WO2024003579A1

WO2024003579A1 - Preventing disease recurrence in mitochondrial replacement therapy

Info

Publication number: WO2024003579A1
Application number: PCT/GB2023/051732
Authority: WO
Inventors: Magomet AUSHEV; Mary Herbert; Gavin HUDSON
Original assignee: University Of Newcastle Upon Tyne
Priority date: 2022-06-30
Filing date: 2023-06-30
Publication date: 2024-01-04
Also published as: GB202209611D0; GB2621813A

Abstract

The present invention relates to a synthetic targeted DNA binding and cleavage complex which is capable of specifically binding to a target sequence within a conserved region of the mitochondrial DNA control region and cleaving mitochondrial DNA comprising the target sequence. The invention also relates to one or more nucleic acid sequences encoding the synthetic targeted DNA binding and cleavage complex and methods of using the same.

Description

Preventing Disease Recurrence in Mitochondrial Replacement Therapy

Field of the invention

Background of the Invention

The following discussion is provided to aid the reader in understanding the disclosure and does not constitute any admission as to the contents or relevance of the prior art.

Mitochondrial diseases are caused by mitochondrial dysfunction. Mitochondrial diseases may be caused by acquired or inherited detrimental mutations in mtDNA or nuclear genes coding for mitochondrial components or by environmental factors. Mitochondria are organelles generating adenosine triphosphate (ATP) in order to power different cellular functions. Unlike most organelles, mitochondria comprise their own genome called mitogenome or mitochondrial DNA (mtDNA) which is most often inherited exclusively from the mother. Each mitochondrion contains between two and ten copies of the mtDNA.

During cell division, mitochondria segregate between the daughter cells and then proliferate in order to replenish the mitochondria reserve in the daughter cells. During the proliferation process, mutations in mtDNA occur more often than in nuclear DNA since mitochondria do not possess all the DNA repair pathways which nuclei do. Accumulation of mutations can lead to heteroplasmy, coexistence of two or more variants of mtDNA. Therefore, mtDNA copies with inherited detrimental mutations or de novo detrimental mutations may coexist with mtDNA copies which are wild type. Over numerous mitochondria proliferation cycles, the proportion of the mtDNA copies with inherited defective mutations or de novo defective mutations may increase and reach the threshold of the disease phenotype once enough mitochondria are affected. Prevalence of mitochondrial diseases in the UK is 20 in 10,000.

Mitochondrial DNA replacement is one of the therapeutic strategies for treatment of mitochondrial disease. Mitochondrial DNA replacement may involve introducing new wild type mitochondrial DNA and optionally elimination of the disease mitochondrial DNA ex vivo. Since mtDNA is most often inherited exclusively from the mother, women whose mtDNA comprises detrimental mutations causing a mitochondrial disease are prone to pass a fraction of the detrimental mtDNA mutations to their offspring. Therefore, women with family history of mitochondrial disease or who have detrimental mutations in their mtDNA have a high risk of transmitting mtDNA disease to their offspring. In order to prevent transmission of mitochondrial disease from mother to child, mitochondrial replacement therapy (MRT) may be used during in vitro fertilisation (IVF).

Mitochondrial replacement therapy allows for the mother’s dysfunctional mitochondria to be replaced with mitochondria from a third party with no mitochondria diseases. Multiple assisted reproductive technologies designed to prevent transmission of pathogenic mtDNA variants exist, including material spindle transfer, pronuclear transfer, pre-nuclear transfer and polar body transfer.

Pronuclear transfer has been licenced for clinical treatment and involves the transfer of the pronuclei from the patient zygote (nuclear donor) to the donor zygote (mitochondrial donor) as shown in Fig. 1. PNT reduces the risk of mitochondrial disease transmission from mother to child but not completely. This is due to the co-transfer (carryover) of small amounts of mitochondria and mtDNA (<2%) from the egg of the nuclear donor together with the pronuclei to the egg of the mitochondrial donor.

Reversion of mitochondrial genome of nuclear donor has been observed in embryonic stem cells derived from embryos generated by mitochondrial replacement as the carried over mtDNA increased over time. This raises possibility of resurgence of mutation-carrying mtDNA following PNT and reversion to disease phenotype.

Various systems have been developed to edit and/or reduce mutated mtDNA. For example, WO2018/093954A1 discloses development of a tool to target mitochondrial DNA by modifying the CRISPR/Cas9 system for genome editing in mitochondria. Reddy et al., 2015 used mito-TALENs designed to target a specific mutant mtDNA to specifically reduce expression of this mutant mtDNA. Rai et al., 2018 also disclose the use of mito-TALENs specific for mutant mtDNA to reduce expression of the mutant mtDNA. However, these approaches have the drawback of having to design, synthesise, pre-screen and test a construct to target each specific mutant mtDNA in each patient.

There is a need for improved tools and methods for removing or eliminating mtDNA from a cell regardless of the mutations present in the mtDNA. These tools and methods may be particularly beneficial in further reducing the chance of reversion to a disease phenotype in PNT. These tools may also be particularly beneficial in mitochondrial DNA replacement therapy.

Summary of the Invention

In a first aspect, there is provided a synthetic targeted DNA binding and cleavage complex, or one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex, wherein the synthetic targeted DNA binding and cleavage complex is capable of specifically binding to a target sequence within a conserved region of the mitochondrial DNA control region and cleaving mitochondrial DNA comprising the target sequence.

The target sequence may be a suitable sequence within SEQ ID NO: 403, SEQ ID NO: 404, SEQ ID NO: 1813 or a homolog of the conserved region of mitochondrial DNA in another organism. SEQ ID NO: 403 is the mouse conserved region of mitochondrial DNA. SEQ ID NO: 404 is the human conserved region of mitochondrial DNA. SEQ ID NO: 1813 is the conserved control region for human mtDNA. ‘N’ indicates positions where there is variability. In some embodiments, the homolog of the conserved region of mitochondrial DNA in another organism may be conserved region of mitochondrial DNA in pig, cow, sheep, hen, camel, dog, horse or a standard model organism such as mouse, rat, drosophila and zebrafish.

In some embodiments, the target sequence may be a sequence having at least 7-30 consecutive nucleotides from SEQ ID NQ:403, SEQ ID NO: 404, SEQ ID NO: 1813 or a homolog of the conserved region of the mitochondrial DNA in another organism. In some embodiments, the target sequence may be a sequence having at least 7, 8, 9, 10, 11 consecutive nucleotides from SEQ ID NQ:403, SEQ ID NO: 404, SEQ ID NO: 1813 or a homolog of the conserved region of the mitochondrial DNA in another organism. In some embodiments, the target sequence may be a sequence having at least 12, 13, 14, 15, 16 consecutive nucleotides from SEQ ID NQ:403, SEQ ID NO: 404, SEQ ID NO: 1813 or a homolog of the conserved region of the mitochondrial DNA in another organism. In some embodiments, the target sequence may be a sequence having at least 17-30, suitably 20-28 consecutive nucleotides from SEQ ID NQ:403, SEQ ID NO: 404, SEQ ID NO: 1813 or a homolog of the conserved region of the mitochondrial DNA in another organism. In some embodiments, wherein the target sequence consists or comprises of any one of SEQ ID NO: 376-378, 1-7, 86-90, 153-155, 196-208, 329-331, 464.

The synthetic targeted DNA binding and cleavage complex does not occur in nature. In some embodiments, the synthetic targeted DNA binding and cleavage complex may be artificial.

The synthetic targeted DNA binding and cleavage complex is capable of specifically binding to a target sequence within a conserved region of the mitochondrial DNA control region and cleaving mitochondrial DNA comprising the target sequence. That is to say that the synthetic targeted DNA binding and cleavage complex is adapted for, designed for or configured to specifically bind to a target sequence within a conserved region of the mitochondrial DNA control region and cleave mitochondrial DNA comprising the target sequence.

In some embodiments, the synthetic targeted DNA binding and cleavage complex comprises at least one DNA binding module and at least one DNA cleavage module.

In some embodiments, the at least one DNA binding module may be linked to the at least one DNA cleavage module.

In some embodiments, the at least one DNA binding module and the at least one DNA cleavage module may be modules which are synthetized or translated separately and subsequently linked by a linker or otherwise by a covalent bond. The linker may be a short non-functional amino acid or nucleotide sequence.

In other embodiments, the at least one DNA binding module and the at least one DNA cleavage module may be synthetized or translated together. In some embodiments, one nucleic acid sequence may encode for the at least one DNA binding module linked to the at least one DNA cleavage module.

In some embodiments, the at least one DNA binding module comprises a DNA binding protein domain. In some embodiments, the at least one DNA binding module comprises at least one Transcription Activator- Like Effector (TALE) domain or at least one zinc finger domain. In some preferred embodiments, the at least one DNA binding module comprises at least one Transcription Activator-Like Effector (TALE) domain. In some embodiments, the at least one TALE domain comprises RVDs according to any one of SEQ ID NQs:379-380, 8-9, 14-15, 20-21, 24-25, 91-92, 97-98, 103-104, 156-157, 160- 161 , 209-210, 215-216, 221-222, 225-226, 229-230, 243-244, 332-333, 338-339.

The skilled person is familiar with the standard RVD code as well as the alternative RVDs, which are less commonly used (Cong et al., 2012 Nature Communications; Miller et al., 2015 Nature Methods). Standard RVDs: A - Nl; T - NG; G - NN; and C - HD. Alternative RVDs: A - HI, Cl, KI; T - HG, VG, IG, EG, MG, YG, QG, KG, RG, NC, EP, AA, VA; G - NH, HN, NK, RH, NQ, QN, GN, VN, LN, DN, EN, AN, FN SN, SS; C - AD, KD, RD, ND, N*;

N - NA, NV, NS, S*, HH, YH, H*, wherein * corresponds to a 33-residue repeat in which the RVD appears to be missing its second residue.

The at least one TALE domain comprises a central repeat domain which allows DNA recognition. In some embodiments, the at least one TALE domain comprises or consists of a central repeat domain according to any one of SEQ ID NO: 383-384, 38-41 , 117-119, 172- 173, 257, 259-263, 352-353, 385-386, 42-45, 120-122, 174-175, 264-269, 354-355, 466, 471 , 506, 511 , 545, 548, 569, 582, 647, 652, 707, 720, 863, 868, 922, 925, 958, 969, 1090, 1095, 1150, 1155, 1210, 1213, 1246, 1249, 1282, 1295, 1438, 1451, 1595, 1600, 1657, 1670. In some embodiments, the at least one TALE domain comprises amino acid sequence according to any one of SEQ ID NO: 383-384, 38-41, 117-119, 172-173, 257, 259-263, 352- 353, 466, 506, 545, 569, 647, 707, 863, 922, 958, 1090, 1150, 1210, 1246, 1282, 1438, 1595, 1657. In some embodiments, the at least one TALE domain is encoded by a nucleotide sequence according to any one of SEQ ID NO: 385-386, 42-45, 120-122, 174- 175, 264-269, 354-355, 471, 511, 548, 582, 652, 720, 868, 925, 969, 1095, 1155, 1213, 1249, 1295, 1451, 1600, 1670.

In some embodiments, the at least one TALE domain is adapted to recognise all four nucleotides at one or more specific locations within the target sequence. Suitably, the at least one TALE domain comprises one or more NA and/or NS RVDs. In some preferred embodiments, the at least one TALE domain comprises one or more NA and/or NS RVDs when the target sequence is human. In some embodiments, the at least one TALE domain comprises RVDs according to any one of SEQ ID NOs: 381-382, 430-431, 10-13, 16-19, 22- 23, 26-37, 93-96, 99-102, 105-116, 158-159, 162-171 , 211-214, 217-220, 223-224, 227-228, 231-242, 245-256, 334-337, 340-351.

In some embodiments, the at least one TALE domain comprises or consists of a central repeat domain according to any one of SEQ ID NO: 467-468, 507-510, 546-547, 570-571 , 648-649, 864-865, 708-709, 923-924, 959-960, 1091-1092, 1151-1152, 1211-1212, 1247- 1248, 1283-1284, 1439-1440, 1596-1597, 1658-1659, 469, 470, 472-475, 512-515, 549-550, 572-581 , 583-594, 650-651 , 653-656, 710-719, 721-732, 866-867, 869-872, 926-927, 961- 968, 970-979, 1093-1094, 1096-1099, 1153-1154, 1156-1159, 1214-1215, 1250-1251 , 1285-1294, 1296-1307, 1441-1450, 1452-1463, 1598-1599, 1601-1604, 1660-1667, 1812, 1669, 1668, 1671-1682, 1814, 1815, 1816, 1817, 1818-1821.

In some embodiments, the at least one TALE domain comprises amino acid sequence according to any one of SEQ ID NO: 467-468, 507-510, 546-547, 570-571 , 572-581, 648- 649, 708-709, 864-865, 923-924, 959-960, 961-968, 1091-1092, 1093-1094, 1151-1152, 1211-1212, 1247-1248, 1283-1284, 1285-1294, 1439-1440, 1441-1450, 1596-1597, 1658- 1659, 1814, 1817, 469, 470, 650-651 , 710-719, 866-867, 1153-1154, 1598-1599, 1660- 1667, 1669, 1812, 1668, 1815, 1816.

In some embodiments, the at least one TALE domain is encoded by a nucleotide sequence according to any one of SEQ ID NO: 512-515, 549-550, 583-594, 653-656, 721-732, 1096- 1099, 1296-1307, 1452-1463, 1601-1604, 1818-1821 , 472-475, 869-872, 926-927, 970- 979,1156-1159, 1214-1215, 1250-1251, 1671-1682.

Suitably, the at least one TALE domain comprises a TALE scaffold.

In some embodiments, the scaffold may be 63aa scaffold described in Miller et al. (2011), wherein there is deletion of 152 residues at N-terminus leaving 136 residues and deletion of 215 residues at C-term leaving 63 residues. In some preferred embodiments, the TALE scaffold comprises or consists of amino acid sequence SEQ ID NO: 436 or nucleotide sequence SEQ ID NO: 442. The central repeat domain according to the present invention is suitably inserted into the designated positions of the TALE scaffold. That is to say that the central repeat domain according to the present invention is flanked by a portion of the TALE scaffold on both sides.

In some embodiments, the TALE scaffold may be a TALE scaffold with different C-terminal truncation. In some embodiments, the TALE scaffold may be a scaffold selected from the group of scaffolds with the following C-terminal truncations: 28aa, 39aa, 50aa, and 79aa (Miller et al., 2011); 2aa, 5aa, and 16aa (Kim et al., 2013); 17aa and 47aa (Mussolino et al., 2011); 230aa (Cermak et al., 2011). Suitably, the DNA cleavage module may be a nuclease or an endonuclease. Suitably, the DNA cleavage module may be configured to create one or more single-stranded breaks (SSBs) or double stranded breaks (DSBs) in the mitochondrial DNA comprising the target sequence. That is to say that the DNA cleavage module is capable of, adapted for, designed to create one or more single-stranded breaks (SSBs) or double stranded breaks (DSBs) in the mitochondrial DNA comprising the target sequence.

In some embodiments, the DNA cleavage module a monomeric or a dimeric nuclease.

In some preferred embodiments, the DNA cleavage module may be made up of two Fokl nuclease subunits. In some embodiments Fokl nuclease subunits may comprise amino acid sequence according to SEQ ID NO: 437 or a nucleotide sequence according to SEQ ID NO: 443.

In some embodiments, the DNA cleavage module may be made up of two restriction endonuclease subunits (e.g. Pvull). In some embodiments, the DNA cleavage module may be a homing endonuclease (e.g. I-Tevl). The DNA cleavage domain may be a monomeric or dimeric nuclease made non-specific by removal of DNA-binding domain. The DNA cleavage domain may be a monomeric or dimeric nuclease which retain specificity such as restriction endonucleases or meganucleases/homing endonucleases. The DNA cleavage module may be selected from the list of: TALE-Pvull (Yanik et al. (2013) PLoS One), homing endonuclease-TALE such as I-Tevl (mitoTev-TALE) (Pereira et al. (2018) EMBO Mol Med), or Mito-meganuclease such as l-Crel (mitoARCUS) (Zekonyte et al. (2021) Nat Commun), mito-restriction endonucleases such as Xhol (Xu et al. (2008) Science), Pstl (Srivastava & Moraes (2001) Human Molecular Genetics; Srivastava & Moraes (2005) Human Molecular Genetics), Smal (Tanaka et al. (2002) Journal of Biomedical Science), Xmal (Alexeyev et al. (2008) Gene Therapy), Seal (Bacman et al. (2007) Gene Therapy; Bacman et al. (2009) Nucleic Acids Research), EcoRI (Tanaka et al. (2002) Journal of Biomedical Science) or ApaLI (Bayona-Bafaluy et al. (2005) PNAS; Bacman et al. (2010) Gene Therapy; Bacman et al. (2012) Gene Therapy; Reddy et al. (2015) Cell). In some embodiments, the DNA cleavage module may be made up of two restriction endonuclease subunits (a dimeric nuclease) selected from Pvull (Yanik et al. (2013) PLoS One), Xhol (Xu et al. (2008) Science), Pstl (Srivastava & Moraes (2001) Human Molecular Genetics; Srivastava & Moraes (2005) Human Molecular Genetics), Smal (Tanaka et al. (2002) Journal of Biomedical Science) and EcoRI (Tanaka et al. (2002) Journal of Biomedical Science). In some embodiments, the DNA cleavage module is be a homing endonuclease. A non-limiting example of a homing endonuclease is I-Tevl (mitoTev-TALE) (Beurdeley et al. (2013) Nat Commun; Pereira et al. (2018) EMBO Mol Med). In some embodiments, the DNA cleavage module is a mito-meganuclease. A non-limiting example of mito-meganuclease is l-Crel (mitoARCUS) (Zekonyte et al. (2021) Nat Commun). In some embodiments, the DNA cleavage module may be selected from Xmal (Alexeyev et al. (2008) Gene Therapy), Seal (Bacman et al. (2007) Gene Therapy; Bacman et al. (2009) Nucleic Acids Research), and ApaLI (Bayona-Bafaluy et al. (2005) PNAS; Bacman et al. (2010) Gene Therapy; Bacman et al. (2012) Gene Therapy; Reddy et al. (2015) Cell).

In some embodiments, the DNA cleavage module with two restriction endonuclease subunits (dimeric nuclease) may be turned into a monomeric nuclease by attaching the restriction endonuclease subunits to each other, such as TALE-Fokl-Fokl (1 TALE domain + 2 Fokl domains).

In some embodiments, the synthetic targeted DNA binding and cleavage complex comprises or consists of a first TALE domain linked to a first DNA cleavage module subunit and a second TALE domain linked to a second DNA cleavage module subunit.

In some embodiments, the DNA cleavage module comprises a dimer of the first and the second DNA cleavage module subunits and wherein the DNA cleavage module is configured to create one or more single-stranded breaks (SSBs) or double stranded breaks (DSBs) in the mitochondrial DNA comprising the target sequence upon dimerization of the first and the second DNA cleavage module subunits.

In some embodiments, the target sequence of the first TALE domain linked to the first DNA cleavage module subunit is the same as the target sequence of the second TALE domain linked to the second DNA cleavage module subunit. That is to say that the synthetic targeted DNA binding and cleavage complex is a homodimer.

In some embodiment, the target sequence of the first TALE domain linked to the first DNA cleavage domain subunit is different to the target sequence of the second TALE domain linked to the second DNA cleavage module subunit. That is to say that the synthetic targeted DNA binding and cleavage complex is a heterodimer.

In some embodiments, the target sequence of the first TALE domain linked to the first DNA cleavage domain subunit is spaced 13 to 23, suitably 14 to 19, suitably 14 to 16, suitably 14 or 16 nucleotides away from the target sequence of the second TALE domain linked to the second DNA cleavage module subunit. In some embodiments, the first DNA cleavage module subunits may comprise of a Fokl nuclease subunit and the second DNA cleavage module subunits may comprise of a Fokl nuclease subunit. That is to say that the DNA cleavage module may be made up of two Fokl nuclease subunits. In some embodiments, the first DNA cleavage module subunits may comprise of a Fokl nuclease subunit comprising or consisting of amino acid sequence according to SEQ ID NO: 437 In some embodiments, the second DNA cleavage module subunits may comprise of a Fokl nuclease subunit comprising or consisting of amino acid sequence according to SEQ ID NO: 437. In some embodiments, the first DNA cleavage module subunits may comprise of a Fokl nuclease subunit comprising or consisting of nucleotide sequence according to SEQ ID NO: 443. In some embodiments, the second DNA cleavage module subunits may comprise of a Fokl nuclease subunit comprising or consisting of nucleotide sequence according to SEQ ID NO: 443. In some embodiments, the DNA cleavage module may be made up of two Pvull endonuclease subunits. In some embodiments, the first DNA cleavage module subunits may comprise of a Pvull nuclease subunit and the second DNA cleavage module subunits may comprise of a Pvull nuclease subunit. That is to say that the DNA cleavage module may be made up of two Pvull endonuclease subunits.

In some embodiments, the first and second DNA cleavage module subunits may comprise of two heterodimeric Fokl nuclease subunits. In some embodiments, the two heterodimeric Fokl nuclease subunits comprise sequences according to SEQ ID NO: 1831 and 1832.

In some embodiments, the synthetic targeted DNA binding and cleavage complex comprises at least one DNA binding module and at least one DNA cleavage module but the DNA binding module and the DNA cleavage module are found within different molecules. That is to say that he DNA binding module and the DNA cleavage module are not part of the same molecule and are not physically linked or connected.

In some embodiments, the DNA binding module may be guide ribonucleic acid (gRNA) and the DNA cleavage domain may be CRISPR-Cas. In some embodiments, the CRISPR-Cas may be Cas9 or Cas12.

In some embodiments, the synthetic targeted DNA binding and cleavage complex, or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex may further comprise at least one mitochondrial targeting signal.

In some embodiments, the at least one mitochondrial targeting signal comprises amino acid sequence according to any out of SEQ ID NO: 434, 435, 447, 448, 451 , 452 or a nucleotide sequence according to any one of SEQ ID NO: 440, 441 , 455, 456, 460, 461. In some embodiments, the synthetic targeted DNA binding and cleavage complex or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex comprises dual tandem mitochondrial targeting signal. The mitochondrial targeting signal directs a protein to the mitochondria.

In some embodiments, the synthetic targeted DNA binding and cleavage complex, or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex may further comprise at least one nuclear export signal. In some embodiments, the at least one nuclear export signal comprises amino acid sequence according to any out of SEQ ID NO: 465 or 453 or a nucleotide sequence according to any one of SEQ ID NO:446. In some embodiments, the synthetic targeted DNA binding and cleavage complex or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex comprises dual tandem nuclear export signals.

In some embodiments, the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex is RNA. In some embodiments, the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex is mRNA. In some embodiments, the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex is DNA.

In some embodiments, the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex further comprise a 5’ UTR and/or a 3’ UTR. In some embodiments, the 5’ UTR may have a nucleotide sequence according to SEQ ID NO: 439, 454, 459. In some embodiments, the 3’ UTR may have a nucleotide sequence according to SEQ ID NO: 444, 457, 462.

In some embodiments, the synthetic targeted DNA binding and cleavage complex comprises or consists of any one of the sequences according to SEQ ID NO: 387-394, 46-57, 70-77, 123-131 , 141-146, 176-181 , 188-191 , 270-287, 306-313, 258, 314-316, 356-361 , 368-371 , 476-490, 516-526, 1830, 527-529, 551-559, 595-607, 657-671, 687-696, 733-771 , Si l- 836, 873-887, 903-912, 928-936, 946-951 , 980-1012, 1046-1067, 1100-1114, 1130-1139, 1160-1174, 1190-1199, 1216-1224, 1234-1239, 1252-1260, 1270-1275, 1308-1346, 1386- 1411 , 1464-1502, 1542-1567, 1580, 1605-1619, 1637-1646, 1683-1709, 1710-1720, 1760- 1767, 1768, 1769-1785, 1822-1825, 1854-1879, 1880-1931. In some embodiments, the nucleic acid sequence encoding a synthetic targeted DNA binding and cleavage complex comprise or consist of any one of SEQ ID NO: 395-402, 58- 69, 78-85, 132-140, 147-152, 182-187, 192-195, 288-305, 317-328, 362-367, 372-375, 491- 505, 530-544, 560-568, 608-646, 672-686, 697-706, 772-810, 837-862, 888-902, 913-921 , 1834, 937-945, 952-957, 1013-1045, 1068-1089, 1115-1129, 1140-1149, 1175-1189, 1200- 1209, 1225-1233, 1240-1245, 1261-1269, 1276-1281 , 1347-1385, 1412-1437, 1503-1541 , 1568-1579, 1581-1594, 1620-1622, 1625, 1626-1636, 1647-1656, 1721-1755, 1756, 1757- 1759, 1786-1811, 1826-1829, 1932-1983.

In a further aspect, there is provided a vector comprising the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to the present invention.

In a further aspect, there is provided a cell comprising a vector according to the present invention.

In some embodiments, the synthetic targeted DNA binding and cleavage complex may be for use in any assisted reproductive technology designed to prevent transmission of pathogenic mtDNA variants. Non-limiting examples include material spindle transfer, pronuclear transfer, pre-nuclear transfer and polar body transfer.

In a further aspect, there is provided a pharmaceutical composition comprising one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to the present invention or a vector according to the present invention and optionally a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier.

In a further aspect, there is provided one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to the present invention, a vector according to the present invention or a pharmaceutical composition according to the present invention for use in medicine. The one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex, the vector or the pharmaceutical composition according to the present invention may be used in mitochondria replacement therapy. The one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex, the vector or the pharmaceutical composition according to the present invention may be used in assisted reproductive technologies. In a further aspect, there is provided an in vitro method of reducing or eliminating mitochondrial DNA in a cell or a sample comprising the step of administering to the cell or the sample:

- a synthetic targeted DNA binding and cleavage complex according to the present invention;

- one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to the present invention; or

- a vector according to the present invention.

In some embodiments, the mitochondrial DNA may comprise one or more mutations associated with mitochondrial disease. In some embodiments, the cell may be an oocyte or a zygote. In some embodiments, the sample may be a tissue sample or a nucleic acid. Suitably the nucleic acid sample may be a sample of purified nucleic acids such as a sample of purified DNA.

In some embodiments, the method may further comprise the step of incubating the cell or sample with the synthetic targeted DNA binding and cleavage complex, the one or more nucleic acid sequences encoding the synthetic targeted DNA binding and cleavage complex, or the vector according to the present invention under suitable conditions for at least thirty minutes, one hour, one hour and thirty minutes or two hours. Suitable conditions may be incubation in water or TE buffer at any temperature between room temperature at 37°C. In some embodiments, the method may further comprise the step of reducing or eliminating the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex from the cell or sample after incubation. In some embodiments, the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex may be RNA. In some embodiments, the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex may be mRNA. In some embodiments, the one or more RNA sequences encoding a synthetic targeted DNA binding and cleavage complex may be reduced or eliminated by addition of guide RNA targeting the RNA sequences encoding the synthetic targeted DNA binding and cleavage complex, and class 2 RNA-targeting CRISPR-Cas system to the cell or the sample. In some embodiments, the class 2 RNA-targeting CRISPR-Cas system may be Cas13b. In some embodiments, the method may further comprise the step of incubating the guide RNA and class 2 RNA- targeting CRISPR-Cas system with the one or more RNA sequences encoding the synthetic targeted DNA binding and cleavage complex under suitable conditions for at least 2 hours. In some embodiments of the method, suitable conditions for complexing gRNA and Cas13 protein in vitro include pre-incubating the Cas13 protein with the gRNA in a tube for 5-15 min at RT or 37°C (Cas13 becomes associated with the gRNA and is ready to be used). In some embodiments, for in vitro digestion, suitable conditions may require water, a buffer (e.g., NEBuffer3.1), Cas13 protein, gRNA, and RNA substrate containing the target sequence together in a tube. The Cas13 protein and gRNA may be pre-incubated for 5-15min at RT, then add RNA substrate and incubate for 15min at 37°C.

In some embodiments of the method, the method comprises co-injection of Cas13 and gRNA into the oocytes/embryos in water. In oocytes, 2-3 hours were allowed for expression of Cas13 (expect expression to start within the first 1-2 hours). In embryos, Cas13, gRNA, and mitoTALEN may be injected at the same time. Cells have the suitable conditions and machinery for expression of Cas13, complexing of Cas13 and gRNA, and activity; suitably minimal conditions would be to inject low concentrations of Cas13 (e.g. ~50 ng/ul) either with mitoTALEN mRNA or shortly before.

In a further aspect, there is provided a method of reducing the likelihood of passing parental mitochondrial DNA to an offspring during assisted reproductive technologies comprising administering to the parental zygote one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to the present invention before pro-nuclear transfer. In some embodiments, administration may be done by microinjection of the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex into the parental zygote. In some embodiments, the one or more nucleic acid sequences encoding the synthetic targeted DNA binding and cleavage complex may be RNA. In some embodiments, the one or more nucleic acid sequences encoding the synthetic targeted DNA binding and cleavage complex may be mRNA. In some embodiments, the method may further comprise reducing or eliminating the one or more mRNA sequences encoding the synthetic targeted DNA binding and cleavage complex before pro nuclear transfer. In some embodiments, the one or more mRNA sequences encoding the synthetic targeted DNA binding and cleavage complex may be reduced by addition of guide RNA targeting the mRNA sequences encoding the synthetic targeted DNA binding and cleavage complex and class 2 RNA-targeting CRISPR-Cas system to the parental zygote. In some embodiments, the class 2 RNA-targeting CRISPR-Cas system may be Cas13b.

In a further aspect, there is provided use of the synthetic targeted DNA binding and cleavage complex according to the present invention or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to the present invention for reducing or eliminating mitochondrial DNA.

In a further aspect, there is provided use of the synthetic targeted DNA binding and cleavage complex according to the present invention or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to the present invention for reducing the likelihood of passing parental mitochondrial DNA to an offspring during assisted reproductive technologies or for mitochondrial DNA replacement therapy. In mitochondrial replacement techniques for prevention of mtDNA disease, transplantation of the nuclear genome may involve co-transfer of a small amount of cytoplasm containing mitochondria. In some embodiments, mitoTALENs could be used to minimise heteroplasmy (maternal mtDNA and donor mtDNA) by greatly reducing the fraction of maternal mtDNA.

Brief Description of the Figures

Figure 1 shows a diagram of pronuclear transfer (PNT).

Figure 2 shows a schematic representation of a TALEN heterodimer. TALEN subunit binding sites are shown (1) with 5’ thymine at position 0 (2) indicated. A TALEN subunit consists of a TALE DNA-binding domain (4) fused to a Fokl nuclease domain (5) at the C-terminus, and a nuclear localization signal (NLS) or mitochondrial targeting signal (MTS) at the N-terminus. The spacer region between the two subunit binding sites is indicated (6). The individual repeat modules contain typically 34 conserved amino acids with hypervariable residues found at positions 12 and 13. These residues, which are referred to as repeat variable diresidues (RVDs), specify the target DNA base. The standard DNA-recognition code of TALEs is indicated (left).

Figure 3 shows mtDNA reduction in mouse zygotes 48 hours after mitoTALEN injection, (a) Schematic representation of mouse mitoTALEN architecture and mouse embryo injection. The architecture consists of a TALE domain fused to a wild-type Fokl nuclease domain at the C-terminus with dual tandem mitochondria-targeting signal (MTS) sequences from Atp5b and Cox8a at the N-terminus. The mRNA was designed to include 5’ and 3’ UTR regions from Atp5b. Mouse mitoTALEN was engineered to target the control region. TALE binding sites (1) with position 0 thymidine (2) are indicated. Position of the target site in mtDNA is indicated, (b) Quantification of mtDNA copy number by qPCR in uninjected (median: 157,608, n = 12) and mitoTALEN-injected (median: 3,516, n = 12) CD-1 zygotes. All samples were frozen for analysis 48 hours after injection with mitoTALEN mRNA (400 - 500 ng/uL). Median (horizontal line) and mean values (plus) in the box plots are indicated. Tukey whiskers extend to data points that are less than 1.5 x IQR away from 1 st/3rd quartile.

Figure 4 shows strong mitochondrial localisation of mitoTALEN-EGFP in mouse zygotes, (a) Schematic representation of mouse mitoTALEN-EGFP architecture. The architecture consists of the mitoTALEN architecture fused to an EGFP domain at the C-terminus, upstream of the C-terminus UTR (SEQ ID NOs: 389, 390, 397, 398). (b) Colocalisation of the mitoTALEN-EGFP (third column) and the fluorescent dye MitoTracker Red which stains mitochondria in live cells (second column) indicates that mitoTALEN-EGFP is localised in the mitochondria (forth column) in injected CD-1 zygotes by live cell imaging using Zeiss LSM880 AiryScan confocal microscope. Uninjected and mitoTALEN-injected CD-1 zygotes were used as controls.

Figure 5 shows mtDNA reduction in mouse zygotes 48 hours after mitoPstl injection, (a) Schematic representation of mitoPstl. The architecture consists of a Pstl restriction endonuclease (recognition sequence 5 -CTGCA/G-3') with dual tandem MTS sequences from Atp5b and Cox8a at the N-terminus. Cleavage occurs at the 7” site. The mRNA was designed to contain 5’ and 3’ UTR regions from Atp5b. MitoPstl targets mouse mtDNA at two sites (mt-Nd5 and mt-Atp6) as these sequences comprise the recognition sequence for Pstl. (b) Quantification of mtDNA copy number by qPCR in uninjected (median: 130,692, n = 12) and mitoPstl-injected (median: 1 ,547, n = 11) CD-1 zygotes. All samples were frozen for analysis 48 hours after injection with mitoPstl mRNA (400 - 500 ng/pL). Median (horizontal line) and mean values (plus) in the box plots are indicated. Tukey whiskers extend to data points that are less than 1.5 x IQR away from 1 st/3rd quartile, (c) Colocalisation of mitoPstl - EGFP (third column) with MitoTracker Red (second column) indicates that mitoPstl-EGFP is localised in the mitochondria (forth column) in injected CD-1 metaphase II oocytes by live cell imaging using Nikon A1R confocal microscope.

Figure 6 shows 95% of mtDNA is depleted 2 hours after mitoTALEN injection (construct: mitoTALEN with WT FoKI (no EGFP, no NES) and standard RVD code: Seq ID 387, 388, 395, 396). (a) Quantification of mtDNA copy number by qPCR in uninjected CD-1 zygotes and zygotes frozen for analysis at varying times after mitoTALEN mRNA injection (400 - 500 ng/pL): 1 hour (control median: 146,529, mitoTALEN median: 127,995, n = 6), 2 hours (control median: 138,326, mitoTALEN median: 6,970, n = 6), 4 hours (control median: 145,149, mitoTALEN median: 3,446, n = 6), 6 hours (control median: 121 ,784, mitoTALEN median: 2,444, n = 6), and 12 hours (control median: 127,043, mitoTALEN median: 2,965, n = 6). Median (horizontal line) and mean values (plus) in the box plots are indicated. Tukey whiskers extend to data points that are less than 1.5 x IQR away from 1 st/3rd quartile, (b) Levels of mtDNA depletion normalised to uninjected controls.

Figure 7 shows visual confirmation of mtDNA depletion in mouse zygotes. Validation of mtDNA reduction at 6 hours after mitoTALEN mRNA injection (construct: mitoTALEN with WT FoKI (no EGFP, no NES) and standard RVD code: SEQ ID NO: 387, 388, 395, 396) by co-staining of (a) uninjected and (b) mitoTALEN-injected mouse zygotes with MitoTracker Red (second column) and PicoGreen which is a fluorescent probe which binds double stranded DNA (third column). Zygotes were analysed by live cell imaging using Zeiss LSM880 AiryScan confocal microscope.

Figure 8 shows high concentrations of mitoTALEN mRNA are needed for efficient mtDNA depletion. Quantification of mtDNA copy number by qPCR in uninjected CD-1 zygotes (median: 162,575, n = 6) and zygotes injected with varying concentrations of mitoTALEN (construct: mitoTALEN with WT FoKI (no EGFP, no NES) and standard RVD code: SEQ ID NO: 387, 388, 395, 396): 50 ng/pL (median: 46,159, n = 6), 100 ng/pL (median: 24,950, n = 6), 250 ng/pL (median: 15,382, n = 6), and 500 ng/pL (median: 2,444, n = 6). All samples were frozen for analysis 6 hours after microinjection. Median (horizontal line) and mean values (plus) in the box plots are indicated. Tukey whiskers extend to data points that are less than 1.5 x IQR away from 1 st/3rd quartile.

Figure 9 shows modified mitoTALEN architecture displays reduced activity and mtDNA depletion, (a) Summary of amino acid mutations in FOKI implemented to generate obligate heterodimeric nuclease variants (ELD/KKR), hyperactive Sharkey nuclease variant, and SunnyTALEN scaffold containing P(-11)H mutation. The modified mitoTALEN architecture (mitoTALEN S-ELD and S-KKR) contained the P(-11 ) H mutation in the TALE scaffold, obligate heterodimeric nuclease mutations, and hyperactive Sharkey nuclease mutations, (b) Quantification of mtDNA copy number by qPCR in uninjected CD-1 zygotes (median: 162,631 , n = 8) and zygotes injected with mitoTALEN with wild-type nuclease domains (WT/WT; median: 3,748, n = 9) or mitoTALEN with modified nuclease domains (S-ELD/S- KKR; median: 15,062, n = 7). All samples were frozen for analysis 6 hours after injection with mitoTALEN mRNA (500 ng/pL). Median (horizontal line) and mean values (plus) in the box plots are indicated. Tukey whiskers extend to data points that are less than 1.5 x IQR away from 1 st/3rd quartile. Top right graph shows the comparison between the data for mitoTALEN with wild-type nuclease domains and mitoTALEN with modified nuclease. Figure 10 shows Cas13b knock-down of mitoTALEN mRNA. (a) Schematic representation of PspCas13b targeting of mitoTALEN mRNA. Cas13b gRNA targets mitoTALEN mRNA at 7 - 8 different sites within the TALE domain, (b) Knock-down of mitoTALEN mRNA was assessed using live cell imaging by injecting CD-1 oocytes with Cas13b mRNA and gRNA, allowing expression of Cas13b and then injecting mitoTALEN-EGFP mRNA. Oocytes were left to mature in vitro and were subsequently imaged. Control oocytes were injected with only mitoTALEN-EGFP and no Cas13b. Images were acquired using Nikon A1R confocal microscope, (c) Quantification of mtDNA copy number by qPCR in uninjected CD-1 zygotes (median: 168,917, n = 11) and zygotes injected with mitoTALEN mRNA (500 ng/pL) and varying concentrations of Cas13b mRNA and gRNA: 0/0 ng/pL (median: 3,534, n = 13), 50/25 ng/pL (median: 2,254, n = 8), 100/50 ng/pL (median: 3,785, n = 13), 250/125 ng/pL (median: 4,847, n = 13), and 500/250 ng/pL (median: 15,763, n = 5). All samples were frozen for analysis 6 hours after microinjection. Median (horizontal line) and mean values (plus) in the box plots are indicated. Tukey whiskers extend to data points that are less than l .5 x IQR away from 1 st/3rd quartile.

Figure 11 shows design of human mitoTALENs. (a) Schematic representation of human mitoTALEN architectures. The architectures consist of a TALE domain fused to a wild-type Fokl nuclease domain at the C-terminus with dual tandem MTS sequences from ATP5B and COX8A (human mitoTALEN ATP5B architecture) or COX8A and SOD2 (human mitoTALEN C0X8A architecture) at the N-terminus. The mRNA was designed to include 5’ and 3’ UTR regions from ATP5B (human mitoTALEN ATP5B architecture) or COX8A (human mitoTALEN C0X8A architecture), (b) A ‘universal’ mitoTALEN was developed by aligning 30,506 human mitochondrial genomes and identifying conserved sites. The conserved sites have a minor allele frequency of 0. (c) Several human mitoTALEN subunits were engineered to target a conserved site in the control region. Several variants (m.16,023G>T, m.16,051A>G, m.16,052C>T, m.16,054A>G, m.16,060G>A, m.16,061T>G) have been identified in the target site. To allow targeting of all genomes regardless of the variants present, promiscuous repeat variable di-residues, RVDs, (NA or NS; 3) were used at the positions showing variation. TALE recognition code is shown. TALE binding sites (1) with position 0 thymidine (2) are indicated. Position of the target site in mtDNA is indicated.

Figure 12 shows an assessment of the effect of promiscuous RVDs on mitoTALEN activity, (a) Schematic representation of mouse mitoTALEN and target site. The promiscuous RVDs NA and NS were incorporated (3 to 4 per subunit) at random positions in the mouse mitoTALEN. The RVDs used in the different mitoTALENs are shown: mitoTALEN with standard recognition code, mitoTALEN with NA RVDs, and mitoTALEN with NS RVDs. TALE binding sites (1) with position 0 thymidine (2) are indicated, (b) Quantification of mtDNA copy number by qPCR in uninjected CD-1 zygotes (median: 118,031 , n = 11), and zygotes injected with mitoTALEN with standard code (median: 1 ,350, n = 9), mitoTALEN with NA (median: 4,557, n = 9) or mitoTALEN with NS RVDs (median: 6,438, n = 9). All samples were frozen for analysis 6 hours after injection with mitoTALEN mRNA (500 ng/pL). Median (horizontal line) and mean values (plus) in the box plots are indicated. Tukey whiskers extend to data points that are less than 1.5 x IQR away from 1 st/3rd quartile. Top right graph shows the comparison of the data for mitoTALEN with standard code, mitoTALEN with NA and mitoTALEN with NS RVDs.

Figure 13 shows mtDNA reduction in human oocytes 6 hours after mitoTALEN injection. Quantification of mtDNA copy number by qPCR in uninjected human metaphase II oocytes (median: 1 ,142,772, n = 5), and oocytes microinjected with 640 ng/pL mitoTALEN(NA) (median: 172,806, n = 5), 640 ng/pL mitoTALEN(NS) (median: 162,822, n = 7), and 1500 ng/pL mitoTALEN(NS) mRNA (median: 24,030, n = 5). All samples were frozen for analysis 6 hours after microinjection. Median (horizontal line) and mean values (plus) in the box plots are indicated. Tukey whiskers extend to data points that are less than 1.5 x IQR away from 1 st/3rd quartile.

Figure 14 shows alignment of mitoTALEN-targeted mtDNA from human oocytes, (a) Sanger sequencing of mitoTALEN target site in mitoTALEN-injected metaphase II oocytes previously used for copy number analysis. MitoTALEN subunit binding sites are highlighted by rectangles, (b) Sequence logo of mitoTALEN target sites.

Figure 15 shows visual confirmation of mtDNA depletion in human metaphase II oocytes. Validation of mtDNA reduction at 6 hours by co-staining of (a) uninjected and (b) mitoTALEN-injected human metaphase II oocytes with MitoTracker Red (second column) and PicoGreen (third column). Oocytes were analysed by live cell imaging using Zeiss LSM880 AiryScan confocal microscope.

Figure 16 shows strong mitochondrial localisation of mitoTALEN-EGFP in human oocytes, (a) Schematic representation of human mitoTALEN-EGFP architecture. The architecture consists of the mitoTALEN architecture fused to an EGFP domain at the C-terminus before the C-terminus UTR. (b) 2D and (c) 3D images showing mitochondrial co-localization of mitoTALEN-EGFP (third column) with MitoTracker Red (second column) in injected human eggs acquired by live cell imaging using Zeiss LSM880 AiryScan confocal microscope. Uninjected human eggs were used as controls. Figure 17 shows that mitoTALEN with NES improves development and reduces mtDNA carryover in mouse embryos, (a) Comparison of blastocyst development of uninjected CD-1 embryos (n = 16), embryos injected with Cas13b (100 ng/pL) and gRNA (50 ng/pL) targeting mitoTALEN mRNA (n = 14), embryos injected with mitoTALEN mRNA without NES (mitoTALEN(-NES); 500 ng/pL of each subunit; n = 19), and embryos injected with mitoTALEN mRNA with NES (mitoTALEN(+NES); 500 ng/pL of each subunit; n = 26).

(b) Schematic representation of mouse mitoTALEN architecture with dual tandem nuclear export signals (NES) from the NS2 protein of minute virus of mice, (c) Schematic representation of mouse pronuclear transfer (PNT), where mitoTALEN and Cas13b-gRNA are injected into CD-1 zygotes, which serve as the karyoplast donors while uninjected C57BL/6 zygotes serve as the cytoplast donors. In control PNT experiments, CD-1 zygotes are uninjected, (d) Chromatograms from Sanger sequencing of mtDNA from CD-1 and C57BL/6 embryos showing a variant at position m.9461. (e) Heteroplasmy analysis (based on levels of m.9461C using pyrosequencing of unmanipulated CD-1 and C57BL/6 embryos, and whole control (C-PNT; n = 4) and mitoTALEN blastocysts (mT-PNT; n = 6) generated from mouse PNT. (f) Sequence pyrograms for m.9461C in unmanipulated CD-1 and C57BL/6 embryos, and control PNT (n = 3) and mitoTALEN PNT blastocysts (n = 3). The level of heteroplasmy is show above.

Figure 18 shows visual confirmation of mtDNA depletion in human metaphase II oocytes, (a) Schematic representation of primary human mitoTALEN architecture with dual tandem nuclear export signals (NES) from the NS2 protein of minute virus of mice. Validation of mtDNA reduction at 6 hours by co-staining of (b) uninjected and (c) mitoTALEN(+NES)- injected human metaphase II oocytes with MitoTracker Red (gray) and PicoGreen (orange). Oocytes were analysed by live cell imaging using Zeiss LSM880 AiryScan confocal microscope. Scale bar is 20 pm.

Detailed Description of Embodiments of the Invention and Examples

While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as "a", "an" and "the" are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.

Definitions and general points

The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge in any country as of the priority date of any of the claims.

Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. All documents cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings or sections of such documents herein specifically referred to are incorporated by reference.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Current Protocols in Molecular Biology (Ausubel, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001 , Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J. Gait ed., 1984); U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (Harries and Higgins eds. 1984);

Transcription and Translation (Hames and Higgins eds. 1984); Culture of Animal Cells (Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells and Enzymes (IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning (1984); the series, Methods in Enzymology (Abelson and Simon, eds. -in-chief, Academic Press, Inc., New York), specifically, Vols.154 and 155 (Wu et al. eds.) and Vol. 185, "Gene Expression Technology" (Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (Miller and Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook of Experimental Immunology, Vols. I-IV (Weir and Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

The terms "comprising", "comprises" and "comprised of" as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited features, elements or method steps.

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

The term "nucleic acid" as used herein typically refers to an oligomer or polymer (preferably a linear polymer) of any length composed essentially of nucleotides. A nucleotide unit commonly includes a heterocyclic base, a sugar group, and at least one, e.g. one, two, or three, phosphate groups, including modified or substituted phosphate groups. Heterocyclic bases may include inter alia purine and pyrimidine bases such as adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (II) which are widespread in naturally-occurring nucleic acids, other naturally-occurring bases (e.g., xanthine, inosine, hypoxanthine) as well as chemically or biochemically modified (e.g., methylated), non-natural or derivatised bases. Sugar groups may include inter alia pentose (pentofuranose) groups such as preferably ribose and/or 2-deoxyribose common in naturally-occurring nucleic acids, or arabinose, 2- deoxyarabinose, threose or hexose sugar groups, as well as modified or substituted sugar groups. Nucleic acids as intended herein may include naturally occurring nucleotides, modified nucleotides or mixtures thereof. A modified nucleotide may include a modified heterocyclic base, a modified sugar moiety, a modified phosphate group or a combination thereof. Modifications of phosphate groups or sugars may be introduced to improve stability, resistance to enzymatic degradation, or some other useful property. The term "nucleic acid" further preferably encompasses DNA, RNA and DNA RNA hybrid molecules, specifically including hnRNA, pre-mRNA, mRNA, cDNA, genomic DNA, amplification products, oligonucleotides, and synthetic (e.g., chemically synthesised) DNA, RNA or DNA RNA hybrids. A nucleic acid can be naturally occurring, e.g., present in or isolated from nature; or can be non-naturally occurring, e.g., recombinant, i.e. , produced by recombinant DNA technology, and/or partly or entirely, chemically or biochemically synthesised. A "nucleic acid" can be double-stranded, partly double stranded, or single-stranded. Where singlestranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear.

The term “synthetic” refers to a nucleic acid or a DNA binding and cleavage complex that does not occur in nature. The DNA binding and cleavage complex may be synthetic if it comprises two or more naturally occurring modules which are not found together in nature. The DNA binding and cleavage complex may be synthetic if an additional module is added to a DNA binding and cleavage complex present in nature. The DNA binding and cleavage complex may be synthetic if an additional sequence (such as e.g. MTS NLS) is added to a DNA binding and cleavage complex present in nature. Synthetic nucleic acid may be produced artificially, typically by recombinant technologies. Such synthetic nucleic acids may contain naturally occurring sequences (e.g. nuclease, UTR, 5’ MTS), but these are present in a non-naturally occurring context. For example, synthetic nucleic acids typically may contain one or more nucleic acid sequences that are present together in nature, and/or may encompass substitutions, insertions, and deletions and combinations thereof.

The term “vector” is well known in the art, and as used herein refers to a nucleic acid molecule, e.g. double-stranded DNA, which may have inserted into it a nucleic acid sequence according to the present invention. A vector is suitably used to transport an inserted nucleic acid molecule into a suitable host cell. A vector typically contains all of the necessary elements that permit transcribing the insert nucleic acid molecule, and, preferably, translating the transcript into a polypeptide. A vector typically contains all of the necessary elements such that, once the vector is in a host cell, the vector can replicate independently of, or coincidental with, the host chromosomal DNA; several copies of the vector and its inserted nucleic acid molecule may be generated. Vectors of the present invention can be episomal vectors (i.e. , that do not integrate into the genome of a host cell), or can be vectors that integrate into the host cell genome. This definition includes both non-viral and viral vectors. Non-viral vectors include but are not limited to plasmid vectors (e.g. pMA-RQ, plIC vectors, bluescript vectors (pBS) and pBR322 or derivatives thereof that are devoid of bacterial sequences (minicircles)) transposons-based vectors (e.g. PiggyBac (PB) vectors or Sleeping Beauty (SB) vectors), etc. Larger vectors such as artificial chromosomes (bacteria (BAG), yeast (YAC), or human (HAG)) may be used to accommodate larger inserts. Viral vectors are derived from viruses and include but are not limited to retroviral, lentiviral, adeno- associated viral, adenoviral, herpes viral, hepatitis viral vectors or the like. Typically, but not necessarily, viral vectors are replication-deficient as they have lost the ability to propagate in a given cell since viral genes essential for replication have been eliminated from the viral vector. However, some viral vectors can also be adapted to replicate specifically in a given cell, such as e.g. a cancer cell, and are typically used to trigger the (cancer) cell-specific (onco)lysis. Virosomes are a non-limiting example of a vector that comprises both viral and non-viral elements, in particular they combine liposomes with an inactivated HIV or influenza virus (Yamada et al., 2003). Another example encompasses viral vectors mixed with cationic lipids.

The term “spacer” as used herein is a nucleic acid sequence that separates two nucleic acid sequences (e.g. two target sites for the DNA binding and cleavage protein). It can have essentially any sequence, provided it does not prevent the function of the flanking nucleic acid sequences (e.g. this could happen if the spacer prevents binding of the targeted DNA binding and cleavage protein, or suchlike). Spacer may be of optimal length such as 10 to 20, suitably 12 to 18, suitably 13 to 17, suitably 16 nucleotides.

The term “synthetic targeted DNA binding and cleavage complex” refers to a combination of at least two proteins, two protein domains or a protein and a nucleic domain which are capable, in combination, to bind to a target sequence and cleave a nucleic acid comprising the target sequence. The synthetic targeted DNA binding and cleavage complex may comprise at least two independent proteins. Suitably one protein may provide the DNA binding module and one protein may provide the DNA cleavage module. The synthetic targeted DNA binding and cleavage complex may comprise at least two proteins domains (within a protein). Suitably one protein domain may provide the DNA binding module and one protein domain may provide the DNA cleavage module. The synthetic targeted DNA binding and cleavage complex may comprise at least one protein and at least one nucleic acid. Suitably the at least one nucleic acid may provide the DNA binding module and the at least one protein may provide the DNA cleavage module.

The term “DNA binding module” refers to a protein, a protein domain or a nucleic acid which recognizes and binds selectively to a particular DNA sequence, i.e. the target sequence. The DNA binding module may recognize and selectively bind to the target sequence independently of the DNA cleavage module or it may require the DNA cleavage module in order to recognize and selectively bind to the target sequence. It may be preferable for the DNA binding module to be as small as possible while still recognizing and binding to a target sequence of sufficient length to allow specificity.

The term “DNA cleavage module” refers to a protein or one or more protein domains which are capable of cleaving a nucleotide sequence. The DNA cleavage module may be an endonuclease. Cleavage may be in or close to the target sequence. The DNA cleavage module may be provided by one protein. The DNA cleavage module may be provided by a dimer of DNA cleavage module subunits such as a dimer of two protein domains.

The term “target sequence” is the nucleic acid sequence which is recognized to and bound by the synthetic targeted DNA binding and cleavage complex or by the DNA binding module. The target sequence can be of any length. Preferably, the target sequence is as short as possible but long enough to provide specificity. Preferably, the target sequence is at least 7 nucleotides in length. The target sequence is within a conserved region of the mitochondrial DNA control region. Two target sequences spaced apart by a spacer may be targeted by two DNA binding modules.

The term “wild type” refers to the typical version or allele of a gene. Having the wild type version of the gene is associated with normal function of the organism and health.

The term “detrimental”, “pathogenic” or “faulty” refers to an atypical, mutant version or allele of a gene as opposed to the wild type version of the gene. Having the pathogenic or faulty version of the gene is associated with abnormal function of the organism and disease (in this case mitochondrial disease).

The term “mitochondrial replacement therapy (MRT)” refers to the replacement of mitochondria in one or more cells to remove non-functional or otherwise defective mitochondria.

The term “mitochondrial DNA (mtDNA)” refers to the DNA naturally present in the mitochondria. The mtDNA may be faulty (e.g., pathogenic) or wild type (e.g., non- pathogenic).

The term “state of heteroplasmy” or “heteroplasmy” refers to the coexistence of two or more variants of mtDNA in the same mitochondrion, same cell, same tissue or the same organism. The two or more variants may be, for example, pathogenic mtDNA and wild type mtDNA. The frequency of each of the variants may range from 0%-100% such that the sum of all variants is equal to 100%. For example, when the two or more variants are pathogenic mtDNA and wild type mtDNA, the pathogenic mtDNA variant may be 25% and the wild type mtDNA variant may be 75%.

The term “threshold of the disease phenotype” refers to the threshold at which a pathogenic variant of mtDNA is at sufficient level to cause the disease phenotype (e.g. disease symptoms). Depending on the defective gene and the tissue, the threshold of the disease phenotype may be reached at frequency of the pathogenic variant equal to any number within the range from 0%-100%. For example, the threshold of the disease phenotype may be reached at frequency of the pathogenic variant equal to 25%.

The term “heteroplasmy shift” refers to changing the proportion of the coexisting two or more variants of mtDNA. When the two or more variants are pathogenic mtDNA and wild type mtDNA, changing the proportion can be done by selecting for the wild type mtDNA and/or by selectively destroying the pathogenic mtDNA.

The term “pronuclear transfer” or “PNT” refers to transfer of the pronuclei from the patient zygote (nuclear donor) to the donor zygote (mitochondrial donor). PNT is used in order to reduce the risk of a mother carrying a pathogenic mtDNA mutation transmitting it to their offspring during in vitro fertilization. Historically, PNT has been carried out around 16-20 h after sperm injection. However, it was discovered that carrying out PNT around 8 h after sperm injection was beneficial for development (often called early PNT, ePNT). Therefore, currently PNT is commonly carried out around around 8 h after sperm injection and “PNT” and “ePNT” are used synonymously.

The term “in vitro fertilization” refers to the process of fertilization where an egg is combined with sperm in vitro.

The terms “mtDNA control region” or “D-loop region” refer to the non-coding area of mtDNA encompassing the D-loop and associated promoters. The displacement loop (D-loop) is a short segment of mtDNA that contains three strands, where the third strand is generated by replication of the heavy strand. According to the reference sequence of the house mouse mitochondrial genome (C57BL/6J: NC_005089.1), the control region (or the D-loop region) is 877 bp in size; positions m. 15,423 to m.16, 299 (15,424 to 16,300 for Crl:CD1(ICR): KC663622.1). This sequence is shown in SEQ ID NO: 403. According to the Revised Cambridge Reference Sequence (rCRS) of the human mitochondrial genome (NC_012920.1), the control region (or the D-loop region) is 1,122 bp in size; positions m.16,024 to m.576. This sequence is shown in SEQ ID NO: 404. The human conserved control region is shown in SEQ ID NO: 1813.

The term “homolog” refers to a gene, a portion of a gene, a nucleic acid or a portion of a nucleic acid which is inherited by a common ancestor in two species. Homologous genes, portion of genes, nucleic acids or portions of nucleic acids are often similar in sequence. The “homolog of the conserved region of the mitochondrial DNA” refers to the conserved region of the mitochondrial DNA in an organism. SEQ ID NO: 403 , SEQ ID NO: 1813 and SEQ ID NO: 404 provide the conserved region of mouse and human so a homolog of the conserved region of the mitochondrial DNA in another organism may be the conserved region of the mitochondrial DNA in e.g. horse, pig, sheep etc.

The term “linked” refers to the connection of the DNA binding module and the DNA cleavage module. The DNA binding module and the DNA cleavage module may be connected by a linker (an amino acid sequence which does not perform function other than to connect the DNA binding module and the DNA cleavage module). The DNA binding module and the DNA cleavage module may be conjugated without a linker, i.e. the DNA binding module starts where the DNA cleavage module finishes or vice versa. The DNA binding module and the DNA cleavage module may be encoded by the same nucleic acid molecule or they may be connected following translation. The DNA binding module and the DNA cleavage module may be separated by another module.

The term “TALE domain” and “TALEN” are well known to the person skilled in the art. TALE domains are proteins which recognize DNA sequences through a central repeat domain consisting of a variable number of ~34 amino acid repeats. Two critical amino acids (called RVDs) in each repeat bind specific DNA bases. The TALE-DNA code is well known to the one skilled in the art. One or more TALE domains may be fused to a DNA cleavage module such as a nuclease to make a TALEN. TALEN technology is described extensively in the literature and, inter alia, in the following patent documents: US8420782, US8470973, US8440431, US8440432, US8450471, US8586363, US8697853, EP2510096, US8586526, US8623618, EP2464750, US2011041195, US2011247089, US2013198878, WO2012/116274, WO2014110552, W02014070887, W02014022120, WO2013192316, and WO2010008562, all of which are incorporated by reference. TALENs can be obtained commercially from Thermo Fisher Scientific, Inc. (Waltham, MA, US) under the GeneArt® TALs branded products and services (formerly marketed under the Life Technologies brand).

The term “Zinc fingers” or “zinc finger domains” and “Zinc finger nuclease” are well known o the person skilled in the art. Zinc fingers are small protein structural motifs which make finger-like protrusions that make tandem contacts with their target molecule. Zinc finger- \dna code is well known to the person skilled in the art. Zinc fingers may be fused to DNA cleavage module such as a nuclease to make a zinc finger nuclease. ZFNs can be engineered to target and induce a DSB at any sequence. ZFN technology is described extensively in the literature and, inter alia, in the following patent documents: US 6,479,626, 6,534,261 , 6,607,882, 6,746,838, 6,794,136, 6,824,978, 6,866,997, 6,933,113, 6,979,539, 7,013,219, 7,030,215, 7,220,719, 7,241,573, 7,241 ,574, 7,585,849, 7,595,376, 6,903,185, 6,479,626, 8,106,255, 20030232410, and 20090203140, all of which are incorporated by reference. ZFNs can be obtained commercially from Sigma-Aldrich (St. Louis, MO, US) under the CompoZr® Zinc Finger Nuclease Technology branded products and services.

The terms “CRISPR -Cas” AND “gRNA” are well known to the person skilled in the art. CRISPR/Cas technology is described extensively in the literature (e.g. Cong et al. ‘Multiplex Genome Engineering Using CRISPR/Cas Systems’, Science, 15 February 2013: Vol. 339 no. 6121 pp. 819-823) and, inter alia, in the following patent documents: US 8,697,359, US2010076057, WO2013/176772, US8,771 ,945, US2010076057, US2014186843, US2014179770, US2014179006, WO2014093712, W02014093701 , WO2014093635, WO2014093694, WO2014093655, W02014093709, WO2013/188638,

WO2013/142578, W02013/141680, WO2013/188522, US8546553, WO2014/089290, and WO2014/093479, all of which are incorporated by reference. CRISPR/Cas systems can be obtained commercially from Sigma-Aldrich (St. Louis, MO, US) under the CRISPR/Cas Nuclease RNA-guided Genome Editing suite of products and services, or from Thermo Fisher Scientific, Inc. (Waltham, MA, US) under the GeneArt® CRISPR branded products and services.

The term “assisted reproductive technologies” or “ART” refers to all fertility treatments in which either eggs or embryos are handled. ART procedures include but are not limited to surgically removing eggs from a woman’s ovaries, combining them with sperm in the laboratory, and returning them to the woman’s body or donating them to another woman.

Examples

Example 1 - mouse mitoTALEN design and experimental validation

Introduction

In some embodiments, the synthetic targeted DNA binding and cleavage complex which is capable of specifically binding to a target sequence within the D-loop of the mitochondrial DNA control region and cleaving a nucleic acid comprising the said target sequence is a transcription activator-like effector nuclease (TALEN). TALENs are artificially engineered dimeric endonucleases that can be programmed to recognize and cleave specific DNA sequences (Miller et al., 2011 ; Mussolino et al., 2011 ; Cermak et al., 2011). A single TALEN subunit includes a TAL effector DNA-binding domain fused (4) to a nuclear localization signal (NLS) or mitochondria-targeting signal (MTS) and a non-specific nuclease domain of the dimeric-type IIS restriction enzyme Fokl (5) (Fig. 2). A mitochondria-targeted TALEN (mitoTALEN) comprises a mitochondrial-targeting signal (MTS) (Bacman et al., 2013) which gets cleaved off after import into the mitochondrial matrix. After dimerization of two TALEN subunits in correct spacing and orientation, the nuclease domain cleaves the target DNA within the spacer region between the subunit binding sites (6). The induction of DNA doublestrand breaks (DSBs) in the nuclear genome activates endogenous DNA repair pathways, resulting in gene disruption via non-homologous end joining (NHEJ) or gene correction via homology-directed repair (HDR) in the presence of a donor template. However, induction of DSBs in the mitochondrial genome (mtDNA) results in the rapid degradation of mtDNA because mammalian mitochondria lack efficient mechanisms for repairing DSBs (Srivastava and Moraes, 2001 ; Bayona-Bafaluy et al., 2005).

TAL effectors were originally identified in plant pathogenic bacteria of the genus Xanthomonas and function as eukaryotic transcription factors modulating host cell gene expression (Boch and Bonas, 2010). The central DNA-binding domain contains a tandem array of repeat modules, each of which recognizes a specific DNA base. Each repeat module consists of typically 34 conserved amino acids (33 to 35) with hypervariable residues found at positions 12 and 13. It is these two residues, termed repeat variable di-residues (RVDs), which specify the target DNA base. Previous studies have deciphered the DNA- binding code (Boch et al., 2009; Moscou and Bogdanove, 2009) allowing the generation of custom TALE proteins for genome engineering applications.

Previous mitoTALEN and mitochondria-targeted zinc finger nuclease architectures have sometimes had the drawback of exhibiting sub-optimal mitochondrial import (Bacman et al., 2013; Reddy et al., 2015; Gammage et al., 2014; Gammage et al., 2016). The mito-nuclease architectures disclosed herein have dual tandem MTS sequences from Atp5b and Cox8a with Atp5b 5’ and 3’ UTR regions in order to improve mitochondrial localization and reduce accumulation of mitochondria-targeted nucleases in the cytoplasm (Fig. 3A). The mitoTALEN architectures disclosed herein have been developed based on the A152/+63 TALE scaffold (deletion of 152 residues at the N-terminus and retention of 63 at the C- terminus) of Miller et al. (2011). Fig 1a of Miller et al. (2011) demonstrates N-terminal truncation resulting in delta152, which means deletion of 152 residues at the N-terminus of the -288 residue TALE domain. Supplementary Figure 1 B and 4 of Miller et al. (2011) shows C-terminal truncations (+28 to +95, which means leaving 28 to 95 residues at the C- terminus). Additionally Fig 3 and 4, Supplementary Figure 8 and 9 of Miller et al. (2011) demonstrate the testing of the two main scaffolds: d152/+28 and d152/+63 (+28/+28 or +63/+63 which refers to left and right subunits with the same C-terminal truncation).

Previous strategies for mtDNA manipulation have largely focused on targeting specific point mutations (Gammage et al., 2014; Reddy et al., 2015; Hashimoto et al., 2015; Bacman et al., 2018; Gammage et al., 2018; Yang et al., 2018) or deletion mutations (Bacman et al., 2013; Gammage et al., 2014) to shift heteroplasmy. However, these approaches have the drawback of having to design, synthesise, pre-screen and test a construct to target each specific mutant mtDNA. Since there are 94 confirmed pathogenic mutations in human mtDNA alone (MITOMAP database) and taking account of all the different combinations (mutation type, position, and neighboring patient-specific variants), it would be very cumbersome and expensive to generate a mitoTALEN for each mutation, as well as the safety data for each mitoTALEN.

To address this problem, our approach aims to eliminate all mtDNA molecules regardless of the mitochondrial genotype using a single universal mitoTALEN or a set of mitoTALENs. For the successful application of mitoTALENs with PNT, in terms of efficacy and cost, it would be more beneficial to develop a single mitoTALEN or a set of mitoTALENs that could be used to target all mitochondrial genomes regardless of the mutation type, position of the mutation within the genome, haplogroup, and non-pathogenic patient-specific variants.

To develop such a universal strategy, a highly conserved region of the mtDNA is required. However, mtDNA is highly polymorphic. The inventors have surprisingly identified highly conserved regions, with fewer positions with variability, within the control region (D-loop and associated promoters) in the mitochondrial genome, which contains the heavy strand origin (OH) of replication from which mtDNA replication is first initiated. The control region is largely unexplored therapeutically as all of the confirmed pathogenic mutations lie outside the control region, either in coding or RNA regions (MITOMAP database). Targeting the control region has primarily been used to investigate mtDNA recombination and the formation of deletion mutations such as the common deletion of 4,977 bp (Phillips et al., 2017; Bacman et al., 2009). When two mitochondrial regions outside the control region are cut at the same time, the two regions often hybridise with each other if there is sequence similarity. On the other hand, when the control region is cut, no replication is possible which leads to fast degradation of the mitochondrial DNA. Therefore, introducing a double-strand break in the control region is expected to inhibit mtDNA replication. Furthermore, introducing a single double-strand break in the control region would decrease the likelihood of deletion formation, as induction of multiple double-strand breaks at different sites in mtDNA (typically outside the control region, in major or minor arcs) has been reported to promote intra- and inter- molecular mtDNA recombination and formation of large deletions (Srivastava and Moraes, 2005; Fukui and Moraes, 2008; Bacman et al., 2009). Finally, in rare cases where a single deletion mutation can be transmitted through the germline (Shanske et al., 2002; Chinnery et al., 2004) targeting the control region would allow elimination of mtDNA with large deletions that can span a large part of the mitochondrial genome (deletions often involve the major arc between the major origins of replication and are outside the control region).

Design of mitoTALEN targeting mouse mtDNA To engineer an efficient and specific mitoTALEN, the following criteria were set. Firstly, each TALE binding site was chosen so that it preceded by a 5' thymine (position 0). Secondly, as many strong repeat-variable diresidues (RVDs) as possible (such as HD and NN) were incorporated with at least 3 to 4 per TALE protein and stretches of weak RVDs (>6) were avoided (Streubel et al., 2012). Strong RVDs have been shown to be key for the generation efficient TALEs as they form hydrogen bonds with the target DNA base, while weak RVDs (such as Nl and NG) that compromise TALE activity make van der Waals contacts (Deng et al., 2012; Mak et al., 2012). Thirdly, low numbers of repeat modules in each TALE protein were avoided; ideally 12.5 or more (13 RVDs) were allowed (Boch et al., 2009; Boch and Bonas, 2010; Reyon et al., 2012; Rinaldi et al., 2017). Finally, the length of the spacer region between the two target half-sites (binding sites of each subunit) was allowed to be within the optimal spacer range (Miller et al., 2011) as TALENs with optimal spacers are more likely to display optimal cleavage efficiency (Mussolino et al., 2014). The optimal spacer range is demonstrated in Miller et., al 2011 , for example Figure 4 14-16bp showing the highest for +63 in cells. The results also reveal clear patterns of activity versus target spacing. Within the +63/+63 TALEN panel, all nuclease pairs separated by 12-21 bp yielded at least 5% modification, whereas no activity (<1 % modification) was detected for any pair of targets with a separation distance lying outside this range (Fig. 4b). Based on these principles, a target site was identified and a mitoTALEN was engineered targeting the control region (Fig. 3A). The left mitoTALEN subunit contained 12.5 repeat modules (13 RVDs in total) with 7xHD and 2xNN RVDs (9 strong RVDs in total) (SEQ ID NO: 387; SEQ ID NO: 395), while the right subunit also contained 12.5 repeat modules with 6xHD and 2xNN RVDs (8 strong RVDs in total) (SEQ ID NO: 388; SEQ ID NO: 396). The two target half-sites were separated by a 16 bp spacer, which is within the optimal spacer range (Miller et al., 2011).

Specific details about the constructs which were used are provided below:

- The mouse mitoTALEN architecture contain (in order) a 5’ UTR region from Atp5b, two tandem MTS sequences from Atp5b and Cox8a, the N-terminal TALE scaffold (A152), the central domain with a tandem array of repeat modules (34 residues per repeat), a half repeat (20 residues), the C-terminal TALE scaffold (+63), a Fokl nuclease domain, and 3’ UTR region from Atp5b.

- The mitoTALEN-EGFP architecture contains a flexible linker (GGSGGS) and an EGFP fused to the C-terminus of the nuclease domain; the N-terminal domain and central domain are unchanged. The modified mitoTALEN architecture comprises two different subunits (S-ELD and S-KKR), which contain mutations in the C-terminal domain and nuclease domain; the N-terminal domain and central domain are unchanged. For S-ELD: P(-11)H in the C- terminal TALE scaffold (SunnyTALEN); S418P and K441E (Sharkey hyperactive nuclease domain); and Q486E, N496D, and I499L (obligate heterodimeric nuclease domain ELD). For S-KKR: P(-11)H in the C-terminal TALE scaffold (SunnyTALEN); S418P and K441 E (Sharkey hyperactive nuclease domain); and E490K, H537R, and l538K(obligate heterodimeric nuclease domain KKR).

- The mitoTALEN with NES architecture contains two tandem nuclear export signals after MTS sequences, the C-terminal domain and central domain are unchanged.

Methods

Methods for all examples are detailed in example 6.

Results

Highly efficient mtDNA depletion in mouse embryos

Imaging of CD-1 zygotes 6 to 12 hours after microinjection with mitoTALEN-EGFP mRNA (SEQ ID NO: 389, 390, 397, 398) showed strong mitochondrial localization as suggested by MitoTracker Red staining (Fig. 4B). No obvious nuclear localization of mitoTALEN-EGFP was observed. Analysis of mtDNA content in CD-1 zygotes 48 hours after microinjection with mitoTALEN mRNA showed a substantial reduction with 98% of mtDNA depleted (Fig. 3B). It has previously been suggested that mitoTALENs are not as robust as mitochondria-targeted restriction endonucleases (Reddy et al., 2015). Therefore, for comparison purposes, a previously reported mito-restriction endonuclease mitoPstl (Srivastava and Moraes, 2001; Srivastava and Moraes, 2005) based on our mito-nuclease architecture was generated and mtDNA depletion efficiency was compared with that of our mitoTALEN (Fig. 5A) SEQ ID NO: 406, 407, 408, 409). Analysis of mtDNA copy number in mitoPstl-injected (SEQ ID NO: 406, 408) CD-1 embryos showed profound reduction suggesting that mitoTALENs can be equally as effective at reducing mtDNA content as mitoPstl (Fig. 5B). The mitoPstl recognition sequence is CTGCAG (SEQ ID NO: 405).

Timing of mtDNA elimination

Early pronuclear transfer (ePNT) is carried out as soon as pronuclei appear at ~8 to 10 hours after intracytoplasmic sperm injection (ICSI) in human eggs (Hyslop et al., 2016). For mitoTALENs to be compatible with ePNT, mtDNA reduction would need to be achieved within the first 8 hours after microinjection. We therefore assessed mtDNA depletion in mouse zygotes at different time points after mitoTALEN mRNA injection. CD-1 zygotes were microinjected with mitoTALEN mRNA (SEQ ID NO: 387, 388, 395, 396) and frozen for analysis 1 , 2, 4, 6, and 12 hours after. Analysis of mtDNA copy number showed that mitoTALENs efficiently depleted mtDNA (~97 to 98%) at 4- to 12-hour time points (Fig. 6A and Fig.6B). Reduced mtDNA depletion was observed at the 2-hour (95%) and 1-hour (-20%) time points.

Visual confirmation of mtDNA depletion in mouse embryos

To visually confirm the analysis of mtDNA copy number, CD-1 zygotes were co-stained with PicoGreen and MitoTracker Red 4 to 6 hours after microinjection with mitoTALEN mRNA (SEQ ID NO: 387, 388, 395, 396). PicoGreen is a DNA dye that can be used to label both nuclear as well as mitochondrial DNA in living cells (Ashley et al., 2005). Imaging of live zygotes showed that the foci corresponding to mtDNA were virtually absent compared to uninjected zygotes, confirming mtDNA elimination (Fig. 7). These findings confirm that mtDNA is rapidly depleted by our mitoTALEN in mouse embryos.

Tuning mitoTALEN concentrations

Initial experiments were carried out using 400 to 500 ng/pL of mRNA of each mitoTALEN subunit (SEQ ID NO: 387, 388, 395, 396). We therefore attempted to identify concentrations of mitoTALEN mRNA needed for optimal efficiency. We tested different concentrations of mitoTALEN subunit mRNA: 50 ng/pL, 100 ng/pL, 250 ng/pL, and 500 ng/pL by injecting CD- 1 zygotes and freezing them 6 hours after. Analysis of mtDNA copy number indicated that 500 ng/pL resulted in highest mtDNA depletion (98%) and lower concentrations compromised mtDNA elimination efficiency (72%, 85% and 90% mtDNA depletion, respectively) (Fig. 8).

Example 2 - mitoTALEN with Heterodimeric Fokl

Methods

Methods for all examples are detailed in example 6.

Results

High concentrations of nucleases are generally associated with genotoxicity (Bohne and Cathomen, 2008). Previous studies have used lower concentrations of nucleases (Reddy et al., 2015 Cell) or inject Cas9 mRNA at 50-75 ng/ul, which normally targets 2 copies (2 alleles) in the nuclear genome to attempt to reduce toxicity. The present study used higher concentrations of nucleases, at 10x the concentration to target >100,000 copies of the mitochondrial genome. We therefore attempted to change the mitoTALEN architecture in order to reduce the risk of off-target activity in the nuclear genome by introducing obligate heterodimeric Fokl nuclease domains (ELD and KKR), which would significantly reduce homodimerization and potential off-target activity in the nuclear genome (Fig. 9a) (Doyon et al., 2011), i.e. reduce the ability of a subunit to dimerize with itself. The trade-off for such modifications to the nuclease domain is that they may have a negative impact on cleavage efficiency (Doyon et al., 2011 ; Sun et al., 2013). To counteract that, we introduced additional variants reported to enhance TALEN cleavage efficiency such as the P(-11)H mutation in the C-terminus of the TALE scaffold (Sun et al., 2013) and hyperactive Sharkey nuclease mutations (Guo et al., 2010). Analysis of mtDNA copy number in injected CD-1 zygotes showed that the new mitoTALEN architecture displayed slightly reduced mtDNA depletion efficiency (91%) compared to the original architecture (98%) containing wild-type Fokl nuclease domains (SEQ ID NO: WT (387, 388, 395, 396), S-ELD (391, 399), S-KKR (392, 400)) (Fig. 9b), suggesting that obligate heterodimeric nuclease variants may have slightly compromised cleavage efficiency. In some circumstances it may be desirable to use mitoTALENs with reduced risk of off-target activity in the nuclear genome even if this is associated with slightly compromised cleavage efficiency. For ease, the original architecture was used for subsequent experiments.

Example 3 - Reducing mitoTALEN activity window using Cas13b

Methods

Methods for all examples are detailed in example 6.

Results

It may be desirable to limit the presence of mitoTALEN mRNA in the ‘patient’ egg during PNT and subsequently in the karyoplast as presence of mitoTALEN mRNA may result in adverse expression of mitoTALEN in the donor egg after PNT. For proof-of-concept, to control the amount of mitoTALEN mRNA present and protein produced in the ‘patient’ egg and reduce transfer of mitoTALEN mRNA to the donor egg we focused on eliminating mitoTALEN mRNA. To this end, we used Cas13 (PspCas13b), an RNA-targeting CRISPR system, for elimination of mitoTALEN mRNA (Cox et al., 2017). To achieve similar rate of translation of Cas13b, we designed Cas13b to include the same 5’ and 3’ UTR regions as the mitoTALEN. We designed a gRNA targeting the TALE domain, so that this gRNA would be specific for the mitoTALEN mRNA and could be used to control the expression of the mouse mitoTALENS and the human mitoTALENs used in later examples. We took advantage of the repetitive nature of the TALE domain and designed a gRNA that targets the domain at 7-8 different sites potentially increasing knock-down efficiency (SEQ ID NO: 389, 390, 397, 398) (Fig. 10a). The gRNA was designed to recognise the repeat modules. The amino acid sequences of the repeat modules are identical (except at 12-13), but the DNA sequences differ at the start and end of the modules (utilise different codons). This is done to improve efficiency of TALE engineering; repetitive DNA sequences are difficult to work with and therefore small changes in the DNA are introduced to improve this. To assess the efficacy of this gRNA, we injected Cas13b mRNA and gRNA into mouse oocytes, allowed expression of Cas13b, and then injected mitoTALEN-EGFP mRNA. In oocytes not injected with Cas13b, expression of mitoTALEN-EGFP was observed (Fig. 10b). However, in oocytes injected with Cas13b prior to mitoTALEN-EGFP, no expression of EGFP was observed suggesting that the gRNA is able guide Cas13b to effectively cleave the mitoTALEN mRNA.

Next, we assessed how much Cas13b mRNA and gRNA are needed to knock-down mitoTALEN mRNA without compromising mitoTALEN activity and mtDNA depletion, i.e. what is the highest concentration of Cas13b mRNA that can be injected without affecting mtDNA depletion. We co-injected CD-1 zygotes with mitoTALEN mRNA, Cas13b mRNA, and gRNA with varying concentrations of Cas13b and gRNA (SEQ ID NO: 387, 388, 395, 396). These experiments indicated that mitoTALEN activity and mtDNA depletion at the 6- hour time point are not compromised by concentrations of approximately 50 or 100 ng/uL Cas13b and 25 or 50 ng/uL gRNA (Fig. 10c). It is believed that mtDNA is reduced even in the highest concentration of cas13 + gRNA as there is still enough mitoTALEN protein being produced to cause the depletion. If more Cas13 + gRNA (e.g. 2-3x the concentration) is injected, or reduced the concentration of mitoTALEN mRNA, or injected Cas13 + gRNA 1-2 hours before injecting mitoTALEN mRNA, then mitoTALEN activity would be greatly reduced.

Example 4 - mouse mitoTALEN with NES

A complimentary strategy to limit the presence of mitoTALEN mRNA in the ‘patient’ egg during PNT and subsequently in the karyoplast is to incorporate dual tandem nuclear export signals (NES). Addition of NES signal would limit adverse expression of mitoTALEN in the donor egg nucleus as the mitoTALEN and/or the mitoTALEN mRNA would be exported out of the nucleus. To compare embryo development, CD-1 zygotes were injected with Cas13b mRNA and gRNA, mitoTALEN mRNA (as described above) or mitoTALEN mRNA with dual tandem NES from the nonstructural protein 2 (NS2) of minute virus of mice (Eichwald et al., 2002; Minczuk et al., 2006) and development was assessed at blastocyst stage. Uninjected zygotes were used as control. Zygotes injected with mitoTALEN (as described above, SEQ ID NO: 387, 388, 395, 396) showed slightly reduced blastocyst development (47%). On the other hand, zygotes injected with Cas13b-gRNA (-93%) and zygotes injected with mRNA encoding mitoTALEN with NES (-92%) (with +NES, SEQ ID NO: (393, 394, 401 , 402)) showed comparable blastocyst development to uninjected controls (-94%) (Fig. 17A).

Pronuclear transfer (PNT) was carried out in mouse embryos (Fig. 17 C) in which mitoTALEN-injected CD-1 zygotes were used as karyoplast donors (nuclear donors) and uninjected C57BL/6 zygotes were used as cytoplast donors (mitochondrial donors), due to the presence of a variant in mtDNA at position m.9461 (Fig. 17 D). CD-1 zygotes were microinjected with mitoTALEN with NES mRNA (500 ng/pL of each subunit) (SEQ ID NO: 393, 394, 401 , 402), Cas13b mRNA (100 ng/pL), and gRNA (50 ng/pL). Pronuclei of injected embryos were transferred into enucleated C57BL/6 zygotes 3 - 4 hours after microinjection and the resulting PNT embryos were left to develop to blastocyst stage at which point they were frozen for mtDNA carryover analysis.

Heteroplasmy analysis using pyrosequencing showed that unmanipulated CD-1 embryos had 100% of C at m.9461 while C57BL/6 embryos had 1% C and 99% T (Fig. 17 E and F). Analysis of control PNT blastocysts from uninjected CD-1 zygotes, showed mtDNA carryover levels ranging from 11 - 27%, while mitoTALEN PNT blastocysts showed low levels of 0 - 2% as expected with a C57BL/6 cytoplast, indicating low or undetectable levels of carryover.

Example 5 - human mitoTALEN design and experimental validation

Methods

Methods for all examples are detailed in example 6.

Results

Development of the human mitoTALEN architecture

Building on our findings in mouse embryos, we developed a human architecture based on the sequences used in the mouse mitoTALEN architecture. Our primary architecture contained ATP5B 5’ UTR, tandem ATP5B and COX8A MTS sequences, and ATP5B 3’ UTR (Fig. 11a). Additionally, we developed a secondary architecture that contained COX8A 5’ UTR, tandem COX8A and SOD2 MTS sequences, and COX8A 3’ UTR, which could be used with the primary architecture if mitochondrial import is sub-optimal, i.e. use different architectures for different subunits of the mitoTALEN.

Comparison of promiscuous RVDs

Since the control region is the most polymorphic region in human mtDNA, the key to targeting the control region would involve targeting conserved sites. We aligned the mtDNA from 30,506 individuals of African, Asian, and European background identifying 9,921 variants (1498 in the control region) and the most conserved sites in the control region (Fig. 11b). However, these sites still contained positions with variants albeit most were infrequent. Therefore, the recognition of all variants and targeting of the conserved sites required the incorporation of promiscuous RVDs (e.g. NS or NA), which recognize all four bases. This would result in a ‘universal’ mitoTALEN or set of mitoTALENs that could be used in theory to target all mitochondrial genomes.

Since there are several promiscuous RVDs, we attempted to determine which RVD(s) would be best to incorporate in mitoTALENs targeting human mtDNA. The RVD NS is found at high frequency in nature but displays low binding affinity, whereas the RVD NA is found at low frequency in nature but displays high binding affinity (Cong et al., 2012). We therefore incorporated 3 to 4 NA or NS RVDs at random positions in each subunit of our benchmark mouse mitoTALEN with standard recognition code (standard RVD: SEQ ID NO: 387, 388, 395, 396; NA RVD: SEQ ID NO: 1822, 1824, 1826, 1828; NS RVD: SEQ ID NO: 1823, 1825, 1827, 1829) (Fig. 12a), mostly substituting strong RVDs to also determine the consequence of strong RVD loss and NS or NA incorporation. Analysis of mtDNA content in microinjected mouse zygotes showed that both mitoTALENs were efficient with mitoTALEN(NS) (-94.5%) being slightly less compared to mitoTALEN(NA) (-96.1%) and our benchmark mitoTALEN (-98.9%) (Fig. 12b). This suggests that both NS and NA could be used in our human universal mitoTALEN, with NA possibly yielding slightly higher efficiencies.

Design of mitoTALEN targeting human mtDNA

Similar to the design of our mouse mitoTALEN, we aimed to maximize the number of strong RVDs, avoid stretches of weak RVDs, and maintain 12.5 repeat modules or more in each TALE protein with an optimal spacer length between the target half-sites. The optimal spacer length is a range or a single length that gives the highest TALEN activity. In some embodiments, the optimal spacer length is between 1-100bp. In some embodiments the optimal space length is 14-16bp. In another embodiment, the optimal spacer length is at least 12-21 bp. In another embodiment the optimal spacer length is at least 14-20bp. We designed and generated several mitoTALEN subunits per site with varying spacer lengths and numbers of repeat modules that followed the standard recognition code, or that incorporated NA or NS RVDs. For the 1st target site, we generated three left subunits (L2, L3, and L4) and one right subunit (R2) that followed the standard recognition code, resulting in three mitoTALENs with 14 bp (L2+R2), 15bp (L3+R2), and 16 bp spacers (L4+R2). L2 had

13.5 repeat modules (14 RVDs) with 3*HD and 3*NN RVDs (6 strong RVDs in total). L3 had

17.5 repeat modules (18 RVDs) with 4*HD and 2*NN RVDs (6 strong RVDs in total). L4 had

16.5 repeat modules (17 RVDs) with 4*HD and 1 *NN RVDs (5 strong RVDs in total). R2 had 12.5 repeat modules (13 RVDs) with 2*HD and 5*NN RVDs (7 strong RVDs in total).

Furthermore, we designed and generated additional left and right subunits that incorporated NH or HN RVDs (alternative to NN) and NA or NS RVDs for universal targeting. For subunit L2, RVD at position 3 can be NA or NS, and RVDs at positions 13 and 14 can be NN, NH, or HN. For subunit L3, RVDs at positions 2, 3, and 8 can be NA or NS, and RVD at position 18 can be NN, NH, or HN. For subunit L4, RVDs at positions 2, 3, and 8 can be NA or NS. For subunit R2, RVDs at position 1, 2, 8, and 11 can be NA or NS, and RVD at position 10 can be NA, NS, NN, or HN.

Target site 1 (m.16,015 to m.16, 062) contained several variants at different positions. Subunits L3 and L4 contained variants at 3 positions: m.16,017T>G (3.28E-05) or T>C (2.89E-03), m.16,018T>C (3.28E-05), and m.16,023G>T (1.64E-04). Subunit L2 contained a variant at a single position: m.16,023G>T (1.64E-04). Subunit R2 contained variants at 5 positions: m.16,051A>G (2.63E-02), m.16,052C>T (1.31E-04), m.16,054A>G (3.28E-05), m.16,060G>A (3.28E-05), and m.16,061T>G (3.28E-05).

Highly efficient mtDNA depletion in human oocytes

We selected four subunits (L2(NA) and R2(NA), L2(NS) and R2(NS)) for the first target sites, which would generate a mitoTALEN with a 14 bp spacer (Fig. 11c) and validated this ‘universal’ mitoTALEN in human metaphase II oocytes that failed to fertilize and were donated for research by patients undergoing infertility treatment. Imaging of human oocytes 6 to 12 hours after microinjection with mitoTALEN-EGFP (NS EGFP (SEQ ID NO: 483, 498, 1856, 623)) mRNA showed strong mitochondrial localisation as suggested by MitoTracker Red staining (Fig. 16). Analysis of mtDNA content in human oocytes injected with 640 ng/pL of each mitoTALEN subunit mRNA (NA or NS; NA (SEQ ID NO: 477, 596, 492, 609), NS (SEQ ID NO: 478, 597, 493, 610)) indicated a substantial reduction of mtDNA copy number (85% or 86%) (Fig. 13). Higher concentrations of each mitoTALEN subunit mRNA (1500 ng/pL) resulted in a more profound reduction in mtDNA (98%). We used the remaining oocyte samples for sequencing of the control region to look for the presence of variants at the mitoTALEN target site. The majority of the oocytes had the reference sequence at the target site with flanking variants (Fig. 14). However, an egg from one donor showed the presence of the variant m.16,051A>G in the target site with minor allele frequency of 2.63E- 02, which was targeted efficiently.

Visual confirmation of mtDNA depletion in human oocytes

To visually confirm the rapid mitoTALEN-induced depletion of mtDNA, human metaphase II oocytes were co-stained with PicoGreen and MitoTracker Red 4 to 6 hours after microinjection with each mitoTALEN subunit mRNA (NA (477, 596, 492, 609)) (1500 ng/pL). Imaging of live oocytes showed that the foci corresponding to mtDNA were virtually absent compared to uninjected oocytes, confirming mtDNA elimination (Fig. 15). Furthermore, building on our findings in mouse embryos (Fig. 17), a variant of the primary architecture (ATP5B) (Fig. 11a) with dual tandem NES sequences (NA NES (SEQ ID NO: 487, 502, 1868 635,)) was developed (Fig. 18a). Imaging of live oocytes confirmed mtDNA elimination (Fig. 18 B and C).

Mitochondrial localization of human mitoTALEN-EGFP

Human metaphase II oocytes that failed to fertilize and were donated for research by patients undergoing infertility treatment were imaged 6 to 12 hours after microinjection with mitoTALEN-EGFP mRNA. mitoTALEN-EGFP showed strong mitochondrial localization as previously suggested by MitoTracker Red staining (Fig. 16).

Example 6 - Materials and methods

Mouse strains and embryos

Oocytes and embryos from the mouse strain CD-1 (Crl:CD1(ICR); Charles River) were used for all mitoTALEN optimisation experiments and as karyoplast donors for mouse PNT experiments. Embryos from the mouse strain C57BL/6 (C57BL/6NCrl; Charles River) were used as cytoplast donors for mouse PNT experiments. GV-stage mouse oocytes were harvested from ovaries of CD-1 mice in M2 medium (Sigma) and subsequently cultured in G- IVF PLUS medium (Vitrolife) under mineral oil (FUJIFILM Irvine Scientific) at 36.6°C, with 6% CO2 and 5% O2. CD-1 and C57BL/6 mice were used as these mice are outbred and display good reproductive performance CD-1 female mice were superovulated with 5 IU PMSG and 5 IU hCG and mated with CD-1 and C57BL/6 males, respectively. Zygotes were harvested from oviducts approximately 0.5 days post coitum (dpc) in M2 medium overlaid with mineral oil. If cumulus cells were intact, zygotes were treated with 80 ILI/mL hyaluronidase (HYASE-10X; Vitrolife) for 3 to 5 minutes on a 36.6°C-heated stage to remove cumulus cells. Embryos were subsequently washed through and cultured in drops of KSOM supplemented with amino acids (Merck Millipore) under mineral oil at 36.6°C, with 6% CO2 and 5% 02. All animal research was performed in compliance with the UK Home Office; license number PDD4CCF4F.

Human oocytes

The study was approved by the Newcastle and North Tyneside 1 Research Ethics Committee (REC reference: 18/NE/0348) and was licensed by the UK Human Fertilisation and Embryology Authority (HFEA) (R0152 HFEA license number). Informed consent was obtained from all donors by research nurses who were not directly involved in the research or in the clinical treatments of women participating in the study. Human metaphase II oocytes that failed to fertilize and were donated for research by women undergoing infertility treatment were included in this study. Oocytes were cultured in drops of G-TL medium (Vitrolife) under OVOIL culture oil (Vitrolife) at 36.6°C, with 6% CO2 and 5% 02.

Sample freezing

Single mouse embryos or human oocytes were washed through Ca^2+/Mg²⁺-free PBS (Thermo Fisher Scientific), transferred with minimal volume to PCR tubes (0.2 mL), and snap frozen on dry ice. Samples were then transferred and stored at -80°C until analysis.

Oligonucleotides, gene fragments and PCR

All DNA oligonucleotides and synthetic gene fragments were synthesized by Integrated DNA Technologies (Table 1). All PCR-amplified DNA fragments for cloning were generated using the Q5 DNA polymerase (New England Biolabs). Primers and probes for qPCR were designed by Newcastle University and the Wellcome Centre for Mitochondrial Research

Table 1. List of primer and gRNA sequences

HEX- Hexachlorofluorescein; BHQ_1 - Black Hole Quencher

Construct generation For the generation of mitoPstl, a synthetic gene fragment containing sequences for Atp5b 5’ UTR, Atp5b MTS, Cox8a MTS (dual tandem MTS), Pstl restriction endonuclease codon- optimized for mammalian translation, and Atp5b 3’ UTR (amplified from mouse genomic DNA) were sub-cloned into an empty pCMV vector using In-Fusion cloning (Takara Bio). The plasmids used for the development of the mitoTALEN architectures and engineering of TALE proteins were part of the TALE Toolbox, and were a gift from Feng Zhang (Addgene kit # 1000000019) (Zhang et al., 2011; Sanjana et al., 2012). For the generation of the mouse mitoTALEN architecture, 3xFLAG and SV40 NLS sequences were removed from pCMV TALEN and Atp5b 5’ and 3’ UTRs, tandem Atp5b and Cox8a MTS sequences were inserted. For the generation of mouse mitoTALEN architecture with NES, dual tandem nuclear export signals (NES) from the NS2 protein of minute virus of mice (Eichwald et al., 2002; Minczuk et al., 2006) were inserted after dual tandem MTS sequences. For the assessment of localization, an additional set of constructs was generated with the EGFP added to the C- terminus of Pstl or TALEN. For the generation of pCMV Cas13b, Cas13b-NES was PCR- amplified from the plasmid pC0046-PspCas13b and sub-cloned into a pCMV vector containing Atp5b 5’ and 3’ UTR sequences. pC0046-EF1a-PspCas13b-NES-HIV was a gift from Feng Zhang (Addgene plasmid # 103862).

For the generation of the primary human mitoTALEN architecture (ATP5B), a synthetic gene fragment containing sequences for ATP5B 5’ UTR, and tandem ATP5B and COX8A MTS, as well as ATP5B 3’ UTR PCR-amplified from human genomic DNA were sub-cloned into pCMV mitoTALEN replacing all corresponding mouse sequences (Atp5b 5’ and 3’ UTR, Atp5b and Cox8a MTS). For the generation of the primary human mitoTALEN architecture with NES, dual tandem nuclear export signals (NES) from the NS2 protein were inserted after dual tandem MTS sequences. To assess localization, an additional set of constructs was generated with the EGFP added to the C-terminus of TALEN. For the generation of the secondary human mitoTALEN architecture (COX8A), a synthetic gene fragment containing sequences for COX8A 5’ UTR, and tandem COX8A and SOD2 MTS, as well as COX8A 3’ UTR PCR-amplified from human genomic DNA were sub-cloned into pCMV mitoTALEN replacing all corresponding ATP5B sequences (ATP5B 5’ and 3’ UTR, ATP5B and COX8A MTS). An additional set of constructs was generated with the mCherry added to the C- terminus of TALEN to assess localization. TALEN target sites for mouse and human mtDNA were identified visually, and the central repeat modules of the TALE protein were assembled using the Golden Gate Assembly method (Sanjana et al., 2012). All constructs were Sanger sequenced to validate correct assembly.

RNA synthesis To produce capped mRNA with 3’ poly(A) tail for mitoTALEN and Cas13b, the pCMV mitoTALEN and pCMV Cas13b plasmids was linearized by restriction digestion with Bglll (New England Biolabs) and used as template for in vitro transcription using the mMESSAGE mMACHINE T7 In Vitro Transcription Kit (Thermo Fisher Scientific) and Poly(A) Tailing Kit (Thermo Fisher Scientific). RNA products were purified with an RNeasy Micro Kit (Qiagen) according to the manufacturer’s protocol.

For gRNA synthesis, the Cas13b crRNA backbone was PCR-amplified from the pC0043- crRNA backbone plasmid and used as a template for in vitro transcription. pC0043- PspCas13b crRNA backbone was a gift from Feng Zhang (Addgene plasmid # 103854). A forward primer containing the T7 promoter, 30 nt gRNA spacer sequence, and gRNA scaffold binding site was used with a gRNA scaffold-specific reverse primer to generate the amplicon. For in vitro transcription the HiScribe T7 Quick High Yield RNA Synthesis Kit was used (New England Biolabs). gRNAs were purified using phenol-chloroform extraction and iso-propanol RNA precipitation.

Cytoplasmic microinjection of mouse zygotes and human eggs

Piezo-assisted microinjection of mouse oocytes, zygotes and human eggs was performed using a PMM-150FU Piezo impact drive (PrimeTech) and IM300 Pneumatic Microinjector (Narishige) on a TE300 microscope (Nikon) fitted with micromanipulators. Injection needles were made from borosilicate glass capillaries using a P-97 micropipette puller (Sutter Instruments). Mouse oocytes or zygotes were transferred to drops of M2 medium overlaid with mineral oil and microinjected with mito-Pstl mRNA (400 - 500 ng/pL), mitoTALEN subunit mRNA (50 - 500 ng/pL), Cas13b mRNA (50 - 500 ng/pL) and gRNA (25 - 250 ng/pL) on a heated stage. After microinjection embryos were washed through and cultured in drops of KSOM supplemented with amino acids under mineral oil at 36.6°C, with 6% CO2 and 5% O2. Human oocytes were transferred to drops of G-MOPS PLUS medium (Vitrolife) overlaid with OVOIL and microinjected with mitoTALEN subunit mRNA (640 or 1500 ng/pL) on a heated stage. After microinjection oocytes were washed through and cultured in drops of G-TL under OVOIL at 36.6°C, with 6% CO₂ and 5% O₂.

Quantification of mtDNA copy number

Absolute mtDNA copy numbers were quantified by quantitative real-time PCR (qPCR), performed on a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad). On day of analysis, samples were subjected to 5 freeze-thaw cycles using LN2 and 37°C, and transferred into lysis buffer (200 mM KOH) and incubated for 10 min at 65°C. The reaction was neutralized by addition of 200 mM HCI. Mouse mitochondrial DNA copy number was calculated by absolute quantification using a singleplex Taqman assay targeting the mitochondrial mt-Nd5 gene. Human mitochondrial DNA copy number was calculated by absolute quantification using a singleplex Taqman assay targeting the mitochondrial MT- ND1 gene. Standard curves using PCR-generated templates (Nd5 or ND1) were used for absolute quantification. Samples and standards were measured in triplicate. The method for mtDNA quantification used in this study was developed by Angela Pyle.

Sequencing of human mtDNA control region

Remaining cell lysates from quantification of mtDNA copy number were used for amplification of the control region and Sanger sequencing. Primers used for amplification are shown in Table 1. Sanger sequencing was performed by Source Bioscience.

Heteroplasmy analysis

Heteroplasmy levels in unmanipulated and PNT embryos was measured by pyrosequencing. Frozen embryos were subjected to 5 freeze-thaw cycles using LN2 and 37°C, transferred into lysis buffer (200 mM KOH) and incubated for 10 min at 65°C. The reaction was neutralized by addition of 200 mM HCI. A large region of mouse mtDNA was first amplified generating a ~5.3 kb amplicon (version 2 amplicon 2) (Morris et al., 2017). The mtDNA amplicon was then used as a template to generate a biotinylated PCR product using pyrosequencing primers (Table 1) designed with PyroMark Assay Design Software (Qiagen). Pyrosequencing was performed on the Q24 PyroMark instrument (Qiagen) using the PyroMark Q24 Advanced Reagents (Qiagen) according to the manufacturer’s instructions.

Live cell imaging

Images of live mouse oocytes, embryos and human oocytes were acquired either with Zeiss LSM880 AiryScan confocal microscope and Zen Black acquisition software or Nikon A1 R confocal microscope and NIS-Elements acquisition software. Mouse embryos and human oocytes were stained in KSOM and G-TL drops, respectively, with 100 nM MitoTracker Red CMXRos (Thermo Fisher Scientific) and 3 pl/mL Quant-iT PicoGreen dsDNA Reagent (Thermo Fisher Scientific) for 1 - 2 h (Ashley et al., 2005). Mouse oocytes and embryos were imaged in drops of of G-IVF PLUS and KSOM supplemented with amino acids, respectively, in glass-bottom dishes (ibidi GmbH) under mineral oil at 36.6°C, with 6% CO2 and 5% O2. Human oocytes were imaged in drops of G-TL in glass-bottom dishes under OVOIL at 36.6°C, with 6% CO2 and 5% O2. Z stacks at 5- or 0.5-pm intervals were acquired with a C-Apochromat 40x/1.2 W Corr M27 objective (Zeiss LSM880) or S Fluor 40x/1.3 Oil objective (Nikon A1 R). Images were analyzed in Zen Black or NIS-Elements and Imaged Fiji software. Example 7 - Targeting the control region using other nucleases

Other nucleases can be used to target sites in the mtDNA control region. Such nucleases include zinc finger nucleases (previously demonstrated to effectively target mtDNA), meganucleases, homing endonucleases (previously demonstrated to effectively target mtDNA), or CRISPR-Cas9 or -Cas12 systems.

Zinc finger nucleases (ZFNs) are artificially engineered dimeric nucleases (two ZFN subunits separated by a spacer; monomeric ZFNs have also been described) composed of a DNA- binding zinc-finger domain and Fokl nuclease domain (Cathomen and Joung, 2008; Kim and Kim, 2014). Each ZFN subunit contains a tandem array of 3 - 6 zinc-fingers where each finger recognises a G-rich 3bp sequence; typically, 5’-GNN-3’ sequences (Cathomen and Joung, 2008; Kim and Kim, 2014). ZFNs can be designed and engineered from large archives of zinc-finger domains with known DNA-binding specificities (Kim and Kim, 2014). Therefore, ZFNs can be designed to target sites with 5’-GNN-3’ repeats using the precharacterized zinc-fingers. A ZFN targeting the mtDNA control region could be generated by targeting the most conserved sites using pre-characterized zinc-fingers recognising the most common variants. Additionally, a library of ZFN subunits recognising all or most common variant combinations could allow the targeting of a broader range of mitochondrial genomes.

Meganucleases or homing endonucleases are naturally occurring endonucleases that tend to be specific and recognise large sites of 12 - 40bp in length that occur rarely in the genome. Through complex protein engineering, meganucleases and homing endonucleases can be engineered to recognise and cleave specific dsDNA sequences (e.g. ARCUS by Precision BioSciences).

For CRISPR-Cas systems, the targetable sequences generally contain a sequence recognised by the gRNA and a sequence recognised by the Cas enzyme (PAM sequence). CRISPR-Cas targeting (e.g. by Cas9 or Cas12) is generally limited by the requirement for the PAM sequence, which is different depending on the Cas enzyme, genus and species of origin (e.g. 5’-NGG-3’ for SpCas9). The targetable sites are further limited by the requirement for at least one G at the 5’ end of the gRNA if the gRNA is expressed from a plasmid or synthesised by in vitro transcription. The length of the gRNA spacer (target sequence) is typically 20 nt long, however, this can vary by several nt (gRNA spacers can be e.g. 16 - 25 nt long; also depends on Cas enzyme, genus and species of origin). Therefore, the design requirement for targeting with e.g. SpCas9 is 5’-GN19NGG-3’ where is 5’-GN19- 3’ is the gRNA spacer sequence and 5’-NGG-3’ is the PAM sequence (first look for PAM sequence, then look for GN 19). A CRISPR-Cas9 system targeting the mtDNA control region could be generated by targeting the most conserved sites using a gRNA recognising the most common variants. Additionally, a library of gRNAs recognising all or most common variant combinations could allow the targeting of a broader range of mitochondrial genomes.

Example 8 - Sequences

- Mouse mtDNA control region (SEQ ID NO: 403) Agtacataaatttacatagtacaacagtacatttatgtatatcgtacattaaactattttccccaagcatataagctagtacattaaatc aatggttcaggtcataaaataatcatcaacataaatcaatatatataccatgaatattatcttaaacacattaaactaatgttataag gacatatctgtgttatctgacatacaccatacagtcataaactcttctcttccatatgactatccccttccccatttggtctattaatctacc atcctccgtgaaaccaacaacccgcccaccaatgcccctcttctcgctccgggcccattaaacttgggggtagctaaactgaaac tttatcagacatctggttcttacttcagggccatcaaatgcgttatcgcccatacgttccccttaaataagacatctcgatggtatcggg tctaatcagcccatgaccaacataactgtggtgtcatgcatttggtatctttttattttggcctactttcatcaacatagccgtcaaggcat gaaaggacagcacacagtctagacgcacctacggtgaagaatcattagtccgcaaaacccaatcacctaaggctaattattca tgcttgttagacataaatgctactcaataccaaattttaactctccaaaccccccaccccctcctcttaatgccaaaccccaaaaac actaagaacttgaaagacatataatattaactatcaaaccctatgtcctgatcaattctagtagttcccaaaatatgacttatattttag tacttgtaaaaattttacaaaatcatgttccgtgaaccaaaactctaatcatactctattacgcaataaacattaacaa

- Human mtDNA control region (SEQ ID NO: 404) Ttctttcatggggaagcagatttgggtaccacccaagtattgactcacccatcaacaaccgctatgtatttcgtacattactgccagc caccatgaatattgtacggtaccataaatacttgaccacctgtagtacataaaaacccaatccacatcaaaaccccctccccatg cttacaagcaagtacagcaatcaaccctcaactatcacacatcaactgcaactccaaagccacccctcacccactaggatacc aacaaacctacccacccttaacagtacatagtacataaagccatttaccgtacatagcacattacagtcaaatcccttctcgtccc catggatgacccccctcagataggggtcccttgaccaccatcctccgtgaaatcaatatcccgcacaagagtgctactctcctcgc tccgggcccataacacttgggggtagctaaagtgaactgtatccgacatctggttcctacttcagggtcataaagcctaaatagcc cacacgttccccttaaataagacatcacgatggatcacaggtctatcaccctattaaccactcacgggagctctccatgcatttggt attttcgtctggggggtatgcacgcgatagcattgcgagacgctggagccggagcaccctatgtcgcagtatctgtctttgattcctg cctcatcctattatttatcgcacctacgttcaatattacaggcgaacatacttactaaagtgtgttaattaattaatgcttgtaggacata ataataacaattgaatgtctgcacagccactttccacacagacatcataacaaaaaatttccaccaaaccccccctcccccgcttc tggccacagcacttaaacacatctctgccaaaccccaaaaacaaagaaccctaacaccagcctaaccagatttcaaattttatct tttggcggtatgcacttttaacagtcaccccccaactaacacattattttcccctcccactcccatactactaatctcatcaatacaacc cccgcccatcctacccagcacacacacaccgctgctaaccccataccccgaaccaaccaaaccccaaagacaccccccac a

Human mtDNA control region (conserved regions with ‘N’ at positions where there is variability):

TTCTTTCATGGGNNNNCNNNTTTNNGTNNCNCCCAANNANTNNNNNNNNCNNCNNNNN NNNCNATGNNNNNNNNNCATNNNNNCNNGNCNNCANGNANNNNGNNCNNNNNNANNN NNNNTNNNNNNNCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNCTNANANNCNNGNNNNNNNNNNNNNNNNNANNNNNNNNNCNTNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNANNNANNNANNNNNNNNNNNNNNNNNNNNCAN

NNNNCATNNNNNNNNNNNNNGNNCATNNNACATNNNANNNANANNNNNNCNNNNCCN NANGNNNGNCCNCNNNNANNTANNNNNNNCTNNNNNACCATCNNCNGTGAAATCAANA TCNNNNNNANGNGNNNNACTNNCCNNGNNNNNNNCCCNTNANNNNNNNNNNTAGNTA

NNNNNNNNNNNATNCNNCATCNGNNNNNNANNTNANGNNNANNNNNNNNANANNNCC CANNNNTNCCCCNNAANNNAGNCNNCACGATGGATCNCANNNNNANCNCNCTANNAAN CACTCANNNNAGNTNNNNNNNCATNNNNNNNNNNNNNNNNNNNNNNNNNNACNCNAT

NNCATNNNNNNNNNNTGNNNCCNNNNCNCNNNATNNCNCAGNNNNTGNNTTTNATNNC TNNCNCATNNNNNNATNNATCGNNCCNACNTNNNATNNNNNNNNNNNNCNNNNNNNNN NNNNNNNNNNNNNTNNNNAANGNTNNNNNGNNATNNNNNNNNNNNNNNNNNNNNTNN

NNNNNNNNNNNCNNNNCANNNANNNNNNNNNNAANNNNNNNNNNNNCCNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNANNNNNTNTNNGNCNNACCCCNAAANCAANNANCN CNNNNNNNNNCNTNNCCAGNNNNNANANTNTNTCTTNNGGNNGNNNNNNNTTNNAACA

GNNNCCCCNNAANTANNNNANNNNNNNNCNNNNNNNNNNNNNNNNNACNNNNNNNAN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTGNTNNNNNNNNNCN CNNNNNNANNNAAACCCNAANNNCNNNNNNNNNN (SEQ ID NO: 1813)

Mouse sequences

- Target site DNA sequence for mouse mitoTALEN (SEQ ID NO: 376)

5’ - TGCCCCTCTTCTCGctccqqqcccattaaaCTTGGGGGTAGCTA - 3’

Underlined uppercase letters indicates left target sequence.

Underlined and bold uppercase letters indicate right target sequence.

Lowercase letters indicate a spacer.

Based on the reference sequence of house mouse mitochondrial genome (C57BL/6J: NC_005089.1), the target site ranges from position m.15, 726 to m.15,769 (m.15,727 to m.15,770 for Crl:CD1 (ICR): KC663622.1).

- Mouse mitoTALEN - left subunit binding site (SEQ ID NO: 377)

5’ - TGCCCCTCTTCTCG - 3’

5’ T (underlined) is position 0 in the subunit binding site and is recognised by the N-terminal

TALE scaffold

Binding site ranges from m.15, 726 to m.15, 739 (m.15, 727 to m.15, 740 for CD1)

- Mouse mitoTALEN - right subunit binding site (SEQ ID NO: 378)

5’ - TAGCTACCCCCAAG - 3’ 5’ T (underlined) is position 0 in the subunit binding site and is recognised by the N-terminal TALE scaffold

Binding site ranges from m.15, 756 to m.15,769 (m.15, 757 to 15,770 for CD1)

- Mouse mitoTALEN RVD sequences

These sequences show amino acids sequences at the repeat variable di-residues (RVDs) (which specify the target DNA base) also known as hypervariable residues. Mouse mitoTALENs with NA or NS RVDs were generated to test the effect of promiscuous RVD incorporation on mitoTALEN activity. NA or NS RVDs are shown in bold. NA and NS are promiscuous.

Left RVD sequence of mouse mitoTALEN with standard code

NN HD HD HD HD NG HD NG NG HD NG HD NN (SEQ ID NO: 379) Right RVD sequence of mouse mitoTALEN with standard code Nl NN HD NG Nl HD HD HD HD HD Nl Nl NN (SEQ ID NO: 380) Left RVD sequence of mouse mitoTALEN with NA RVDs

NN NA HD NA HD NG NA NG NG HD NG NA NN (SEQ ID NO: 381) Right RVD sequence of mouse mitoTALEN with NA RVDs

Nl NN NA NG Nl HD NA HD NA HD Nl Nl NN (SEQ ID NO: 382)

Left RVD sequence for mouse mitoTALEN with NS RVDs

NN NS HD NS HD NG NS NG NG HD NG NS NN (SEQ ID NO: 430) Right RVD sequence for mouse mitoTALEN with NS RVDs Nl NN NS NG Nl HD NS HD NS HD Nl Nl NN (SEQ ID NO: 431)

Human sequences

Target site 1 DNA sequence

- 5’ TCTN3TTCTTTCATGGaaaaacaaatttaaGTN4N5CN6CCCAAN7N₈A 3’ (L2- 14bp spacer-R2) (SEQ ID NO: 1) Target site ranges from m.16, 020 to m.16,062

- 5’ TANiN2CTCTN3TTCTTTCATGaaaaaacaaatttaaGTN4N5CN₆CCCAAN7N8A 3’ (L3- 15bp spacer-R2) (SEQ ID NO: 2) Target site ranges from m.16, 015 to m.16, 062

- 5’ TANiN2CTCTN3TTCTTTCATaaaaaaacaaatttaaGTN4N5CN6CCCAAN7N₈A 3’ (L4- 16bp spacer-R2) (SEQ ID NO: 3) Target site ranges from m.16, 015 to m.16, 062 mitoTALEN subunit binding sites

- Left subunit (L2 - 13.5 repeats) binding site: 5’ T-CTN3TTCTTTCATGG 3’ (SEQ ID NO: 4) binding site ranges from m.16,020 to m.16,034 (with R2 produces a 14 bp spacer); N3 at position 3 is G or T (m.16023) - Left subunit (L3 - 17.5 repeats) binding site: 5’ T-AN1N2CTCTN3TTCTTTCATG 3’ (SEQ ID NO: 5) binding site ranges from m.16,015 to m.16, 033 (with R2 produces a

15 bp spacer); N1 at position 2 is T, G or C (m.16017); N2 at position 3 is T or C (m.16018); N3 at position 8 is G or T (m.16023)

- Left subunit (L4 - 16.5 repeats) binding site: 5’ T-AN1N2CTCTN3TTCTTTCAT 3’ (SEQ ID NO: 6) binding site ranges from m.16,015 to m.16, 032 (with R2 produces a

16 bp spacer); N1 at position 2 is T, G or C (m.16017); N2 at position 3 is T or C (m.16018); N3 at position 8 is G or T (m.16023)

- Right subunit (R2 - 12.5 repeats) binding site (reverse complement): 5’ T- N8N7TTGGGN6GN5N4AC 3’ (SEQ ID NO: 7) binding site ranges from m.16,049 to m.16,062; Ns at position 1 is A or C (m.16061T>G); N7 at position 2 is C or T (m.16060G>A); Ns at position 8 is T or C (m.16054A>G); N5 at position 10 is G or A (m.16052C>T); N4 at position 11 is T or C (m.16051 A>G)

- Left subunit variants (minor allele frequency based on 30,506 human mitochondrial genomes):

- N1 : m.16017T>G (3.28E-05), m.16017T>C (2.89E-03) - recognised by NA or NS

- N2: m.16018T>C (3.28E-05) - recognised by NA or NS

- N3: m.16023G>T (1.64E-04) - recognised by NA or NS

- Right subunit variants (minor allele frequency based on 30,506 human mitochondrial genomes):

- N8: m.16061T>G (3.28E-05) - recognised by NA or NS

- N7: m.16060G>A (3.28E-05) - recognised by NA or NS

- N6: m.16054A>G (3.28E-05) - recognised by NA or NS

- N5: m.16052C>T (1.31E-04) - recognised by NA, NS, NN or HN

- N4: m.16051A>G (2.63E-02) - recognised by NA or NS

Human mitoTALEN subunit RVD sequences

- Human mitoTALEN left (L2) subunit - standard code

HD NG NN NG NG HD NG NG NG HD Nl NG NN NN (SEQ ID NO: 8)

- Human mitoTALEN left (L2) subunit - improved G specificity

HD NG NH NG NG HD NG NG NG HD Nl NG NH NH (SEQ ID NO: 9)

- Human mitoTALEN left (L2) subunit - with promiscuous RVDs

HD NG NA NG NG HD NG NG NG HD Nl NG NN NN (SEQ ID NO: 10)

HD NG NS NG NG HD NG NG NG HD Nl NG NN NN (SEQ ID NO: 11)

- Human mitoTALEN left (L2) subunit - combined HD NG NA NG NG HD NG NG NG HD Nl NG NH NH (SEQ ID NO: 12)

HD NG NS NG NG HD NG NG NG HD Nl NG NH NH (SEQ ID NO: 13)

- Human mitoTALEN left (L3) subunit - standard code

Nl NG NG HD NG HD NG NN NG NG HD NG NG NG HD Nl NG NN (SEQ ID NO: 14)

- Human mitoTALEN left (L3) subunit - improved G specificity

Nl NG NG HD NG HD NG NH NG NG HD NG NG NG HD Nl NG NH (SEQ ID NO: 15)

- Human mitoTALEN left (L3) subunit - with promiscuous RVDs

Nl NA NA HD NG HD NG NA NG NG HD NG NG NG HD Nl NG NN (SEQ ID NO: 16)

Nl NS NS HD NG HD NG NS NG NG HD NG NG NG HD Nl NG NN (SEQ ID NO: 17)

- Human mitoTALEN left (L3) subunit -combined

Nl NA NA HD NG HD NG NA NG NG HD NG NG NG HD Nl NG NH (SEQ ID NO: 18)

Nl NS NS HD NG HD NG NS NG NG HD NG NG NG HD Nl NG NH (SEQ ID NO: 19)

- Human mitoTALEN left (L4) subunit - standard code:

Nl NG NG HD NG HD NG NN NG NG HD NG NG NG HD Nl NG (SEQ ID NO: 20)

- Human mitoTALEN left (L4) subunit - improved G specificity

Nl NG NG HD NG HD NG NH NG NG HD NG NG NG HD Nl NG (SEQ ID NO: 21)

- Human mitoTALEN left (L4) subunit - promiscuous RVDs

Nl NA NA HD NG HD NG NA NG NG HD NG NG NG HD Nl NG (SEQ ID NO: 22)

Nl NS NS HD NG HD NG NS NG NG HD NG NG NG HD Nl NG (SEQ ID NO: 23)

- Human mitoTALEN right (R2) subunit - standard code:

Nl HD NG NG NN NN NN NG NN NN NG Nl HD (SEQ ID NO: 24)

- Human mitoTALEN right (R2) subunit - improved G specificity:

Nl HD NG NG NH NH NH NG NH NH NG Nl HD (SEQ ID NO: 25)

- Human mitoTALEN right (R2) subunit - with promiscuous RVDs:

NA NA NG NG NN NN NN NA NN NA NA Nl HD (SEQ ID NO: 26)

NS NS NG NG NN NN NN NS NN NS NS Nl HD (SEQ ID NO: 27)

NA NA NG NG NN NN NN NA NN NN NA Nl HD (SEQ ID NO: 28)

NS NS NG NG NN NN NN NS NN NN NS Nl HD (SEQ ID NO: 29) NA NA NG NG NN NN NN NA NN HN NA Nl HD (SEQ ID NO: 30)

NS NS NG NG NN NN NN NS NN HN NS Nl HD (SEQ ID NO: 31)

- Human mitoTALEN right (R2) subunit - Combined:

NA NA NG NG NH NH NH NA NH NA NA Nl HD (SEQ ID NO: 32)

NS NS NG NG NH NH NH NS NH NS NS Nl HD (SEQ ID NO: 33)

NA NA NG NG NH NH NH NA NH NN NA Nl HD (SEQ ID NO: 34)

NS NS NG NG NH NH NH NS NH NN NS Nl HD (SEQ ID NO: 35)

NA NA NG NG NH NH NH NA NH HN NA Nl HD (SEQ ID NO: 36)

NS NS NG NG NH NH NH NS NH HN NS Nl HD (SEQ ID NO: 37)

Target sequence 2

- 5’TNiCCCCN2N3AAN4N5N6AGN7CatcacaatggatcaCAN8N9NioNnNi2ANi3CNi4CNi5CT A 3’ (L1-14bp spacer-R1) (SEQ ID NO: 86) Target site ranges from m.16,544 to m.21

- S’TNiCCCC^NsAA^NsNsAGacatcacqatggatcaCANsNQNioNnN^AN CNuCNisCTA 3’ (L2-16bp spacer-R1) (SEQ ID NO: 87) Target site ranges from m.16,544 to m.21

- Left subunit (L1 - 15.5 repeats) binding site: 5’ T-N1CCCCN2N3AAN4N5N6AGN7C 3’ Binding site ranges from m.16, 544 to m.16,560 (with R1 produces a 14 bp spacer) (SEQ ID NO: 88); N1 at position 1 is T or C (m.16545); N2 at position 6 is T or C

(m.16550); N3 at position 7 is T or A (m.16551); N4 at position 10 is A or T (m.16554); N5 at position 11 is T, G or C (m.16555); Ne at position 12 is A or G (m.16556); N7 at position 15 is A or G (m.16559)

- Left subunit (L2 - 13.5 repeats) binding site: 5’ T-N1CCCCN2N3AAN4N5N6AG 3’ binding site ranges from m.16,544 to m.16,558 (with R1 produces a 16 bp spacer)(SEQ ID NO: 89); N1 at position 1 is T or C (m.16545); N2 at position 6 is T or C (m.16550); N3 at position 7 is T or A (m.16551); N4 at position 10 is A or T

(m.16554); N5 at position 11 is T, G or C (m.16555); Ne at position 12 is A or G (m.16556)

- Right subunit (R1 - 14.5 repeats) binding site (reverse complement): 5’ T- AGN15GN14GN13TN12N11N10N9N8TG 3’ binding site ranges from m.6 to m.21 (SEQ ID NO: 90); N15 at position 3 is G or A (m.18C>T); N14 at position 5 is T or A (m.16A>T); N13 at position 7 is A or G (m.14T>C); N12 at position 9 is A or G (m.12T>C); N11 at position 10 is G or A (m.11C>T); N10 at position 11 is A or G (m.10T>C); Ng at position 12 is C, T or A (m.9G>A, m.9G>T); Ns at position 13 is C or A (m.8G>T)

- N1 : m.16545T>C (1.97E-04) - recognised by NA or NS

- N2: m.16550T>C (3.28E-05) - recognised by NA or NS

- N3: m.16551T>A (6.56E-05) - recognised by NA or NS

- N4: m.16554A>T (9.83E-05) - recognised by NA or NS - N5: m.16555T>G (6.56E-05), m.16555T>C (3.28E-05) - recognised by NA or NS

- N6: m.16556A>G (3.28E-05) - recognised by NA or NS

- N7: m.16559A>G (6.56E-05) - recognised by NA or NS

- N15: m.18C>T (3.28E-05) - recognised by NA, NS, NN or HN

- N14: m.16A>T (9.83E-04) - recognised by NA or NS

- N13: m.14T>C (9.83E-05) - recognised by NA or NS

- N12: m.12T>C (9.83E-05) - recognised by NA or NS

- N11: m.11C>T (3.28E-05) - recognised by NA, NS, NN or HN

- N10: m.10T>C (5.57E-04) - recognised by NA or NS

- N9: m.9G>A (2.30E-04), m.9G>T (3.28E-05) - recognised by NA or NS

- N8: m.8G>T (9.83E-05) - recognised by NA or NS

Human mitoTALEN subunit RVD sequences

- Human mitoTALEN left (L1) subunit - standard code

NG HD HD HD HD NG NG Nl Nl Nl NG Nl Nl NN Nl HD (SEQ ID NO: 91)

- Human mitoTALEN left (L1) subunit - with improved G specificity

NG HD HD HD HD NG NG Nl Nl Nl NG Nl Nl NH Nl HD (SEQ ID NO: 92)

- Human mitoTALEN left (L1) subunit - with promiscuous RVDs

NA HD HD HD HD NA NA Nl Nl NA NA NA Nl NN NA HD (SEQ ID NO: 93)

NS HD HD HD HD NS NS Nl Nl NS NS NS Nl NN NS HD (SEQ ID NO: 94)

- Human mitoTALEN left (L1) subunit - combined

NA HD HD HD HD NA NA Nl Nl NA NA NA Nl NH NA HD (SEQ ID NO: 95)

NS HD HD HD HD NS NS Nl Nl NS NS NS Nl NH NS HD (SEQ ID NO: 96)

- Human mitoTALEN left (L2) subunit - standard code:

NG HD HD HD HD NG NG Nl Nl Nl NG Nl Nl NN (SEQ ID NO: 97)

- Human mitoTALEN left (L2) subunit - with improved G specificity:

NG HD HD HD HD NG NG Nl Nl Nl NG Nl Nl NH (SEQ ID NO: 98) - Human mitoTALEN left (L2) subunit -with promiscuous RVDs:

NA HD HD HD HD NA NA Nl Nl NA NA NA Nl NN (SEQ ID NO: 99)

NS HD HD HD HD NS NS Nl Nl NS NS NS Nl NN (SEQ ID NO: 100)

- Human mitoTALEN left (L2) subunit - combined

NA HD HD HD HD NA NA Nl Nl NA NA NA Nl NH (SEQ ID NO: 101)

NS HD HD HD HD NS NS Nl Nl NS NS NS Nl NH (SEQ ID NO: 102)

- Human mitoTALEN right (R1) subunit - standard cod

Nl NN NN NN NG NN Nl NG Nl NN Nl HD HD NG NN (SEQ ID NO: 103)

- Human mitoTALEN right (R1) subunit - with improved G specificity

Nl NH NH NH NG NH Nl NG Nl NH Nl HD HD NG NH (SEQ ID NO: 104)

- Human mitoTALEN right (R1) subunit - with promiscuous RVDs

Nl NN NA NN NA NN NA NG NA NA NA NA NA NG NN (SEQ ID NO: 105)

Nl NN NS NN NS NN NS NG NS NS NS NS NS NG NN (SEQ ID NO: 106)

Nl NN NN NN NA NN NA NG NA NN NA NA NA NG NN (SEQ ID NO: 107)

Nl NN NN NN NS NN NS NG NS NN NS NS NS NG NN (SEQ ID NO: 108)

Nl NN HN NN NA NN NA NG NA HN NA NA NA NG NN (SEQ ID NO: 109)

Nl NN HN NN NS NN NS NG NS HN NS NS NS NG NN (SEQ ID NO: 110)

- Human mitoTALEN right (R1) subunit - combined

Nl NH NA NH NA NH NA NG NA NA NA NA NA NG NH (SEQ ID NO: 111)

Nl NH NS NH NS NH NS NG NS NS NS NS NS NG NH (SEQ ID NO: 112)

Nl NH NN NH NA NH NA NG NA NN NA NA NA NG NH (SEQ ID NO: 113)

Nl NH NN NH NS NH NS NG NS NN NS NS NS NG NH (SEQ ID NO: 114)

Nl NH HN NH NA NH NA NG NA HN NA NA NA NG NH (SEQ ID NO: 115)

Nl NH HN NH NS NH NS NG NS HN NS NS NS NG NH (SEQ ID NO: 116)

Target sequence 3

- 5’TCNiCAN2N3N4N5N6AN7CN8CN9CTattaaccactcacgggAGNioTNiiNi2Ni3Ni4NisNi6Ni ?CA 3’ (L-16bp spacer-R) (SEQ ID NO: 153) Target site ranges from m.3 to m.49

- Left subunit (L - 16.5 repeats) binding site:

5’T-CNiCAN2N3N4N5N6AN7CN8CNgCT 3’ binding site ranges from m.3 to m.20 (SEQ ID NO: 154; N1 at position 2 is A or C (m.5); N2 at position 5 is G or T (m.8); N3 at position 6 is G, A or T (m.9); N4 at position 7 is T or C (m.10); N5 at position 8 is C or T (m.11); Ne at position 9 is T or C (m.12); N? at position 11 is T or C (m.14); Ns at position 13 is A or T (m.16); Ng at position 15 is C or T (m.18)

- Right subunit (R - 11.5 repeats) binding site (reverse complement):

5’ T-GN17N16N15N14N13N12N11AN10CT 3’ binding site ranges from m.37 to m.49 (SEQ ID NO: 155); N17 at position 2 is C or T (m.47G>A); N at position 3 is A, G or C (m.46T>C, m.46T>G); N15 at position 4 is T or C (m.45A>G); N14 at position 5 is G or A (m.44C>T); N13 at position 6 is G or A (m.43C>T); N12 at position 7 is A or G (m.42T>C); N11 at position 8 is G or A (m.41C>T); N10 at position 10 is G or A (m.39C>T)

- N m.5A>C (6.56E-05) - recognised by NA or NS

- N2: m.8G>T (9.83E-05) - recognised by NA or NS

- N₃: m.9G>A (2.30E-04), m.9G>T (3.28E-05) - recognised by NA or NS

- N4: m.10T>C (5.57E-04) - recognised by NA or NS

- N5: m.11C>T (3.28E-05) - recognised by NA or NS

- Ne: m.12T>C (9.83E-05) - recognised by NA or NS

- N7: m.14T>C (9.83E-05) - recognised by NA or NS

- Ns: m.16A>T (9.83E-04) - recognised by NA or NS

- Ng: m.18C>T (3.28E-05) - recognised by NA or NS

- N17: m.47G>A (6.88E-04) - recognised by NA or NS

- NI₆: m.46T>C (6.56E-05), m.46T>G (6.56E-05) - recognised by NA or NS

- N15: m.45A>G (3.28E-05) - recognised by NA or NS

- N14: m.44C>T (3.28E-05) - recognised by NA, NS, NN or HN

- N13: m.43C>T (3.28E-05) - recognised by NA, NS, NN or HN

- N12: m.42T>C (9.83E-05) - recognised by NA or NS

- N11 : m.41C>T (1.97E-03) - recognised by NA, NS, NN or HN

- N : m.39C>T (6.56E-05) - recognised by NA, NS, NN or HN

- Human mitoTALEN left subunit -standard code:

HD Nl HD Nl NN NN NG HD NG Nl NG HD Nl HD HD HD NG (SEQ ID NO: 156) - Human mitoTALEN left subunit - with improved G specificity:

HD Nl HD Nl NH NH NG HD NG Nl NG HD Nl HD HD HD NG (SEQ ID NO: 157)

- Human mitoTALEN left subunit - with promiscuous RVDs:

HD NA HD Nl NA NA NA NA NA Nl NA HD NA HD NA HD NG (SEQ ID NO: 158)

HD NS HD Nl NS NS NS NS NS Nl NS HD NS HD NS HD NG (SEQ ID NO: 159)

- Human mitoTALEN right subunit - standard code:

NN HD Nl NG NN NN Nl NN Nl NN HD NG (SEQ ID NO: 160)

- Human mitoTALEN right subunit - with improved G specificity:

NH HD Nl NG NH NH Nl NH Nl NH HD NG (SEQ ID NO: 161)

- Human mitoTALEN right subunit - with promiscuous RVDs:

NN NA NA NA NA NA NA NA Nl NA HD NG (SEQ ID NO: 162)

NN NS NS NS NS NS NS NS Nl NS HD NG (SEQ ID NO: 163)

NN NA NA NA NN NN NA NN Nl NN HD NG (SEQ ID NO: 164)

NN NS NS NS NN NN NS NN Nl NN HD NG (SEQ ID NO: 165)

NN NA NA NA HN HN NA HN Nl HN HD NG (SEQ ID NO: 166)

NN NS NS NS HN HN NS HN Nl HN HD NG (SEQ ID NO: 167)

- Human mitoTALEN right subunit - combined:

NH NA NA NA NN NN NA NN Nl NN HD NG (SEQ ID NO: 168)

NH NS NS NS NN NN NS NN Nl NN HD NG (SEQ ID NO: 169)

NH NA NA NA HN HN NA HN Nl HN HD NG (SEQ ID NO: 170)

NH NS NS NS HN HN NS HN Nl HN HD NG (SEQ ID NO: 171)

Target sequence 4

- 5’ TGNi N2TTTN3ATN4NsCTN6N7CN8CatcctattatttatCGN9NioCCNi 1 ACN12TN13N14N15A 3’ (L1-14bp spacer-R1) (SEQ ID NO: 196) Target site ranges from m.129 to m.176

- 5’ TGNiN2TTTN3ATN4NsCTN6N7CN8CatcctattatttatcGN9NioCCNnACNi2TNi3Ni4Ni5A 3’ (L1-15bp spacer-R2) (SEQ ID NO: 197) Target site ranges from m.129 to m.176

- 5’ TGNiN2TTTN3ATN4NsCTN6N7CtcatcctattatttatCGN9NioCCNnACNi2TNi3Ni4Ni5A 3’ (L2-16bp spacer-R1) (SEQ ID NO: 198) Target site ranges from m.129 to m.176

- 5’ TGNiN2TTTN3ATN4NsCTN6N7CtcatcctattatttatcGN9NioCCNnACNi2TNi3Ni4Ni5A 3’ (L2-17bp spacer-R2) (SEQ ID NO: 199) Target site ranges from m.129 to m.176

- 5’ TTTN3ATN4NsCTN6N7CN8CatcctattatttatCGN9NioCCNnACNi2TNi3Ni4Ni5A 3’ (L3- 14bp spacer-R1) (SEQ ID NO: 200) Target site ranges from m.133 to m.176 - 5’ TTTN3ATN4NsCTN6N7CN8CatcctattatttatcGN9NioCCNnACNi2TNi3Ni4Ni5A 3’ (L3- 15bp spacer-R2) (SEQ ID NO: 201) Target site ranges from m.133 to m.176

- 5’ TTN3ATN4NsCTN6N7CN8CatcctattatttatCGN9NioCCNnACNi2TNi3Ni4Ni5A 3’ (L4- 14bp spacer-R1) (SEQ ID NO: 464) Target site ranges from m.134 to m.176

- 5’ TTN3ATN4NsCTN6N7CN8CatcctattatttatcGN9NioCCNnACNi2TNi3Ni4Ni5A 3’ (L4- 15bp spacer-R2) (SEQ ID NO: 202) Target site ranges from m.134 to m.176

- Left subunit (L1 - 17.5 repeats) binding site: 5’ T-GN1N2TTTN3ATN4N5CTN6N7CN8C 3’ (14 or 15 bp spacer) m.129 to m.147 (SEQ ID NO: 203); N1 at position 2 is T or C (m.131); N2 at position 3 is C or T (m.132); N3 at position 7 is G or C (m.136); N4 at position 10 is T or C (m.139); N5 at position 11 is C or T (m.140); Ne at position 14 is G, A or T (m.143); N7 at position 15 is C, A or T (m.144); Ns at position 17 is T, C or A (m.146)

- Left subunit (L2 - 15.5 repeats) binding site: 5’ T-GN1N2TTTN3ATN4N5CTN6N7C 3’ (16 or 17 bp spacer) m.129 to m.145 (SEQ ID NO: 204); N1 at position 2 is T or C (m.131); N2 at position 3 is C or T (m.132); N3 at position 7 is G or C (m.136); N4 at position 10 is T or C (m.139); N5 at position 11 is C or T (m.140); Ne at position 14 is G, A or T (m.143); N7 at position 15 is C, A or T (m.144)

- Left subunit (L3 - 13.5 repeats) binding site: 5’ T-TTN3ATN4N5CTN6N7CN8C 3’ (14 or 15 bp spacer) m.133 to m.147 (SEQ ID NO: 205); N3 at position 3 is G or C (m.136); N4 at position 6 is T or C (m.139); N5 at position 7 is C or T (m.140); Ne at position 10 is G, A or T (m.143); N7 at position 11 is C, A or T (m.144); Ns at position 13 is T, C or A (m.146)

- Left subunit (L4 - 12.5 repeats) binding site: 5’ T-TN3ATN4N5CTN6N7CN8C 3’ (14 or 15 bp spacer) m.134 to m.147 (SEQ ID NO: 206); N3 at position 2 is G or C (m.136); N4 at position 5 is T or C (m.139); N5 at position 6 is C or T (m.140); Ne at position 9 is G, A or T (m.143); N7 at position 10 is C, A or T (m.144); Ns at position 12 is T, C or A (m.146)

- Right subunit (R1 - 13.5 repeats) binding site (reverse complement): 5’ T- N15N14N13AN12GTN11GGN10N9CG 3’ (14 or bp 16 spacer) m.162 to m.176 (SEQ ID NO: 207); N15 at position 1 is T or G (m.175A>C); N14 at position 2 is G or A (m.174C>T); N13 at position 3 is A or G (m.173T>C)

N12 at position 5 is C or T (m.171G>A); N11 at position 8 is A or G (m.168T>C); N10 at position 11 is T or C (m.165A>G); Ng at position 12 is G or A (m.164C>T)

- Right subunit (R2 - 12.5 repeats) binding site (reverse complement): 5’ T- Ni₅Ni4Ni3ANi₂GTNiiGGNioN₉C 3’ (15 or 17 bp spacer) m.163 to m.176 (SEQ ID NO: 208); N15 at position 1 is T or G (m.175A>C); N14 at position 2 is G or A (m.174C>T); N13 at position 3 is A or G (m.173T>C); N12 at position 5 is C or T (m.171G>A); N11 at position 8 is A or G (m.168T>C); N10 at position 11 is T or C (m.165A>G); Ng at position 12 is G or A (m.164C>T)

Human mitoTALEN subunit RVD sequences

- Human mitoTALEN left (L1) subunit - standard code

NN NG HD NG NG NG NN Nl NG NG HD HD NG NN HD HD NG HD (SEQ ID NO: 209)

- Human mitoTALEN left (L1) subunit - with improved G specificity NH NG HD NG NG NG NH Nl NG NG HD HD NG NH HD HD NG HD (SEQ ID NO: 210)

- Human mitoTALEN left (L1) subunit - with promiscuous RVDs

NN NA NA NG NG NG NA Nl NG NA NA HD NG NA NA HD NA HD (SEQ ID NO: 211)

NN NS NS NG NG NG NS Nl NG NS NS HD NG NS NS HD NS HD (SEQ ID NO: 212)

- Human mitoTALEN left (L1) subunit - combined

NH NA NA NG NG NG NA Nl NG NA NA HD NG NA NA HD NA HD (SEQ ID NO: 213)

NH NS NS NG NG NG NS Nl NG NS NS HD NG NS NS HD NS HD (SEQ ID NO: 214)

- Human mitoTALEN left (L2) subunit - standard code

NN NG HD NG NG NG NN Nl NG NG HD HD NG NN HD HD (SEQ ID NO: 215)

- Human mitoTALEN left (L2) subunit - with improved G specificity

NH NG HD NG NG NG NH Nl NG NG HD HD NG NH HD HD (SEQ ID NO: 216)

- Human mitoTALEN left (L2) subunit - with promiscuous RVDs

NN NA NA NG NG NG NA Nl NG NA NA HD NG NA NA HD (SEQ ID NO: 217)

NN NS NS NG NG NG NS Nl NG NS NS HD NG NS NS HD (SEQ ID NO: 218)

- Human mitoTALEN left (L2) subunit - combined

NH NA NA NG NG NG NA Nl NG NA NA HD NG NA NA HD (SEQ ID NO: 219)

NH NS NS NG NG NG NS Nl NG NS NS HD NG NS NS HD (SEQ ID NO: 220)

- Human mitoTALEN left (L3) subunit - standard code

NG NG NN Nl NG NG HD HD NG NN HD HD NG HD (SEQ ID NO: 221)

- Human mitoTALEN left (L3) subunit - with improved G specificity

NG NG NH Nl NG NG HD HD NG NH HD HD NG HD (SEQ ID NO: 222)

- Human mitoTALEN left (L3) subunit - with promiscuous RVDs

NG NG NA Nl NG NA NA HD NG NA NA HD NA HD (SEQ ID NO: 223)

NG NG NS Nl NG NS NS HD NG NS NS HD NS HD (SEQ ID NO: 224)

- Human mitoTALEN left (L4) subunit - standard code

NG NN Nl NG NG HD HD NG NN HD HD NG HD (SEQ ID NO: 225)

- Human mitoTALEN left (L4) subunit - with improved G specificity NG NH Nl NG NG HD HD NG NH HD HD NG HD (SEQ ID NO: 226)

- Human mitoTALEN left (L4) subunit - with promiscuous RVDs

NG NA Nl NG NA NA HD NG NA NA HD NA HD (SEQ ID NO: 227)

NG NS Nl NG NS NS HD NG NS NS HD NS HD (SEQ ID NO: 228)

- Human mitoTALEN left (R1) subunit - standard code

NG NN Nl Nl HD NN NG Nl NN NN NG NN HD NN (SEQ ID NO: 229)

- Human mitoTALEN left (R1) subunit - with improved G specificity:

NG NH Nl Nl HD NH NG Nl NH NH NG NH HD NH (SEQ ID NO: 230)

- Human mitoTALEN left (R1) subunit - with promiscuous RVDs

NA NA NA Nl NA NN NG NA NN NN NA NA HD NN (SEQ ID NO: 231)

NS NS NS Nl NS NN NG NS NN NN NS NS HD NN (SEQ ID NO: 232)

NA NN NA Nl NA NN NG NA NN NN NA NN HD NN (SEQ ID NO: 233)

NS NN NS Nl NS NN NG NS NN NN NS NN HD NN (SEQ ID NO: 234)

NA HN NA Nl NA NN NG NA NN NN NA HN HD NN (SEQ ID NO: 235)

NS HN NS Nl NS NN NG NS NN NN NS HN HD NN (SEQ ID NO: 236)

- Human mitoTALEN left (R1) subunit - combined

NA NA NA Nl NA NH NG NA NH NH NA NA HD NH (SEQ ID NO: 237)

NS NS NS Nl NS NH NG NS NH NH NS NS HD NH (SEQ ID NO: 238)

NA NN NA Nl NA NH NG NA NH NH NA NN HD NH (SEQ ID NO: 239)

NS NN NS Nl NS NH NG NS NH NH NS NN HD NH (SEQ ID NO: 240)

NA HN NA Nl NA NH NG NA NH NH NA HN HD NH (SEQ ID NO: 241)

NS HN NS Nl NS NH NG NS NH NH NS HN HD NH (SEQ ID NO: 242)

- Human mitoTALEN left (R2) subunit -standard code

NG NN Nl Nl HD NN NG Nl NN NN NG NN HD (SEQ ID NO: 243)

- Human mitoTALEN left (R2) subunit - with improved G specificity

NG NH Nl Nl HD NH NG Nl NH NH NG NH HD (SEQ ID NO: 244)

- Human mitoTALEN left (R2) subunit - with promiscuous RVDs

NA NA NA Nl NA NN NG NA NN NN NA NA HD (SEQ ID NO: 245) NS NS NS Nl NS NN NG NS NN NN NS NS HD (SEQ ID NO: 246)

NA NN NA Nl NA NN NG NA NN NN NA NN HD (SEQ ID NO: 247)

NS NN NS Nl NS NN NG NS NN NN NS NN HD (SEQ ID NO: 248)

NA HN NA Nl NA NN NG NA NN NN NA HN HD (SEQ ID NO: 249)

NS HN NS Nl NS NN NG NS NN NN NS HN HD (SEQ ID NO: 250)

- Human mitoTALEN left (R2) subunit - combined

NA NA NA NI NA NH NG NA NH NH NA NA HD (SEQ ID NO: 251)

NS NS NS Nl NS NH NG NS NH NH NS NS HD (SEQ ID NO: 252)

NA NN NA Nl NA NH NG NA NH NH NA NN HD (SEQ ID NO: 253)

NS NN NS Nl NS NH NG NS NH NH NS NN HD (SEQ ID NO: 254)

NA HN NA Nl NA NH NG NA NH NH NA HN HD (SEQ ID NO: 255)

NS HN NS Nl NS NH NG NS NH NH NS HN HD (SEQ ID NO: 256)

Target site 5

- 5’TNiTN2N3GN4CN5N6ACCCCaaaaacaaaaaaccCN7N8N9NioNnNi2Ni3Ni4Ni5CNi6TNi 7N18CCA 3’ (L-14 bp spacer-R) (SEQ ID NO: 329) Target site ranges from m.342 to m.388

- Left subunit (L - 13.5 repeats) binding site: 5’ T-N1TN2N3GN4CN5N6ACCCC 3’ binding site ranges from m.342 to m.356 (SEQ ID NQ:330); N1 at position 1 is C or T (m.343); N2 at position 3 is C, T or A (m.345); N3 at position 4 is T or C (m.346); N4 at position 6 is C, T or G (m.348);Ns at position 8 is A or T (m.350); Ne at position 9 is A or C (m.351)

- Right subunit (R - 16.5 repeats) binding site (reverse complement): 5’ T-

GGN18N17AN16GN15N14N13N12N11N10N9N8N7G 3’ binding site ranges from m.371 to m.388 (SEQ ID NO: 331); N at position 3 is T, C or A (m.385A>G, m.385A>T); N17 at position 4 is T or C (m.384A>G); N at position 6 is G, A or C (m.382C>T, m.382C>G); N15 at position 8 is C, T or G (m.380G>A, m.380G>C); N14 at position 9 is T, C or G (m.379A>G, m.379A>C); N13 at position 10 is G or A (m.378C>T); N12 at position 11 is G or A (m.377C>T); N11 at position 12 is T or A (m.376A>T); N10 at position 13 is G, A or C (m.375C>T, m.375C>G); Ng at position 14 is T, A or C (m.374A>T, m.374A>G); Ns at position 15 is T, C or A (m.373A>G, m.373A>T); N7 at position 16 is A or G (m.372T>C)

Human mitoTALEN subunit RVD sequences

- Human mitoTALEN left subunit - standard code:

HD NG HD NG NN HD HD Nl Nl Nl HD HD HD HD (SEQ ID NO: 332)

- Human mitoTALEN left subunit - with improved G specificity:

HD NG HD NG NH HD HD Nl Nl Nl HD HD HD HD (SEQ ID NO: 333) - Human mitoTALEN left subunit - with promiscuous RVDs:

NA NG NA NA NN NA HD NA NA Nl HD HD HD HD (SEQ ID NO: 334)

NS NG NS NS NN NS HD NS NS Nl HD HD HD HD (SEQ ID NO: 335)

- Human mitoTALEN left subunit - combined:

NA NG NA NA NH NA HD NA NA Nl HD HD HD HD (SEQ ID NO: 336)

NS NG NS NS NH NS HD NS NS Nl HD HD HD HD (SEQ ID NO: 337)

- Human mitoTALEN right subunit - standard code

NN NN NG NG Nl NN NN HD NG NN NN NG NN NG NG Nl NN (SEQ ID NO: 338)

- Human mitoTALEN right subunit - with improved G specificity

NH NH NG NG Nl NH NH HD NG NH NH NG NH NG NG Nl NH (SEQ ID NO: 339)

- Human mitoTALEN right subunit - with promiscuous RVDs

NN NN NA NA Nl NA NN NA NA NA NA NA NA NA NA NA NN (SEQ ID NO: 340)

NN NN NS NS Nl NS NN NS NS NS NS NS NS NS NS NS NN (SEQ ID NO: 341)

NN NN NA NA Nl NA NN NA NA NN NN NA NA NA NA NA NN (SEQ ID NO: 342)

NN NN NS NS Nl NS NN NS NS NN NN NS NS NS NS NS NN (SEQ ID NO: 343)

NN NN NA NA Nl NA NN NA NA HN HN NA NA NA NA NA NN (SEQ ID NO: 344)

NN NN NS NS Nl NS NN NS NS HN HN NS NS NS NS NS NN (SEQ ID NO: 345)

- Human mitoTALEN right subunit -combined

NH NH NA NA N I NA NH NA NA NA NA NA NA NA NA NA NH (SEQ ID NO: 346)

NH NH NS NS N I NS NH NS NS NS NS NS NS NS NS NS NH (SEQ ID NO: 347)

NHNHNANANINANHNANANNNNNANANANANA NH (SEQ ID NO: 348)

NHNHNSNSNINSNHNSNSNNNNNSNSNSNSNS NH (SEQ ID NO: 349)

NHNHNANANINANHNANAHNHNNANANANANA NH (SEQ ID NO: 350)

NHNHNSNSNINSNHNSNSHNHNNSNSNSNSNS NH (SEQ ID NO: 351) eGFP amino acid sequence is presented in SEQ ID NO: 438 and eGFP nucleotide sequence is presented in SEQ ID NO: 445. mCherry amino acid sequence is presented in SEQ ID NO: 458 and mCherry nucleotide sequence is presented in SEQ ID NO: 463.

Fokl mutated sequence S-ELD (Sharkey and obligate heterodimer variant of Fokl) QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNPTQDRILEMKVMEFFMKVYGYRGEHLGG SRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMERYVEENQTRDKHLNPNEWW KVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEV RRKFNNGEINF (SEQ ID NO: 1831)

Fokl mutated sequence S-KKR (Sharkey and obligate heterodimer variant of Fokl)

QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNPTQDRILEMKVMEFFMKVYGYRGEHLGG SRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVKENQTRNKHINPNEWWK VYPSSVTEFKFLFVSGHFKGNYKAQLTRLNRKTNCNGAVLSVEELLIGGEMIKAGTLTLEEV RRKFNNGEINF (SEQ ID NO: 1832)

Bibliography

Alexeyev MF, Venediktova N, Pastukh V, Shokolenko I, Bonilla G, Wilson GL. (2008)

Selective elimination of mutant mitochondrial genomes as therapeutic strategy for the treatment of NARP and MILS syndromes. Gene Ther, 15, 516 - 23.

Ashley N, Harris D, Poulton J. (2005) Detection of mitochondrial DNA depletion in living human cells using PicoGreen staining. Exp Cell Res, 303, 432-46.

Bacman SR, Williams SL, Duan D, Moraes CT. (2012) Manipulation of mtDNA heteroplasmy in all striated muscles of newborn mice by AAV9- mediated delivery of a mitochondria- targeted restriction endonuclease. Gene Ther, 19, 1101-6.

Bacman SR, Williams SL, Hernandez D, Moraes CT. (2007) Modulating mtDNA heteroplasmy by mitochondria-targeted restriction endonucleases in a ‘differential multiple cleavage-site’ model. Gene Ther, 14, 1309-18.

Bacman SR, Williams SL, Garcia S, Moraes CT. (2010) Organ-specific shifts in mtDNA heteroplasmy following systemic delivery of a mitochondria- targeted restriction endonuclease. Gene Ther, 17, 713-20.

Bacman SR, Kauppila JHK, Pereira CV, Nissanka N, Miranda M, Pinto M, et al. (2018)

MitoTALEN reduces mutant mtDNA load and restores tRNAAIa levels in a mouse model of heteroplasmic mtDNA mutation. Nat Med, 24, 1696-1700.

Bacman SR, Williams SL, Moraes CT. (2009) Intra- and inter-molecular recombination of mitochondrial DNA after in vivo induction of multiple double-strand breaks. Nucleic Acids Res, 37, 4218-26.

Bacman SR, Williams SL, Pinto M, Peralta S, Moraes CT. (2013) Specific elimination of mutant mitochondrial genomes in patient-derived cells by mitoTALENs. Nat Med, 19, 1111— 3.

Bayona-Bafaluy MP, Blits B, Battersby BJ, Shoubridge EA, Moraes CT. (2005) Rapid directional shift of mitochondrial DNA heteroplasmy in animal tissues by a mitochondrially targeted restriction endonuclease. Proc Natl Acad Sci U S A, 102, 14392-7. Boch J, Bonas II. (2010) Xanthomonas AvrBs3 Family-Type III Effectors: Discovery and Function. Annu Rev Phytopathol, 48, 419-36.

Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S, et al. (2009) Breaking the code of DNA binding specificity of TAL-type III effectors. Science, 326, 1509-12.

Bohne J, Cathomen T. (2008) Genotoxicity in gene therapy: an account of vector integration and designer nucleases. Curr Opin Mol Ther, 10, 214-23.

Cermak T, Doyle EL, Christian M, Wang L, Zhang Y, Schmidt C, et al. (2011) Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res, 39, e82.

Chinnery PF, DiMauro S, Shanske S, Schon EA, Zeviani M, Mariotti C, et al. (2004) Risk of developing a mitochondrial DNA deletion disorder. Lancet, 364, 592-6.

Cong L, Zhou R, Kuo YC, Cunniff M, Zhang F. (2012) Comprehensive interrogation of natural TALE DNA-binding modules and transcriptional repressor domains. Nat Commun, 3, 968.

Cox DBT, Gootenberg JS, Abudayyeh GO, Franklin B, Kellner MJ, Joung J, et al. (2017) RNA editing with CRISPR-Cas13. Science, 358, 1019-27.

Deng D, Yan C, Pan X, Mahfouz M, Wang J, Zhu JK, et al. (2012) Structural basis for sequence-specific recognition of DNA by TAL effectors. Science, 335, 720-3.

Doyon Y, Vo TD, Mendel MC, Greenberg SG, Wang J, Xia DF, et al. (2011) Enhancing zinc- finger-nuclease activity with improved obligate heterodimeric architectures. Nat Methods, 8, 74-9.

Eichwald V, Daeffler L, Klein M, Rommelaere J, Salome N. (2002) The NS2 proteins of parvovirus minute virus of mice are required for efficient nuclear egress of progeny virions in mouse cells. J Virol, 76, 10307-19.

Fukui H, Moraes CT. (2008) Mechanisms of formation and accumulation of mitochondrial DNA deletions in aging neurons. Hum Mol Genet, 18,1028-36.

Gammage PA, Gaude E, Van Haute L, Rebelo-Guiomar P, Jackson CB, Rorbach J, et al. (2016) Near-complete elimination of mutant mtDNA by iterative or dynamic dose-controlled treatment with mtZFNs. Nucleic Acids Res, 44, 7804-16.

Gammage PA, Rorbach J, Vincent Al, Rebar EJ, Minczuk M. (2014) Mitochondrially targeted ZFNs for selective degradation of pathogenic mitochondrial genomes bearing large-scale deletions or point mutations. EMBO Mol Med, 6, 458-66.

Gammage PA, Viscomi C, Simard ML, Costa ASH, Gaude E, Powell CA, et al. (2018) Genome editing in mitochondria corrects a pathogenic mtDNA mutation in vivo. Nat Med, 24, 1691-95.

Guo J, Gaj T, Barbas CF 3rd. (2010) Directed Evolution of an Enhanced and Highly Efficient Fokl Cleavage Domain for Zinc Finger Nucleases. J Mol Biol, 400, 96-107. Hashimoto M, Bacman SR, Peralta S, Falk MJ, Chomyn A, Chan DC, et al. (2015) MitoTALEN: A General Approach to Reduce Mutant mtDNA Loads and Restore Oxidative Phosphorylation Function in Mitochondrial Diseases. Mol Ther, 23, 1592-9.

Hyslop LA, Blakeley P, Craven L, Richardson J, Fogarty NME, Fragouli E, et al. (2016) Towards clinical application of pronuclear transfer to prevent mitochondrial DNA disease. Nature, 534, 383-6.

Kim H, Kim J-S. (2014) A guide to genome engineering with programmable nucleases. Nat Rev Genet, 15, 321-34.

Kim Y, Kweon J, Kim A, Chon JK, Yoo JY, Kim HJ, et al. (2013) A library of TAL effector nucleases spanning the human genome. Nat Biotechnol, 31 , 251 - 8.

Mak AN, Bradley P, Cernadas RA, Bogdanove AJ, Stoddard BL. (2012) The crystal structure of TAL effector PthXol bound to its DNA target. Science, 335, 716-9.

Marc P, Margeot A, Devaux F, Blugeon C, Corral-Debrinski M, Jacq C. (2002) Genome-wide analysis of mRNAs targeted to yeast mitochondria. EMBO Rep, 3, 159-64.

Mert Yanik M, Alzubi J, Lahaye T, Cathomen T, Pingoud A, Wende W. (2013) TALE-Pvull fusion proteins-novel tools for gene targeting. PLoS One, 8, e82539.

Miller JC, Tan S, Qiao G, Barlow KA, Wang J, Xia DF, et al. (2011) A TALE nuclease architecture for efficient genome editing. Nat Biotechnol, 29, 143-8.

Minczuk M, Papworth MA, Kolasinska P, Murphy MP, Klug A. (2006) Sequence-specific modification of mitochondrial DNA using a chimeric zinc finger methylase. Proc Natl Acad Sci U S A, 103, 19689-94.

Morris J, Na Y-J, Zhu H, Lee J-H, Giang H, Ulyanova AV, et al. (2017) Pervasive within- Mitochondrion Single-Nucleotide Variant Heteroplasmy as Revealed by SingleMitochondrion Sequencing. Cell Rep, 21 , 2706-13.

Moscou MJ, Bogdanove AJ. (2009) A simple cipher governs DNA recognition by TAL effectors. Science, 326, 1501.

Mussolino C, Alzubi J, Fine EJ, Morbitzer R, Cradick TJ, Lahaye T, et al. (2014) TALENs facilitate targeted genome editing in human cells with high specificity and low cytotoxicity. Nucleic Acids Res, 42, 6762-73.

Mussolino C, Morbitzer R, Lutge F, Dannemann N, Lahaye T, Cathomen T. (2011) A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity. Nucleic Acids Res, 39, 9283-93.

Pereira CV, Bacman SR, Arguello T, Zekonyte U, Williams SL, Edgell DR. et al. (2018) mitoTev-TALE: a monomeric DNA editing enzyme to reduce mutant mitochondrial DNA levels. EMBO Mol Med, 10, e8084. Phillips AF, Millet AR, Tigano M, Dubois SM, Crimmins H, Babin L, et al. (2017) SingleMolecule Analysis of mtDNA Replication Uncovers the Basis of the Common Deletion. Mol Cell, 65, 527-38. e6.

Rai, P. K. et al. (2018) ‘Advances in methods for reducing mitochondrial DNA disease by replacing or manipulating the mitochondrial genome’, Essays in Biochemistry, 62(3), pp. 455-465. doi: 10.1042/EBC20170113.

Reddy, P. et al. (2015) ‘Selective elimination of mitochondrial mutations in the germline by genome editing’, Cell, 161(3), pp. 459-469. doi: 10.1016/j. cell.2015.03.051.

Reyon D, Tsai SQ, Khayter C, Foden JA, Sander JD, Joung JK. (2012) FLASH assembly of TALENs for high-throughput genome editing. Nat Biotechnol, 30, 460-5.

Rinaldi FC, Doyle LA, Stoddard BL, Bogdanove AJ. The effect of increasing numbers of repeats on TAL effector DNA binding specificity. Nucleic Acids Res, 45, 6960-70.

Sanjana NE, Cong L, Zhou Y, Cunniff MM, Feng G, Zhang F. (2012) A transcription activator-like effector toolbox for genome engineering. Nature Protoc, 7, 171-92.

Shanske S, Tang Y, Hirano M, Nishigaki Y, Tanji K, Bonilla E, et al. (2002) Identical mitochondrial DNA deletion in a woman with ocular myopathy and in her son with pearson syndrome. Am J Hum Genet, 71 , 679-83.

Srivastava S, Moraes CT. (2001) Manipulating mitochondrial DNA heteroplasmy by a mitochondrially targeted restriction endonuclease. Hum Mol Genet, 10, 3093-9.

Srivastava S, Moraes CT. (2005) Double-strand breaks of mouse muscle mtDNA promote large deletions similar to multiple mtDNA deletions in humans. Hum Mol Genet, 14, 893- 902.

Streubel J, Blucher C, Landgraf A, Boch J. (2012) TAL effector RVD specificities and efficiencies. Nat Biotechnol, 30, 593-5.

Sun N, Bao Z, Xiong X, Zhao H. (2013) SunnyTALEN: A Second-Generation TALEN System for Human Genome Editing. Biotechnol Bioeng, 111, 683-91.

Tanaka M, Borgeld H-J, Zhang J, Muramatsu S, Gong J-S, Yoneda M, et al. (2002) Gene therapy for mitochondrial disease by delivering restriction endonuclease Smal into mitochondria. J Biomed Sci, 9, 534-41.

Yang Y, Wu H, Kang X, Liang Y, Lan T, Li T, et al. (2018) Targeted elimination of mutant mitochondrial DNA in MELAS-iPSCs by mitoTALENs. Protein Cell, 9, 283-97.

Xu H, DeLuca SZ, O’Farrell PH. (2008) Manipulating the Metazoan Mitochondrial Genome with Targeted Restriction Enzymes. Science, 321 , 575-7.

Zekonyte U, Bacman SR, Smith J, Shoop W, Pereira CV, Tomberlin G, et al. (2021) Mitochondrial targeted meganuclease as a platform to eliminate mutant mtDNA in vivo. Nat Commun, 12, 3210. Zhang F, Cong L, Lodato S, Kosuri S, Church GM, Arlotta P. (2011) Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat Biotechnol, 29, 149-53.

Claims

1. A synthetic targeted DNA binding and cleavage complex, or one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex, wherein the synthetic targeted DNA binding and cleavage complex is capable of specifically binding to a target sequence within a conserved region of the mitochondrial DNA control region and cleaving mitochondrial DNA comprising the target sequence.

2. The synthetic targeted DNA binding and cleavage complex, or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex, according to claim 1 wherein the target sequence is a suitable sequence within SEQ ID NO: 403, SEQ ID NO: 404, SEQ ID NO: 1813 or a homolog of the conserved region of mitochondrial DNA in another organism.

3. The synthetic targeted DNA binding and cleavage complex, or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex, according to any preceding claim wherein the target sequence is a sequence having at least 7, more preferably 8, consecutive nucleotides from SEQ ID NQ:403, SEQ ID NO: 404; SEQ ID NO: 1813 or a homolog of the conserved region of the mitochondrial DNA in another organism.

4. The synthetic targeted DNA binding and cleavage complex, or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex, according to any preceding claim wherein the target sequence is a sequence having at least 14 consecutive nucleotides, more preferably at least 16, from SEQ ID NQ:403, SEQ ID NO: 404, SEQ ID NO: 1813 or a homolog of the conserved region of the mitochondrial DNA in another organism.

5. The synthetic targeted DNA binding and cleavage complex, or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex, according to any preceding claim wherein the target sequence consists or comprises of any one of SEQ ID NO: 376-378, 1-7, 86-90, 153-155, 196-208, 329-331 , 464.

6. The synthetic targeted DNA binding and cleavage complex, or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex, according to any preceding claim wherein the synthetic targeted DNA binding and cleavage complex comprises at least one DNA binding module and at least one DNA cleavage module.

7. The synthetic targeted DNA binding and cleavage complex or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to claim 6 wherein the at least one DNA binding module is linked to the at least one DNA cleavage module.

8. The synthetic targeted DNA binding and cleavage complex or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to claim 7 wherein one nucleic acid sequence encodes for the at least one DNA binding module linked to the at least one DNA cleavage module.

9. The synthetic targeted DNA binding and cleavage complex or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to claim 7 or 8 wherein the at least one DNA binding module comprises at least one Transcription Activator- Like Effector (TALE) domain or at least one zinc finger domain.

10. The synthetic targeted DNA binding and cleavage complex or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to claim 9 wherein the at least one TALE domain comprises RVDs according to any one of SEQ ID NOs:379-380, 8-9, 14-15, 20-21, 24-25, 91-92, 97-98, 103-104, 156-157, 160-161 , 209-210, 215-216, 221-222, 225-226, 229-230, 243-244, 332-333, 338-339

11. The synthetic targeted DNA binding and cleavage complex or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to claim 9 or 10 wherein the at least one TALE domain comprises amino acid sequence according to any one of SEQ ID NO: 383-384, 38-41 , 117-119, 172-173, 257, 259-263, 352-353, 466, 506, 545, 569, 647, 707, 863, 922, 958, 1090, 1150, 1210, 1246, 1282, 1438, 1595, 1657 or is encoded by a nucleotide sequence according to any one of SEQ ID NO: 385-386, 42-45, 120-122, 174-175, 264-269, 354-355, 471, 511, 548, 582, 652, 720, 868, 925, 969, 1095, 1155, 1213, 1249, 1295, 1451, 1600, 1670

12. The synthetic targeted DNA binding and cleavage complex or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to claim 9 wherein the at least one TALE domain is adapted to recognise all four nucleotides at one or more specific locations within the target sequence.

13. The synthetic targeted DNA binding and cleavage complex or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to claim 12 wherein the at least one TALE domain comprises NA and/or NS RVDs.

14. The synthetic targeted DNA binding and cleavage complex or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to any one of claim 12 or 13 wherein the at least one TALE domain comprises RVDs according to any one of SEQ ID NOs: 381-382, 430-431, 10-13, 16-19, 22-23, 26-37, 93-96, 99-102, 105-116, 158-159, 162- 171 , 211-214, 217-220, 223-224, 227-228, 231-242, 245-256, 334-337, 340-351.

15. The synthetic targeted DNA binding and cleavage complex or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to any one of claim 12-14 wherein the at least one TALE domain comprises a sequence according to any one of SEQ ID NO: 467-468, 507-510, 546-547, 570-571 , 648-649, 864-865, 708-709, 923-924, 959- 960, 1091-1092, 1151-1152, 1211-1212, 1247-1248, 1283-1284, 1439-1440, 1596-1597, 1658-1659, , 469, 470, 472-475, ,, 512-515, 549-550, 572-581 , 583-594, 650-651 , 653-656, 710-719, 721-732, 866-867, 869-872, 926-927, 961-968, 970-979, 1093-1094, 1096-1099, 1153-1154, 1156-1159, 1214-1215, 1250-1251 , 1285-1294, 1296-1307, 1441-1450, 1452- 1463, 1598-1599, 1601-1604, 1660-1667, 1812, 1669, 1668, 1671-1682, 1814, 1815, 1816, 1817, 1818-1821.

16. The synthetic targeted DNA binding and cleavage complex or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to any one of claims 6 to 15 wherein the DNA cleavage module is configured to create one or more single-stranded breaks (SSBs) or double stranded breaks (DSBs) in the mitochondrial DNA comprising the target sequence.

17. The synthetic targeted DNA binding and cleavage complex or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to any one of claims 9 to 16 wherein the synthetic targeted DNA binding and cleavage complex comprises or consists of a first TALE domain linked to a first DNA cleavage module subunit and a second TALE domain linked to a second DNA cleavage module subunit.

18. The synthetic targeted DNA binding and cleavage complex or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to claim 17 wherein the DNA cleavage module comprises a dimer of the first and the second DNA cleavage module subunits and wherein the DNA cleavage module is configured to create one or more single-stranded breaks (SSBs) or double stranded breaks (DSBs) in the mitochondrial DNA comprising the target sequence upon dimerization of the first and the second DNA cleavage module subunits.

19. The synthetic targeted DNA binding and cleavage complex or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to claim 17 or 18 wherein the target sequence of the first TALE domain linked to the first DNA cleavage module subunit is the same as the target sequence of the second TALE domain linked to the second DNA cleavage module subunit (homodimer).

20. The synthetic targeted DNA binding and cleavage complex or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to claim 17 or 18 wherein the target sequence of the first TALE domain linked to the first DNA cleavage domain subunit is different to the target sequence of the second TALE domain linked to the second DNA cleavage module subunit (heterodimer).

21. The synthetic targeted DNA binding and cleavage complex or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to claim 20 wherein the target sequence of the first TALE domain linked to the first DNA cleavage domain subunit is spaced 13 to 23, suitably 13 to 17, suitably 14-16, suitably 14 or 16 nucleotides away from the target sequence of the second TALE domain linked to the second DNA cleavage module subunit.

22. The synthetic targeted DNA binding and cleavage complex or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to any one of claims 18 to 22 wherein the first DNA cleavage module subunits comprises of a Fokl nuclease subunit and the second DNA cleavage module subunits comprises of a Fokl nuclease subunit.

23. The synthetic targeted DNA binding and cleavage complex or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to claim 22 wherein the Fokl nuclease subunit comprises amino acid sequence according to SEQ ID NO: 437 or a nucleotide sequence according to SEQ ID NO: 443.

24. The synthetic targeted DNA binding and cleavage complex or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to any one of claims 20 to 21 the first and second DNA cleavage module subunits comprise of two heterodimeric Fokl nuclease subunits.

25. The synthetic targeted DNA binding and cleavage complex or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to any claim 24 wherein the two heterodimeric Fokl nuclease subunits comprise sequences according to SEQ ID NO: 1831 and SEQ ID NO:1832.

26. The synthetic targeted DNA binding and cleavage complex, or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex, according to claim 4 wherein the synthetic targeted DNA binding and cleavage complex comprises at least one DNA binding module and at least one DNA cleavage module.

27. The synthetic targeted DNA binding and cleavage complex, or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex, according to claim 26 wherein the DNA binding module and the DNA cleavage module are found within different molecules.

28. The synthetic targeted DNA binding and cleavage complex, or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to claim 27 wherein the DNA binding module is guide ribonucleic acid (gRNA) and the DNA cleavage domain is CRISPR-Cas, suitably Cas9.

29. The synthetic targeted DNA binding and cleavage complex, or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex, according to any preceding claim wherein the synthetic targeted DNA binding and cleavage complex further comprises at least one mitochondrial targeting signal.

30. The synthetic targeted DNA binding and cleavage complex, or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex, according to claim 29 wherein the at least one mitochondrial targeting signal comprises amino acid sequence according to any out of SEQ ID NO: 434, 435, 447, 448, 451, 452 or a nucleotide sequence according to any one of SEQ ID NO:.440, 441 , 455, 456, 460, 461.

31. The synthetic targeted DNA binding and cleavage complex, or one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex, according to any one of claims 29 to 30 wherein the synthetic targeted DNA binding and cleavage complex comprises dual tandem mitochondrial targeting signal.

32. The synthetic targeted DNA binding and cleavage complex, or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex, according to any preceding claim, wherein the synthetic targeted DNA binding and cleavage complex further comprises at least one nuclear export signal.

33. The synthetic targeted DNA binding and cleavage complex, or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex, according to claim 32 wherein the at least one nuclear export signal comprises amino acid sequence according to any out of SEQ ID NO: 465 or 453 or a nucleotide sequence according to any one of SEQ ID NO: 446.

34. The synthetic targeted DNA binding and cleavage complex, or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex, according to claim 32 or 33 wherein the synthetic targeted DNA binding and cleavage complex comprises dual tandem nuclear export signals.

35. The synthetic targeted DNA binding and cleavage complex, or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex, according to any preceding claim wherein the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex is RNA, suitably mRNA.

36. The synthetic targeted DNA binding and cleavage complex, or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex, according to any one of claims 1-34 wherein the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex is DNA.

37. The one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according any preceding claim wherein the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex further comprise a 5’ UTR and/or a 3’ UTR.

38. The one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to claim 37 wherein the 5’ UTR has a nucleotide sequence according to SEQ ID NO: 439, 454, 459 and the 3’ UTR has a sequence according to SEQ ID NO: 444, 457, 462.

39. The synthetic targeted DNA binding and cleavage complex according to claim 1 wherein the synthetic targeted DNA binding and cleavage complex comprises or consists of any one of the sequences according to SEQ ID NO: 387-394, 46-57, 70-77, 123-131 , 141- 146, 176-181 , 188-191, 270-287, 306-313, 314-316, 356-361 , 368-371 , 258, 476-490, 516- 526, 1830, 527-529, 551-559, 595-607, 657-671 , 687-696, 733-771 , 811-836, 873-887, 903- 912, 928-936, 946-951, 980-1012, 1046-1067, 1100-1114, 1130-1139, 1160-1174, 1190- 1199, 1216-1224, 1234-1239, 1252-1260, 1270-1275, 1308-1346, 1386-1411, 1464-1502, 1542-1567, 1580, 1605-1619, 1637-1646, 1683-1709, 1710-1720, 1760-1767, 1768, 1769- 1785, 1822-1825, 1854-1879, 1880-1931.

40. The one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to claim 1 wherein the nucleic acid sequence encoding a synthetic targeted DNA binding and cleavage complex comprise or consist of any one of SEQ ID NO: 395-402, 58-69, 78-85, 132-140, 147-152, 182-187, 192-195, 288-305, 317- 328, 362-367, 372-375, 491-505, 530-544, 560-568, 608-646, 672-686, 697-706, 772-810, 837-862, 888-902, 913-921 , 1834, 937-945, 952-957, 1013-1045, 1068-1089, 1115-1129, 1140-1149, 1175-1189, 1200-1209, 1225-1233, 1240-1245, 1261-1269, 1276-1281 , 1347- 1385, 1412-1437, 1503-1541 , 1568-1579, 1581-1594, 1620-1622, 1625, 1626-1636, 1647- 1656, 1721-1755, 1756, 1757-1759, 1786-1811 , 1826-1829, 1932-1983.

41. A vector comprising the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to any one of claims 1 to 38, 40.

42. A cell comprising a vector according to claim 41.

43. A pharmaceutical composition comprising one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to any one of claims 1 to 38, 40 or a vector according to claim 41 and optionally a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier.

44. One or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to any one of claims 1 to 38, 40, a vector according to claim 38 or, a pharmaceutical composition according to claim 43 for use in medicine.

45. In vitro method of reducing or eliminating mitochondrial DNA in a cell or a sample comprising the step of administering to the cell or the sample:

- a synthetic targeted DNA binding and cleavage complex according to any one of claims 1 to 39; - one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to any one of claims 1-38 or 40;

- a vector according to claim 41 ; or

- a pharmaceutical composition according to claim 43.

46. The method of claim 45 wherein the mitochondrial DNA comprises one or more mutations associated with mitochondrial disease.

47. The method of any one of claims 45 or 46 wherein the cell is an oocyte or a zygote.

48. The method of any one of claims 45 to 47 further comprising the step of incubating the cell or sample with the synthetic targeted DNA binding and cleavage complex, the one or more nucleic acid sequences encoding the synthetic targeted DNA binding and cleavage complex, the vector, or the pharmaceutical composition under suitable conditions for at least thirty minutes.

49. The method of claim 48 further comprising the step of reducing or eliminating the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex from the cell or sample after incubation.

50. The method of claim 49 wherein the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex are RNA, suitably mRNA.

51.The method of claim 50 wherein the one or more RNA sequences encoding a synthetic targeted DNA binding and cleavage complex are reduced or eliminated by addition of guide RNA targeting the RNA sequences encoding the synthetic targeted DNA binding and cleavage complex, and class 2 RNA-targeting CRISPR-Cas system such as Cas13b to the cell or the sample.

52. The method of claim 51 further comprising the step of incubating the guide RNA and class 2 RNA-targeting CRISPR-Cas system with the one or more RNA sequences encoding the synthetic targeted DNA binding and cleavage complex under suitable conditions for at least 2 hours.

53. A method of reducing the likelihood of passing parental mitochondrial DNA to an offspring during assisted reproductive technologies comprising administering to the parental zygote one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to any of one of claims 1-38 or 40 before pro-nuclear transfer.

54. The method of claim 53 wherein administration is done by microinjection of the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex into the parental zygote.

55. The method of claim 53 or 54 wherein one or more nucleic acid sequences encoding the synthetic targeted DNA binding and cleavage complex is mRNA.

56. The method of claim 55 further comprising reducing or eliminating the one or more mRNA sequences encoding the synthetic targeted DNA binding and cleavage complex before pro nuclear transfer.

57. The method of claim 56 wherein the one or more mRNA sequences encoding the synthetic targeted DNA binding and cleavage complex are reduced by addition of guide RNA targeting the mRNA sequences encoding the synthetic targeted DNA binding and cleavage complex and class 2 RNA-targeting CRISPR-Cas system such as Cas13b to the parental zygote.

58. Use of the synthetic targeted DNA binding and cleavage complex according to any one of claims 1 to 39 or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to any one of claims 1-38 or 40 for reducing or eliminating mitochondrial DNA.

59. Use of the synthetic targeted DNA binding and cleavage complex according to any one of claims 1 to 39 or the one or more nucleic acid sequences encoding a synthetic targeted DNA binding and cleavage complex according to any one of claims 1-38 or 40 for reducing the likelihood of passing parental mitochondrial DNA to an offspring during assisted reproductive technologies or for mitochondrial DNA replacement therapy.