctggttattgctg ataaatotggagccggtgagcgtggaagccgcgglatcaltgcagcactggggccagatggtaagccctcccgtatcgta gtt.atotacacgacggggagtoaggcaactotggatgaacgaaatagacagatogct.gagataggt.gcctcact.gat.taag catiggtaactgtcagaccaagtttactcatatatacttiagattgatttaaaacttoatttttaattaaaaggalctaggtgaagat cct.ttgataatctcatgaccaaaatccctaacgtgagt.ttogttccactgagcgtcagaccccglagaaaagatcaaagg atctcttgagatccttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctoccagcggtggtttgtttgccgg atoaagagctaccaactot.lttccgaaggtaactggctcagcagagcgcagataccaaataetgt.ctctagtgtagccgt agttaggccaccacttcaagaactctgtagcaccgcctacatacctogctolgctaalccigttaccagtggctgctgccagt ggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggg gttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctocagcgigagctoigagaaagcgc cacgctcccgaagggagaaaggcggacaggtatecggtaagcggcagggteggaacaggagagcgcacgagggag c!.tccagggggaaacgcclggta!.clt.tatagt.cctgtcgggtUcgccacc!.cigactgagcgicgat.tltgigatgetogt caggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacat gt.gagggcctot.tcccatgatlccttoatot.lgcato.lacgatacaaggcigtagagagataattggaattoattgactgta aacacaaagaiatiag!.acaaaatacgtgacglagaaaglaataaiti.cttgggtagt!.tgcagtiltaaaa£iatgltttaaaalg gactatc;itatgcttaccgtoacttgaaagtatitogatttot.ggcttatatatottgtggaaaggacgaaacaccgacctacg agtacaccgactagttttagagctagaaatagcaagttaaaataaggciagtccgttatcaacttgaaaaagtggcaccgagt cggtgcttttt.gtttagagctagaaalagcaagttoaaatoaggctagtccgttttogcgcgigcgccaat.tctgcagacaa atggctctagaggtaccagaagaagtgacggctgggggcacagtgggctgggcgcccctgcagaacatgaaccttccg ct.cctggct.gccacagggtcctocgatgctggcctt.gcgcctotagaggcagccactcatggattoaagtcctggctccgc ctctccatcaggaccactctagagggcagagcgcacatcgcccacagiccccgagaagtggggggaggggtoggca attgatccggtgcctagagaagglggcgcggggtaaactgggaaagtgatgtcgtgtaciggctccgcclttttcccgagg gt.gggggagaaccgtatataagtgcagtagtcgccgtgaacgttcttttcgcaacgggttgccgccagaacacaggacc ggtggatcccgccaccatggtgagcaagggcgaggaggaiaacatggccatcatcaaggagltcatgcgcttcaaggtg cacatggagggctccgtgaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacgagggcaccc agaccgccaagctgaaggtgaccaagggtggccccctgcccttcgcctgggacaicctgtcccctcagttcatgtacggct ccaaggcctacgtgaagcaccccgccgacatccccgactactgaagctgtccttccccgagggcttcaagtgggagcgc gtgatgaacltcgaggacggcggcgtggtgaccgtgacccaggactcctccctgcaggacggcgagttcatctacaaggi gaagctgcgcggcaccaacttcccctccgacggccccgtaatgcagaagaagaccatgggctgggaggcctcctccga gcggatgtaccccgaggacggcgccctgaagggcgagatcaagcagcggctgaagctgaaggacggcggccactac gacgctgaggtcaagaccacctacaaggccaagaagcccgtgcagctgcccggcgcctacaacgtcaacatcaagttgg acatcacctcccacaacgaggactacaccaicgtggaacagiacgaacgcgccgagggccgccactccaccggcggca tggacgagctgtacaagiaag SEQ ID NO: 19 - pSP-COX3gRNA aattctaactagagctcgctgatcagcctcgactgtgccttctagttgccagccatctgttgtitgcccctcccccgtgccttcct tgaccctggaaggtgccactoccactgtcctttcciaataaaaigaggaaattgeatcgcatgtcigaglaggtgtcattetat tctggggggtggggtggggcaggacagcaagggggaggattgggaagagaatagcaggcatgctggggagcggccg caggaacccctagtgatggagtggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgc ccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggggcgcctgatgcggt attltclccttacgcatclgtgcggtatttcacaccgeatacgtcaaagcaaccalagtacgcgccctgtagcggcgcattaag cgcggcgggtgtggtggttacgcgcagcgtgaccgciacacttgccagcgccttagcgcccgctcctttcgctttcttccctt ccttctcgccacgtcgccggctttccccgtcaagctctaaatcgggggctccctttagggtccgattagtgcttacggca cctcgaccccaaaaaacttgatttgggigatggttcacgtagigggccaicgcccigatagacggtttticgcccittgacgit ggagtccacgltctttaatagtggacictlgttccaaactggaacaacactcaactctatctcgggctattcttttgatttaiaagg gatttt.gccgattcggtctattggttaaaaaatgagct.gatttaacaaaaattaacgcgaatt.ttaacaaaatattaacgtt.tac aatittatgglgcactctcagtacaatctgciclgatgccgcatagttaagccagccccgacacccgccaacacccgctgac gcgcccigacgggcttgtct.gctcccggeatccgcttacagacaagctgtgaccgtetccgggagctgcatglgtcagagg tttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctattttataggttaatgtcatgataataatg glttcttagacgtcaggtggcactttcggggaaatgtgcgcggaacccctatu.gtt.tattttcta:iaiacaticaaatatgiatc cgctcalgagacaataaccctgataaaigcucaataatatigaaaaaggaagaglatgagtateaacautccgtgtcgccc ttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgg gtgcacgagtgggttacatcgaaciggatctcaacagcggtaagaiccttgagagiticgccccgaagaacgtitccaatg atgagcactttaaagttctgctatgtggcgcggtatatcccgtattgacgccgggcaagagcaactcggtcgccgcataca ctaitctcagaatga£ttggttgagtactcaccagicacagaaaagcatcttacggaiggcatgacagiaagagaatatgca gtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgctt ttitgc:Mjaacatgggggatcalgtaactcgccttgatcgtgggaaccggagctgaat.gaagccataccaaacgacgage gtgacaccacgaigcetgtagcaatggcaacaacgitgcgcaaaciaUaaclggcgaaciacitactctagctlcccggca acaattaatagactggat.ggaggcggataaagltgcaggaccacttctgcgcicggeccttccggctggctggtta.ttgct.g ataaatctggagccggtgagcgtggaagccgcggtatcattgcagcaciggggccagatggtaagccctcccgiatcgta gttatctaeacgacggggagtcaggcaactat.ggatgaacgaaaiagacagatcgctgagataggigcctcactgattaag cattggtaactgtcagaccaagtttactcatalatactttagaltgaittaaaacitcatttttaatttaaaaggatctaggigaagat cctttttgataatctcatgaccaaaatcccttaacgtgagtttcgttccactgagcgtcagaa'ccgtagaaaagatcaaagg atcitcitgagatcctttitttctgcgcgtaatcigcigctigcaaacaaaaaaaccaccgctaccagcggtggtitgtitgccgg atcaagagctaccaactcttutccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgt agtta.ggecaccacttcaagaacictgtagcaccgcctacatacetcgctctgctaatcctgttaccagi.ggct.gctgccagt ggcgataagtcgtgtctaccgggtiggactcaagacgatagitaccggataaggcgcagcggtcgggctgaacggggg gttcgtgcacacagcccagctggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgc cacgctcccgaagggagaaaggcggacaggiatccggiaagcggcagggtcggaacaggagagcgcacgagggag cttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgt caggggggcggagcctaiggaaaaacgccagcaacgcggccitUtacggltcctggcctittgctggccUtlgctcacat gtgagggcctatttcccatgatccttcatattgcatatacgatacaaggctgttagagagataattggaattaatttgactgta aacacaaagat.attagtacaaaatacgtgacgtagaaagtaataatitcttgggiagtttgcagtttiaaaattaigttttaaaatg gactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccgcagccca lgacccctaacaggtttlagagctagaaatagcaagtlaaaataaggctagtccgtiatcaactigaaaaagt.ggcaccgagi cggtgctttttgtttagagctagaaatagcaagtaaaataaggctagtccgtttttagcgcgtgcgccaatctgcagacaa atggctclagaggtaccagaagaagtgacggctgggggcacaglgggctgggcgcccctgcagaacatgaaccticcg ctcctggclgccacagggtcciccgatgctggccttgcgcciclagaggcagccactcatggatlcaagtcctggctccgc ctcttccatcaggaccactctagagggcagagegcacatcgeccacagtccccgagaagttggggggaggggtcggca atgatccggtgcctagagaaggtggcgcggggiaaactgggaaagtgatgtcgtgtactggctccgccttttcccgagg glgggggagaaccgtatataagt.gcagtagtcgccgtgaacgltcittitcgcaacgggittgccgccagaacacaggacc ggtggatcccgccaccatggtgagcaagggcgaggaggataacatggccaicatcaaggagttcatgcgcttcaaggig cacatggagggctccgtgaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacgagggcaccc agaccgccaagcigaaggtgaccaagggiggccccctgccciicgccigggaeatcctgtccccicagtcatgtacggci cvaaggcclacgtgaagcaccccgccgacaiccccgaciacltgaagclgtccltccvcgagggcltcaagtgggagcgc gtgatgaacttcgaggacggcggcgtggtgaccgtgacccaggactcctecctgcaggacggcgagttcatctacaaggt gaagctgcgcggcaccaacttcccctccgacggccccgtaatgcagaagaagaccatgggctgggaggcctcctccga gcggatgtaccccgaggacggcgecctgaagggcgagatcaagcagcggctgaagctgaaggacggcggccactae gacgctgaggtcaagaccacctacaaggccaagaagcccgtgcagctgcccggcgcclacaacgtcaacatcaagttgg acatcacctcccacaacgaggactacaccatcgtggaacagtacgaacgcgccgagggccgccactccaccggcggca tggacgagctgtacaagtaag SEQ ID NO: 20 - sc-U6-muthTLl ccaigtgagggcctaittcceatgattccitcatattigcaiaiacgatacaaggcigttagagagaiaattggaati.aattigact gtaaacacaaagatatagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagtttaaaattatgtttaaa atggaciatcatatgcttaccgtaacitgaaagtalttcgatttct.tggctttaiatatct.tgiggaaaggacgaaacaccgtaag atggcagggcccggtaatcgcataaaactlaaaactttacagtcagaggttcaattcctcttcttaacagacaaaiggctctag aggtaccagaagaagigacggctgggggcacagtgggcigggcgcccctgcagaacatgaacettccgctxictggctg ccacagggicctccgatgctggcctitgcgcctctagaggcagccactcalggattcaagtcclggctccgcctctlccatca ggaccactctagagggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgatccggt gcciagagaaggtggcgcggggtaaaclgggaaagtgatgicgtgtaetggctccgccttutccegagggtgggggaga accgtatataagtgcagtagtcgccgtgaacgttcttttcgcaacgggttgccgccagaacacaggaccggtggatcccg ccaccaiggtgagcaagggcgaggaggalaacatggccatcatcaaggagticatgcgctcaaggtgcacatggaggg ctccgtgaacggccacgagtcgagatcgagggcgagggcgagggccgcccctacgagggcacccagaccgccaag ctgaaggigaccaagggtggccccctgcccticgcctgggacatcctgtcccctcagttcatgtacggctccaaggcctac gtgaagcaccccgccgacatccccgactacttgaagctgtccttccccgagggcttcaagtgggagcgcgtgatgaactc gaggacggcggegtggtgaccgtgacccaggactcclccct.gcaggacggcgagttcatctacaaggtgaagctgcgc ggcaccaacticccctccgacggccccgtaalgcagaagaagaccatgggctgggaggcctcctccgagcggatgtacc ccgaggacggcgccctgaagggcgagatcaagcagcggctgaagctgaaggacggcggccactacgacgctgaggt caagaccacciacaaggccaagaagcccgtgcagctgcccggcgcctacaacgtcaacaicaagtiggacatcacctcc cacaacgaggactacaccatcgtggaacagtacgaacgcgccgagggccgccactccaccggcggcatggacgagct gtacaagtaagaaticiaactagagctcgctgaicagcctcgactgtgcct.tctagttgccagccatctgtgtitgcccctccc ccgtgcctccttgaccctggaaggtgccactcccactgtccttcciaataaaatgaggaaatgcatcgcattgtctgagta ggtgtcaitctattciggggggtggggtggggcaggacagcaagggggaggatlgggaagacaalagcaggaacccca ctccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggc ctcagtgagcgagcgagcgcgcagctgctgcataatgaatcggccaacgcgcggggagaggcggttigcgtatgggc gctcttccgctcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggt aalacggt.tatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccg taaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagag glggcgaaacccgacaggactaiaaagataccaggcgtitccccctgg:iagctccctcglgcgctctcctgtccgaccct gccgcttaccggataccigtccgcctltctcccticgggaagcgtggcgctltcicatagctcacgctgtaggtatctcagttc ggtgtaggtcgttcgctceaagctgggctgtgtgc^gaaccccccgttcagcccgaccgctgcgccttatccggtaactat cgtcttgagtccaacccggtaagacacgacttatcgccaciggcagcagccactggtaacaggatagcagagcgaggta tgtaggcggtgctacagagttcttgaagtgglggcciaactacggctacacLagaaggacagtaittggtaictgcgctctgc tgaagccagttaccttcggaaaaagagitgglagctcltgaiccggcaaacaaaccaccgctggtagcggtggtt.lttltgtit. gcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaa cgaaaactcacgttaagggatutggicatgagattatcaaaaaggatcticacctagatccttitaaaitaaaaatgaagttia aatcaalctaaagtataialgagtaaacttggtctgacagttaccaatgcltaatcaglgaggcacctalclcagcgatctglcl ait.tcgttcatccalagtigccigactccccgtcglgtagataactacgaiacgggagggcttaccatctggccccagigctgc aatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcag aagiggtcclgcaacttatccgcctccatccagtctaitaattgttgecgggaagctagagtaagtagticgccagiiaatagi ttgcgcaacgttgitgccattgciacaggcatcgtgglgtcacgctcgtcgttggtalggcltcattcagctccggttcccaac gatcaaggcgagttacatgatcccccat.gttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaag tggccgcagtgliaicactcatggttatggcagcactgcaiaattctcitactgtcaigccatccglaagatgcttiteigtgact ggt.gagtactcaaccaagtcattcigagaatagtglatgcggcgaccgagtigclctigcccggcgtcaalacgggataata ccgcgccacatagcagaactlaaaagtgclcatcattggaaaacgttcttcggggcgaaaactctcaaggatctaccgci gltgagatccagtcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactit.caccagcgt.tlctgggtgagca aaaacaggaaggcaaaatgccgcaaaaaagggaaiaagggcgacacggaaatgttgaalactcatactcitcctattcaat attattgaagcattatcagggttattgtctcatgagcggatacatattgaatgtatttagaaaaataaacaaataggggttccg cgcacatttccccgaaaagtgccacctgacgtctaagaaaceattataicatgacatiaacciataaaaataggcgtatcac gaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagctgt ctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagegggtgtiggcgggtgtcggggctggcitaac tatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaatac cgcatcaggaaaiccaacatccaataaatcatacaggcaaggcaaagaatiagcaaaataagcaataaagccicagagc ataaagctaaalcggttgtaccaaaaacatatgaccctgtaaiacttUgcgggagaagccttulitcaacgcaaggataaa aatittiagaaccclcaiatattttaaatgcaatgccigaglaatgt.gtaggt;iaagattcaaacgggtgagaaaggccggaga cagtcaaatcaccaicaatatgatattcaaccgtictagcigataaattcatgccggagagggtagctattttgagaggtctct acaaaggclatcagglcatt.gcctgagaglctggagcaaacaagagaatcgatgaacggtaatcgtaaaactagcatgica atcatatgtaccccggttgataatcagaaaagccccaaaaacaggaagattgtataagcaaatatttaaattgtaaacgttaat atttt.gttaaaattcgcgttaaatitttgttaaatcagctcatttttiaaccaataggccgaaatcggcaaaatcccttataaatcaa aagaatagaccgagatagggttgagtgttgttccagttgga£icaagagtccactattaaagaacgtggactccaacgtcaa agggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaaicaagtlttitgggglcgaggtgccg taaagcactaaatcggaaccctaaagggagcccccgat.tlagagctgacggggaaagccggcgaacgtggcgagaaa ggaagggaagaaagcgaaaggagcgggcgctagggcgciggcaagtgiagcggtcacgctgcgcgtaaccaccaca cccgccgcgcttaatgcgccgctacagggcgcgtactatggttgcttgacgagcacgtataacgtgcttcctcgttagaat cagagcgggagctaaacaggaggccgattaaagggaltitagacaggaacgglacgccagaaiccigagaagigtttttat. aatcagtgaggccaccgagtaaaagagtctgtccatcacgcaaataaccgtgtcgcaatactcttgattagtaataacat cacttgcagagiagaagaaclcaaactaicggcctigctggiaataiccagaacaatatlacegccagccaUgcaacagg aaaaacgctcatggaaatacctacattttgacgctcaatcgtctggaaatccattcgccattcaggctgcgcaactgttggga agggcgatcggtgcgggccicticgctaitacgccagctggcgcgcicgctcgct.cacigaggccgcccgggcaaagcc cgggcgtcgggcgaccttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatca ctaggggttcctgiagLtaatgaitaacccgecaigciacLtatciacgiagccalgcictaggaictgaaLtcggtac SEQ ID NO: 21 - sc-U6-wthTLl ccatgtgagggcctattcccatgattccttcatattgcatatacgatacaaggctgttagagagataattggaataattgact giaaacacaaagatat.tagtacaaaatacgtgacgtagaaagiaaiaatticttgggtagtttgcagttttaaaat.latgtttiaaa atggactatcatatgcttaccgtaacttgaaagtatttegatttcttggctttatatatcttgtggaaaggacgaaacaccgttaag atggcagagcccggtaategcataaaacttaaaacttUicagtcagaggit.caat.tcctctctlaacagacaaatggctctag aggtaccagaagaagigacggctgggggcacaglgggctgggcgcccctgcagaacatgaaccttccgctcctggctg ccacagggtcctccgatgciggcctttgcgcctctagaggcagccact,catggai.tcaagtcct.ggctccgcctcttccatca ggaccactctagagggcagagcgcacalcgcccacagtccccgagaagttggggggaggggtcggcaattgatccggt gcctagagaagglggcgcggggtaaactgggaaagtgaigtcgtgtaclggctccgkXtitttcccgagggtgggggaga accgtatataagtgcagtaglcgccgtgaacgttctttttcgcaacgggttigccgccagaacacaggaccggtggatcccg ccaccatggtgagcaagggcgaggagctgtcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacg gccacaagUcagcglgiccggcgagggcgagggcgatgecacctacggcaagctgaccctgaagttcatctgcaccac cggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgctcagccgctaccccgac cacatgaagcagcacgacttcttcaagtccgccaigcccgaaggciacgtccaggagcgcaccatcttcticaaggacga cggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcga cticaaggaggacggcaacaLcclggggcacaagctggaglaeaactacaacagccacaacgtciatat.catggccgaca agcagaagaacggcatcaaggtgaactcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccact accagcagaacacccccatcggcgacggccccgtgcigctgcccgacaaccactacctgagcacccagtccgccctga gcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatgga cgagctgtacaagta.agaaitctaactagagetcgctgatcagcetcgacigtgcctictagtigccagccalctgitgttt.ge ccctcccccgtgccitcctgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcaicgcatgt ctgagia.ggtgtcatictattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcagga accccactccctclctgcgcgctcgctcgclcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccg ggcggcctcagtgagcgagcgagcgcgcagctgctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgt atigggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgtcggctgcggcgagcggtatcagctcactcaa aggcggiaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggcca ggaaccgtaaaaaggccgegttgctggcgttttccataggctccgccccccigacgagcalcacfiaaaatcgacgctcaa gtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttc cgaccctgccgcitaccggataccigtccgcctttctccctlcgggaagcgiggcgcttclcaiagcicacgctgtaggtatc tcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgcctatccgg taactatcgicttgagiccaacccggtaagaeacgacltaicgccactggeagcagccactggtaacaggaitagcagagc gaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgc gctctgctgaagccagttaccttcggaaaaagagttggtitgctcttgatccggcaaacaaaccaccgctggtagcggtggtt ttittgtttgcaagcagcagatiacgcgeagaaaaaaaggatctcaagaagatcctitgaiclttictacggggtctgaegctc agtggaacgaaaacteacgtaagggattttggteatgagattatcaaaaaggatctcacctagatcctttaaattaaaaatg aagtittaaatcaatctaaagiaiatatgagtaaactggtcigacagttaccaatgcttaatcagtgaggcacctatctcagcg atctgtctatttcgttcatccatagtgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggcccca gtgctgcaalgataccgcgagacccacgctcaccggctccagatttalcagcaataaaccagccagccggaagggccga gcgcagaagtggtectgcaactttatccgcctccatccagtctattaattgtgccgggaagctagagtaagtagttcgccag ttaatagttgcgcaacgttgitgccattgctacaggcatcgtggtgtcacgctcgtcgttiggtatggcttcaticagctccggt tcccaacgatcaaggcgagttacatgatcccccatgtgtgcaaaaaagcggttagctccttcggtcctccgatcgtgtcag aagtaagttggccgcagigttatcactcaiggttatggcagcactgcataattctctiactgtcatgccatccgtaagatgciti ctgtgactggtgagtactcaaccaagtcattctgagaatagtgtaigcggcgaccgagttgctcttgcccggcgtcaatacg ggataataccgcgccacatagcagaacttaaaagtgctcatcattggaaa^gttcttcggggcgaaaactctcaaggatc ttaccgctgtigagatccagltcgatgtaacccacicglgcacccaactgatcltcagcatcitiactttcaccagcgatctgg gtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgt.tgaata.ctcatactctte cttttcaatattatgaagcatttatcagggttattgtctcatgagcggatacatatitgaatgtatttagaaaaataaacaaatag gggitccgcgcacatttccccgaaaa.gigcc^ct.gacgtctaagaaaccattatlatcat.gacaitaacctataaaaatagg cgtaicacgaggccctttcgtctcgcgcgttLcggigatgacggtgaaaaccictgacacatgcagctcccggagacggtc acagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggegggtgtcggggc tggcttaaciatgcggcatcagagcagattgtactgagagtgcaccataigcggtgtgaaataccgcacagatgcgtaagg agaaaalaccgcatcaggaaatccaacalccaataaalcaiacaggcaaggcaaagaaUagcaaaaUaagcaataaag cctcagagcaiaaagctaaatcggttgiaccaaaaacattatgaccctgtaaiacttttgcgggagaagcctitatttcaacgc aaggataaaaattttagaaccctcatatattttaaatgcaatgcctgagtaatgtgtaggtaaagattcaaacgggtgagaaa ggccggagacagtcaaatcaccatcaatatgatattcaaccgitctagctgataaaticatgccggagagggtagctat.lttig agaggtctctacaaaggctatcaggtcattgcctgagagtctggagcaaacaagagaatcgatgaacggtaatcgtaaaac tagcatgtcaatcatatgtaceccggttgataatcagaaaagccccaaaaacaggaagattgtataagcaaatatttaaattgt aaacgttaaiattlgttaaaattcgcgttaaatttttgitaaatcagctcattttaaccaataggccgaaatcggcaaaaiccct taiaaatcaaaagaatagaccgagatagggtigagtgttgttccagttggaacaagagtccactaUaaagaacgtggacic caacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtc gaggtgcx:gtaaagcactaaaicggaac,eclaaagggagcccccgatltagagcti.gacggggaaagccggcgaacgt ggcgagaaaggaagggaagaaagcgaaaggagcgggcgclagggcgctggcaagtgtagcggicacgcigcgcgta accaccacacccgccgcgcttaatgcgccgctacagggcgcgtactatggttgctttgacgagcacgtataacgtgctttcc tcgtagaatcagagcgggagctaaacaggaggccgattaaagggattiagacaggaacggtacgccagaatcctgaga agtgtttataatcagtgaggccaccgagtaaaagagtctgtccatcacgcaaataaccgtgtcgcaatacttctttgatta gtaaiaacaicactgcctgagiagaagaactcaaactalcggccttgctggtaatatccagaacaatataccgccagccaU gcaacaggaaaaacgctcatggaaatacctacatttgacgctcaatcgtctggaaatccattcgccattcaggctgcgcaa ctgtgggaagggcgatcggtgcgggcctct.tegctatiacgccagctggcgcgctegclcgctcactgaggccgcccgg gcaaagcccgggcglcgggcgaccttiggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggcca actccatcactaggggltcci.tgiagttaatgattaacccgccatgciacttatctacgtagccatgcictaggatctgaattcg gtac

[0130] SEQ ID NO: 22 cct act cat tea acc aat age cct ggc cgt acg cct aa SEQ ID NO: 23 tt agg cgt acg gcc agg get att ggt tga atg agt agg

[0131] SEQ ID NO: 24 cct act cat tea acc aat age ccg ggc cgt acg cct aa

[0132] SEQ ID NO: 25 cca tac tag tta tta teg aaa cca tea gcc taC tea ttc aac caa taG ccC ggg ccg tac gcc taa ccg c

[0133] SEQ ID NO: 26 g egg tta ggc gta egg ccc ggg eta ttg gtt gaa tga gta ggc tga tgg ttt ega taa taa eta gta tgg

[0134] SEQ ID NO: 27

[0135] CCA TAC TAG TTA TTA TCG AAA CCA TCA GCC TAC TCA TTC AAC CAA TAG CCC TGG CCG TAC GCC TAA CCG C

[0136] SEQ ID NO: 28 cgcatcataatcctctctca

[0137] SEQ ID NO: 29 gca etc aca gtc aca tea taa tee tet etc aAg gac ttc aaa c

[0138] SEQ ID NO: 30 g ttt gaa gtc ett gag aga gga tta tga tgt gac tgt gag tgc

[0139] SEQ ID NO: 31 acgcactcacagtcacatcataatcctctctcaaggacttcaaactctactccc

[0140] SEQ ID NO: 32 acgcactcacagtcacatcgtaatcctctctcaaggacttcaaactctactccc

[0141] SEQ ID NO: 33 acgcactcacagtcacgtcataatcctctctcaaggacttcaaactctactccc

[0142] SEQ ID NO: 34 acgcactcacagtcgcatcataatcctctctcaaggacttcaaactctactccc

[0143] SEQ ID NO: 35 acgcactcacagtcgcgtcataatcctctctcaaggacttcaaactctactccc

[0144] SEQ ID NO: 36 acgcactcacagtcgcatcgtaatcctctctcaaggacttcaaactctactccc

[0145] SEQ ID NO: 37 acgcactcacagtcacattataatcctctctcaaggacttcaaactctactccc

[0146] SEQ ID NO: 38 acgcactcacagtcatattataatcctctctcaaggacttcaaactctactccc

[0147] SEQ ID NO: 39 ctcacagtcgcatcataatcctctctcaaggacttc

[0148] SEQ ID NO: 40 attctactatcaacattactaataagtggctcc

[0149] SEQ ID NO: 41 attctactattaacattactaataagtggctcc SEQ ID NO: 42 tcttatccatcatagcaggcagttgaggtggat

[0150] SEQ ID NO: 43 atagaaaaatccaccccttacgagtgcggcttcg

[0151] SEQ ID NO: 44 atagaaaaatttaccccttacgagtgcggcttcg

[0152] SEQ ID NO: 45 aaacgcctggcagccggaagcctattcgcaggatttctc

[0153] SEQ ID NO: 46 tgaaatgccccaactaaatactaccgtatggccca

[0154] SEQ ID NO: 47 tggtactgaacctacgagtacaccgactacggcgga

[0155] SEQ ID NO: 48 agtaaaacccagcccatgacccctaacaggggccctc

[0156] SEQ ID NO: 49 aatgtctgcacagccactttccacacagacatcataacaaaaaatttccaccaaaccccccc tccccc gcttctggccacagcacttaaacacatctctgccaaaccccaaaaacaaag

[0157] SEQ ID NO: 50 aatgtctgcacagccgctttccacacaaacatcataacaaaaaatttccaccaaacccccccctccccccgcttctggccacagcactta aacacatctctaccaaaccccaaaaacaaag

[0158] SEQ ID NO: 51 ctttgtttttggggtttggtagagatgtgtttaagtgctgtggccagaagcggggggaggggggggtttggtggaaattttttgttatgatg tttgtgtggaaagcggctgtgcagacatt

[0159] SEQ ID NO: 52 aatgtctacacaaccactttccacacaaacatcataacaaaaaatttccaccaaacccccccctccccccacttctaaccacaacactta aacacatctctaccaaaccccaaaaacaaag

[0160] SEQ ID NO: 53 ctttgtttttggggtttggtagagatgtgtttaagtgttgtggttagaagtggggggaggggggggtttggtggaaattttttgttatgatgttt gtgtggaaagtggttgtgtagacatt

Claims

CLAIMSWhat is claimed is:

1. A chimeric nucleic acid comprising a first nucleic acid sequence operably fused to a second nucleic acid sequence; wherein the second nucleic acid sequence comprises a mitochondrial targeting sequence (MTS) comprising a mitochondrial ribosomal protein S12 (MRPS12) gene, or a fragment thereof.

2. The chimeric nucleic acid of claim 1 , wherein the MTS comprises at least 50% identity with SEQ ID NO: 1.

3. The chimeric nucleic acid of claim 1 , wherein the MTS comprises at least 60% identity with SEQ ID NO: 1.

4. The chimeric nucleic acid of claim 1 , wherein the MTS comprises at least 70% identity with SEQ ID NO: 1.

5. The chimeric nucleic acid of claim 1 , wherein the MTS comprises at least 80% identity with SEQ ID NO: 1.

6. The chimeric nucleic acid of claim 1 , wherein the MTS comprises at least 90% identity with SEQ ID NO: 1.

7. The chimeric nucleic acid of claim 1, wherein the MTS comprises SEQ ID NO: 1.

8. The chimeric nucleic acid of any one of claims 1-7, wherein the MTS encodes at least 25 nucleotides.

9. The chimeric nucleic acid of any one of claims 1-8, wherein the MTS encodes about 163 nucleotides.

10. The chimeric nucleic acid of any one of claims 1-9, wherein the first nucleic acid comprises a CRISPR nuclease.

11. The chimeric nucleic acid of any one of claims 1-10, wherein the first nucleic acid comprises a ten-eleven translocation (TET) enzyme.

12. The chimeric nucleic acid of any one of claims 1-11, wherein the first nucleic acid comprises a reporter gene.

13. The chimeric nucleic acid of claim 12, wherein the reporter gene is either a monomeric red fluorescent protein Cherry (mCherry) gene or a green fluorescent protein (GFP) gene.

14. A mitochondrial genome editing system comprising the chimeric nucleic acid of claims 1-13 and an RNA-guided nuclease.

15. The mitochondrial genome editing system of claim 14, wherein the MTS directs the chimeric nucleic acid into a mitochondrion.

16. The mitochondrial genome editing system of claims 14 or 15, wherein the first nucleic acid sequence is integrated into a mitochondrial genome in the presence of the RNA- guided nuclease.

17. The mitochondrial genome editing system of any one of claims 14-16, wherein the first nucleic acid sequence comprises a targeted gene modification.

18. The mitochondrial genome editing system of claim 17, wherein the targeted gene modification is selected from the group consisting of: insertions, deletions, base pair conversions, methylation, and any variations thereof.

19. A method of treating a mitochondrial disease in a subject, wherein the method comprises administering the mitochondrial genome editing system of any one of claims 14-18 to the subject.

20. The method of claim 19, wherein the mitochondrial genome editing system is imported into a cell and further imported into a mitochondrion.

21. The method of claim 19 or 20, wherein the mitochondrial disease comprises a mitochondrial DNA (mtDNA) mutation.

22. The method of any one of claims 19-21, wherein the mitochondrial disease is selected from the group consisting of: oxidative phosphorylation defects, Barth’s syndrome, Complex I deficiency, Complex II deficiency, Complex III deficiency, Complex IV deficiency Complex V deficiency, cytochrome c oxidase (COX) deficiency, Leigh Disease, Leiber’s Hereditary Optic Neuropathy, and any related diseases thereof.

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