Engineered meganucleases having specificity for recognition sequences in the c9ORF72 gene

Abstract

The present disclosure encompasses engineered meganucleases that bind and cleave recognition sequences within a C9Orf72 gene. The present disclosure also encompasses methods of using such engineered meganucleases to make genetically modified cells. Further, the disclosure encompasses pharmaceutical compositions comprising engineered meganuclease proteins, or polynucleotides encoding engineered meganucleases of the disclosure, and the use of such compositions for the modification of a C9Orf72 gene in a subject, or for treatment of amyotrophic lateral sclerosis (ALS) and / or frontotemporal dementia (FTD).

Description

[0001] ENGINEERED MEGANUCLEASES HAVING SPECIFICITY FOR RECOGNITION

[0002] SEQUENCES IN THE C9ORF72 GENE

[0003] FIELD OF THE INVENTION

[0004] The application relates to the field of engineered meganucleases, molecular biology and recombinant nucleic acid technology. In particular aspects, the invention relates to engineered meganucleases useful for the removal of non-coding nucleotide sequences from the C90rf72 gene and for the treatment of subjects having a neurological disorder such as amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD).

[0005] REFERENCE TO A SEQUENCE LISTING SUBMITTED AS AN XML ST.26 FILE

[0006] The instant application contains a Sequence Listing which has been submitted in XML ST.26 format and is hereby incorporated by reference in its entirety. Said XML ST.26 copy, created on July 31, 2025, is named P89339_2060WO_ST.26.txt, and is 212 kb in size.

[0007] BACKGROUND OF THE INVENTION

[0008] Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized clinically by progressive paralysis leading to death from respiratory failure, typically within two to three years of symptom onset (Rowland and Shneider, N. Engl. J. Med., 2001, 344, 1688-1700). ALS is the third most common neurodegenerative disease in the Western world (Hintz et al., Neurology, 2007, 68, 326-337), and there are currently no effective therapies. Approximately 10% of cases are familial in nature, whereas the bulk of patients diagnosed with the disease are classified as sporadic as they appear to occur randomly throughout the population (Chio et al., Neurology, 2008, 70, 533-537). There is growing recognition, based on clinical, genetic, and epidemiological data, that ALS and frontotemporal dementia (FTD) represent an overlapping continuum of disease, characterized pathologically by the presence of TDP-43 positive inclusions throughout the central nervous system (Lillo and Hodges, J. Clin. Neurosci., 2009, 16, 1131-1135; Neumann et al., Science, 2006, 314, 130-133).

[0009] To date, a number of genes have been discovered as causative for classical familial ALS, for example, SOD1, TARDBP, FUS, OPTN, and VCP (Johnson et al., Neuron, 2010, 68, 857-864; Kwiatkowski et al., Science, 2009, 323, 1205-1208; Maruyama et al., Nature, 2010, 465, 223-226; Rosen et al., Nature, 1993, 362, 59-62; Sreedharan et al., Science, 2008, 319, 1668-1672; Vance et al., Brain, 2009, 129, 868-876). Recently, linkage analysis of kindreds involving multiple cases of ALS, FTD, and ALS-FTD had suggested that there was an important locus for the disease on the short arm of chromosome 9 (Boxer et al., J. Neurol. Neurosurg. Psychiatry, 2011, 82, 196-203; Morita et al., Neurology, 2006, 66, 839-844; Pearson et al. J. Nerol., 2011, 258, 647-655; Vance et al., Brain, 2006, 129, 868-876). The chromosome 9p21ALS-FTD locus in the last major autosomal- dominant gene whose mutation is causative of ALS. The ALS-FTD causing mutation is a large hexanucleotide (GGGGCC) repeat expansion in the first intron of the C90rf72 gene (Renton et al., Neuron, 2011, 72, 257-268; De Jesus-Hernandez et al., Neuron, 2011, 72, 245-256). A founder haplotype, covering the C90rf72 gene, is present in the majority of cases linked to this region (Renton et al., Neuron, 2011, 72, 257-268). This locus on chromosome 9p21 accounts for nearly half of familial ALS and nearly one-quarter of all ALS cases in a cohort of 405 Finnish patients (Laaksovirta et al, Lancet Neurol., 2010, 9, 978-985). A founder haplotype, covering the C90rf72 gene, is present in the majority of cases linked to this region. There are currently no effective therapies to treat such neurodegenerative diseases. Therefore, it is an object to provide compositions and methods for the treatment of such neurodegenerative diseases stemming from hexanucleotide repeat expansions within the C90rf72 gene.

[0010] The present disclosure provides compositions and methods for treatment of neurodegenerative disease, such as ALS and FTD. The invention is a permanent treatment for ALS and FTD that involves the excision of specific non-coding sequence (i.e., hexanucleotide repeat region) located 5’ to the C90rf72 coding sequence using a pair of engineered, site-specific homing endonucleases, often referred to as meganucleases. By targeting a pair of such endonucleases to the hexanucleotide repeat region in the C90rf72 gene, it is possible to permanently remove the intervening fragment from the genome. The resulting cell, and its progeny, will express a modified C90rf72 RNA in which a portion of the non-coding sequence comprising the sequence susceptible to mutation (i.e., hexanucleotide repeat expansion) that is associated with disease is removed.

[0011] Homing endonucleases, or meganucleases, are a group of naturally-occurring nucleases that recognize 15-40 base-pair cleavage sites commonly found in the genomes of plants and fungi. They are frequently associated with parasitic DNA elements, such as group 1 self-splicing introns and inteins. They naturally promote homologous recombination or gene insertion at specific locations in the host genome by producing a double- stranded break in the chromosome, which recruits the cellular DNA-repair machinery (Stoddard (2006) Q. Rev. Biophys. 38:49-95). Homing endonucleases are commonly grouped into four families: the LAGLID ADG family, the GIY-YIG family, the His-Cys box family and the HNH family. These families are characterized by structural motifs, which affect catalytic activity and recognition sequence. For instance, members of the LAGLID ADG family are characterized by having either one or two copies of the conserved LAGLID ADG motif (see, Chevalier et al. (2001) Nucleic Acids Res. 29:3757-74). The LAGLID ADG homing endonucleases with a single copy of the LAGLID ADG motif form homodimers, whereas members with two copies of the LAGLID ADG motif are found as monomers.

[0012] I-Crel (SEQ ID NO: 1) is a member of the LAGLID ADG family of homing endonucleases that recognizes and cuts a 22 basepair recognition sequence in the chloroplast chromosome of the algae Chlamydomonas reinhardtii . Genetic selection techniques have been used to modify the wild-type I-Crel cleavage site preference (Sussman et al. (2004) J. Mol. Biol. 342:31-41; Chames et al. (2005) Nucleic Acids Res. 33:el78; Seligman et al. (2002) Nucleic Acids Res. 30:3870-79, Amould et al. (2006) J. Mol. Biol. 355:443-58). Methods of rationally-designing mono- LAGLIDADG homing endonucleases have been described which are capable of comprehensively redesigning I-Crel and other homing endonucleases to target widely-divergent DNA sites, including sites in mammalian, yeast, plant, bacterial, and viral genomes (WO 2007 / 047859).

[0013] As first described in WO 2009 / 059195, I-Crel and its engineered derivatives are normally dimeric but can be fused into a single polypeptide using a short peptide linker that joins the C- terminus of a first subunit to the N-terminus of a second subunit (Li et al. (2009) Nucleic Acids Res. 37: 1650-62; Grizot et a / . (2009) Nucleic Acids Res. 37:5405-19). Thus, a functional “single-chain” meganuclease can be expressed from a single transcript. By delivering genes encoding two different single-chain meganucleases to the same cell, it is possible to simultaneously cut two different sites. This, coupled with the extremely low frequency of off-target cutting observed with engineered meganucleases makes them the preferred endonuclease for the present disclosure.

[0014] SUMMARY OF THE INVENTION

[0015] The present disclosure provides engineered meganucleases that bind and cleave recognition sequences in a C90rf72 gene, as well as compositions comprising such engineered meganucleases and methods of their use. The recognition sequences targeted by the disclosed engineered meganucleases are selected to have identical four base pair center sequences, such that the first and second cleavage sites will have complementary four base pair 3 ’ overhangs that can perfectly ligase to one another (i.e., each base pair of one overhang pairs with its complement on the other overhang).

[0016] Thus, in one aspect the invention provides an engineered meganuclease that binds and cleaves a recognition sequence comprising SEQ ID NO: 3 within a C90rf72 gene, wherein said engineered meganuclease comprises a first subunit and a second subunit, wherein said first subunit binds to a first recognition half-site of said recognition sequence and comprises a first hypervariable (HVR1) region, and wherein said second subunit binds to a second recognition halfsite of said recognition sequence and comprises a second hypervariable (HVR2) region.

[0017] In some such embodiments, the HVR1 region comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of any one of SEQ ID NOs: 7-18.

[0018] In some embodiments, the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any one of SEQ ID NOs: 7- 18. In some embodiments, the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any one of SEQ ID NOs: 7-18.

[0019] In some embodiments, the HVR1 region comprises a residue corresponding to residue 29 of any one of SEQ ID NOs: 7-11. In some embodiments, the HVR1 region comprises a residue corresponding to residue 41 of any one of SEQ ID NOs: 7-11. In some embodiments, the HVR1 region comprises a residue corresponding to residue 48 of SEQ ID NO: 18. In some embodiments, the HVR1 region comprises a residue corresponding to residue 50 of any one of SEQ ID NOs: 7- 11. In some embodiments, the HVR1 region comprises a residue corresponding to residue 59 of any one of SEQ ID NOs: 7-11. In some embodiments, the HVR1 region comprises a residue corresponding to residue 64 of any one of SEQ ID NOs: 7-11 or 9-17. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of any one of SEQ ID NOs: 7-11. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of any one of SEQ ID NOs: 7-11.

[0020] In some embodiments, the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of any one of SEQ ID NOs: 7-18. In some embodiments, the HVR1 region comprises residues 24-79 of any one of SEQ ID NOs: 7-18.

[0021] In some embodiments, the HVR1 region comprises residues 24-79 of any one of SEQ ID NOs: 7-18 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of any one of SEQ ID NOs: 7-18.

[0022] In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of any one of SEQ ID NOs: 7-18. In some embodiments, the first subunit is an N-terminal subunit that comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of any one of SEQ ID NOs: 7-18. In some embodiments, the first subunit is an N-terminal subunit that comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 6- 153 of any one of SEQ ID NOs: 7-18. In some embodiments, the first subunit is an N-terminal subunit that comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 5-153 of any one of SEQ ID NOs: 7-18. In some embodiments, the first subunit is an N-terminal subunit that comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 4-153 of any one of SEQ ID NOs: 7-18. In some embodiments, the first subunit is an N-terminal subunit that comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 3-153 of any one of SEQ ID NOs: 7-18. In some embodiments, the first subunit is an N-terminal subunit that comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-153 of any one of SEQ ID NOs: 7-18. In some embodiments, the first subunit is an N-terminal subunit and the residue at position 1 of any one of SEQ ID NOs: 7-18 is modified from M to another amino acid. In some embodiments, the residue at position 1 is modified from M to A. In some embodiments, the first subunit is an N-terminal subunits that comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of any one of SEQ ID NOs: 7-18. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 to SEQ ID NO: 195.

[0023] In some embodiments, the first subunit comprises a residue corresponding to residue 19 of any one of SEQ ID NOs: 7-18. In some embodiments, the first subunit comprises a residue corresponding to residue 80 of any one of SEQ ID NOs: 8-12, 14, or 1418. In some embodiments, the first subunit comprises a residue corresponding to residue 139 of SEQ ID NO: 18. In some embodiments, the first subunit comprises a residue corresponding to residue 142 of SEQ ID NO: 9. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of any one of SEQ ID NOs: 7-18. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of any one of SEQ ID NOs: 7-18.

[0024] In some embodiments, the first subunit is an N-terminal subunit that comprises residues 7- subunit that comprises residues 6-153 of any one of SEQ ID NOs: 7-18. In some embodiments, the first subunit is an N-terminal subunit that comprises residues 5-153 of any one of SEQ ID NOs: 7- 18. In some embodiments, the first subunit is an N-terminal subunit that comprises residues 4-153 of any one of SEQ ID NOs: 7-18. In some embodiments, the first subunit is an N-terminal subunit that comprises residues 3-153 of any one of SEQ ID NOs: 7-18. In some embodiments, the first subunit is an N-terminal subunit that comprises residues 2-153 of any one of SEQ ID NOs: 7-18. In some embodiments, the first subunit is an N-terminal subunit that comprises residues 1-153 of any one of SEQ ID NOs: 7-18. In some embodiments, the first subunit comprises residues 1-153 of any one of SEQ ID NOs: 7-18.

[0025] In some embodiments, the first subunit comprises residues 7-153 of any one of SEQ ID NOs: 7-18 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 1-153 of any one of SEQ ID NOs: 7-18.

[0026] In some such embodiments, the HVR2 region comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of any one of SEQ ID NOs: 7-18.

[0027] In some embodiments, the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of any one of SEQ ID NOs: 7-18. In some embodiments, the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of any one of SEQ ID NOs: 7-18.

[0028] In some embodiments, the HVR2 region comprises a residue corresponding to residue 241 of any one of SEQ ID NOs: 7-18. In some embodiments, the HVR2 region comprises a residue corresponding to residue 258 of SEQ ID NO: 8. In some embodiments, the HVR2 region comprises a residue corresponding to residue 263 of any one of SEQ ID NOs: 7-18. In some embodiments, the HVR2 region comprises a residue corresponding to residue 265 of SEQ ID NO: 18. In some embodiments, the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of any one of SEQ ID NOs: 7-18.

[0029] In some embodiments, the HVR2 region comprises residues 215-270 of any one of SEQ ID NOs: 7-18. In some embodiments, the HVR2 region comprises residues 215-270 of any one of SEQ ID NOs: 7-18 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions.

[0030] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of any one of SEQ ID NOs: 7-18. In some embodiments, the second subunit is a C-terminal subunit that comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of any one of SEQ ID NOs: 7-18.

[0031] In some embodiments, the second subunit comprises a residue corresponding to residue 271 of any one of SEQ ID NOs: 8, 10, or 12-14. In some embodiments, the second subunit comprises a residue corresponding to residue 330 of any one of SEQ ID NOs: 7, 10-12, 14, or 18. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of any one of SEQ ID NOs: 7-18. In some embodiments, the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of any one of SEQ ID NOs: 7-18.

[0032] In some embodiments, the second subunit comprises residues 198-344 of any one of SEQ ID NOs: 7-18. In some embodiments, the second subunit comprises residues 198-344 of any one of SEQ ID NOs: 7-18 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 196-354 of any one of SEQ ID NOs: 7-18. In some embodiments, the second subunit comprises residues 196-354 of any one of SEQ ID NOs: 7-18 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions.

[0033] In some embodiments, the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins the first subunit and the second subunit. In some embodiments, the linker comprises an amino acid sequence according to SEQ ID NO: 196.

[0034] In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs: 7-18. In some embodiments, the engineered meganuclease comprises an amino acid sequence of any one of SEQ ID NOs: 7-18.

[0035] In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-354 of any one of SEQ ID NOs: 7-18. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 6-354 of any one of SEQ ID NOs: 7-18. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 5-354 of any one of SEQ ID NOs: 7-18. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 4-354 of any one of SEQ ID NOs: 7-18. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 3-354 of any one of SEQ ID NOs: 7-18. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of any one of SEQ ID NOs: 7-18.

[0036] In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 195. In some embodiments, the engineered meganuclease comprises an amino acid sequence according to SEQ ID NO: 195.

[0037] In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 67-78. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence of any one of SEQ ID NOs: 67-78.

[0038] In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 7. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 7. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 7. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 7.

[0039] In some embodiments, the engineered meganuclease comprises a nuclear localization signal. In some embodiments, the nuclear localization signal is at the N-terminus of the engineered meganuclease. In some embodiments, the nuclear localization signal is at the C-terminus of the engineered meganuclease. In some embodiments, the nuclear localization signal comprises an amino acid sequence having at least 80% or at least 90% sequence identity to SEQ ID NO: 133 or 134. In some embodiments, the nuclear localization signal comprises SEQ ID NO: 133 or 134. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs: 145-148. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence of any one of SEQ ID NOs: 145-148.

[0040] In another aspect, provided herein is an engineered meganuclease that binds and cleaves a recognition sequence comprising SEQ ID NO: 5 within a C90rf72 gene, wherein the engineered meganuclease comprises a first subunit and a second subunit, wherein the first subunit binds to a first recognition half-site of the recognition sequence and comprises a first hypervariable (HVR1) region, and wherein the second subunit binds to a second recognition half-site of the recognition sequence and comprises a second hypervariable (HVR2) region.

[0041] In some such embodiments, the HVR1 region comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of any one of SEQ ID NOs: 79-87.

[0042] In some embodiments, the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any one of SEQ ID NOs: 79-87. In some embodiments, the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any one of SEQ ID NOs: 79-87.

[0043] In some embodiments the HVR1 region comprises a residue corresponding to residue 50 of any one of SEQ ID NOs: 79-87. In some embodiments the HVR1 region comprises a residue corresponding to residue 59 of any one of SEQ ID NOs: 79-83 or 85. In some embodiments the HVR1 region comprises a residue corresponding to residue 72 of any one of SEQ ID NOs: 79-87. In some embodiments the HVR1 region comprises a residue corresponding to residue 73 of any one of SEQ ID NOs: 79-87. In some embodiments the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of any one of SEQ ID NOs: 79-87.

[0044] In some embodiments, the HVR1 region comprises 24-79 of any one of SEQ ID NOs: 79- 87. In some embodiments, the HVR1 region comprises residues 24-79 of any one of SEQ ID NOs: 79-87 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions.

[0045] In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of any one of SEQ ID NOs: 79-87. In some embodiments, the first subunit is an N-terminal subunit that comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of any one of SEQ ID NOs: 79-87. In some embodiments, the first subunit is an N-terminal subunit that comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 6-153 of any one of SEQ ID NOs: 79-87. In some embodiments, the first subunit is an N- terminal subunit that comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 5-153 of any one of SEQ ID NOs: 79-87. In some embodiments, the first subunit is an N-terminal subunit that comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 4-153 of any one of SEQ ID NOs: 79-87. In some embodiments, the first subunit is an N-terminal subunit that comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 3-153 of any one of SEQ ID NOs: 79-87. In some embodiments, the first subunit is an N-terminal subunit that comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-153 of any one of SEQ ID NOs: 79-87. In some embodiments, the first subunit is an N- terminal subunit and the residue at position 1 of any one of SEQ ID NOs: 79-87 is modified from M to another amino acid. In some embodiments, the residue at position 1 is modified from M to A. In some embodiments, the first subunit is an N-terminal subunits that comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of any one of SEQ ID NOs: 79-87.

[0046] In some embodiments, the first subunit comprises a residue corresponding to residue 19 of any one of SEQ ID NOs: 79-87. In some embodiments, the first subunit comprises a residue corresponding to residue 80 of any one of SEQ ID NOs: 81, 82, 85, or 87. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of any one of SEQ ID NOs: 79-87. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of any one of SEQ ID NOs: 79-87.

[0047] In some embodiments, the first subunit comprises residues 7-153 of any one of SEQ ID NOs: 79-87. In some embodiments, the first subunit comprises residues 7-153 of any one of SEQ ID NOs: 79-87 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions.

[0048] In some embodiments, the first subunit is an N-terminal subunit that comprises residues 7- 153 of any one of SEQ ID NOs: 79-87. In some embodiments, the first subunit is an N-terminal subunit that comprises residues 6-153 of any one of SEQ ID NOs: 79-87. In some embodiments, the first subunit is an N-terminal subunit that comprises residues 5-153 of any one of SEQ ID NOs: 79-87. In some embodiments, the first subunit is an N-terminal subunit that comprises residues 4- 153 of any one of SEQ ID NOs: 79-87. In some embodiments, the first subunit is an N-terminal subunit that comprises residues 3-153 of any one of SEQ ID NOs: 79-87. In some embodiments, the first subunit is an N-terminal subunit that comprises residues 2-153 of any one of SEQ ID NOs: 79-87. In some embodiments, the first subunit is an N-terminal subunit that comprises residues 1- 153 of any one of SEQ ID NOs: 79-87. In some embodiments, the first subunit comprises residues 1-153 of any one of SEQ ID NOs: 79-87.

[0049] In some such embodiments, the HVR2 region comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of any one of SEQ ID NOs: 79-87.

[0050] In some embodiments, the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of any one of SEQ ID NOs: 79-87. In some embodiments, the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of any one of SEQ ID NOs: 79-87.

[0051] In some embodiments, the HVR2 region comprises a residue corresponding to residue 241 of any one of SEQ ID NOs: 79-87. In some embodiments, the HVR2 region comprises a residue corresponding to residue 263 of any one of SEQ ID NOs: 79-87. In some embodiments, the HVR2 region comprises a residue corresponding to residue 264 of any one of SEQ ID NOs: 79-87. In some embodiments, the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of any one of SEQ ID NOs: 79-87.

[0052] In some embodiments, the HVR2 region comprises residues 215-270 of any one of SEQ ID NOs: 79-87. In some embodiments, the HVR2 region comprises residues 215-270 of any one of SEQ ID NOs: 79-87 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions.

[0053] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of any one of SEQ ID NOs: 79-87. In some embodiments, the second subunit is a C-terminal subunit that comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more sequence identity to residues 196-354 of any one of SEQ ID NOs: 79-87.

[0054] In some embodiments, the second subunit comprises a residue corresponding to residue 271 of any one of SEQ ID NOs: 79, 80, 81, 83, 84, or 87. In some embodiments, the second subunit comprises a residue corresponding to residue 330 of SEQ ID NO: 81 or 86. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of any one of SEQ ID NOs: 79-87. In some embodiments, the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of any one of SEQ ID NOs: 79-87.

[0055] In some embodiments, the second subunit comprises residues 198-344 of any one of SEQ ID NOs: 79-87. In some embodiments, the second subunit comprises residues 198-344 of any one of SEQ ID NOs: 79-87 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 196-354 of any one of SEQ ID NOs: 79-87. In some embodiments, the second subunit comprises residues 196-354 of any one of SEQ ID NOs: 79-87 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions.

[0056] In some embodiments, the engineered meganuclease is a single chain meganuclease comprising a linker that covalently joins the first subunit and the second subunit. In some embodiments, the engineered meganuclease linker comprises an amino acid sequence according to SEQ ID NO: 196.

[0057] In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs: 79-87. In some embodiments, the engineered meganuclease comprises an amino acid sequence of any one of SEQ ID NOs: 79-87.

[0058] In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-354 of any one of SEQ ID NOs: 79-87. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 6-354 of any one of SEQ ID NOs: 79-87. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 5-354 of any one of SEQ ID NOs: 79-87. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 4-354 of any one of SEQ ID NOs: 79-87. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 3-354 of any one of SEQ ID NOs: 79-87. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of any one of SEQ ID NOs: 79-87.

[0059] In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs: 124-132. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence of any one of SEQ ID NOs: 124-132.

[0060] In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 80. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 80. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 80. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 80.

[0061] In some embodiments, the engineered meganuclease comprises a nuclear localization signal. In some embodiments, the nuclear localization signal is at the N-terminus of the engineered meganuclease. In some embodiments, the nuclear localization signal is at the C-terminus of the engineered meganuclease. In some embodiments, the nuclear localization signal comprises an amino acid sequence having at least 80% or at least 90% sequence identity to SEQ ID NO: 133 or 134. In some embodiments, the nuclear localization signal comprises SEQ ID NO: 133 or 134.

[0062] In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 149. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence according to SEQ ID NO: 149.

[0063] In another aspect, provided herein is a polynucleotide comprising a nucleic acid sequence encoding an engineered meganuclease described herein. In some embodiments, the polynucleotide comprises a promoter operably linked to the nucleic acid sequence encoding the engineered meganuclease. In some embodiments, the promoter is a CNS cell-specific promoter. In some embodiments, the promoter is active in cortex cells, upper motor neurons, spinal cells, lower motor neurons, neurons, neuron progenitor cells, astrocytes, excitatory neurons, inhibitory neurons, oligodendrocytes or motor neuron progenitor cells. In some embodiments, the promoter is a CAG promoter or a human synapsin 1 (Syn-1) promoter.

[0064] In another aspect, provided herein is a polynucleotide comprising a first nucleic acid sequence encoding a first engineered meganuclease described herein that binds and cleaves a recognition sequence comprising SEQ ID NO: 3, and a second nucleic acid sequence encoding a second engineered meganuclease described herein that binds and cleaves a recognition sequence comprising SEQ ID NO: 5.

[0065] In some embodiments, the polynucleotide comprises, from 5’ to 3’, (i) the first nucleic acid sequence encoding the first engineered meganuclease, and (ii) the second nucleic acid sequence encoding the second engineered meganuclease.

[0066] In some embodiments, the polynucleotide comprises, from 5’ to 3’, (i) the second nucleic acid sequence encoding the second engineered meganuclease and (ii) the first nucleic acid sequence encoding the first engineered meganuclease.

[0067] In some embodiments, the polynucleotide comprises a promoter enhancer. In some embodiments, the promoter enhancer is a neuron-specific promoter enhancer.

[0068] In some embodiments, the first nucleic acid sequence and / or the second nucleic acid sequence is codon modified to reduce the percent sequence identity between the first nucleic acid sequence and the second nucleic acid sequence, wherein the codon modification does not alter the amino acid sequence of the first engineered meganuclease or the second engineered meganuclease. In some embodiments, the first nucleic acid sequence has no more than about 40% to about 80% sequence identity to the second nucleic acid sequence. In some embodiments, the first nucleic acid sequence has no more than about 60% sequence identity to the second nucleic acid sequence.

[0069] In some embodiments, the first engineered meganuclease comprises a nuclear localization sequence (NLS). In some embodiments, the NLS is attached to the N-terminus of the first engineered meganuclease. In some embodiments, the NLS is attached to the C-terminus of the second engineered meganuclease. In some embodiments, the NLS comprises an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 133 or 134. In some embodiments, the NLS comprises an amino acid sequence set forth in SEQ ID NO: 133 or 134.

[0070] In some embodiments, the engineered meganuclease comprises a first NLS attached at the N-terminus and a second NLS attached at the C-terminus. In some embodiments, the first NLS and the second NLS are identical. In some embodiments, the first NLS and the second NLS comprise an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 133 or 134. In some embodiments, the first NLS and the second NLS comprise an amino acid sequence set forth in SEQ ID NO: 133 or 134.

[0071] In some embodiments, the first NLS and the second NLS are not identical. In some embodiments, the NLS comprises an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 133 and the second NLS comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 134. In some embodiments, the first NLS comprises an amino acid sequence set forth in SEQ ID NO: 133 and the second NLS comprises an amino acid sequence set forth in SEQ ID NO: 134. In some embodiments, the first NLS comprises an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 133 and the second NLS comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 134. In some embodiments, the first NLS comprises an amino acid sequence set forth in SEQ ID NO: 133 and the second NLS comprises an amino acid sequence set forth in SEQ ID NO: 134.

[0072] In some embodiments, the second engineered meganuclease comprises a third NLS attached at the N-terminus and a fourth NLS attached at the C-terminus. In some embodiments, the third and fourth NLS are identical. In some embodiments, the third NLS and the fourth NLS comprise an amino acid sequence having at least 80% sequence identity to a second set forth in SEQ ID NO: 133 or 134. In some embodiments, the third and the fourth NLS comprise an amino acid sequence set forth in SEQ ID NO: 133 or 134.

[0073] In some embodiments, the third NLS and the fourth NLS are not identical. In some embodiments, the third NLS comprises an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 133 and the fourth NLS comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 134. In some embodiments, the third NLS comprises an amino acid sequence set forth in SEQ ID NO: 133 and the fourth NLS comprises an amino acid sequence set forth in SEQ ID NO: 134. In some embodiments, the third NLS comprises an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 133 and the fourth NLS comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 134. In some embodiments, the third NLS comprises an amino acid sequence set forth IN SEQ ID NO: 133 and the fourth NLS comprises an amino acid sequence set forth in SEQ ID NO: 134.

[0074] In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are separated by an internal ribosome entry site (IRES) element or a nucleic acid sequence encoding a 2A peptide. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are separated by a nucleic acid sequence encoding a furin cleavage motif and a nucleic acid sequence encoding a 2A peptide. In some such embodiments, the first nucleic acid sequence and the second nucleic acid sequence are separated by a nucleic acid sequence encoding a furin cleavage motif, a nucleic acid sequence encoding a polypeptide linker, and a nucleic acid sequence encoding a 2A peptide. In certain embodiments, the 2A sequence is a T2A, P2A, E2A, or F2A sequence. In some embodiments, the 2A peptide is a P2A peptide. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are separated by a nucleic acid sequence encoding a P2A / furin peptide comprising an amino acid sequence set forth in SEQ ID NO: 135.

[0075] In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are operably linked to a promoter. In some embodiments, the promoter is a CNS cell-specific promoter. In some embodiments, the promoter is active in cortex cells, upper motor neurons, spinal cells, lower motor neurons, neurons, neuron progenitor cells, astrocytes, excitatory neurons, inhibitory neurons, oligodendrocytes or motor neuron progenitor cells. In some embodiments, the promoter is a CAG promoter or a human Syn-1 promoter.

[0076] In some such embodiments, the first nucleic acid sequence is operably linked to a first promoter and the second nucleic acid sequence is operably linked to a second promoter. In some embodiments, the first and second promoters are identical. In some embodiments, the first and the second promoter is a CNS cell-specific promoter. In some embodiments, the first and the second promoter is active in cortex cells, upper motor neurons, spinal cells, lower motor neurons, neurons, neuron progenitor cells, astrocytes, excitatory neurons, inhibitory neurons, oligodendrocytes or motor neuron progenitor cells. In some embodiments, the first promoter and the second promoter are a CAG promoter or a human Syn-1 promoter. In some embodiments, the first promoter and the second promoter are a CNS cell-specific promoter. In some embodiments, the first promoter and the second promoter are active in cortex cells, upper motor neurons, spinal cells, lower motor neurons, neurons, neuron progenitor cells, astrocytes, excitatory neurons, inhibitory neurons, oligodendrocytes or motor neuron progenitor cells. In some embodiments, the first promoter is a CAG promoter, and the second promoter is a human Syn-1 promoter. In some embodiments, the first promoter is a human Syn-1 promoter, and the second promoter is a CAG promoter.

[0077] In some embodiments, the polynucleotide comprises a woodchuck hepatitis virus post- transcriptional regulatory element (WPRE). In some embodiments, the polynucleotide comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to according to SEQ ID NO: 194.

[0078] In some embodiments, the polynucleotide comprises an intron sequence. In some such embodiments, the intron sequence is a chimeric or synthetic intron sequence. In some embodiments, the intron sequence comprises a nucleic acid sequence set forth in SEQ ID NO: 136.

[0079] In some embodiments, the polynucleotide comprises a termination sequence. In some embodiments, the termination sequence is a polyA sequence. In some such embodiments, the polyA sequence comprises a nucleic acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 137. In some embodiments, the polyA sequence comprises a nucleic acid sequence set forth in SEQ ID NO: 137.

[0080] In some embodiments, the polynucleotide comprises, from 5' to 3': (a) a neuron-specific promoter enhancer; (b) a human Syn-1 promoter; (c) an intron sequence; (d) the first nucleic acid sequence encoding the first engineered meganuclease, wherein the first engineered meganuclease comprises an NLS; (e) a nucleic acid sequence encoding a P2A / furin peptide; (f) the second nucleic acid sequence encoding the second engineered meganuclease, wherein the second engineered meganuclease comprises an NLS; (g) a WPRE sequence; and (h) a polyA sequence.

[0081] In some embodiments, the polynucleotide comprises, from 5' to 3': (a) a neuron-specific promoter enhancer; (b) a human Syn-1 promoter comprising a nucleic acid sequence set forth in SEQ ID NO: 138; (c) an intron sequence comprising a nucleic acid sequence set forth in SEQ ID NO: 136; (d) the first nucleic acid sequence encoding the first engineered meganuclease, wherein the first engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 7 or residues 2-354 of SEQ ID NO: 7, and wherein the first engineered meganuclease comprises an NLS; (e) a nucleic acid sequence encoding a P2A / furin peptide comprising an amino acid sequence set forth in SEQ ID NO: 135; (f) the second nucleic acid sequence encoding the second engineered meganuclease, wherein said second engineered meganuclease comprises an amino acid sequence set forth in SEQ ID NO: 80 or residues 2-354 of SEQ ID NO: 80, and wherein said second engineered meganuclease comprises an NLS; (g) a WPRE sequence comprising a nucleic acid sequence set forth in SEQ ID NO: 194; and (h) a polyA sequence.

[0082] In some embodiments, the polynucleotide comprises, from 5' to 3': (a) a neuron-specific promoter enhancer; (b) a human Syn-1 promoter; (c) an intron sequence; (d) the second nucleic acid sequence encoding the second engineered meganuclease, wherein the second engineered meganuclease comprises an NLS; (e) a nucleic acid sequence encoding a P2A / furin peptide; (f) the first nucleic acid sequence encoding the first engineered meganuclease, wherein the first engineered meganuclease comprises an NLS; (g) a WPRE sequence; and (h) a polyA sequence.

[0083] In some embodiments, the polynucleotide comprises, from 5' to 3': (a) a neuron-specific promoter enhancer; (b) a human Syn-1 promoter comprising a nucleic acid sequence set forth in SEQ ID NO: 138; (c) an intron sequence comprising a nucleic acid sequence set forth in SEQ ID NO: 136; (d) the second nucleic acid sequence encoding the second engineered meganuclease, wherein said second engineered meganuclease comprises an amino acid sequence set forth in SEQ ID NO: 80 or residues 2-354 of SEQ ID NO: 80, and wherein said second engineered meganuclease comprises an NLS; (e) a nucleic acid sequence encoding a P2A / furin peptide comprising an amino acid sequence set forth in SEQ ID NO: 135; (f) the first nucleic acid sequence encoding the first engineered meganuclease, wherein the first engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 7 or residues 2-354 of SEQ ID NO: 7, and wherein the first engineered meganuclease comprises an NLS; (g) a WPRE sequence comprising a nucleic acid sequence set forth in SEQ ID NO: 194; and (h) a polyA sequence.

[0084] In some embodiments, the first engineered meganuclease comprises a first NLS attached at its C-terminus. In some such embodiments, the first NLS comprises a nucleic acid sequence set forth in SEQ ID NO: 134. In some embodiments, the second engineered meganuclease comprises a second NLS attached at its N-terminus. In some embodiments, the second NLS comprises a nucleic acid sequence set forth in SEQ ID NO: 133.

[0085] In some embodiments, the polynucleotide is an mRNA.

[0086] In some embodiments, the polynucleotide comprises a first nucleic acid sequence encoding a first engineered meganuclease and a second nucleic acid sequence encoding a second engineered meganuclease, wherein: (a) the first engineered meganuclease binds and cleaves a recognition sequence comprising SEQ ID NO: 3 and comprises an amino acid sequence of SEQ ID NO: 7 or residues 2-354 of SEQ ID NO: 7; and (b) the second engineered meganuclease binds and cleaves a recognition sequence comprising SEQ ID NO: 5 and comprises an amino acid sequence of SEQ ID NO: 80 or residues 2-354 of SEQ ID NO: 80.

[0087] In another aspect, provided herein is a recombinant DNA construct comprising a polynucleotide described herein. In some embodiments, the recombinant DNA construct is a plasmid DNA.

[0088] In some embodiments, the recombinant DNA construct encodes a recombinant virus comprising the polynucleotide. In some embodiments, the recombinant virus is a recombinant adenovirus, a recombinant lentivirus, a recombinant retrovirus, or a recombinant adeno-associated virus (AAV). In some embodiments, the recombinant virus is a recombinant AAV. In some embodiments, the recombinant AAV has an AAV9 capsid.

[0089] In some embodiments, the polynucleotide comprises a promoter operably linked to the nucleic acid sequence encoding the engineered meganuclease. In some embodiments, the promoter is a CNS cell-specific promoter. In some embodiments, the promoter is active in cortex cells, upper motor neurons, spinal cells, lower motor neurons, neurons, neuron progenitor cells, astrocytes, excitatory neurons, inhibitory neurons, oligodendrocytes or motor neuron progenitor cells. In some embodiments, the promoter is a CAG promoter or a Syn-1 promoter.

[0090] In another aspect, the disclosure provides a recombinant virus comprising a polynucleotide described herein. In some embodiments, the polynucleotide is a polynucleotide described herein that comprises a first nucleic acid sequence encoding a first engineered meganuclease and a second nucleic acid sequence encoding a second engineered meganuclease. In some such embodiments, the polynucleotide comprises a promoter operably linked to the first nucleic acid sequence and the second nucleic acid sequence. In some such embodiments, the first nucleic acid sequence and the second nucleic acid sequence are separated by an IRES or 2A sequence. In certain embodiments, the 2A sequence is a T2A, P2A, E2A, or F2A sequence.

[0091] In another aspect, the disclosure provides a lipid nanoparticle composition comprising lipid nanoparticles comprising a polynucleotide described herein.

[0092] In some embodiments, the polynucleotide is an mRNA described herein. In some embodiments, the polynucleotide is a recombinant DNA construct described herein.

[0093] In another aspect, the disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a polynucleotide described herein.

[0094] In some embodiments, the polynucleotide comprises an mRNA described herein. In some embodiments, the polynucleotide comprises a recombinant DNA construct described herein. In some embodiments, the pharmaceutical composition comprises a recombinant virus described herein. In some embodiments, the pharmaceutical composition comprises a lipid nanoparticle composition described herein.

[0095] In another aspect, provided herein is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a recombinant DNA construct described herein.

[0096] In another aspect, provided herein is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a recombinant virus described herein.

[0097] In another aspect, provided herein is a host cell comprising a polynucleotide described herein.

[0098] In another aspect, provided herein is a host cell comprising an engineered meganuclease described herein.

[0099] In another aspect, provided herein is a method for producing a genetically-modified eukaryotic cell having a modified target sequence in a C90rf72 gene of the genetically-modified eukaryotic cell, wherein the method comprises introducing into a eukaryotic cell a polynucleotide comprising a nucleic acid sequence encoding an engineered meganuclease described herein, wherein the engineered meganuclease is expressed in the eukaryotic cell, and wherein the engineered meganuclease produces a cleavage site in the C90rf72 gene at a recognition sequence comprising SEQ ID NO: 3 or SEQ ID NO: 5.

[0100] In some such embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a CNS cell. In some embodiments, the CNS cell is a neuron or a motor neuron progenitor cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a recombinant virus, a lipid nanoparticle, or by an mRNA.

[0101] In another aspect, provided herein is a method for producing a genetically-modified eukaryotic cell having a modified target sequence in a C90rf72 gene of the genetically-modified eukaryotic cell, wherein the method comprises introducing into a eukaryotic cell an engineered meganuclease described herein, wherein the engineered meganuclease produces a cleavage site in the C90rf72 gene at a recognition sequence comprising SEQ ID NO: 3 or SEQ ID NO: 5.

[0102] In some such embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a CNS cell. In some embodiments, the CNS cell is a neuron or a motor neuron progenitor cell. In some embodiments, the mammalian cell is a human cell.

[0103] In another aspect, provided herein is a method for producing a genetically-modified eukaryotic cell comprising an exogenous sequence of interest inserted into a C90rf72 gene of the genetically-modified eukaryotic cell, the method comprising introducing into a eukaryotic cell one or more polynucleotides comprising a first nucleic acid sequence encoding an engineered meganuclease described herein and a second nucleic acid sequence comprising the sequence of interest, wherein the engineered meganuclease produces a cleavage site in the C90rf72 gene at a recognition sequence comprising SEQ ID NO: 3 or SEQ ID NO: 5, wherein the sequence of interest is inserted into the C90rf72 gene at the cleavage site.

[0104] In some such embodiments, the second nucleic acid sequence further comprises nucleic acid sequences homologous to nucleic acid sequences flanking the cleavage site and the sequence of interest is inserted at the cleavage site by homologous recombination.

[0105] In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a CNS cell. In some embodiments, the CNS cell is a neuron or a motor neuron progenitor cell. In some embodiments, the mammalian cell is a human cell.

[0106] In some embodiments, the first and / or second polynucleotide is introduced into the eukaryotic cell by a recombinant virus, a lipid nanoparticle, or by an mRNA.

[0107] In another aspect, provided herein is a method for producing a genetically-modified eukaryotic cell comprising an exogenous sequence of interest inserted into a C90rf72 gene of the genetically-modified eukaryotic cell, the method comprising introducing into a eukaryotic cell an engineered meganuclease described herein and a polynucleotide comprising the sequence of interest, wherein the engineered meganuclease produces a cleavage site in the C90rf72 gene at a recognition sequence comprising SEQ ID NO: 3 or SEQ ID NO: 5, wherein the sequence of interest is inserted into the C90rf72 gene at the cleavage site. In some such embodiments, the polynucleotide further comprises nucleic acid sequences homologous to nucleic acid sequences flanking the cleavage site and the sequence of interest is inserted at the cleavage site by homologous recombination.

[0108] In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a CNS cell. In some embodiments, the CNS cell is a neuron or a motor neuron progenitor cell. In some embodiments, the mammalian cell is a human cell.

[0109] In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a recombinant virus, a lipid nanoparticle, or by an mRNA.

[0110] In another aspect, provided herein is a method for producing a genetically-modified eukaryotic cell comprising a modified C90rf72 gene, the method comprising introducing into the eukaryotic cell one or more polynucleotides comprising a first nucleic acid sequence encoding a first engineered meganuclease and a second nucleic acid sequence encoding a second engineered meganuclease, wherein the first engineered meganuclease and the second engineered meganuclease are expressed in the eukaryotic cell, wherein the first engineered meganuclease produces a first cleavage site in an endogenous C90rf72 gene at a recognition sequence comprising SEQ ID NO: 3, wherein the second engineered meganuclease produces a second cleavage site in the C90rf72 gene at a recognition sequence comprising SEQ ID NO: 5, wherein an intervening genomic DNA between the first cleavage site and the second cleavage site is excised from the C90rf72 gene, and wherein the C90rf72 gene is annealed to generate the modified C90rf72.

[0111] In some embodiments, the modified C90rf72 gene comprises a reduced number of GGGGCC hexanucleotide repeats relative to the endogenous C90rf72 gene.

[0112] In some embodiments, the method comprises introducing into the eukaryotic cell a polynucleotide described herein comprising a first nucleic acid encoding the first engineered meganuclease and a second nucleic acid sequence encoding the second engineered meganuclease. In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a recombinant virus described herein. In some embodiments, the recombinant virus is a recombinant AAV described herein. In some embodiments, the polynucleotide is a recombinant DNA construct. In some embodiments, the polynucleotide is an mRNA described herein. In some embodiments, the polynucleotide is introduced into the eukaryotic cell is a lipid nanoparticle. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the eukaryotic cell is a human cell. In some embodiments, the eukaryotic cell is a CNS cell. In some embodiments, the CNS cell is a neuron or a motor neuron progenitor cell.

[0113] In some embodiments, the method comprises introducing into the eukaryotic cell a first polynucleotide described herein comprising a first nucleic acid sequence encoding the first engineered meganuclease and a second polynucleotide described herein comprising the second nucleic acid sequence encoding the second engineered meganuclease.

[0114] In some such embodiments, the first polynucleotide comprises a first promoter operably linked to the first nucleic acid sequence encoding the first engineered meganuclease and / or the second polynucleotide comprises a second promoter operably linked to the second nucleic acid sequence encoding the second engineered meganuclease. In some embodiments, the first promoter and / or the second promoter is a CNS cell-specific promoter. In some embodiments, the first promoter and / or the second promoter is active in cortex cells, upper motor neurons, spinal cells, lower motor neurons, neurons, neuron progenitor cells, astrocytes, excitatory neurons, inhibitory neurons, oligodendrocytes or motor neuron progenitor cells. In some embodiments, the first promoter and / or the second promoter is a CAG promoter or a human Syn-1 promoter. In some embodiments, the first polynucleotide comprises a first promoter enhancer and / or the second polynucleotide comprises a second promoter enhancer. In some embodiments, the first and / or the second promoter enhancer is a neuron-specific promoter enhancer. In some embodiments, the first engineered meganuclease and / or the second engineered meganuclease comprises an NLS. In some embodiments, the NLS is attached to the N-terminus of the first engineered meganuclease and / or the second engineered meganuclease. In some embodiments, the NLS is attached to the C-terminus of the first engineered meganuclease and / or the second engineered meganuclease. In some embodiments, the NLS comprises an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 133 or 134. In some embodiments, the NLS comprises an amino acid sequence set forth in SEQ ID NO: 133 or 134.

[0115] In some embodiments, the first engineered meganuclease and / or the second engineered meganuclease comprises a first NLS attached at the N-terminus and a second NLS attached at the C-terminus. In some embodiments, the first NLS and the second NLS are identical. In some embodiments, the first NLS and the second NLS comprise an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 133 or 134. In some embodiments, the first NLS and the second NLS comprise an amino acid sequence set forth in SEQ ID NO: 133 or 134.

[0116] In some embodiments, the first NLS and the second NLS are not identical. In some embodiments, the first NLS comprises an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 133 and the second NLS comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO; 134. In some embodiments, the first NLS comprises an amino acid sequence set forth in SEQ ID NO: 133 and the second NLS comprises an amino acid sequence set forth in SEQ ID NO: 134. In some embodiments, the first polynucleotide and / or the second polynucleotide comprises a WPRE. In some embodiments, the first polynucleotide and / or the second polynucleotide comprises an intron sequence. In some embodiments, the intron sequence is a chimeric or synthetic intron sequence. In some embodiments, the intron sequence comprises a nucleic acid sequence set forth in SEQ ID NO: 136. In some embodiments, the first polynucleotide and / or the second polynucleotide sequence comprises a termination sequence. In some embodiments, the termination sequence is a polyA sequence. In some embodiments, the polyA sequence comprises a nucleic acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 137. In some embodiments, the polyA sequence comprises a nucleic acid sequence set forth in SEQ ID NO: 137. In some embodiments, the first polynucleotide is introduced into the eukaryotic cell by a first recombinant virus. In some embodiments, the second polynucleotide is introduced into the eukaryotic cell by a second recombinant virus. In some embodiments, the first recombinant virus is a first recombinant AAV and / or the second recombinant virus is a second recombinant AAV. In some embodiments, the first recombinant and / or the second recombinant AAV has an AAV9 capsid. In some embodiments, the first polynucleotide is a first mRNA. In some embodiments, the second polynucleotide is a second mRNA. In some embodiments, the first polynucleotide is a first recombinant DNA construct. In some embodiments, the second polynucleotide is a second recombinant DNA construct. In some embodiments, the first polynucleotide and the second polynucleotide are introduced into the eukaryotic cell by one or more lipid nanoparticles. In some embodiments, the first polynucleotide is introduced into the eukaryotic cell by a first lipid nanoparticle. In some embodiments, the second polynucleotide is introduced into the eukaryotic cell by a second lipid nanoparticle.

[0117] In another aspect, provided herein is a method for modifying a C90rf72 gene in a target cell in a subject, the method comprising delivering to the target cell one or more polynucleotides comprising a first nucleic acid sequence encoding a first engineered meganuclease and a second nucleic acid sequence encoding a second engineered meganuclease, wherein the first and the second engineered meganucleases are expressed in the target cell, wherein the first meganuclease produces a first cleavage site in the C90rf72 gene at a recognition sequence comprising SEQ ID NO: 3, wherein the second engineered meganuclease produces a second cleavage site in the C90rf72 gene at a recognition sequence comprising SEQ ID NO: 5, wherein the intervening genomic DNA between the first cleavage site and the second cleavage site is excised from the C90rf72 gene, and wherein the C90rf72 gene is annealed to generate a modified C90rf72gene.

[0118] In some embodiments, the modified C90rf72 gene comprises a reduced number of GGGCC hexanucleotide repeats relative to the endogenous C90rf72 gene. In some embodiments, the method comprises introducing into the target cell a polynucleotide described herein comprising a first nucleic acid sequence encoding the first engineered meganuclease and a second nucleic acid sequence encoding the second engineered meganuclease. In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a recombinant virus. In some embodiments, the recombinant virus is a recombinant AAV. In some embodiments, the polynucleotide is a recombinant DNA construct. In some embodiments, the polynucleotide is an mRNA. In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a lipid nanoparticle. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the eukaryotic cell is a human cell. In some embodiments, the eukaryotic cell is a CNS cell. In some embodiments, the CNS cell is a neuron or a motor neuron progenitor cell.

[0119] In some such embodiments, the method comprises introducing into the target cell a first polynucleotide comprising a first nucleic acid sequence encoding the first engineered meganuclease and a second polynucleotide comprising the second nucleic acid sequence encoding the second engineered meganuclease.

[0120] In some such embodiments, the first polynucleotide comprises a first promoter operably linked to the first nucleic acid sequence encoding the first engineered meganuclease and / or the second polynucleotide comprises a second promoter operably linked to the second nucleic acid sequence encoding the second engineered meganuclease. In some embodiments, the first promoter and / or the second promoter is a CNS cell-specific promoter. In some embodiments, the first promoter and / or the second promoter is active in cortex cells, upper motor neurons, spinal cells, lower motor neurons, neurons, neuron progenitor cells, astrocytes, excitatory neurons, inhibitory neurons, oligodendrocytes or motor neuron progenitor cells. In some embodiments, the first promoter and / or the second promoter is a CAG promoter or a human Syn-1 promoter. In some embodiments, the first polynucleotide comprises a first promoter enhancer and / or the second polynucleotide comprises a second promoter enhancer. In some embodiments, the first and / or the second promoter enhancer is a neuron-specific promoter enhancer. In some embodiments, the first engineered meganuclease and / or the second engineered meganuclease comprises an NLS. In some embodiments, the NLS is attached to the N-terminus of the first engineered meganuclease and / or the second engineered meganuclease. In some embodiments, the NLS is attached to the C-terminus of the first engineered meganuclease and / or the second engineered meganuclease. In some embodiments, the NLS comprises an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 133 or 134. In some embodiments, the NLS comprises an amino acid sequence set forth in SEQ ID NO: 133 or 134. In some embodiments, the first engineered meganuclease and / or the second engineered meganuclease comprises a first NLS attached at the N-terminus and a second NLS attached at the C-terminus. In some embodiments, the first NLS and the second NLS are identical. In some embodiments, the first NLS and the second NLS comprise an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 133 or 134. In some embodiments, the first NLS and the second NLS comprise an amino acid sequence set forth in SEQ ID NO: 133 or 134.

[0121] In some embodiments, the first NLS and the second NLS are not identical. In some embodiments, the first NLS comprises an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 133 and the second NLS comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO; 134. In some embodiments, the first NLS comprises an amino acid sequence set forth in SEQ ID NO: 133 and the second NLS comprises an amino acid sequence set forth in SEQ ID NO: 134.

[0122] In some embodiments, the first polynucleotide and / or the second polynucleotide comprises a WPRE. In some embodiments, the first polynucleotide and / or the second polynucleotide comprises an intron sequence. In some embodiments, the intron sequence is a chimeric or synthetic intron sequence. In some embodiments, the intron sequence comprises a nucleic acid sequence set forth in SEQ ID NO: 136. In some embodiments, the first polynucleotide and / or the second polynucleotide sequence comprises a termination sequence. In some embodiments, the termination sequence is a polyA sequence. In some embodiments, the polyA sequence comprises a nucleic acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 137. In some embodiments, the polyA sequence comprises a nucleic acid sequence set forth in SEQ ID NO: 137. In some embodiments, the first polynucleotide is introduced into the eukaryotic cell by a first recombinant virus. In some embodiments, the second polynucleotide is introduced into the eukaryotic cell by a second recombinant virus. In some embodiments, the first recombinant virus is a first recombinant AAV and / or the second recombinant virus is a second recombinant AAV. In some embodiments, the first recombinant and / or the second recombinant AAV has an AAV9 capsid. In some embodiments, the first polynucleotide is a first mRNA. In some embodiments, the second polynucleotide is a second mRNA. In some embodiments, the first polynucleotide is a first recombinant DNA construct. In some embodiments, the second polynucleotide is a second recombinant DNA construct. In some embodiments, the first polynucleotide and the second polynucleotide are introduced into the eukaryotic cell by one or more lipid nanoparticles. In some embodiments, the first polynucleotide is introduced into the eukaryotic cell by a first lipid nanoparticle. In some embodiments, the second polynucleotide is introduced into the eukaryotic cell by a second lipid nanoparticle.

[0123] In another aspect, provided herein is a method for treating a neurological disorder in a subject in need thereof, wherein the neurological disorder is characterized by a mutation in a C90rf72 gene that increases the number of GGGGCC hexanucleotide repeats relative to a full- length wild-type C90rf72 gene, the method comprising: administering to the subject an effective amount of one or more polynucleotides comprising a first nucleic acid sequence encoding a first engineered meganuclease and a second nucleic acid sequence encoding a second engineered meganuclease, wherein the one or more polynucleotides are delivered to a target cell in the subject, wherein the first engineered meganuclease and the second engineered meganuclease are expressed in the target cell, wherein the first engineered meganuclease produces a first cleavage site in the C90rf72 gene at a recognition sequence comprising SEQ ID NO:3, wherein the second engineered meganuclease produces a second cleavage site in the C90rf72 gene at a recognition sequence comprising SEQ ID NO: 5, wherein intervening genomic DNA between the first cleavage site and the second cleavage site is excised from the C90rf72 gene, and wherein the C90rf72 gene is annealed to generate a modified C90rf72 gene.

[0124] In some embodiments, the neurological disorder is amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD). In some embodiments, the modified C9ORf72 gene comprises a reduced number of GGGGCC hexanucleotide repeats relative to the endogenous C90rf72 gene.

[0125] In some embodiments, the method comprises delivering to the target cell a polynucleotide described herein comprising a first nucleic acid encoding the first engineered meganuclease and a second nucleic acid sequence encoding the second engineered meganuclease. In some embodiments, the polynucleotide is introduced into the eukaryotic cell by a recombinant virus. In some embodiments, the recombinant virus is an AAV. In some embodiments, the polynucleotide is a recombinant DNA construct. In some embodiments, the polynucleotide is an mRNA. In some embodiments, the polynucleotide is delivered to the target cell by a lipid nanoparticle. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the target cell is a CNS cell. In some embodiments, the CNS cell is a neuron or a motor neuron.

[0126] In some such embodiments, the method comprises introducing into the target cell a first polynucleotide comprising a first nucleic acid sequence encoding the first engineered meganuclease and a second polynucleotide comprising the second nucleic acid sequence encoding the second engineered meganuclease. In some such embodiments, the first polynucleotide comprises a first promoter operably linked to the first nucleic acid sequence encoding the first engineered meganuclease and / or the second polynucleotide comprises a second promoter operably linked to the second nucleic acid sequence encoding the second engineered meganuclease. In some embodiments, the first promoter and / or the second promoter is a CNS cell-specific promoter. In some embodiments, the first promoter and / or the second promoter is active in cortex cells, upper motor neurons, spinal cells, lower motor neurons, neurons, neuron progenitor cells, astrocytes, excitatory neurons, inhibitory neurons, oligodendrocytes or motor neuron progenitor cells. In some embodiments, the first promoter and / or the second promoter is a CAG promoter or a human Syn-1 promoter. In some embodiments, the first polynucleotide comprises a first promoter enhancer and / or the second polynucleotide comprises a second promoter enhancer. In some embodiments, the first and / or the second promoter enhancer is a neuron-specific promoter enhancer. In some embodiments, the first engineered meganuclease and / or the second engineered meganuclease comprises an NLS. In some embodiments, the NLS is attached to the N-terminus of the first engineered meganuclease and / or the second engineered meganuclease. In some embodiments, the NLS is attached to the C-terminus of the first engineered meganuclease and / or the second engineered meganuclease. In some embodiments, the NLS comprises an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 133 or 134. In some embodiments, the NLS comprises an amino acid sequence set forth in SEQ ID NO: 133 or 134.

[0127] In some embodiments, the first engineered meganuclease and / or the second engineered meganuclease comprises a first NLS attached at the N-terminus and a second NLS attached at the C-terminus. In some embodiments, the first NLS and the second NLS are identical. In some embodiments, the first NLS and the second NLS comprise an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 133 or 134. In some embodiments, the first NLS and the second NLS comprise an amino acid sequence set forth in SEQ ID NO: 133 or 134.

[0128] In some embodiments, the first NLS and the second NLS are not identical. In some embodiments, the first NLS comprises an amino acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 133 and the second NLS comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 134. In some embodiments, the first NLS comprises an amino acid sequence set forth in SEQ ID NO: 133 and the second NLS comprises an amino acid sequence set forth in SEQ ID NO: 134.

[0129] In some embodiments, the first polynucleotide and / or the second polynucleotide comprises a WPRE. In some embodiments, the first polynucleotide and / or the second polynucleotide comprises an intron sequence. In some embodiments, the intron sequence is a chimeric or synthetic intron sequence. In some embodiments, the intron sequence comprises a nucleic acid sequence set forth in SEQ ID NO: 136. In some embodiments, the first polynucleotide and / or the second polynucleotide sequence comprises a termination sequence. In some embodiments, the termination sequence is a polyA sequence. In some embodiments, the polyA sequence comprises a nucleic acid sequence having at least 80% sequence identity to a sequence set forth in SEQ ID NO: 137. In some embodiments, the polyA sequence comprises a nucleic acid sequence set forth in SEQ ID NO: 137. In some embodiments, the first polynucleotide is introduced into the eukaryotic cell by a first recombinant virus. In some embodiments, the second polynucleotide is introduced into the eukaryotic cell by a second recombinant virus. In some embodiments, the first recombinant virus is a first recombinant AAV and / or the second recombinant virus is a second recombinant AAV. In some embodiments, the first recombinant and / or the second recombinant AAV has an AAV9 capsid. In some embodiments, the first polynucleotide is a first mRNA. In some embodiments, the second polynucleotide is a second mRNA. In some embodiments, the first polynucleotide is a first recombinant DNA construct. In some embodiments, the second polynucleotide is a second recombinant DNA construct. In some embodiments, the first polynucleotide and the second polynucleotide are introduced into the eukaryotic cell by one or more lipid nanoparticles. In some embodiments, the first polynucleotide is introduced into the eukaryotic cell by a first lipid nanoparticle. In some embodiments, the second polynucleotide is introduced into the eukaryotic cell by a second lipid nanoparticle.

[0130] In some embodiments, the rate of motor function decline according to the ALS or FTD Functional Rating Scale is reduced compared to the decline prior to treatment or compared to an untreated subject having ALS or FTD. In some embodiments, the neurofilament light chain levels in the cerebral spinal fluid are reduced. In some embodiments, the method reduces the number of GGGGCC dipeptides in the CSF and / or neurons of the subject compared to an untreated subject having ALS or FTD. In some embodiments, the method reduces the number of GGGGCC RNA Foci in neurons of the subject compared to an untreated subject having ALS or FTD.

[0131] In another aspect, the disclosure provides engineered meganucleases described herein, or polynucleotides described herein encoding engineered meganucleases, or cells described herein expressing engineered meganucleases, for use as a medicament.

[0132] In some embodiments, the medicament is useful for producing a modified C90rf72 gene in a subject. In some embodiments, the medicament is useful for the treatment of ALS or FTD.

[0133] In another aspect, the disclosure provides the use of engineered meganucleases described herein, or polynucleotides disclosed herein encoding engineered meganucleases, or cells described herein expressing engineered meganucleases, in the manufacture of a medicament for treating ALS or FTD, for increasing levels of a modified C90rf72 gene (i.e., lacking the GGGGCC hexanucleotide repeats relative to the endogenous C90rf72 gene), or reducing the symptoms associated with ALS or FTD.

[0134] BRIEF DESCRIPTION OF THE FIGURES

[0135] Figure 1. Schematic providing the approximate location of the CNR1-2 and CNR 21-22 meganuclease recognition sequences and illustrating the dual meganuclease approach for excising a region of DNA from the C90rf72 gene using either an upstream or downstream excision strategy. As shown, a pair of engineered meganucleases that bind and cleave either upstream sites and the CNR 1-2 site (upstream excision approach) or that bind and cleave downstream CNR 21-22 sites and the CNR 1-2 site (downstream excision approach) are used. The resultant C90rf72 transcript from the upstream excision approach is expressed from exon lb, thereby any mRNA lacks the hexanucleotide repeat region. The resultant C90rf72 transcript from the downstream excision approach is expressed from exon la, but with an excised hexanucleotide repeat region.

[0136] Figure 2. A schematic showing exemplary recognition sequences of the disclosure, which includes sense and anti-sense sequences for CNR 1-2 (SEQ ID NOs: 3 and 4) and CNR 21-22 (SEQ ID NOs: 5 and 6). Each CNR recognition sequence targeted by engineered meganucleases described herein comprises two recognition half-sites. Each recognition half-site comprises 9 base pairs, separated by a 4 base pair central sequence. For example, the CNR 1-2 recognition sequence has a 5’ CNR1 half-site and a 3’ CNR2 half-site with a four base pair center sequence ATAA.

[0137] Figure 3. The engineered meganucleases described herein comprise two subunits, with the first subunit having a hyper variable region (HVR), HVR1, that binds to a first recognition half-site and the second subunit having a HVR2 that binds to a second recognition half-site. In embodiments wherein the engineered meganuclease is a single chain meganuclease, the first subunit comprising HVR1 can be positioned as either the N-terminal or C-terminal subunit connected via a linker to the second subunit comprising HVR2. Likewise, the second subunit comprising the HVR2 can be positioned as either the N-terminal or C-terminal subunit connected via a linker to the first subunit comprising HVR1.

[0138] Figures 4A-4B. Figure 4 A provides an alignment of the sequences of the CNR 1-2 meganucleases described herein. Figure 4B provides an alignment of the sequences of the CNR 21-22 meganucleases described herein. Asterisks indicate conserved residues amongst all aligned nucleases, and a space or colon indicates that at least one amino acid differed amongst the meganucleases. Figure 5. Schematic of a reporter assay in CHO cells for evaluating engineered meganucleases targeting recognition sequences. For the engineered meganucleases described herein, a CHO cell line was produced in which a reporter cassette was integrated stably into the genome of the cell. The reporter cassette comprised, in 5’ to 3’ order: an SV40 Early Promoter, the 5’ 2 / 3 of the green fluorescence protein (GFP) gene, the recognition sequence for an engineered meganuclease described herein (e.g., CNR 1-2 or CNR 21-22), the recognition sequence for the CHO-23 / 24 meganuclease (WO 2012 / 167192), and the 3’ 2 / 3 of the GFP gene. Cells stably transfected with this cassette did not express GFP in the absence of a DNA break-inducing agent. Meganucleases were introduced by transduction of an mRNA encoding each meganuclease. When a DNA break was induced at either of the meganuclease recognition sequences, the duplicated regions of the GFP gene recombined with one another to produce a functional GFP gene. The percentage of GFP-expressing cells could then be determined by flow cytometry as an indirect measure of the frequency of genome cleavage by the meganucleases.

[0139] Figures 6A-6J. Figure 6A, Figure 6B, Figure 6C, Figure 6D, Figure 6E, and Figure 6F provide the efficiency of engineered meganucleases for binding and cleaving the CNR 1-2 recognition sequence expressed in the CHO cell reporter assay. Figure 6G, Figure 6H, Figure 61, and Figure 6J provide the efficiency of engineered meganucleases for binding and cleaving the CNR 21-22 recognition sequence expressed in the CHO cell reporter assay. The relative activity index represents the %GFP positive cells for each cell line expressing the test meganuclease normalized to the cell line expressing the CHO-23 / 24 meganuclease, accounting for the toxicity of the meganuclease.

[0140] Figure 7. A bar graph showing the percentage frequency of insertions and deletions (indels) of the tested meganucleases targeting the indicated recognition sequences in HEK293 cells. Each meganuclease was tested at two time points (days 2 and 6) following transfection of the meganuclease.

[0141] Figure 8. Provides a schematic of the oligocapture assay used to determine off-target effects of an engineered nuclease (e.g., an engineered meganuclease described herein). As shown, the integration cassette or oligo anneals with a double-strand break (DSB) in the gnome that may be due to engineered nuclease cleavage. The DNA is then sheared by sonication, adapters are ligated, and PCR amplified, followed by sequence analysis to determine location of the DSB.

[0142] Figure 9. Provides a graph depicting results from an oligo capture assay to identify off target cutting induced by the CNRl-2xl, CNRl-2xl7, CNRl-2x25, CNRl-2x80, CNRl-2x87, CNRl-2x88, CNRl-2x91, and CNRl-2x93 meganucleases transfected in HEK293 cells. The circled dots indicate the on-target site, and the non-circled dots indicate off-target sites, with the X axis representing the number of sequencing reads for each detected off-target site. The shade of the dot indicates the number of base pair mismatches between the on-target site and each of the detected off target sites.

[0143] Figures 10A-10B. Provide bar graphs showing the percentage of indels in a HEK293 cell line as assessed by digital PCR (Figure 10A) or NGS targeted sequencing (Figure 10B). The percentage of indels were assessed at day 2 and day 5 post transfection.

[0144] Figure 11. Provides a graph depicting results from an oligo capture assay to identify off target cutting induced by the CNR1-2L.15, CNR1-2L.18, or CNR1-2L.56 meganucleases transfected in HEK293 cells. The circled dots indicate the on-target site, and the non-circled dots indicate off-target sites, with the X axis representing the number of sequencing reads for each detected off-target site. The shade of the dot indicates the number of base pair mismatches between the on-target site and each of the detected off target sites.

[0145] Figures 12A-12B. Provides bar graphs showing the percentage of excision and ligation in the C90rf72 gene as assessed by digital droplet PCR (ddPCR). Figure 12A provides data using the downstream excision strategy (see Figure 1), with the combination of CNR1-2L.15 and CNR19- 20x.l l meganucleases, the CNR1-2L.98 and CNR19-20x.l l meganucleases, the CNR1-2L.100 and CNR19-20x. l l meganucleases, the CNR1-2L.104 and CNR19-20x.l l meganucleases, the CNR1- 2L.108 and CNR19-20x.l l meganucleases, the CNR1-2L.140 and CNR19-20x.l l meganucleases, and a mock or GFP control. Figure 12B provides data using the upstream excision strategy (see Figure 1), with the combination of CNR1-2L.15 and CNR17-18x.85 meganucleases, the CNR1- 2L.98 and CNR17-18x.85 meganucleases, the CNR1-2L.100 and CNR17-18x.85 meganucleases, the CNR1-2L.104 and CNR17-18x.85 meganucleases, the CNR1-2L.108 and CNR17-18x.85 meganucleases, the CNR1-2L.140 and CNR17-18x.85 meganucleases, and a mock or GFP control.

[0146] Figure 13. Provides a graph depicting results from an oligo capture assay to identify off target cutting induced by the CNR1-2L100, CNR1-2L104, CNR1-2L108, CNR1-2L126, CNR1- 2L140, CNR1-2L141, or CNR1-2L198 meganucleases transfected in HEK293 cells. The circled dots indicate the on-target site, and the non-circled dots indicate off-target sites, with the X axis representing the number of sequencing reads for each detected off-target site. The shade of the dot indicates the number of base pair mismatches between the on-target site and each of the detected off target sites.

[0147] Figure 14A-14B. Provide bar graphs showing the percentage of excision and ligation in the C90rf72 gene as assessed by digital droplet PCR (ddPCR). Figure 14A provides data using the upstream excision strategy (see Figure 1), with the combination of CNR1-2L.98 and CNR17- 18L.12 meganucleases, CNR1-2L.211 and CNR17-18L.12 meganucleases, CNR1-2L.213 and CNR17-18L.12 meganucleases, the CNR1-2L.217 and CNR17-18L.12 meganucleases, or mock or GFP controls. Figure 14B provides data using the downstream excision strategy (see Figure 1), with the combination of CNR1-2L.98 and CNR19-20L.71 meganucleases, the CNR1-2L.211 and CNR19-20L.71 meganucleases, the CNR1-2L.213 and CNR19-20L.71 meganucleases, the CNR1- 2L.217 and CNR19-20L.71 meganucleases, or mock or GFP controls.

[0148] Figure 15. Provides a graph depicting results from an oligo capture assay to identify off target cutting induced by the CNR1-2L.211, CNR1-2L.213, or CNR1-2L.217 meganucleases transfected in HEK293 cells. The circled dots indicate the on-target site, and the non-circled dots indicate off-target sites, with the X axis representing the number of sequencing reads for each detected off-target site. The shade of the dot indicates the number of base pair mismatches between the on-target site and each of the detected off target sites.

[0149] Figure 16. Provides a bar graph depicting multiplex targeted amplicon sequencing results of the intended site (chr9_27573614_27573672) and several top off-target sites as identified by oligocapture assays for CNR1-2L.211, CNR1-2L.213, CNR1-2L.217, or CNR1-2L.98.

[0150] Figure 17A-17B. Provide bar graphs showing the percentage of excision and ligation in the C90rf72 gene as assessed by digital droplet PCR (ddPCR). Figure 17A provides data using the upstream excision strategy (see Figure 1), with the indicated CNR 1-2 meganucleases with GFP as a control or the combinations of indicated CNR1-2 and CNR17-18 engineered meganucleases and a mock or GFP controls. Figure 17b provides data using the downstream excision strategy (see Figure 1), with the indicated CNR 1-2 meganucleases with GFP as a control or the combinations of indicated CNR1-2 and CNR19-20 engineered meganucleases and a mock or GFP controls.

[0151] Figure 18. Provides a graph depicting results from an oligo capture assay to identify off target cutting induced by the CNR1-2L.277, CNR1-2L.280, CNR1-2L.284, CNR1-2L.295, CNR1- 2L.297, CNR1-2L.304, CNR1-2L.320, CNR1-2L.321, CNR1-2L.326, CNR1-2L.327, CNR1- 2L.335, CNR1-2L.343, CNR1-2L.350, CNR1-2L.356, or CNR1-2L.368 meganucleases transfected in HEK293 cells. The circled dots indicate the on-target site, and the non-circled dots indicate off- target sites, with the X axis representing the number of sequencing reads for each detected off- target site. The shade of the dot indicates the number of base pair mismatches between the on-target site and each of the detected off target sites.

[0152] Figure 19. Provides a bar graph depicting multiplex targeted amplicon sequencing results of the intended site (9_27573614_27573635) and several top off-target sites as identified by oligocapture assays for CNR1-2L.284 or CNR1-2L.297 meganucleases.

[0153] Figures 20A-20B. Provide bar graphs showing the percentage of excision and ligation in the C90rf72 gene as assessed by digital droplet PCR (ddPCR). Figure 20A provides data using the upstream excision strategy (see Figure 1), with the combination of indicated CNR 1-2 and CNR 17- 18 meganucleases mock, or GFP controls. Figure 20B provides data using the downstream excision strategy (see Figure 1), with the combination of indicated CNR1-2 and CNR21-22 meganucleases mock, or GFP controls.

[0154] Figure 21. Provides a bar graph showing the percentage of excision and ligation in the C90rf72 gene as assessed by digital droplet PCR (ddPCR) for the indicated combinations of CNR1-2 and CNR21-22 meganucleases or a mock control.

[0155] Figure 22. Provides a graph depicting results from an oligo capture assay to identify off target cutting induced by the CNR1-2L.371, CNR1-2L.403, or CNR1-2L.425 meganucleases transfected in HEK293 cells. The circled dots indicate the on-target site, and the non-circled dots indicate off-target sites, with the X axis representing the number of sequencing reads for each detected off-target site. The shade of the dot indicates the number of base pair mismatches between the on-target site and each of the detected off target sites.

[0156] Figure 23. Provides a bar graph depicting multiplex targeted amplicon sequencing results of the intended site (9_27573614_27573635) and several top off-target sites as identified by oligocapture assays for CNR1-2L.297, CNR1-2L.371, CNR1-2L.403, CNR1-2L.425 meganucleases or GFP control.

[0157] Figure 24A-24B. Provide bar graphs showing the percentage of excision and ligation in the C90rf72 gene as assessed by digital droplet PCR (ddPCR). Figure 24A provides data using the upstream excision strategy (see Figure 1), with the combination of the indicated CNR1-2 and CNR 17-18 meganucleases, mock, or GFP controls. Figure 24B provides data using the downstream excision strategy (see Figure 1), with the combination of CNR1-2 and CNR21-22 meganucleases mock, or GFP controls.

[0158] Figure 25. Provides a graph depicting results from an oligo capture assay to identify off target cutting induced by the CNR1-2L.468 or CNR1-2L.531 meganucleases transfected in HEK293 cells. The circled dots indicate the on-target site, and the non-circled dots indicate off- target sites, with the X axis representing the number of sequencing reads for each detected off- target site. The shade of the dot indicates the number of base pair mismatches between the on-target site and each of the detected off target sites.

[0159] Figure 26. Provides a bar graph depicting multiplex targeted amplicon sequencing results of the intended site (9_27573614_27573635) and several top off-target sites as identified by oligocapture assays for the CNR1-2L.425 or CNR1-2L.531 meganucleases or GFP control at the 5ng, 25ng, or 200ng dose. Figure 27. Provides a bar graph showing the percentage of excision and ligation in the C90rf72 gene as assessed by ddPCR with the indicated combinations of CNR 1-2 and CNR 21-22 meganucleases or GFP control.

[0160] Figure 28A-28C. Provide graphs depicting results from an oligo capture assay to identify off target cutting induced by the CNR1-2L.425 (Figure 28A), CNR1-2L.531 (Figure 28B), or CNR1-2L.532 (Figure 28C) meganucleases transfected in HEK293 cells. The circled dots indicate the on-target site, and the non-circled dots indicate off-target sites, with the X axis representing the number of sequencing reads for each detected off-target site. The shade of the dot indicates the number of base pair mismatches between the on-target site and each of the detected off target sites.

[0161] Figure 29. Provides a graph showing the percentage of indel formation in the C90rf72 gene as assessed by ddPCR for the CNR1-2L.425 (circle), CNR1-2L.531 (square), and CNR1-2L.532 (triangle) meganucleases at concentrations ranging from 0.39 ng to 400 ng.

[0162] Figure 30. Provides the percentage of indels at on-target (intended target) and off-target sites as assessed by MTA for CNR1-2L.425 and CNR1-2L.532 at concentrations ranging from 6.25 ng to 400 ng.

[0163] Figure 31 A-3 IB. Provides bar graphs showing the percentage of excision and ligation in the C90rf72 gene in either healthy cells (Figure 31 A) or C9 patient-derived motor neuron progenitor cells (MNPCs) (Figure 3 IB) using the dual meganuclease approach as shown in Figure 1. Cells were transfected with the combination of CNR1-2L.98 and CNR19-20L.71 meganucleases, the CNR1-2L.98 and CNR21-22L.38 meganucleases, the CNR1-2L.98 and CNR23-24L.35 meganucleases, the CNR3-4L.111 and CNR35-36L.81 meganucleases, the CNR1-2L.98 and CNR17-18L.12 meganucleases, the CNR3-4L.111 and CNR29-30L.16 meganucleases, the CNR3- 4L.111 and CNR25-26L.17 meganucleases, or the CNR3-4L.111 and CNR31-32L.4 meganucleases.

[0164] Figure 32A-32B. Provide bar graphs showing the percentage of excision and ligation in the C90rf72 gene as assessed by digital droplet PCR (ddPCR). Figure 32A provides data using the upstream excision strategy (see Figure 1), with the combination of CNR1-2L.211 and CNR17- 18L.120 meganucleases, the CNR1-2L.211 and CNR17-18L.132 meganucleases, the CNR9- 10L.257 and CNR17-18L.120 meganucleases, the CNR9-10L.257 and CNR17-18L.132 meganucleases, the CNR3-4L.212 and CNR29-30L.107 meganucleases, the CNR3-4L.212 and CNR25-26L.103 meganucleases, the CNR3-4L.212 and CNR25-26L.174 meganucleases, or the CNR3-4L.212 and CNR31-32L.103 meganucleases. Figure 32B provides data using the downstream excision strategy (see Figure 1), with the combination of CNR1-2L.211 and CNR19- 20L.93 meganucleases, the CNR1-2L.211 and CNR21-22L.139 meganucleases, the CNR1-2L.211 and CNR21-22L.169 meganucleases, or the CNR3-4L.212 and CNR35-36L.176 meganucleases.

[0165] Figure 33. Provides a bar graph showing the percentage of excision and ligation in the C90rf72 gene as assessed by digital droplet PCR for the combination of CNR1-2L.15 and CNR21- 22x.l3 meganucleases, the CNR1-2L.15 and CNR21-22x.45 meganucleases, the CNR1-2L.15 and CNR21-22x.84 meganucleases, mock, GFP, and single meganuclease controls.

[0166] Figure 34. Provides a graph depicting results from an oligo capture assay to identify off target cutting induced by the CNR21-22xl3, CNR21-22x45, and CNR21-22x84 meganucleases transfected in HEK293 cells. The circled dots indicate the on-target site, and the non-circled dots indicate off-target sites, with the X axis representing the number of sequencing reads for each detected off-target site. The shade of the dot indicates the number of base pair mismatches between the on-target site and each of the detected off target sites.

[0167] Figure 35. Provides a bar graph showing the percentage of excision and ligation in the C90rf72 gene as assessed by digital droplet PCR for the indicated combinations of CNR 21-22 and CNR 1-2 meganucleases, mock, or GFP controls.

[0168] Figure 36. Provides a graph depicting results from an oligo capture assay to identify off target cutting induced by the CNR21-22L.13, CNR21-22L.27, CNR21-22L.34, CNR21-22L.37, CNR21-22L.38, and CNR21-22L.45 meganucleases transfected in HEK293 cells. The circled dots indicate the on-target site, and the non-circled dots indicate off-target sites, with the X axis representing the number of sequencing reads for each detected off-target site. The shade of the dot indicates the number of base pair mismatches between the on-target site and each of the detected off target sites.

[0169] Figure 37. Provides a bar graph showing the percentage of excision and ligation in the C90rf72 gene as assessed by digital droplet PCR for the indicated combinations of CNR21-22 and CNR1-2 meganucleases, CNR 21-22 meganuclease with GFP, mock, or GFP alone controls.

[0170] Figure 38. Provides a graph depicting results from an oligo capture assay to identify off target cutting induced by the CNR21-22L.109, CNR21-22L.117, CNR21-22L.121, CNR21- 22L.122, CNR21-22L.124, CNR21-22L.139, CNR21-22L.140, CNR21-22L.148, CNR21-22L.152, CNR21-22L.153, CNR21-22L.157, CNR21-22L.158, CNR21-22L.165, CNR21-22L.167, CNR21- 22L.169, CNR21-22L.176, and CNR21-22L.181 meganucleases transfected in HEK293 cells. The circled dots indicate the on-target site, and the non-circled dots indicate off-target sites, with the X axis representing the number of sequencing reads for each detected off-target site. The shade of the dot indicates the number of base pair mismatches between the on-target site and each of the detected off target sites. Figure 39. Provides a bar graph showing the percentage of excision and ligation in the C90rf72 gene as assessed by digital droplet PCR for the indicated combinations of CNR 21-22 and CNR 1-2 meganucleases, CNR 21-22 meganuclease with GFP, mock, or GFP controls.

[0171] Figure 40A-40B. Provide graphs depicting results from an oligo capture assay to identify off target cutting induced by meganucleases described herein. Figure 40A depicts results for the CNR21-22L.197, CNR21-22L.198, CNR21-22L.228, CNR21-22L.243, CNR21-22L.250, and CNR21-22L.274 meganucleases transfected in HEK293 cells. Figure 40B depicts results for the CNR21-22L.212 and CNR21-22L.206 meganucleases transfected in HEK293 cells. The circled dots indicate the on-target site, and the non-circled dots indicate off-target sites, with the X axis representing the number of sequencing reads for each detected off-target site. The shade of the dot indicates the number of base pair mismatches between the on-target site and each of the detected off target sites.

[0172] Figure 41. Provides the percentage of indels at on-target (9_27571934_27571955; intended target) and off-target sites as assessed by MTA for CNR21-22L.197, CNR21-22L.212, and CNR21-22L.274 meganucleases.

[0173] Figure 42. Provides the percentage of indels at on-target (9_27571934_27571955; intended target) and off-target sites as assessed by MTA for CNR21-22L.197, CNR21-22L.212, and CNR21-22L.274 meganucleases at 5 ng, 25ng, and 200ng doses.

[0174] Figure 43. Provides an enlarged perspective of the percentage of indels at off-target sites of the meganucleases as shown in Figure 42.

[0175] Figure 44. Provides a bar graph showing the percentage of excision and ligation in the C90rf72 gene as assessed by digital droplet PCR for the indicated CNR 21-22 meganucleases mock, or GFP controls in combination with meganucleases CNR1-2L.425 (black bar), CNR1- 2L.531 (light grey bar), or CNR1-2L.532 (dark grey bar).

[0176] Figure 45. Provides a graph depicting results from an oligo capture assay to identify off target cutting induced by the CNR21-22L.277, CNR21-22L.279, CNR21-22L.281, CNR21- 22L.285, CNR21-22L.286, CNR21-22L.289, or CNR21-22L.290 meganucleases transfected in HEK293 cells. The circled dots indicate the on-target site, and the non-circled dots indicate off- target sites, with the X axis representing the number of sequencing reads for each detected off- target site. The shade of the dot indicates the number of base pair mismatches between the on-target site and each of the detected off target sites.

[0177] Figure 46. Provides a graph showing the percentage of indels in the C90rf72 gene as assessed by ddPCR for the CNR21-22L.212 (diamond), CNR21-22L.286 (circle), and CNR21- 22L.290 (square) meganucleases at concentrations ranging from 0.39 ng to 400 ng. Figure 47. Provides the percentage of indels at the on target (9:27571934-27571955) and one-off target site as assessed by MTA for CNR21-22L.286, CNR21-22L.290, and CNR21- 22L.212 at concentrations ranging from 6.25 ng to 400 ng.

[0178] Figures 48A-48D. Provide bar graphs depicting the percentage of excision and ligation observed in an in vivo assay using a transgenic mouse line C9BAC. Mice were injected with one of six different constructs encapsulated by AAV9 or AAVrhlO. Figure 48A depicts the percentage of excision and ligation in the cortex for Group 1 to Group 7 (G1 - G7). Figure 48B depicts the percentage of excision and ligation in the brain stem for G1 to G7. Figure 48C depicts the percentage of excision and ligation in the cervical spine for G1 to G7. Figure 48D depicts the percentage of excision and ligation in the thoracic spine for G1 to G7. The vector for G1 comprises a CAG promoter, a CNR1-2L.98 and a CNR21-22L.38 meganuclease, and a WPRE 3’UTR encapsulated in an AAV9 particle; the vector for G2 comprises a Synl promoter, a CNR1-2L.98 and a CNR21-22L.38 meganuclease, and a WPRE 3’UTR encapsulated in an AAV9 particle; the vector for G3 comprises a CAG promoter, a CNR1-2L.98 and a CNR21-22L.38 meganuclease encapsulated in an AAV9 particle; the vector for G4 comprises a Synl promoter, a CNR1-2L.98 and a CNR21-22L.38 meganuclease encapsulated in an AAV9 particle; the vector for G5 comprises a CAG promoter, a CNR1-2L.98 and a CNR21-22L.38 meganuclease encapsulated in an AAVrhlO particle; the vector for G6 comprises a CAG promoter, a CNR1 -2L.15 and a CNR21 -22x.13 meganuclease, and a WPRE 3’ UTR encapsulated in an AAV9 particle. Group 7 is treated with PBS control.

[0179] Figures 49A-49B. Provide bar graphs depicting the percentage of excision and ligation observed in an in vivo assay using a transgenic mouse line C9BAC. Mice were injected with one of six different constructs encapsulated by AAV9 or AAVrhlO. Figure 49A depicts the percentage of excision and ligation in the liver for Group 1 to Group 7 (G1 - G7). Figure 49B depicts the percentage of excision and ligation in the heart for G1 to G7. The vector for G1 comprises a CAG promoter, a CNR1-2L.98 and a CNR21-22L.38 meganuclease, and a WPRE 3’UTR encapsulated in an AAV9 particle; the vector for G2 comprises a Synl promoter, a CNR1-2L.98 and a CNR21- 22L.38 meganuclease, and a WPRE 3’UTR encapsulated in an AAV9 particle; the vector for G3 comprises a CAG promoter, a CNR1-2L.98 and a CNR21-22L.38 meganuclease encapsulated in an AAV9 particle; the vector for G4 comprises a Synl promoter, a CNR1-2L.98 and a CNR21-22L.38 meganuclease encapsulated in an AAV9 particle; the vector for G5 comprises a CAG promoter, a CNR1-2L.98 and a CNR21-22L.38 meganuclease encapsulated in an AAVrhlO particle; the vector for G6 comprises a CAG promoter, a CNR1-2L.15 and a CNR21-22x.l3 meganuclease, and a WPRE 3’ UTR encapsulated in an AAV9 particle. Group 7 is treated with PBS control. Figure 50A-50D. Provide bar graphs depicting the percentage of excision and ligation observed in an in vivo assay in non-human primates (cynomolgus macaques). NHP were injected with one of three different constructs encapsulated by AAV9 or AAVrhlO or a control of PBS. Figure 50A depicts the percentage of excision and ligation in the target CNS. Figure 50B depicts the percentage of excision and ligation in the other CNS. Figure 50C depicts the percentage of excision and ligation in the peripheral nervous system. Figure 50D depicts the percentage of excision and ligation in the peripheral tissues. AAV9-CAG represents a viral vector comprising a CAG promoter, a CNR1-2L.98 and a CNR21-22L.38 meganuclease, and a WPRE 3’UTR encapsulated in an AAV9 particle. AAV9-Synl represents a viral vector comprising a Synl promoter, a CNR1-2L.98 and a CNR21-22L.38 meganuclease, and a WPRE 3’ UTR encapsulated in an AAV9 particle. AAV4hlO-Synl represents a viral vector comprising a Synl promoter, a CNR1-2L.98 and a CNR21-22L.38 meganuclease, and a WPRE 3’UTR encapsulated in an AAVrhlO particle.

[0180] Figure 51A-51B. Figure 51 A provides RNAscope staining of lower motor neurons from the spine with the CHAT marker in NHPs treated with either AAV vectors (upper left and right panel) or PBS control (lower left and lower right panel). CHAT expression is provided in the upper and lower left panels and meganuclease staining is provided in the upper right and lower right panels. Figure 5 IB provides quantification of meganuclease positive motor neurons from the entire spine from RNAscope staining and analysis.

[0181] Figure 52A-52B. Figure 52A provides RNA scope staining of upper motor neurons from the motor cortex of NHPs treated with either AAV vectors or PBS control. The left panel provides staining of upper motor neurons, and the right panel shows staining of upper motor neurons expressing the meganuclease. Figure 52B provides quantification of the RNAscope staining of meganuclease positive upper motor neurons from RNAscope staining and analysis.

[0182] Figure 53A-53B. Figure 53A provides RNA scope staining of motor neurons from the medulla in NHPs treated with AAV vectors that are positive for meganuclease expression. Figure 53B provides quantification of the RNAscope staining of meganuclease motor neurons from RNAscope staining and analysis.

[0183] Figure 54. Provides a bar graph depicting the percentage of excision and ligation observed in an in vivo assay using a transgenic mouse line C9BAC across different tissue types. Mice were injected with one of four different constructs encapsulated by AAV9. Group 1 was treated with a viral vector comprising a CNR1-2L.98 and CNR19-20L.71 meganuclease encapsulated in an AAV9 particle; Group 2 was treated with a viral vector comprising a CNR1-2L.98 and CNR21- 22L.38 meganuclease encapsulated in an AAV9 particle; Group 3 was treated with a viral vector comprising a CNR1-2L.98 and CNR23-24L.35 meganuclease encapsulated in an AAV9 particle; and Group 4 was treated with a viral vector comprising a CNR1-2L.98 and CNR17-18L.12 meganuclease encapsulated in an AAV9 particle.

[0184] Figure 55A-55E. Provide survival curves depicting the percent survival of a transgenic mouse line C9-500 treated as follows. Mice were injected with one of four different constructs encapsulated by AAV9. Group 1 was a control group treated with PBS. Group 2 was treated with a viral vector comprising a CAG promoter, and a CNR1-2L.211 and a CNR21-22L.129 meganuclease encapsulated in an AAV9 particle. Group 3 was treated with a viral vector comprising a Synl promoter, and a CNR1-2L.211 and a CNR21-22L.139 meganuclease encapsulated in an AAV9 particle. Group 4 was treated with a viral vector comprising a CAG promoter, and a CNR3-4L.212 and a CNR29-30L.107 meganuclease encapsulated in an AAV9 particle. Group 5 was treated with a viral vector comprising a Synl promoter, and a CNR3-4L.212 and a CNR29-30L.107 meganuclease encapsulated in an AAV9 particle. Figure 55A shows the survival curve for Group 1 (control); Figure 55B shows the survival curve for Group 2; Figure 55C shows the survival curve for Group 3; Figure 55D shows the survival curve for Group 4; and Figure 55E shows the survival curve for Group 5.

[0185] Figure 56. Provides bar graphs showing the excision and ligation of regions as detected by ddPCR in the spine and cortex of the surviving mice in group 1 (labelled as PBS), group 2 ( labelled as CAG CNR1 -2x21-22), group 3 (labelled as Synl CNR1 -2x21-22), and group 4 (labelled as CNR3-4x29-30) corresponding to those groups described in Figure 55A-55E.

[0186] Figure 57. Provides a bar graph showing expression levels of polyGP in the cortex of mice from Group 1 (control; circle), Group 2 (square), Group 3 (triangle) and Group 4 (inverted triangle) corresponding to those groups described in Figure 55A-55E.

[0187] Figures 58A-58B. Provide bar graphs showing the number of RNA foci in the motor cortex (Figure 58A) or the lumbar spine (Figure 58B) of mice from Group 1 (control; circle), Group 2 (square), Group 3 (triangle), and Group 4 (inverted triangle) corresponding to those groups described in Figure 55A-55E.

[0188] Figures 59A-59D. Provide bar graphs showing the expression of C90rf72 mRNA f RNA foci in the cortex (Figure 59A) or the cervical spine (Figure 59B). Figure 59A depicts expression results for all the C90rf72 isoforms for mice from Group 1 (control; circle), Group 2 (square), Group 3 (triangle), and Group 4 (inverted triangle). Figure 59B depicts expression results for all the C90rf72 isoforms for mice from group 1 (control; circle), Group 2 (square), Group 3 (triangle), and Group 4 (inverted triangle) . Figure 59C depicts expression results for the C90rf72 long isoforms (isoform variant 2 and isoform variant 3 denoted as V23) for mice from group 1 (control; circle), Group 2 (square), Group 3 (triangle), and Group 4 (inverted triangle). Figure 59D depicts expression results for the C90rf72 short isoform variant 1 (denoted as VI) for mice from group 1 (control; circle), Group 2 (square), Group 3 (triangle), and Group 4 (inverted triangle) corresponding to those groups described in Figure 55A-55E.

[0189] Figures 60A-60B. Provide bar graphs showing the percentage of excision and ligation of the C90rf72 locus and RNA foci in motor neuron progenitor cells (MNPCs) derived from a C9-ALS patient or a healthy donor. Cells were treated with a viral vector comprising a neuron specific enhancer, a Synl promoter, a CNR21-22L.286 and a CNR1-2L.532 meganuclease encapsulated in an AAV9 particle. Figure 60A depicts the percentage of percentage of excision and ligation of the C90rf72 in treated cells (square) vs untreated control (circle). Figure 60B depicts the number of RNA Foci between healthy donor cells, untreated C9-ALS-derived cells, and treated C9-ALS- derived cells.

[0190] Figures 61 A-61B. Provide bar graphs showing the percentage of excision and ligation of the C90rf72 locus and RNA foci in motor neuron progenitor cells (MNPCs) derived from a C9-ALS patient or a healthy donor. Cells were treated with a viral vector comprising a neuron specific enhancer, a Synl promoter, a CNR 1-2L.425 and a CNR 21-22L.212 meganuclease encapsulated in an AAV9 particle. Figure 61 A depicts the percentage of excision and ligation of the C90rf72 in treated cells vs untreated control. Figure 61B depicts the number of RNA Foci between healthy donor cells, untreated C9-ALS-derived cells, and treated C9-ALS-derived cells.

[0191] BRIEF DESCRIPTION OF THE SEQUENCES

[0192] SEQ ID NO: 1 sets forth amino acid sequence of the wild-type I-Crel meganuclease from Chlamydomonas reinhardtii.

[0193] SEQ ID NO: 2 sets forth the amino acid sequence of the LAGLID ADG motif.

[0194] SEQ ID NO: 3 sets forth the nucleic acid sequence of the sense strand of the CNR 1-2 recognition sequence.

[0195] SEQ ID NO: 4 sets forth the nucleic acid sequence of the antisense strand of the CNR 1-2 recognition sequence.

[0196] SEQ ID NO: 5 sets forth the nucleic acid sequence of the sense strand of the CNR 21-22 recognition sequence.

[0197] SEQ ID NO: 6 sets forth the nucleic acid sequence of the antisense strand of the CNR 21-22 recognition sequence.

[0198] SEQ ID NO: 7 sets forth the amino acid sequence of the CNR 1-2L.532 engineered meganuclease. SEQ ID NO: 8 sets forth the amino acid sequence of the CNR 1-2L.468 engineered meganuclease.

[0199] SEQ ID NO: sets forth the amino acid sequence of the CNR 1-2L.531 engineered meganuclease.

[0200] SEQ ID NO: 10 sets forth the amino acid sequence of the CNR 1-2L.371 engineered meganuclease.

[0201] SEQ ID NO: 11 sets forth the amino acid sequence of the CNR 1-2L.425 engineered meganuclease.

[0202] SEQ ID NO: 12 sets forth the amino acid sequence of the CNR 1-2L.284 engineered meganuclease.

[0203] SEQ ID NO: 13 sets forth the amino acid sequence of the CNR 1-2L.297 engineered meganuclease.

[0204] SEQ ID NO: 14 sets forth the amino acid sequence of the CNR 1-2L.211 engineered meganuclease.

[0205] SEQ ID NO: 15 sets forth the amino acid sequence of the CNR 1-2L.98 engineered meganuclease.

[0206] SEQ ID NO: 16 sets forth the amino acid sequence of the CNR 1-2L.108 engineered meganuclease.

[0207] SEQ ID NO: 17 sets forth the amino acid sequence of the CNR 1-2L15 engineered meganuclease.

[0208] SEQ ID NO: 18 sets forth the amino acid sequence of the CNR l-2x.88 engineered meganuclease.

[0209] SEQ ID NO: 19 sets forth the amino acid sequence of the CNR 1-2L.532 engineered meganuclease CNR1 binding subunit.

[0210] SEQ ID NO: 20 sets forth the amino acid sequence of the CNR 1-2L.468 engineered meganuclease CNR1 binding subunit.

[0211] SEQ ID NO: 21 sets forth the amino acid sequence of the CNR 1-2L.531 engineered meganuclease CNR1 binding subunit.

[0212] SEQ ID NO: 22 sets forth the amino acid sequence of the CNR 1-2L.371 engineered meganuclease CNR1 binding subunit.

[0213] SEQ ID NO: 23 sets forth the amino acid sequence of the CNR 1-2L.425 engineered meganuclease CNR1 binding subunit.

[0214] SEQ ID NO: 24 sets forth the amino acid sequence of the CNR 1-2L.284 engineered meganuclease CNR1 binding subunit. SEQ ID NO: 25 sets forth the amino acid sequence of the CNR 1-2L.297 engineered meganuclease CNR1 binding subunit.

[0215] SEQ ID NO: 26 sets forth the amino acid sequence of the CNR 1-2L.211 engineered meganuclease CNR1 binding subunit.

[0216] SEQ ID NO: 27 sets forth the amino acid sequence of the CNR 1-2L.98 engineered meganuclease CNR1 binding subunit.

[0217] SEQ ID NO: 28 sets forth the amino acid sequence of the CNR 1-2L.108 engineered meganuclease CNR1 binding subunit.

[0218] SEQ ID NO: 29 sets forth the amino acid sequence of the CNR 1-2L15 engineered meganuclease CNR1 binding subunit.

[0219] SEQ ID NO: 30 sets forth the amino acid sequence of the CNR l-2x.88 engineered meganuclease CNR1 binding subunit.

[0220] SEQ ID NO: 31 sets forth the amino acid sequence of the CNR 1-2L.532 engineered meganuclease CNR2 binding subunit.

[0221] SEQ ID NO: 32 sets forth the amino acid sequence of the CNR 1-2L.468 engineered meganuclease CNR2 binding subunit.

[0222] SEQ ID NO: 33 sets forth the amino acid sequence of the CNR 1-2L.531 engineered meganuclease CNR2 binding subunit.

[0223] SEQ ID NO: 34 sets forth the amino acid sequence of the CNR 1-2L.371 engineered meganuclease CNR2 binding subunit.

[0224] SEQ ID NO: 35 sets forth the amino acid sequence of the CNR 1-2L.425 engineered meganuclease CNR2 binding subunit.

[0225] SEQ ID NO: 36 sets forth the amino acid sequence of the CNR 1-2L.284 engineered meganuclease CNR2 binding subunit.

[0226] SEQ ID NO: 37 sets forth the amino acid sequence of the CNR 1-2L.297 engineered meganuclease CNR2 binding subunit.

[0227] SEQ ID NO: 38 sets forth the amino acid sequence of the CNR 1-2L.211 engineered meganuclease CNR2 binding subunit.

[0228] SEQ ID NO: 39 sets forth the amino acid sequence of the CNR 1-2L.98 engineered meganuclease CNR2 binding subunit.

[0229] SEQ ID NO: 40 sets forth the amino acid sequence of the CNR 1-2L.108 engineered meganuclease CNR2 binding subunit.

[0230] SEQ ID NO: 41 sets forth the amino acid sequence of the CNR 1-2L15 engineered meganuclease CNR2 binding subunit. SEQ ID NO: 42 sets forth the amino acid sequence of the CNR l-2Lx.88 engineered meganuclease CNR2 binding subunit.

[0231] SEQ ID NO: 43 sets forth the amino acid sequence of the CNR 1-2L.532 engineered meganuclease CNR1 binding subunit HVR1 region.

[0232] SEQ ID NO: 44 sets forth the amino acid sequence of the CNR 1-2L.468 engineered meganuclease CNR1 binding subunit HVR1 region.

[0233] SEQ ID NO: 45 sets forth the amino acid sequence of the CNR 1-2L.531 engineered meganuclease CNR1 binding subunit HVR1 region.

[0234] SEQ ID NO: 46 sets forth the amino acid sequence of the CNR 1-2L.371 engineered meganuclease CNR1 binding subunit HVR1 region.

[0235] SEQ ID NO: 47 sets forth the amino acid sequence of the CNR 1-2L.425 engineered meganuclease CNR1 binding subunit HVR1 region.

[0236] SEQ ID NO: 48 sets forth the amino acid sequence of the CNR 1-2L.284 engineered meganuclease CNR1 binding subunit HVR1 region.

[0237] SEQ ID NO: 49 sets forth the amino acid sequence of the CNR 1-2L.297 engineered meganuclease CNR1 binding subunit HVR1 region.

[0238] SEQ ID NO: 50 sets forth the amino acid sequence of the CNR 1-2L.211 engineered meganuclease CNR1 binding subunit HVR1 region.

[0239] SEQ ID NO: 51 sets forth the amino acid sequence of the CNR 1-2L.98 engineered meganuclease CNR1 binding subunit HVR1 region.

[0240] SEQ ID NO: 52 sets forth the amino acid sequence of the CNR 1-2L.108 engineered meganuclease CNR1 binding subunit HVR1 region.

[0241] SEQ ID NO: 53 sets forth the amino acid sequence of the CNR 1-2L15 engineered meganuclease CNR1 binding subunit HVR1 region.

[0242] SEQ ID NO: 54 sets forth the amino acid sequence of the CNR l-2x.88 engineered meganuclease CNR1 binding subunit HVR1 region.

[0243] SEQ ID NO: 55 sets forth the amino acid sequence of the CNR 1-2L.532 engineered meganuclease CNR2 binding subunit HVR2 region.

[0244] SEQ ID NO: 56 sets forth the amino acid sequence of the CNR 1-2L.468 engineered meganuclease CNR2 binding subunit HVR2 region.

[0245] SEQ ID NO: 57 sets forth the amino acid sequence of the CNR 1-2L.531 engineered meganuclease CNR2 binding subunit HVR2 region.

[0246] SEQ ID NO: 58 sets forth the amino acid sequence of the CNR 1-2L.371 engineered meganuclease CNR2 binding subunit HVR2 region. SEQ ID NO: 59 sets forth the amino acid sequence of the CNR 1-2L.425 engineered meganuclease CNR2 binding subunit HVR2 region.

[0247] SEQ ID NO: 60 sets forth the amino acid sequence of the CNR 1-2L.284 engineered meganuclease CNR2 binding subunit HVR2 region.

[0248] SEQ ID NO: 61 sets forth the amino acid sequence of the CNR 1-2L.297 engineered meganuclease CNR2 binding subunit HVR2 region.

[0249] SEQ ID NO: 62 sets forth the amino acid sequence of the CNR 1-2L.211 engineered meganuclease CNR2 binding subunit HVR2 region.

[0250] SEQ ID NO: 64 sets forth the amino acid sequence of the CNR 1-2L.98 engineered meganuclease CNR2 binding subunit HVR2 region.

[0251] SEQ ID NO: 64 sets forth the amino acid sequence of the CNR 1-2L.108 engineered meganuclease CNR2 binding subunit HVR2 region.

[0252] SEQ ID NO: 65 sets forth the amino acid sequence of the CNR 1-2L15 engineered meganuclease CNR2 binding subunit HVR2 region.

[0253] SEQ ID NO: 66 sets forth the amino acid sequence of the CNR l-2x.88 engineered meganuclease CNR2 binding subunit HVR2 region.

[0254] SEQ ID NO: 67 sets forth a nucleic acid sequence encoding the CNR 1-2L.532 engineered meganuclease.

[0255] SEQ ID NO: 68 sets forth a nucleic acid sequence encoding the CNR 1-2L.468 engineered meganuclease.

[0256] SEQ ID NO: 69 sets forth a nucleic acid sequence encoding the CNR 1-2L.531 engineered meganuclease.

[0257] SEQ ID NO: 70 sets forth a nucleic acid sequence encoding the CNR 1-2L.371 engineered meganuclease.

[0258] SEQ ID NO: 71 sets forth a nucleic acid sequence encoding the CNR 1-2L.425 engineered meganuclease.

[0259] SEQ ID NO: 72 sets forth a nucleic acid sequence encoding the CNR 1-2L.284 engineered meganuclease.

[0260] SEQ ID NO: 73 sets forth a nucleic acid sequence encoding the CNR 1-2L.297 engineered meganuclease.

[0261] SEQ ID NO: 74 sets forth a nucleic acid sequence encoding the CNR 1-2L.211 engineered meganuclease.

[0262] SEQ ID NO: 75 sets forth a nucleic acid sequence encoding the CNR 1-2L.98 engineered meganuclease. SEQ ID NO: 76 sets forth a nucleic acid sequence encoding the CNR 1-2L.108 engineered meganuclease.

[0263] SEQ ID NO: 77 sets forth a nucleic acid sequence encoding the CNR 1-2L15 engineered meganuclease.

[0264] SEQ ID NO: 78 sets forth a nucleic acid sequence encoding the CNR l-2x.88 engineered meganuclease.

[0265] SEQ ID NO: 79 sets forth the amino acid sequence of the CNR 21-22L.290 engineered meganuclease.

[0266] SEQ ID NO: 80 sets forth the amino acid sequence of the CNR 21-22L.286 engineered meganuclease.

[0267] SEQ ID NO: 81 sets forth the amino acid sequence of the CNR 21-22L.274 engineered meganuclease.

[0268] SEQ ID NO: 82 sets forth the amino acid sequence of the CNR 21-22L.212 engineered meganuclease.

[0269] SEQ ID NO: 83 sets forth the amino acid sequence of the CNR 21-22L.197 engineered meganuclease.

[0270] SEQ ID NO: 84 sets forth the amino acid sequence of the CNR 21-22L.169 engineered meganuclease.

[0271] SEQ ID NO: 85 sets forth the amino acid sequence of the CNR 21-22L.139 engineered meganuclease.

[0272] SEQ ID NO: 86 sets forth the amino acid sequence of the CNR 21-22L.38 engineered meganuclease.

[0273] SEQ ID NO: 87 sets forth the amino acid sequence of the CNR 21-22x.13 engineered meganuclease.

[0274] SEQ ID NO: 88 sets forth the amino acid sequence of the CNR 21-22L.290 engineered meganuclease CNR21 binding subunit.

[0275] SEQ ID NO: 89 sets forth the amino acid sequence of the CNR 21-22L.286 engineered meganuclease CNR21 binding subunit.

[0276] SEQ ID NO: 90 sets forth the amino acid sequence of the CNR 21-22L.274 engineered meganuclease CNR21 binding subunit.

[0277] SEQ ID NO: 91 sets forth the amino acid sequence of the CNR 21-22L.212 engineered meganuclease CNR21 binding subunit.

[0278] SEQ ID NO: 92 sets forth the amino acid sequence of the CNR 21-22L.197 engineered meganuclease CNR21 binding subunit. SEQ ID NO: 93 sets forth the amino acid sequence of the CNR 21-22L.169 engineered meganuclease CNR21 binding subunit.

[0279] SEQ ID NO: 94 sets forth the amino acid sequence of the CNR 21-22L.139 engineered meganuclease CNR21 binding subunit.

[0280] SEQ ID NO: 95 sets forth the amino acid sequence of the CNR 21-22L.38 engineered meganuclease CNR21 binding subunit.

[0281] SEQ ID NO: 96 sets forth the amino acid sequence of the CNR 21-22x.13 engineered meganuclease CNR21 binding subunit.

[0282] SEQ ID NO: 97 sets forth the amino acid sequence of the CNR 21-22L.290 engineered meganuclease CNR22 binding subunit.

[0283] SEQ ID NO: 98 sets forth the amino acid sequence of the CNR 21-22L.286 engineered meganuclease CNR22 binding subunit.

[0284] SEQ ID NO: 99 sets forth the amino acid sequence of the CNR 21-22L.274 engineered meganuclease CNR22 binding subunit.

[0285] SEQ ID NO: 100 sets forth the amino acid sequence of the CNR 21-22L.212 engineered meganuclease CNR22 binding subunit.

[0286] SEQ ID NO: 101 sets forth the amino acid sequence of the CNR 21-22L.197 engineered meganuclease CNR22 binding subunit.

[0287] SEQ ID NO: 102 sets forth the amino acid sequence of the CNR 21-22L.169 engineered meganuclease CNR22 binding subunit.

[0288] SEQ ID NO: 103 sets forth the amino acid sequence of the CNR 21-22L.139 engineered meganuclease CNR22 binding subunit.

[0289] SEQ ID NO: 104 sets forth the amino acid sequence of the CNR 21-22L.38 engineered meganuclease CNR22 binding subunit.

[0290] SEQ ID NO: 105 sets forth the amino acid sequence of the CNR 21-22Lx. l3 engineered meganuclease CNR22 binding subunit.

[0291] SEQ ID NO: 106 sets forth the amino acid sequence of the CNR 21-22L.290 engineered meganuclease CNR21 binding subunit HVR1 region.

[0292] SEQ ID NO: 107 sets forth the amino acid sequence of the CNR 21-22L.286 engineered meganuclease CNR21 binding subunit HVR1 region.

[0293] SEQ ID NO: 108 sets forth the amino acid sequence of the CNR 21-22L.274 engineered meganuclease CNR21 binding subunit HVR1 region.

[0294] SEQ ID NO: 109 sets forth the amino acid sequence of the CNR 21-22L.212 engineered meganuclease CNR21 binding subunit HVR1 region. SEQ ID NO: 110 sets forth the amino acid sequence of the CNR 21-22L.197 engineered meganuclease CNR21 binding subunit HVR1 region.

[0295] SEQ ID NO: 111 sets forth the amino acid sequence of the CNR 21-22L.169 engineered meganuclease CNR21 binding subunit HVR1 region.

[0296] SEQ ID NO: 112 sets forth the amino acid sequence of the CNR 21-22L.139 engineered meganuclease CNR21 binding subunit HVR1 region.

[0297] SEQ ID NO: 113 sets forth the amino acid sequence of the CNR 21-22L.38 engineered meganuclease CNR21 binding subunit HVR1 region.

[0298] SEQ ID NO: 114 sets forth the amino acid sequence of the CNR 21-22x.l3 engineered meganuclease CNR21 binding subunit HVR1 region.

[0299] SEQ ID NO: 115 sets forth the amino acid sequence of the CNR 21-22L.290 engineered meganuclease CNR22 binding subunit HVR2 region.

[0300] SEQ ID NO: 116 sets forth the amino acid sequence of the CNR 21-22L.286 engineered meganuclease CNR22 binding subunit HVR2 region.

[0301] SEQ ID NO: 117 sets forth the amino acid sequence of the CNR 21-22L.274 engineered meganuclease CNR22 binding subunit HVR2 region.

[0302] SEQ ID NO: 118 sets forth the amino acid sequence of the CNR 21-22L.212 engineered meganuclease CNR22 binding subunit HVR2 region.

[0303] SEQ ID NO: 119 sets forth the amino acid sequence of the CNR 21-22L.197 engineered meganuclease CNR22 binding subunit HVR2 region.

[0304] SEQ ID NO: 120 sets forth the amino acid sequence of the CNR 21-22L.169 engineered meganuclease CNR22 binding subunit HVR2 region.

[0305] SEQ ID NO: 121 sets forth the amino acid sequence of the CNR 21-22L.139 engineered meganuclease CNR22 binding subunit HVR2 region.

[0306] SEQ ID NO: 122 sets forth the amino acid sequence of the CNR 21-22L.38 engineered meganuclease CNR22 binding subunit HVR2 region.

[0307] SEQ ID NO: 123 sets forth the amino acid sequence of the CNR 21-22x.l3 engineered meganuclease CNR22 binding subunit HVR2 region.

[0308] SEQ ID NO: 124 sets forth a nucleic acid sequence encoding the CNR 21-22L.290 engineered meganuclease.

[0309] SEQ ID NO: 125 sets forth a nucleic acid sequence encoding the CNR 21-22L.286 engineered meganuclease.

[0310] SEQ ID NO: 126 sets forth a nucleic acid sequence encoding the CNR 21-22L.274 engineered meganuclease. SEQ ID NO: 127 sets forth a nucleic acid sequence encoding the CNR 21-22L.212 engineered meganuclease.

[0311] SEQ ID NO: 128 sets forth a nucleic acid sequence encoding the CNR 21-22L.197 engineered meganuclease.

[0312] SEQ ID NO: 129 sets forth a nucleic acid sequence encoding the CNR 21-22L.169 engineered meganuclease.

[0313] SEQ ID NO: 130 sets forth a nucleic acid sequence encoding the CNR 21-22L.139 engineered meganuclease.

[0314] SEQ ID NO: 131 sets forth a nucleic acid sequence encoding the CNR 21-22L.38 engineered meganuclease.

[0315] SEQ ID NO: 132 sets forth a nucleic acid sequence encoding the CNR 21-22x.l3 engineered meganuclease.

[0316] SEQ ID NO: 133 sets forth the amino acid sequence of a SV40 nuclear localization signal.

[0317] SEQ ID NO: 134 sets forth the amino acid sequence of a CMYC nuclear localization signal.

[0318] SEQ ID NO: 135 sets forth the amino acid sequence of a P2A / furin peptide.

[0319] SEQ ID NO: 136 sets forth the nucleic acid sequence of a chimeric intron.

[0320] SEQ ID NO: 137 sets forth the nucleic acid sequence of a poly A tail.

[0321] SEQ ID NO: 138 sets forth the nucleic acid sequence of a hSynl promoter.

[0322] SEQ ID NO: 139 sets forth the nucleic acid sequence of a CAG promoter.

[0323] SEQ ID NO: 140 sets forth the nucleic acid sequence of a SV40 nuclear localization signal.

[0324] SEQ ID NO: 141 sets forth the nucleic acid sequence of a CMV enhancer.

[0325] SEQ ID NO: 142 sets forth the nucleic acid sequence of a CMYC nuclear localization signal.

[0326] SEQ ID NO: 143 sets forth the nucleic acid sequence of a SV40 nuclear localization signal that has been codon modified.

[0327] SEQ ID NO: 144 sets forth the nucleic acid sequence of a CMYC nuclear localization signal that has been codon modified.

[0328] SEQ ID NO: 145 sets forth the nucleic acid sequence of a CNR 1-2L.425 engineered meganuclease that has been codon modified.

[0329] SEQ ID NO: 146 sets forth the nucleic acid sequence of a CNR 1-2L.211 engineered meganuclease that has been codon modified.

[0330] SEQ ID NO: 147 sets forth the nucleic acid sequence of a CNR 1-2L.98 engineered meganuclease that has been codon modified. SEQ ID NO: 148 sets forth the nucleic acid sequence of a CNR 1-2L15 engineered meganuclease that has been codon modified.

[0331] SEQ ID NO: 149 sets forth the nucleic acid sequence of a CNR 21-22L.286 engineered meganuclease that has been codon modified.

[0332] SEQ ID NO: 150 sets forth the nucleic acid sequence of a CNR1-2 Fl primer, SEQ ID NO: 151 sets forth the nucleic acid sequence of a CNR1-2 F2 primer, SEQ ID NO: 152 sets forth the nucleic acid sequence of a CNR1-2 F3 primer, SEQ ID NO: 153 sets forth the nucleic acid sequence of a CNR1-2 F4 primer, SEQ ID NO: 154 sets forth the nucleic acid sequence of a CNR1-2 R1 primer, SEQ ID NO: 155 sets forth the nucleic acid sequence of a CNR1-2 R2 primer, SEQ ID NO: 156 sets forth the nucleic acid sequence of a CNR1-2 R3 primer, SEQ ID NO: 157 sets forth the nucleic acid sequence of a CNR1-2 R4 primer, SEQ ID NO: 158 sets forth the nucleic acid sequence of a CNR1-2 R5 primer, SEQ ID NO: 159 sets forth the nucleic acid sequence of a CNR1-2 R6 primer, SEQ ID NO: 160 sets forth the nucleic acid sequence of a CNR1-2 R7 primer, SEQ ID NO: 161 sets forth the nucleic acid sequence of a CNR1-2 R8 primer, SEQ ID NO: 162 sets forth the nucleic acid sequence of a CNR1-2 Pl probe, SEQ ID NO: 163 sets forth the nucleic acid sequence of a DWN Fl primer, SEQ ID NO: 164 sets forth the nucleic acid sequence of a UP R1 primer, SEQ ID NO: 165 sets forth the nucleic acid sequence of a Pl DWN probe, SEQ ID NO: 166 sets forth the nucleic acid sequence of a CNR19-20 R1 primer, SEQ ID NO: 167 sets forth the nucleic acid sequence of a Pl UP probe. SEQ ID NO: 168 sets forth the nucleic acid sequence of a CNR17-18 Fl primer, SEQ ID NO: 169 sets forth the nucleic acid sequence of a P2 probe. SEQ ID NO: 170 sets forth the nucleic acid sequence of a F2 primer, SEQ ID NO: 171 sets forth the nucleic acid sequence of a R2 primer. SEQ ID NO: 172 sets forth the nucleic acid sequence of a CNR21-22 R1 primer, SEQ ID NO: 173 sets forth the nucleic acid sequence of a CNR23-24 R1 primer, SEQ ID NO: 174 sets forth the nucleic acid sequence of a CNR35-36 R1 primer, SEQ ID NO: 175 sets forth the nucleic acid sequence of a CNR29-30 Fl primer, SEQ ID NO: 176 sets forth the nucleic acid sequence of a CNR25-26 Fl primer, SEQ ID NO: 177 sets forth the nucleic acid sequence of a CNR31-32 Fl primer, SEQ ID NO: 178 sets forth the nucleic acid sequence of a CNR21-22 Fl primer, SEQ ID NO: 179 sets forth the nucleic acid sequence of a CNR21-22 R2 primer. SEQ ID NO: 180 sets forth the nucleic acid sequence of a CNR21-22 Pl primer.

[0333] SEQ ID NO: 181 sets forth the nucleic acid sequence of a CNR21-22 R3 primer.

[0334] SEQ ID NO: 182 sets forth the nucleic acid sequence of an APC Fl primer.

[0335] SEQ ID NO: 183 sets forth the nucleic acid sequence of an APC F2 primer.

[0336] SEQ ID NO: 184 sets forth the nucleic acid sequence of an APC Pl primer.

[0337] SEQ ID NO: 185 sets forth the nucleic acid sequence of a region of the C90rf72 gene that has been re-ligated following excision with a CNR1-2 and CNR21-22 engineered meganuclease described herein.

[0338] SEQ ID NO: 186 sets forth the nucleic acid sequence of a region of the C90rf72 gene that has been re-ligated following excision with a CNR1-2 and CNR21-22 engineered meganuclease described herein.

[0339] SEQ ID NO: 187 sets forth the nucleic acid sequence of a region of the C90rf72 gene that has been re-ligated following excision with a CNR1-2 and CNR21-22 engineered meganuclease described herein.

[0340] SEQ ID NO: 188 sets forth the nucleic acid sequence of a region of the C90rf72 gene that has been re-ligated following excision with a CNR1-2 and CNR21-22 engineered meganuclease described herein.

[0341] SEQ ID NO: 189 sets forth the nucleic acid sequence of a region of the C90rf72 gene that has been re-ligated following excision with a CNR1-2 and CNR21-22 engineered meganuclease described herein.

[0342] SEQ ID NO: 190 sets forth the nucleic acid sequence of a minimal promoter region of the C90rf72 gene that is immediately 5' upstream from exon la of the C90rf72 gene.

[0343] SEQ ID NO: 191 sets forth the nucleic acid sequence of exon la of the C90rf72 gene corresponding to mRNA variant 3.

[0344] SEQ ID NO: 192 sets forth the nucleic acid sequence of an exon la of the C90rf72 gene corresponding to mRNA variant 1.

[0345] SEQ ID NO: 193 sets forth the nucleic acid sequence of an exon lb of the C90rf72 gene corresponding to mRNA variant 2.

[0346] SEQ ID NO: 194 sets forth the nucleic acid sequence of a WPRE sequence.

[0347] SEQ ID NO: 195 sets forth an alternative amino acid sequence of the CNR 1-2L.15 engineered meganuclease when the methionine start codon of the engineered meganuclease is replaced with an alanine residue. The methionine start codon is a part of an N-terminal domain such as an NLS. SEQ ID NO: 196 sets forth the amino acid sequence of a peptide linker that is useful for linking a first subunit and a second subunit of the engineered meganucleases described herein.

[0348] DETAILED DESCRIPTION OF THE INVENTION

[0349] 1.1 References and Definitions

[0350] The patent and scientific literature referred to herein establishes knowledge that is available to those of skill in the art. The issued US patents, allowed applications, published foreign applications, and references, including GenBank database sequences, which are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference.

[0351] The present disclosure can be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. For example, features illustrated with respect to one embodiment can be incorporated into other embodiments, and features illustrated with respect to a particular embodiment can be deleted from that embodiment. In addition, numerous variations and additions to the embodiments suggested herein will be apparent to those skilled in the art in light of the present disclosure, which do not depart from the present invention.

[0352] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0353] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference herein in their entirety.

[0354] As used herein, “a,” “an,” or “the” can mean one or more than one. For example, “a” cell can mean a single cell or a multiplicity of cells.

[0355] As used herein, unless specifically indicated otherwise, the word “or” is used in the inclusive sense of “and / or” and not the exclusive sense of “either / or.”

[0356] As used herein, the terms “nuclease” and “endonuclease” are used interchangeably to refer to naturally-occurring or engineered enzymes, which cleave a phosphodiester bond within a polynucleotide chain. Engineered nucleases can include, without limitation, engineered meganucleases, zinc finger nucleases, TALENs, compact TALENs, CRISPR system nucleases, and megaTALs. In addition, any engineered nuclease is envisioned that is capable of generating overhangs at its cleavage site. As used herein, the terms “cleave” or “cleavage” refer to the hydrolysis of phosphodiester bonds within the backbone of a recognition sequence within a target sequence that results in a double-stranded break within the target sequence, referred to herein as a “cleavage site”.

[0357] As used herein, the term “meganuclease” refers to an endonuclease that binds doublestranded DNA at a recognition sequence that is greater than 12 base pairs. In some embodiments, the recognition sequence for a meganuclease of the present disclosure is 22 base pairs. A meganuclease can be an endonuclease that is derived from I-Crel (SEQ ID NO: 1), and can refer to an engineered variant of I-Crel that has been modified relative to natural I-Crel with respect to, for example, DNA-binding specificity, DNA cleavage activity, DNA-binding affinity, or dimerization properties. Methods for producing such modified variants of I-Crel are known in the art (e.g., WO 2007 / 047859, incorporated by reference in its entirety). A meganuclease as used herein binds to double-stranded DNA as a heterodimer. A meganuclease may also be a “single-chain meganuclease” in which a pair of DNA-binding domains is joined into a single polypeptide using a peptide linker. The term “homing endonuclease” is synonymous with the term “meganuclease.” Meganucleases of the present disclosure are substantially non-toxic when expressed in the targeted cells as described herein such that cells can be transfected and maintained at 37°C without observing deleterious effects on cell viability or significant reductions in meganuclease cleavage activity when measured using the methods described herein.

[0358] As used herein, the term “single-chain meganuclease” refers to a polypeptide comprising a pair of nuclease subunits joined by a linker. A single-chain meganuclease has the organization: N- terminal subunit - Linker - C-terminal subunit. The two meganuclease subunits will generally be non-identical in amino acid sequence and will bind non-identical DNA sequences. Thus, singlechain meganucleases typically cleave pseudo-palindromic or non-palindromic recognition sequences. A single-chain meganuclease may be referred to as a “single-chain heterodimer” or “single-chain heterodimeric meganuclease” although it is not, in fact, dimeric. For clarity, unless otherwise specified, the term “meganuclease” can refer to a dimeric or single-chain meganuclease.

[0359] As used herein, the term “linker” refers to an exogenous peptide sequence used to join two nuclease subunits into a single polypeptide. A linker may have a sequence that is found in natural proteins or may be an artificial sequence that is not found in any natural protein. A linker may be flexible and lacking in secondary structure or may have a propensity to form a specific three- dimensional structure under physiological conditions. A linker can include, without limitation, those encompassed by U.S. Patent Nos. 8,445,251, 9,340,777, 9,434,931, and 10,041,053, each of which is incorporated by reference in its entirety. In some embodiments, a linker may have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 196, which sets forth residues 154-195 of any one of SEQ ID NOs: 7-18 or 79-87.

[0360] As used herein, the terms “recombinant” or “engineered,” with respect to a protein, means having an altered amino acid sequence as a result of the application of genetic engineering techniques to nucleic acids that encode the protein and cells or organisms that express the protein. With respect to a nucleic acid, the term “recombinant” or “engineered” means having an altered nucleic acid sequence as a result of the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning technologies; transfection, transformation, and other gene transfer technologies; homologous recombination; site- directed mutagenesis; and gene fusion. In accordance with this definition, a protein having an amino acid sequence identical to a naturally-occurring protein but produced by cloning and expression in a heterologous host, is not considered recombinant or engineered.

[0361] As used herein, the term “wild-type” refers to the most common naturally occurring allele (z.e., polynucleotide sequence) in the allele population of the same type of gene, wherein a polypeptide encoded by the wild-type allele has its original functions. The term “wild-type” also refers to a polypeptide encoded by a wild-type allele. Wild-type alleles (z.e., polynucleotides) and polypeptides are distinguishable from mutant or variant alleles and polypeptides, which comprise one or more mutations and / or substitutions relative to the wild-type sequence(s). Whereas a wildtype allele or polypeptide can confer a normal phenotype in an organism, a mutant or variant allele or polypeptide can, in some instances, confer an altered phenotype. Wild-type nucleases are distinguishable from recombinant or non-naturally-occurring nucleases. The term “wild-type” can also refer to a cell, an organism, and / or a subject which possesses a wild-type allele of a particular gene, or a cell, an organism, and / or a subject used for comparative purposes.

[0362] As used herein, the term “genetically modified” refers to a cell or organism in which, or in an ancestor of which, a genomic DNA sequence has been deliberately modified by recombinant technology. As used herein, the term “genetically modified” encompasses the term “transgenic.”

[0363] As used herein, the term with respect to recombinant proteins, the term “modification” means any insertion, deletion, or substitution of an amino acid residue in the recombinant sequence relative to a reference sequence (e.g., a wild-type or a native sequence).

[0364] As used herein, the terms “recognition sequence” or “recognition site” refers to a DNA sequence that is bound and cleaved by a nuclease. In the case of a meganuclease, a recognition sequence comprises a pair of inverted, 9 basepair “half-sites,” which are separated by four basepairs. In the case of a single-chain meganuclease, the N-terminal domain of the protein contacts a first half-site and the C-terminal domain of the protein contacts a second half-site. Cleavage by a meganuclease produces four basepair 3' overhangs. “Overhangs,” or “sticky ends” are short, single-stranded DNA segments that can be produced by endonuclease cleavage of a double-stranded DNA sequence. In the case of meganucleases and single-chain meganucleases derived from I-Crel, the overhang comprises bases 10-13 of the 22 basepair recognition sequence.

[0365] As used herein, the terms “target site” or “target sequence” refers to a region of the chromosomal DNA of a cell comprising a recognition sequence for a nuclease.

[0366] As used herein, the terms “DNA-binding affinity” or “binding affinity” means the tendency of a nuclease to non-covalently associate with a reference DNA molecule (e.g., a recognition sequence or an arbitrary sequence). Binding affinity is measured by a dissociation constant, Kd. As used herein, a nuclease has “altered” binding affinity if the Kd of the nuclease for a reference recognition sequence is increased or decreased by a statistically significant percent change relative to a reference nuclease.

[0367] As used herein, the term “specificity” means the ability of a nuclease to bind and cleave double-stranded DNA molecules only at a particular sequence of base pairs referred to as the recognition sequence, or only at a particular set of recognition sequences. The set of recognition sequences will share certain conserved positions or sequence motifs but may be degenerate at one or more positions. A highly-specific nuclease is capable of cleaving only one or a very few recognition sequences. Specificity can be determined by any method known in the art.

[0368] As used herein, the term “C90rf72 gene” refers to the gene associated with National Center for Biotechnology Information (NCBI) gene ID 203228, as well as naturally occurring variants thereof. The term “C90rf72 polypeptide” refers to a polypeptide encoded by the C90rf72 gene. In some embodiments described herein, the C90rf72 gene is edited with a pair of engineered meganucleases, resulting in the excision of the hexanucleotide repeat region and exon lb and subsequent perfect ligation of the C90rf72 gene.

[0369] As used herein, the term “re-ligation” or “re-ligate” refers to the ligation (z.e., annealing) of a portion or all of the bases of a 3' overhang of a first cleavage site with bases of a 3' overhang of a second cleavage site in a C90rf72 gene following cleavage by a pair of engineered meganucleases described herein. In some embodiments, the four base pair center sequence of the pair of recognition sequences selected are 100%, 75%, 50%, or 25% complementary to each other. In some embodiments, a first recognition sequence comprises a four base pair center sequence that is 75% complementary to a four base pair center sequence of a second recognition sequence. In certain embodiments a first recognition sequence comprises the four base pair center sequence ATAA and a second recognition sequence comprises the four base pair center sequence ATAT. In some embodiments, the recognition sequences targeted by the disclosed engineered meganucleases have identical (i.e., 100% complementarity), such that the first and second cleavage sites will have complementary four basepair 3' overhangs. Accordingly, each basepair of the first 3' overhang pairs with its complement basepair on the second 3' overhang, and ligation occurs through a DNA ligase enzyme.

[0370] “C90rf72 associated disease” means any disease associated with any C90rf72 nucleic acid or expression product thereof. Such diseases may include a neurodegenerative disease. Such neurodegen erative diseases may include ALS and FTD.

[0371] “C90rf72 hexanucleotide repeat expansion associated disease” means any disease associated with a C90rf72 nucleic acid containing a hexanucleotide repeat expansion. In certain embodiments, the hexanucleotide repeat expansion may comprise GGGGCC, GGGGGG, GGGGGC, or GGGGCG repeated at least 30 times. Such diseases may include a neurodegenerative disease. Such neurodegenerative diseases may include ALS and FTD.

[0372] “C90rf72 nucleic acid” means any nucleic acid encoding C90rf72. For example, in certain embodiments, a C90rf72 nucleic acid includes a DNA sequence encoding C90rf72, an RNA sequence transcribed from DNA encoding C90rf72 (including genomic DNA comprising introns and exons), and an mRNA sequence encoding C90rf72. “C90rf72 mRNA” means an mRNA encoding a C90rf72 protein.

[0373] “Hexanucleotide repeat expansion” (also GGGGCCfn] RNA repeat) means a series of six bases (for example, GGGGCC, GGGGGG, GGGGCG, or GGGGGC) repeated at least twice. In certain embodiments, the hexanucleotide repeat expansion may be located in intron 1 of a C90rf72 nucleic acid. In certain embodiments, a pathogenic hexanucleotide repeat expansion includes at least 30 repeats of GGGGCC, GGGGGG, GGGGCG, or GGGGGC in a C90rf72 nucleic acid and is associated with disease. In certain embodiments, the repeats are consecutive. In certain embodiments, the repeats are interrupted by 1 or more nucleobases. In certain embodiments, a wildtype hexanucleotide repeat expansion includes 23 or fewer repeats of GGGGCC, GGGGGG, GGGGCG, or GGGGGC in a C90rf72 nucleic acid. In certain embodiments, the repeats are consecutive. In certain embodiments, the repeats are interrupted by 1 or more nucleobases.

[0374] As used herein, the term “exon la” when in reference to the C90rf72 gene refers to a noncoding exon that is transcribed as a part of mRNA variants 1 and 3 of the C90rf72 gene. In some embodiments, exon la comprises a sequence according to mRNA variant 3 having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 191. In some embodiments, exon la comprises a sequence according to SEQ ID NO: 191. In some embodiments, exon la comprises a sequence according to mRNA variant 1 having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity SEQ ID NO: 192. In some embodiments, exon la comprises a sequence according to SEQ ID NO: 192.

[0375] As used herein, the term “exon lb” when in reference to the C90rf72 gene refers to a noncoding exon that is transcribed as a part of mRNA variants 2 of the C90rf72 gene. In some embodiments, exon lb comprises a sequence according to mRNA variant 2 having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 193. In some embodiments, exon lb comprises a sequence according to SEQ ID NO: 191.

[0376] As used herein, the term “homologous recombination” or “HR” refers to the natural, cellular process in which a double-stranded DNA-break is repaired using a homologous DNA sequence as the repair template (see, e.g., Cahill et al. (2006) Front. Biosci. 11 : 1958-76). The homologous DNA sequence may be an endogenous chromosomal sequence or an exogenous nucleic acid that was delivered to the cell.

[0377] As used herein, the term “non-homologous end-joining” or “NHEJ” refers to the natural, cellular process in which a double-stranded DNA-break is repaired by the direct joining of two non- homologous DNA segments (see, e.g., Cahill et al. (2006)). DNA repair by non-homologous endjoining is error-prone and frequently results in the untemplated addition or deletion of DNA sequences at the site of repair. In some instances, cleavage at a target recognition sequence results in NHEJ at a target recognition site. Nuclease-induced cleavage of a target site in the coding sequence of a gene followed by DNA repair by non-homologous end joining (NHEJ can introduce mutations into the coding sequence, such as frameshift mutations, which disrupt gene function. Thus, engineered nucleases can be used to effectively knock-out a gene in a population of cells.

[0378] As used herein, the term “homology arms” or “sequences homologous to sequences flanking a nuclease cleavage site” refer to sequences flanking the 5' and 3' ends of a nucleic acid molecule, which promote insertion of the nucleic acid molecule into a cleavage site generated by a nuclease. In general, homology arms can have a length of at least 50 base pairs, preferably at least 100 base pairs, and up to 2000 base pairs or more, and can have at least 90%, preferably at least 95%, or more, sequence homology to their corresponding sequences in the genome. In some embodiments, the homology arms are about 500 base pairs.

[0379] As used herein, the term with respect to both amino acid sequences and nucleic acid sequences, the terms “percent identity,” “sequence identity,” “percentage similarity,” “sequence similarity” and the like refer to a measure of the degree of similarity of two sequences based upon an alignment of the sequences that maximizes similarity between aligned amino acid residues or nucleotides, and which is a function of the number of identical or similar residues or nucleotides, the number of total residues or nucleotides, and the presence and length of gaps in the sequence alignment. A variety of algorithms and computer programs are available for determining sequence similarity using standard parameters. As used herein, sequence similarity is measured using the BLASTp program for amino acid sequences and the BLASTn program for nucleic acid sequences, both of which are available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ), and are described in, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-10; Gish & States (1993) Nature Genet. 3:266-72; Madden et al. (1996) Meth. Enzymol. 266: 131-41; Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402; and Zhang et al. (2000) J. Comput. Biol. 7:203-14. As used herein, percent similarity of two amino acid sequences is the score based upon the following parameters for the BLASTp algorithm: word size=3; gap opening penalty=-l 1; gap extension penalty=-l; and scoring matrix=BLOSUM62. As used herein, percent similarity of two nucleic acid sequences is the score based upon the following parameters for the BLASTn algorithm: word size=l l; gap opening penalty=-5; gap extension penalty=-2; match reward=l; and mismatch penalty=-3.

[0380] As used herein, the term “corresponding to” with respect to modifications of two proteins or amino acid sequences is used to indicate that a specified modification in the first protein is a substitution of the same amino acid residue as in the modification in the second protein, and that the amino acid position of the modification in the first protein corresponds to or aligns with the amino acid position of the modification in the second protein when the two proteins are subjected to standard sequence alignments (e.g., using the BLASTp program). Thus, the modification of residue “X” to amino acid “A” in the first protein will correspond to the modification of residue “Y” to amino acid “A” in the second protein if residues X and Y correspond to each other in a sequence alignment and despite the fact that X and Y may be different numbers.

[0381] As used herein, the term “recognition half-site,” “recognition sequence half-site,” or simply “half-site” means a nucleic acid sequence in a double-stranded DNA molecule that is recognized and bound by a monomer of a homodimeric or heterodimeric meganuclease or by one subunit of a single-chain meganuclease or by one subunit of a single-chain meganuclease.

[0382] As used herein, the term post-transcriptional regulatory element (PTRE) refers to a nucleotide sequence, which functions to increase the stability of mRNA and cytoplasmic transport of the mRNA. Stability is of an intron-less gene is typically increased by promoting mRNA exportation from the nucleus to the cytoplasm, enhancing 3' end processing and stability. Suitable PTRE(s) include PTREs derived from viruses including but not limited to the Hepatitis B virus (HPRE) and Woodchuck Hepatitis virus (WPRE). In some embodiments described herein, the PTRE is a WPRE. In some embodiments the WPRE comprises a nucleotide sequence according to a sequence set forth in SEQ ID NO: 194 As used herein, the term “hypervariable region” refers to a localized sequence within a meganuclease monomer or subunit that comprises amino acids with relatively high variability. A hypervariable region can comprise about 50-60 contiguous residues, about 53-57 contiguous residues, or preferably about 56 residues. In some embodiments, the residues of a hypervariable region may correspond to positions 24-79 or positions 215-270 of any one of SEQ ID NOs: 7-18 or 79-87. A hypervariable region can comprise one or more residues that contact DNA bases in a recognition sequence and can be modified to alter base preference of the monomer or subunit. A hypervariable region can also comprise one or more residues that bind to the DNA backbone when the meganuclease associates with a double-stranded DNA recognition sequence. Such residues can be modified to alter the binding affinity of the meganuclease for the DNA backbone and the target recognition sequence. In different embodiments of the disclosure, a hypervariable region may comprise between 1-20 residues that exhibit variability and can be modified to influence base preference and / or DNA-binding affinity. In particular embodiments, a hypervariable region comprises between about 15-20 residues that exhibit variability and can be modified to influence base preference and / or DNA-binding affinity. In some embodiments, variable residues within a hypervariable region correspond to one or more of positions 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any one of SEQ ID NOs: 7-18 or 79-87. In certain embodiments, variable residues within a hypervariable region can further correspond to residues 48, 50, and 71-73 of any one of SEQ ID NOs: 7-18 or 79-87. In other embodiments, variable residues within a hypervariable region correspond to one or more of positions 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 239, 241, 259, 261, 262, 263, 264, 266, and 268 of any one of SEQ ID NOs: 7- 18 or 79-87. In certain embodiments, variable residues within a hypervariable region can further correspond to residues 239, 241, and 263-265 of any one of SEQ ID NOs: 7-18 or 79-87.

[0383] The terms “increase” in the context of C90rf72 gene or mRNA levels refers to any increase in the levels of C90rf72 gene or mRNA expression relative to a reference level including an increase of C90rf72 gene or mRNA expression of at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or more, when compared to a reference level or control. In some embodiments, an increase in C90rf72 gene or mRNA levels refers to an increase in a shortened C90rf72 gene or mRNA transcript, for example, missing a portion of the gene encoded by at least one intron (e.g., a portion comprising the hexanucleotide repeat region) or missing a portion of mRNA corresponding to the hexanucleotide repeat region and exon lb compared to the wild-type C90rf72 polypeptide or gene.

[0384] As used herein, the term “reference level” in the context of C90rf72 gene or mRNA levels refers to a level of C90rf72 gene or mRNA as measured in, for example, a control cell, control cell population or a control subject, at a previous time point in the control cell, the control cell population or the subject undergoing treatment (e.g., a pre-dose baseline level obtained from the control cell, control cell population or subject), or a pre-defined threshold level of C90rf72 gene or mRNA (e.g., a threshold level identified through previous experimentation).

[0385] As used herein, the term “a control” or “a control cell” refers to a cell that provides a reference point for measuring changes in genotype or phenotype of a genetically modified cell. A control cell may comprise, for example: (a) a wild-type cell, i.e., of the same genotype as the starting material for the genetic alteration which resulted in the genetically modified cell; (b) a cell of the same genotype as the genetically modified cell but which has been transformed with a null construct (i.e., with a construct which has no known effect on the trait of interest); or, (c) a cell genetically identical to the genetically modified cell but which is not exposed to conditions or stimuli or further genetic modifications that would induce expression of altered genotype or phenotype. A control subject may comprise, for example: a wild-type subject, i.e., of the same genotype as the starting subject for the genetic alteration which resulted in the genetically modified subject (e.g., a subject having the same mutation in a C90rf72 gene), which is not exposed to conditions or stimuli or further genetic modifications that would induce expression of altered genotype or phenotype in the subject.

[0386] As used herein, the term “recombinant DNA construct,” “recombinant construct,” “expression cassette,” “expression construct,” “chimeric construct,” “construct,” and “recombinant DNA fragment” are used interchangeably herein and are single or double-stranded polynucleotides. A recombinant construct comprises an artificial combination of nucleic acid fragments, including, without limitation, regulatory and coding sequences that are not found together in nature. For example, a recombinant DNA construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source and arranged in a manner different than that found in nature. Such a construct may be used by itself or may be used in conjunction with a vector.

[0387] As used herein, the term “vector” or “recombinant DNA vector” may be a construct that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell. If a vector is used, then the choice of vector is dependent upon the method that will be used to transform host cells as is well known to those skilled in the art. Vectors can include, without limitation, plasmid vectors and recombinant AAV vectors, or any other vector known in the art suitable for delivering a gene to a target cell. The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells comprising any of the isolated nucleotides or nucleic acid sequences of the disclosure. In some embodiments, a “vector” also refers to a viral vector. Viral vectors can include, without limitation, retroviral vectors, lentiviral vectors, adenoviral vectors, and AAV.

[0388] As used herein, the term “operably linked” is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a nucleic acid sequence encoding a nuclease as disclosed herein and a regulatory sequence (e.g., a promoter) is a functional link that allows for expression of the nucleic acid sequence encoding the nuclease. Operably linked elements may be contiguous or non-contiguous. When used to refer to the joining of two protein coding regions, by operably linked is intended that the coding regions are in the same reading frame.

[0389] As used herein, the terms “treatment” or “treating a subject” refers to the administration of an engineered meganuclease described herein, or a polynucleotide encoding an engineered meganuclease described herein, or a pair of such engineered meganucleases or polynucleotides, to a subject having ALS or FTD, or having increased susceptibility to ALS or FTD, for the purpose of increasing levels of a modified C90rf72 gene or mRNA in the subject. In some embodiments, expression of a shortened version (e.g., missing amino acids encoded by multiple exons) of the C90rf72 gene or mRNA is increased. In some embodiments, expression of a version of the C90rf72 gene or mRNA, lacking the non-coding sequence comprising the hexanucleotide repeat region is increased. Such treatment, in some embodiments, prevents development of a neurological condition or neurodegenerative disorder, such as ALS and / or FTD.

[0390] As used herein, the term “gc / kg” or “gene copies / kilogram” refers to the number of copies of a nucleic acid sequence encoding an engineered meganuclease described herein per weight in kilograms of a subject that is administered a polynucleotide comprising the nucleic acid sequence.

[0391] As used herein, the term “effective amount” or “therapeutically effective amount” refers to an amount sufficient to effect beneficial or desirable biological and / or clinical results. The therapeutically effective amount will vary depending on the formulation or composition used, the disease and its severity and the age, weight, physical condition and responsiveness of the subject to be treated. In specific embodiments, an effective amount of an engineered meganuclease or pair of engineered meganucleases described herein, or polynucleotide or pair of polynucleotides encoding the same, or pharmaceutical compositions disclosed herein, increases the level of expression of a modified, shortened C90rf72 gene or mRNA (e.g., a shortened C90rf72 gene or RNA lacking the non-coding sequence comprising the hexanucleotide repeat region) and ameliorates at least one symptom associated with development of FTD or ALS.

[0392] As used herein, the term “lipid nanoparticle” refers to a lipid composition having a typically spherical structure with an average diameter between 10 and 1000 nm. In some formulations, lipid nanoparticles can comprise at least one cationic lipid, at least one non-cationic lipid, and at least one conjugated lipid. Lipid nanoparticles known in the art that are suitable for encapsulating nucleic acids, such as mRNA, are contemplated for use in the embodiments described herein. A polynucleotide or nucleic acid, such as an mRNA, may be encapsulated in the lipid portion of the lipid nanoparticle or aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle. This affords protection from enzymatic degradation or other undesired effects induced by a cell, an organism, and / or a subject contacted with said lipid nanoparticle. Lipid nanoparticles may further be conjugated with a targeting moiety, such as an antibody or a ligand, to direct said lipid nanoparticle to a target cell or target tissue.

[0393] As used herein, the recitation of a numerical range for a variable is intended to convey that the present disclosure may be practiced with the variable equal to any of the values within that range. Thus, for a variable which is inherently discrete, the variable can be equal to any integer value within the numerical range, including the end-points of the range. Similarly, for a variable that is inherently continuous, the variable can be equal to any real value within the numerical range, including the end-points of the range. As an example, and without limitation, a variable which is described as having values between 0 and 2 can take the values 0, 1 or 2 if the variable is inherently discrete, and can take the values 0.0, 0.1, 0.01, 0.001, or any other real values ^0 and =2 if the variable is inherently continuous.

[0394] 2, 1 Principle of the Invention

[0395] The present disclosure is based, in part, on the hypothesis that certain mutations located in the non-coding portion of the C90rf72 gene associated with neurodegenerative disorders such as frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) can be removed by utilizing pairs of endonucleases to strategically delete exons and / or introns, or portions of both, within the C90rf72 gene in order to remove and / or prevent transcription of hexanucleotide repeat mutation(s) associated with neurological disorders including FTD and ALS.

[0396] The discovery that repeat expansions of the hexanucleotide repeat in the C90rf72 gene are a frequent cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) has revolutionized understanding of these diseases. Three main disease mechanisms have been proposed: loss of function of the C90rf72 protein, toxic gain of function from C90rf72 repeat RNA, or from dipeptide repeat proteins produced by repeat-associated non- ATG translation (Balendra, Rubika, and Adrian M Isaacs. “C90rf72-mediated ALS and FTD: multiple pathways to disease.” Nature reviews. Neurology vol. 14,9 (2018): 544-558). The hexanucleotide repeat is located between non-coding C90rf72 exons la and lb, and expansion of the hexanucleotide repeat has been regarded as the most frequent cause of sporadic ALS and sporadic FTD identified (Majounie, Elisa et al. “Frequency of the C90rf72 hexanucleotide repeat expansion in patients with amyotrophic lateral sclerosis and frontotemporal dementia: a cross-sectional study.” The Lancet. Neurology vol. 11,4 (2012): 323-30. doi: 10.1016 / S 1474-4422(12)70043-1; DeJesus-Hemandez, Mariely et al. “Expanded GGGGCC hexanucleotide repeat in noncoding region of C90rf72 causes chromosome 9p-linked FTD and ALS.” Neuron vol. 72,2 (2011): 245-56. doi : 10.1016 / j . neuron.2011.09. Oi l; and Renton, Alan E et al. “A hexanucleotide repeat expansion in C90rf72 is the cause of chromosome 9p21-linked ALS-FTD.” Neuron vol. 72,2 (2011): 257-68. doi: 10.1016 / j. neuron.2011.09.010). C90rf72 repeat expansions have also been identified as a rare cause of other neurodegenerative diseases, including Parkinson’s disease, progressive supranuclear palsy, ataxia, corticobasal syndrome, Huntington’s disease-like syndrome, Creutzfeldt- Jakob disease and Alzheimer’s disease.

[0397] Thus, in particular embodiments of the disclosure, the intron, or portions thereof, containing the hexanucleotide repeat of the C90rf72 gene will be removed in order to prevent disease- associated outcomes associated with expansion of this non-coding region within C90rf72 RNA. In particular embodiments, the intron comprising the hexanucleotide repeat region as well as exon lb are excised from the C90rf72 gene by utilizing a recognition sequence that is 5’ upstream from the hexanucleotide repeat region and a recognition sequence that is 3’ downstream of exon lb. This excision strategy referred to herein as “downstream” excision allows for the C90rf72 coding sequence to be under control of genetic regulatory elements that are present 5’ of the hexanucleotide repeat region in wild-type C90rf72 gene (e.g., exon la). Following excision of the hexanucleotide repeat region, C90rf72 transcription will be controlled by exon la, and the RNA produced will lack the hexanucleotide repeat region as well as non-coding exon lb (Figure 1).

[0398] In other embodiments, recognition sequences located 5’ upstream from a minimal promoter and exon la of the C90rf72 gene are utilized in combination with recognition sequences 3’ downstream of exon la. The resulting RNA lacks the minimal promoter and exon la, which prevents transcription of the hexanucleotide repeat region. Transcription then proceeds normally from exon lb. This excision strategy as described herein is referred to as “upstream” excision. Without wishing to be bound by theory, since the hexanucleotide repeat region is associated with disease mechanisms, its removal from C90rf72 RNA may function to prevent development of neurogenerative disease, due to any toxicity, or loss of function of the C90rf72 protein, associated with hexanucleotide repeats forming RNA foci or di-peptide repeat proteins (DPRs) being present in C90rf72 RNA. Accordingly, the absence of the hexanucleotide repeat region from C90rf72 gene, and mRNA associated with expression of the gene, following these strategies may function to prevent toxic gain of function from sense and antisense C90rf72 repeat RNA or from DPRs, ultimately preventing signs, symptoms or development of these neurogenerative disorders.

[0399] As disclosed herein, removal of introns and / or exons, or portions of the same, is achieved by the expression of a pair of engineered meganucleases in neural cells or neural precursor cells that generate a pair of cleavage sites in the non-coding sequence of the COrf72 gene, 5’ upstream of the hexanucleotide repeat (e.g., CNR 1-2 recognition sequence; SEQ ID NO: 3) and downstream of exon lb (e.g., CNR 21-22 recognition sequence; SEQ ID NO: 5), allowing for excision of the intervening genomic region (i.e., hexanucleotide repeat and exon lb). Following this approach, a genetically modified cell e.g., a neural cell in a treated subject) will comprise a modified COrf72 gene lacking the hexanucleotide repeat region that is susceptible to extension mutation that is correlated with neurological disease, such as FTD and ALS. This removal of the hexanucleotide repeat mutation may be sufficient to rescue disease permanently by removing the sequence susceptible to mutation.

[0400] In some embodiments described herein is a genetically modified cell having a modified C90rf72 gene, wherein a minimal promoter sequence upstream of exon la of the C90rf72 gene has been excised. In some embodiments, the minimal promoter sequence comprises SEQ ID NO: 190. In some embodiments, exon la comprises SEQ ID NO: 191. In some embodiments, the minimal promoter sequence having a sequence comprising SEQ ID NO: 190 and exon la having a sequence comprising SEQ ID NO: 191 of the C90rf72 gene has been excised. In some embodiments, the minimal promoter region and exon la is excised from the C90rf72 gene utilizing a first engineered nuclease that targets a recognition sequence 5’ upstream from the minimal promoter region and second engineered nuclease that targets a recognition sequence 3’ downstream from exon lb of the C90rf72 gene.

[0401] In some embodiments described herein is a genetically modified cell having a modified C90rf72 gene, wherein a G4C2 hexanucleotide repeat region of the C90rf72 gene upstream of exon lb of the C90rf72 gene has been excised. In some embodiments, a G4C2 hexanucleotide repeat region and exon lb having a sequence comprising SEQ ID NO: 192 of the C90rf72 gene has been excised. In some embodiments, the hexanucleotide repeat region and exon lb is excised from the C90rf72 gene utilizing a first engineered nuclease that targets a recognition sequence 5’ upstream from the G4C2 hexanucleotide repeat region and second engineered nuclease that targets a recognition sequence 3’ downstream from exon lb of the C90rf72 gene. In some embodiments the first engineered nuclease targets a CNR 1-2 recognition sequence comprising SEQ ID NO: 3 and the second engineered nuclease targets a CNR 21-22 recognition sequence comprising SEQ ID NO: 5. Accordingly, it is envisioned that a single treatment will permanently delete the hexanucleotide repeat mutation from a percentage of cells in a subject. In some embodiments, these cells will be neurons (z.e., neuronal cells) or neuroglia, or neural precursor cells or central nervous system (CNS) cells that are capable of replicating and giving rise to the C90rf72 polypeptide.

[0402] 2,2 Meganucleases that Bind and Cleave Recognition Sequences Within a C90rf72 Gene

[0403] Recognition Sequences

[0404] It is known in the art that it is possible to use a site-specific nuclease to make a DNA break in the genome of a living cell, and that such a DNA break can result in permanent modification of the genome via mutagenic NHEJ repair or via homologous recombination with a transgenic DNA sequence. NHEJ can produce mutagenesis at the cleavage site, resulting in inactivation of the allele. NHEJ-associated mutagenesis may inactivate an allele via generation of early stop codons, frameshift mutations producing aberrant non-functional proteins, or could trigger mechanisms such as nonsense-mediated mRNA decay. The use of nucleases to induce mutagenesis via NHEJ can be used to target a specific mutation or a sequence present in a wild-type allele. Further, the use of nucleases to induce a double-strand break in a target locus is known to stimulate homologous recombination, particularly of transgenic DNA sequences flanked by sequences that are homologous to the genomic target. In this manner, exogenous polynucleotides can be inserted into a target locus. Such exogenous polynucleotides can encode any sequence or polypeptide of interest.

[0405] In particular embodiments, engineered meganucleases of the disclosure have been designed to bind and cleave a CNR 1-2 recognition sequence (SEQ ID NO: 3), or a CNR 21-22 recognition sequence (SEQ ID NO: 5). In some embodiments, engineered meganucleases have been designed to bind and cleave a recognition sequence upstream of a minimal promoter / exon la. Exemplary meganucleases that bind and cleave the CNR 1-2 recognition sequence are provided in SEQ ID NOs: 7-18. Exemplary meganucleases that bind and cleave the CNR 21-22 recognition sequence are provided in SEQ ID NOs: 79-87.

[0406] In some embodiments, the first engineered meganuclease is an engineered meganuclease described herein that binds and cleaves a recognition sequence comprising SEQ ID NO: 3, and the second engineered meganuclease is an engineered meganuclease described herein that binds and cleaves a recognition sequence comprising SEQ ID NO: 5. In some embodiments, the first engineered meganuclease and the second engineered meganuclease are selected from the combinations of meganucleases (and variants thereof described herein) provided in Table 1.

[0407] Table 1.

[0408] The sequence of each recognition sequence, and the four base pair 3' overhang produced when cleaved by an engineered meganuclease described herein, is provided in Table 2 below. Table 2. Engineered Meganuclease Recognition Sequences

[0409] In embodiments of the present disclosure, a pair of engineered meganucleases described herein are utilized together in the same cell to modify the C90rf72 gene. Such pairs of engineered meganucleases were designed to generate a first cleavage site in an intron upstream of exon lb (i.e., at a site between exon la and exon lb) and a second cleavage site downstream of exon lb (i.e., at a site between exon lb and C90rf72 coding sequence), allowing for removal of the intervening genomic sequence (i.e., a portion of the nucleotide sequence between exon la and the C90rf2 coding sequence). It was observed that excision of this genomic region from the C90rf72 gene could be accomplished with high efficiency. The meganuclease recognition sequences have 75% complementary four basepair 3' overhangs following cleavage. It was observed that the C90rf72 gene could be repaired at high frequency by a re-ligation of the 3' overhangs of the two cleavage sites. The most frequent re-ligation events are provided in Table 3 below. Because these religation events take place in the intron of the C90rf72 gene, there is no change to the coding sequence of C90rf72.

[0410] Table 3. Ligated Recognition Sequences of CNR1-2 and CNR21-22 Recognition Sites These recognition sequences are located within non-coding nucleotide sequences. The excision of these selected sequences controls the regulatory sequences controlling transcription of C90rf72. Removal of the hexanucleotide repeat region and exon lb prevents the possible integration of hexanucleotide repeat extension mutations in C90rf72 mRNA that is transcribed from the minimal promoter located 5’ to exon la. Following excision of this intergenic region, transcription of C90rf72 RNA lacking the hexanucleotide repeat region and exon lb will be under the transcriptional control of exon la. Removal of the mutation prone hexanucleotide repeat region reduces the likelihood of mutations and increases the likelihood of full-length, wild-type C90rf72 polypeptide.

[0411] It is contemplated here that the disclosed engineered meganucleases can be used to generate genetically modified eukaryotic cells having a modified C90rf72 locus according to formula I below. Accordingly, in some embodiments described herein, is a genetically modified eukaryotic cell comprising a contiguous nucleic acid sequence within its genome according to Formula I:

[0412] AACAAGAAAAGACCX1X2X3X4X5X6X7X8AACTTCACCTTCCAG, wherein Xi-Xs is individually absent or is selected from A, T, G, C.

[0413] In some embodiments Xi-Xs is absent. In some embodiments Xi is selected from A, T, G, or C and X2-X8 is absent. In some embodiments X1-X2 is selected from A, T, G, or C and X3-X8 is absent. In some embodiments X1-X3 is selected from A, T, G, or C and X4-X8 is absent. In some embodiments X1-X4 is selected from A, T, G, or C and X5-X8 is absent. In some embodiments Xi- X5 is selected from A, T, G, or C and Xe-Xs is absent. In some embodiments X1-X7 is selected from A, T, G, or C and Xs is absent.

[0414] In some embodiments, Xi is a T, X2 is a G, X3 is an A, X4 is a T, X5 is an A, Xe is a T, X7 is a G, and Xs is absent. In some embodiments, Xi is a T, X2 is a G, X3 is an A, X4 is a T, X5 is an A, Xe is an A, X7 is a T, and Xs is a G. In some embodiments, Xi is a T, X2 is a G, and X3-X8 is absent. In some embodiments, Xi is a T, X2 is a G, X3 is an A, X4 is a T, X5 is a G, and Xe-Xs is absent. In some embodiments, Xi is a T, X2 is a G, X3 is an A, X4 is a T, and Xs-Xs is absent.

[0415] In some embodiments, the genetically modified eukaryotic cell comprises a contiguous nucleic acid sequence comprising SEQ ID NO: 185. In some embodiments, the genetically modified eukaryotic cell comprises a contiguous nucleic acid sequence comprising SEQ ID NO: 186. In some embodiments, the genetically modified eukaryotic cell comprises a contiguous nucleic acid sequence comprising SEQ ID NO: 187. In some embodiments, the genetically modified eukaryotic cell comprises a contiguous nucleic acid sequence comprising SEQ ID NO: 188. In some embodiments, the genetically modified eukaryotic cell comprises a contiguous nucleic acid sequence comprising SEQ ID NO: 189. Exemplary Engineered Meganucleases

[0416] Engineered meganucleases of the disclosure comprise a first subunit, comprising a HVR1 region, and a second subunit, comprising a HVR2 region. Further, the first subunit binds to a first recognition half-site in the recognition sequence (e.g., the CNR1 half-site), and the second subunit binds to a second recognition half-site in the recognition sequence (e.g., the CNR2 half-site).

[0417] In particular embodiments, the meganucleases used to practice the disclosure are singlechain meganucleases. A single-chain meganuclease comprises an N-terminal subunit and a C- terminal subunit (z.e., the first and second subunits discussed above) joined by a linker peptide. Each of the two subunits recognizes and binds to a half-site of the recognition sequence and the site of DNA cleavage is at the middle of the recognition sequence near the interface of the two subunits. As discussed, DNA strand breaks are offset by four base pairs such that DNA cleavage by a meganuclease generates a pair of four basepair 3' single-strand overhangs.

[0418] In embodiments where the engineered meganuclease is a single-chain meganuclease, the first and second subunits can be oriented such that the first subunit, which comprises the HVR1 region and binds the first half-site, is positioned as the N-terminal subunit, and the second subunit, which comprises the HVR2 region and binds the second half-site, is positioned as the C-terminal subunit. In alternative embodiments, the first and second subunits can be oriented such that the first subunit, which comprises the HVR1 region and binds the first half-site, is positioned as the C- terminal subunit, and the second subunit, which comprises the HVR2 region and binds the second half-site, is positioned as the N-terminal subunit.

[0419] Exemplary CNR meganucleases of the disclosure are provided in SEQ ID NOs:7=18 and 79-87 and are summarized below in Tables 4 and 5.

[0420] Table 4. Exemplary engineered meganucleases that bind and cleave the CNR 1-2 recognition sequence (SEQ ID NO: 3).

[0421] “CNR1 Subunit %” and “CNR2 Subunit %” represent the amino acid sequence identity between the CNR1 -binding and CNR2 -binding subunit regions of each meganuclease and the CNR1- binding and CNR2-binding subunit regions, respectively, of the CNR 1-2L.532 meganuclease.

[0422] Table 5. Exemplary engineered meganucleases that bind and cleave the CNR21-22 recognition sequence (SEQ ID NO: 5).

[0423] “CNR21 Subunit %” and “CNR22 Subunit %” represent the amino acid sequence identity between the CNR21 -binding and CNR22-binding subunit regions of each meganuclease and the CNR21- binding and CNR22 -binding subunit regions, respectively, of the CNR 21-22L.286 meganuclease.

[0424] In certain embodiments of the disclosure, the engineered meganuclease binds and cleaves a recognition sequence comprising SEQ ID NO: 3 (z.e., the CNR 1-2 recognition sequence) within a C90rf72gene, wherein the engineered meganuclease comprises a first subunit and a second subunit, wherein the first subunit binds to a first recognition half-site of the recognition sequence and comprises a HVR1 region, and wherein the second subunit binds to a second recognition halfsite of the recognition sequence and comprises a HVR2 region. Exemplary CNR 1-2 meganucleases are described below.

[0425] CNR 1-2L.532 (SEQ ID NO: 7)

[0426] In some embodiments, the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 7. In some embodiments, the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 7. In some embodiments, the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 7. In some embodiments, the HVR1 region comprises a residue corresponding to residue 29 of SEQ ID NO: 7. In some embodiments, the HVR1 region comprises a residue corresponding to residue 41 of SEQ ID NO: 7. In some embodiments, the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 7. In some embodiments, the HVR1 region comprises a residue corresponding to residue 64 of SEQ ID NO: 7. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 7. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 7. In some embodiments, the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 7. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 7 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 7.

[0427] In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 7. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 7. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 7. In some embodiments, the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 7. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 7. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 7 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 7. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 7.

[0428] In some embodiments, the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 7. In some embodiments, the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 7. In some embodiments, the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 7. In some embodiments, the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 7. In some embodiments, the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 7. In some embodiments, the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 7. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 7 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 7.

[0429] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 7. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 7. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 7. In some embodiments, the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 7. In some embodiments, the second subunit comprises a residue corresponding to residue 330 of SEQ ID NO: 7. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 7 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 7. In some embodiments, the second subunit comprises residues 196-354 of SEQ ID NO: 7.

[0430] In some embodiments, the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 7. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 7. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 7. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 7. In some embodiments, the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 67. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 67.

[0431] CNR 1-2L.468 (SEQ ID NO: 8) In some embodiments, the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 8. In some embodiments, the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 8. In some embodiments, the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 8. In some embodiments, the HVR1 region comprises a residue corresponding to residue 29 of SEQ ID NO: 8. In some embodiments, the HVR1 region comprises a residue corresponding to residue 41 of SEQ ID NO: 8. In some embodiments, the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 8. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 8. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 8. In some embodiments, the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 8. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 8 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 8.

[0432] In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 8. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 8. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 8. In some embodiments, the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 8. In some embodiments, the first subunit comprises a residue corresponding to residue 80 of SEQ ID NO: 8. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 8. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 8 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 8. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 8.

[0433] In some embodiments, the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 8. In some embodiments, the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 8. In some embodiments, the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 8. In some embodiments, the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 8. In some embodiments, the HVR2 region comprises a residue corresponding to residue 258 of SEQ ID NO: 8. In some embodiments, the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 8. In some embodiments, the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 8. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 8 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 8.

[0434] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 8. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 8. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 8. In some embodiments, the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 8. In some embodiments, the second subunit comprises a residue corresponding to residue 271 of SEQ ID NO: 8. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 8 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 8. In some embodiments, the second subunit comprises residues 196-354 of SEQ ID NO: 8.

[0435] In some embodiments, the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 8. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 8. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 8. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 8. In some embodiments, the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 68. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 68.

[0436] CNR 1-2L.531 (SEQ ID NO: 9)

[0437] In some embodiments, the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 9. In some embodiments, the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 9. In some embodiments, the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 9. In some embodiments, the HVR1 region comprises a residue corresponding to residue 29 of SEQ ID NO: 9. In some embodiments, the HVR1 region comprises a residue corresponding to residue 41 of SEQ ID NO: 9. In some embodiments, the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 9. In some embodiments, the HVR1 region comprises a residue corresponding to residue 64 of SEQ ID NO: 9. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 9. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 9. In some embodiments, the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 9. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 9 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 9.

[0438] In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 9. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 9. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 9. In some embodiments, the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 9. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 9. In some embodiments, the first subunit comprises a residue corresponding to residue 80 of SEQ ID NO: 9. In some embodiments, the first subunit comprises a residue corresponding to residue 142 of SEQ ID NO: 9. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 9 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 9. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 9.

[0439] In some embodiments, the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 9. In some embodiments, the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 9. In some embodiments, the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 9. In some embodiments, the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 9. In some embodiments, the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 9. In some embodiments, the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 9. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 9 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 9.

[0440] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 9. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 9. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 9. In some embodiments, the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 9. In some embodiments, the second subunit comprises residues 198- 344 of SEQ ID NO: 9 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 9. In some embodiments, the second subunit comprises residues 196-354 of SEQ ID NO: 9.

[0441] In some embodiments, the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 9. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 9. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 9. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 9. In some embodiments, the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 69. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 69.

[0442] CNR 1-2L.371 (SEQ ID NO: 10)

[0443] In some embodiments, the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 10. In some embodiments, the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 10. In some embodiments, the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 10. In some embodiments, the HVR1 region comprises a residue corresponding to residue 29 of SEQ ID NO: 10. In some embodiments, the HVR1 region comprises a residue corresponding to residue 41 of SEQ ID NO: 10. In some embodiments, the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 10. In some embodiments, the HVR1 region comprises a residue corresponding to residue 59 of SEQ ID NO: 10. In some embodiments, the HVR1 region comprises a residue corresponding to residue 64 of SEQ ID NO: 10. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 10. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 10. In some embodiments, the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 10. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 10 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 10.

[0444] In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 10. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 10. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 10. In some embodiments, the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 10. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 10. In some embodiments, the first subunit comprises a residue corresponding to residue 80 of SEQ ID NO: 10. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 10 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 10. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 10.

[0445] In some embodiments, the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 10. In some embodiments, the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 10. In some embodiments, the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 10. In some embodiments, the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 10. In some embodiments, the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 10. In some embodiments, the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 10. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 10 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 10.

[0446] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 10. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 10. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 10. In some embodiments, the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 10. In some embodiments, the second subunit comprises a residue corresponding to residue 271 of SEQ ID NO: 10. In some embodiments, the second subunit comprises a residue corresponding to residue 330 of SEQ ID NO: 10. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 10 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 10. In some embodiments, the second subunit comprises residues 196-354 of SEQ ID NO: 10.

[0447] In some embodiments, the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 10. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 10. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 10. In some embodiments, the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ ID NOs: 70. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 70.

[0448] CNR 1-2L.425 (SEQ ID NO: 11)

[0449] In some embodiments, the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 11. In some embodiments, the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 11. In some embodiments, the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 11. In some embodiments, the HVR1 region comprises a residue corresponding to residue 29 of SEQ ID NO: 11. In some embodiments, the HVR1 region comprises a residue corresponding to residue 41 of SEQ ID NO: 11. In some embodiments, the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 11. In some embodiments, the HVR1 region comprises a residue corresponding to residue 59 of SEQ ID NO: 11. In some embodiments, the HVR1 region comprises a residue corresponding to residue 64 of SEQ ID NO: 11. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 11. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 11. In some embodiments, the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 11. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 11 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 11.

[0450] In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 11. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 11. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 11. In some embodiments, the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 11. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 11. In some embodiments, the first subunit comprises a residue corresponding to residue 80 of SEQ ID NO: 11. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 11 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 11. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 11.

[0451] In some embodiments, the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 11. In some embodiments, the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 11. In some embodiments, the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 11. In some embodiments, the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 11. In some embodiments, the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 11. In some embodiments, the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 11. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 11 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 11. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 11. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 11. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 11. In some embodiments, the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 11. In some embodiments, the second subunit comprises a residue corresponding to residue 330 of SEQ ID NO: 11. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 11 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 11. In some embodiments, the second subunit comprises residues 196-354 of SEQ ID NO: 11.

[0452] In some embodiments, the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 11. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 11. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 11. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 11. In some embodiments, the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 71. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 71.

[0453] CNR 1-2L.284 (SEQ ID NO: 12)

[0454] In some embodiments, the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 12. In some embodiments, the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 12. In some embodiments, the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 12. In some embodiments, the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 12. In some embodiments, the HVR1 region comprises a residue corresponding to residue 59 of SEQ ID NO: 12. In some embodiments, the HVR1 region comprises a residue corresponding to residue 64 of SEQ ID NO: 12. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 12. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 12. In some embodiments, the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 12. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 12 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 12.

[0455] In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 12. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 12. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 12. In some embodiments, the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 12. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 12. In some embodiments, the first subunit comprises a residue corresponding to residue 80 of SEQ ID NO: 12. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 12 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 12. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 12.

[0456] In some embodiments, the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 12. In some embodiments, the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 12. In some embodiments, the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 12. In some embodiments, the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 12. In some embodiments, the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 12. In some embodiments, the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 12. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 12 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 12.

[0457] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 12. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 12. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 12. In some embodiments, the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 12. In some embodiments, the second subunit comprises a residue corresponding to residue 271 of SEQ ID NO: 12. In some embodiments, the second subunit comprises a residue corresponding to residue 330 of SEQ ID NO: 12. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 12 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 12. In some embodiments, the second subunit comprises residues 196-354 of SEQ ID NO: 12.

[0458] In some embodiments, the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 12. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 12. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 12. In some embodiments, the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 72. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 72. CNR 1-2L.297 (SEQ ID NO: 13)

[0459] In some embodiments, the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 13. In some embodiments, the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 13. In some embodiments, the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 13. In some embodiments, the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 13. In some embodiments, the HVR1 region comprises a residue corresponding to residue 59 of SEQ ID NO: 13. In some embodiments, the HVR1 region comprises a residue corresponding to residue 64 of SEQ ID NO: 13. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 13. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 13. In some embodiments, the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 13. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 13 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 13.

[0460] In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 13. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 13. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 13. In some embodiments, the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 13. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 13. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 13 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 13. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 13.

[0461] In some embodiments, the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 13. In some embodiments, the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 13. In some embodiments, the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 13. In some embodiments, the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 13. In some embodiments, the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 13. In some embodiments, the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 13. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 13 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 13.

[0462] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 13. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 13. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 13. In some embodiments, the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 13. In some embodiments, the second subunit comprises a residue corresponding to residue 271 of SEQ ID NO: 13. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 13 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 13. In some embodiments, the second subunit comprises residues 196-354 of SEQ ID NO: 13.

[0463] In some embodiments, the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 13. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 13. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 13. In some embodiments, the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 73. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 73.

[0464] CNR 1-2L.211 (SEQ ID NO: 14)

[0465] In some embodiments, the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 14. In some embodiments, the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 14. In some embodiments, the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 14. In some embodiments, the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 14. In some embodiments, the HVR1 region comprises a residue corresponding to residue 59 of SEQ ID NO: 14. In some embodiments, the HVR1 region comprises a residue corresponding to residue 64 of SEQ ID NO: 14. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 14. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 14. In some embodiments, the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 14. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 14 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 14.

[0466] In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 14. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 14. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 14. In some embodiments, the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 14. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 14. In some embodiments, the first subunit comprises a residue corresponding to residue 80 of SEQ ID NO: 14. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 43 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 14. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 14.

[0467] In some embodiments, the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 14. In some embodiments, the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 14. In some embodiments, the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 14. In some embodiments, the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 14. In some embodiments, the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 14. In some embodiments, the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 14. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 14 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 14.

[0468] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 14. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 14. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 14. In some embodiments, the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 14. In some embodiments, the second subunit comprises a residue corresponding to residue 271 of SEQ ID NO: 14. In some embodiments, the second subunit comprises a residue corresponding to residue 330 of SEQ ID NO: 14. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 14 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 14. In some embodiments, the second subunit comprises residues 196-354 of SEQ ID NO: 14.

[0469] In some embodiments, the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 14. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 14. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 14. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 14. In some embodiments, the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 74. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 74.

[0470] CNR 1-2L.98 (SEQ ID NO: 15)

[0471] In some embodiments, the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 15. In some embodiments, the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 15. In some embodiments, the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 15. In some embodiments, the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 15. In some embodiments, the HVR1 region comprises a residue corresponding to residue 59 of SEQ ID NO: 15. In some embodiments, the HVR1 region comprises a residue corresponding to residue 64 of SEQ ID NO: 15. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 15. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 15. In some embodiments, the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 15. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 15 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 15.

[0472] In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 15. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 15. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 15. In some embodiments, the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 15. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 15. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 15 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 15. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 15.

[0473] In some embodiments, the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 15. In some embodiments, the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 15. In some embodiments, the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 15. In some embodiments, the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 15. In some embodiments, the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 15. In some embodiments, the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 15. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 15 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 15.

[0474] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 15. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 15. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 15. In some embodiments, the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 15. In some embodiments, the second subunit comprises residues 198- 344 of SEQ ID NO: 15 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 15. In some embodiments, the second subunit comprises residues 196-354 of SEQ ID NO: 15.

[0475] In some embodiments, the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 15. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 15. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 15. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 15. In some embodiments, the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 75. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 75.

[0476] CNR 1-2L.108 (SEQ ID NO: 16)

[0477] In some embodiments, the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 16. In some embodiments, the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 16. In some embodiments, the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 16. In some embodiments, the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 16. In some embodiments, the HVR1 region comprises a residue corresponding to residue 59 of SEQ ID NO: 16. In some embodiments, the HVR1 region comprises a residue corresponding to residue 64 of SEQ ID NO: 16. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 16. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 16. In some embodiments, the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 16. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 16 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 16.

[0478] In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 16. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 16. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 16. In some embodiments, the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 16. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 16. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 16 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 16. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 16.

[0479] In some embodiments, the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 16. In some embodiments, the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 16. In some embodiments, the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 16. In some embodiments, the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 16. In some embodiments, the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 16. In some embodiments, the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 16. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 16 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 16.

[0480] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 16. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 16. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 16. In some embodiments, the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 16. In some embodiments, the second subunit comprises residues 198- 344 of SEQ ID NO: 16 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 16. In some embodiments, the second subunit comprises residues 196-354 of SEQ ID NO: 16.

[0481] In some embodiments, the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 16. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 16. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 16. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 16. In some embodiments, the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 76. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 76.

[0482] CNR 1-2L15 (SEQ ID NO: 17)

[0483] In some embodiments, the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 17. In some embodiments, the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 17. In some embodiments, the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 17. In some embodiments, the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 17. In some embodiments, the HVR1 region comprises a residue corresponding to residue 59 of SEQ ID NO: 17. In some embodiments, the HVR1 region comprises a residue corresponding to residue 64 of SEQ ID NO: 17. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 17. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 17. In some embodiments, the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 17. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 17 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 17.

[0484] In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 17. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 17. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 17. In some embodiments, the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 17. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 17. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 17 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 17. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 17.

[0485] In some embodiments, the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 17. In some embodiments, the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 17. In some embodiments, the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 17. In some embodiments, the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 17. In some embodiments, the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 17. In some embodiments, the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 17. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 17 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 17. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 17. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 17. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 17. In some embodiments, the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 17. In some embodiments, the second subunit comprises residues 198- 344 of SEQ ID NO: 17 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 17. In some embodiments, the second subunit comprises residues 196-354 of SEQ ID NO: 17.

[0486] In some embodiments, the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 17. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 17. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 17. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 17. In some embodiments, the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 77. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 77.

[0487] CNR l-2x.88 (SEQ ID NO: 18)

[0488] In some embodiments, the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 18. In some embodiments, the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 18. In some embodiments, the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 18. In some embodiments, the HVR1 region comprises a residue corresponding to residue 48 of SEQ ID NO: 18. In some embodiments, the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 18. In some embodiments, the HVR1 region comprises a residue corresponding to residue 59 of SEQ ID NO: 18. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 18. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 18. In some embodiments, the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 42. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 18 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 18.

[0489] In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 18. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 18. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 18. In some embodiments, the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 18. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 18. In some embodiments, the first subunit comprises a residue corresponding to residue 80 of SEQ ID NO: 18. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 18 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 18. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 18.

[0490] In some embodiments, the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 18. In some embodiments, the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 18. In some embodiments, the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 18. In some embodiments, the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 18. In some embodiments, the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 18. In some embodiments, the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 18. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 18 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 18.

[0491] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 18. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 18. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 18. In some embodiments, the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 18. In some embodiments, the second subunit comprises a residue corresponding to residue 330 of SEQ ID NO: 18. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 18 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,

[0492] 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 18. In some embodiments, the second subunit comprises residues 196-354 of SEQ ID NO: 18.

[0493] In some embodiments, the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 18. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 18. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 18. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 18. In some embodiments, the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 78. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 78.

[0494] In each of the embodiments above, the engineered meganuclease can comprise a nuclear localization signal. In some embodiments, the nuclear localization signal is at the N-terminus of the engineered meganuclease. In some embodiments, the nuclear localization signal comprises an amino acid sequence having at least 80% or at least 90% sequence identity to SEQ ID NO: 133 or 134. In some embodiments, the nuclear localization signal comprises SEQ ID NO: 133 or 134.

[0495] In certain embodiments of the disclosure, the engineered meganuclease binds and cleaves a recognition sequence comprising SEQ ID NO: 5 (z.e., the CNR 21-22 recognition sequence) within a C90rf72 gene, wherein the engineered meganuclease comprises a first subunit and a second subunit, wherein the first subunit binds to a first recognition half-site of the recognition sequence and comprises a first hypervariable (HVR1) region, and wherein the second subunit binds to a second recognition half-site of the recognition sequence and comprises a second hypervariable (HVR2) region. Exemplary CNR 21-22 meganucleases are described below.

[0496] CNR 21-22L.290 (SEQ ID NO: 79)

[0497] In some embodiments, the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 79. In some embodiments, the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 79. In some embodiments, the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 79. In some embodiments, the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 79. In some embodiments, the HVR1 region comprises a residue corresponding to residue 48 of SEQ ID NO: 79. In some embodiments, the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 79. In some embodiments, the HVR1 region comprises a residue corresponding to residue 59 of SEQ ID NO: 79. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 79. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 79. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 79 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 79.

[0498] In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 79. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 79. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 79. In some embodiments, the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 79. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 79. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 79 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 79. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 79.

[0499] In some embodiments, the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 79. In some embodiments, the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 79. In some embodiments, the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 79. In some embodiments, the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 79. In some embodiments, the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 79. In some embodiments, the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 79. In some embodiments, the HVR2 region comprises a residue corresponding to residue 264 of SEQ ID NO: 79. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 79 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215- 270 of SEQ ID NO: 79.

[0500] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 79. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 79. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 79. In some embodiments, the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 79. In some embodiments, the second subunit comprises a residue corresponding to residue 271 of SEQ ID NO: 79. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 79 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 79. In some embodiments, the second subunit comprises residues 196-354 of SEQ ID NO: 79.

[0501] In some embodiments, the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 79. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 79. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 79. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 79. In some embodiments, the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 124. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 124.

[0502] CNR 21-22L.286 (SEQ ID NO: 80)

[0503] In some embodiments, the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 80. In some embodiments, the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 80. In some embodiments, the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 80. In some embodiments, the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 80. In some embodiments, the HVR1 region comprises a residue corresponding to residue 48 of SEQ ID NO: 80. In some embodiments, the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 80. In some embodiments, the HVR1 region comprises a residue corresponding to residue 59 of SEQ ID NO: 80. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 80. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 80. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 80 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 80.

[0504] In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 80. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 80. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 80. In some embodiments, the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 80. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 80. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 80 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 80. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 80.

[0505] In some embodiments, the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 80. In some embodiments, the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 80. In some embodiments, the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 80. In some embodiments, the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 80. In some embodiments, the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 80. In some embodiments, the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 80. In some embodiments, the HVR2 region comprises a residue corresponding to residue 264 of SEQ ID NO: 80. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 80 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215- 270 of SEQ ID NO: 80.

[0506] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 80. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 80. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 80. In some embodiments, the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 80. In some embodiments, the second subunit comprises a residue corresponding to residue 271 of SEQ ID NO: 80. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 80 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 80. In some embodiments, the second subunit comprises residues 196-354 of SEQ ID NO: 80.

[0507] In some embodiments, the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 80. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 80. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 80. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 80. In some embodiments, the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ IDNO: 125. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 125. In some embodiments, the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 80. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 80. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 80. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 80. In some embodiments, the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ IDNO: 125. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 125.

[0508] CNR 21-22L.274 (SEQ ID NO: 81)

[0509] In some embodiments, the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 81. In some embodiments, the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 81. In some embodiments, the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 81. In some embodiments, the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 81. In some embodiments, the HVR1 region comprises a residue corresponding to residue 48 of SEQ ID NO: 81. In some embodiments, the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 81. In some embodiments, the HVR1 region comprises a residue corresponding to residue 59 of SEQ ID NO: 81. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 81. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 81. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 81 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 81.

[0510] In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 81. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 81. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 81. In some embodiments, the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 81. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 81. In some embodiments, the first subunit comprises a residue corresponding to residue 80 of SEQ ID NO: 81. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 81 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 81. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 81.

[0511] In some embodiments, the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 81. In some embodiments, the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 81. In some embodiments, the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 81. In some embodiments, the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 81. In some embodiments, the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 81. In some embodiments, the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 81. In some embodiments, the HVR2 region comprises a residue corresponding to residue 264 of SEQ ID NO: 81. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 81 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215- 270 of SEQ ID NO: 81.

[0512] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 81. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 81. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 81. In some embodiments, the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 81. In some embodiments, the second subunit comprises a residue corresponding to residue 271 of SEQ ID NO: 81. In some embodiments, the second subunit comprises a residue corresponding to residue 330 of SEQ ID NO: 81. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 81 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 81. In some embodiments, the second subunit comprises residues 196-354 of SEQ ID NO: 81.

[0513] In some embodiments, the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 81. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 81. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 81. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 81. In some embodiments, the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ IDNO: 126. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 126.

[0514] CNR 21-22L.212 (SEQ ID NO: 82)

[0515] In some embodiments, the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 82. In some embodiments, the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 82. In some embodiments, the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 82. In some embodiments, the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 82. In some embodiments, the HVR1 region comprises a residue corresponding to residue 48 of SEQ ID NO: 82. In some embodiments, the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 82. In some embodiments, the HVR1 region comprises a residue corresponding to residue 59 of SEQ ID NO: 82. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 82. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 82. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 82 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 82.

[0516] In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 82. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 82. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 82. In some embodiments, the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 82. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 82. In some embodiments, the first subunit comprises a residue corresponding to residue 80 of SEQ ID NO: 82. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 82 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 82. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 82.

[0517] In some embodiments, the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 82. In some embodiments, the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 82. In some embodiments, the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 82. In some embodiments, the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 82. In some embodiments, the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 82. In some embodiments, the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 82. In some embodiments, the HVR2 region comprises a residue corresponding to residue 264 of SEQ ID NO: 82. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 82 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215- 270 of SEQ ID NO: 82.

[0518] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 82. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 82. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 82. In some embodiments, the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 82. In some embodiments, the second subunit comprises residues 198- 344 of SEQ ID NO: 82 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 82. In some embodiments, the second subunit comprises residues 196-354 of SEQ ID NO: 82.

[0519] In some embodiments, the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 82. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 82. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 82. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 82. In some embodiments, the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 127. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 127.

[0520] CNR 21-22L.197 (SEQ ID NO: 83) In some embodiments, the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 83. In some embodiments, the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 83. In some embodiments, the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 83. In some embodiments, the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 83. In some embodiments, the HVR1 region comprises a residue corresponding to residue 48 of SEQ ID NO: 83. In some embodiments, the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 83. In some embodiments, the HVR1 region comprises a residue corresponding to residue 59 of SEQ ID NO: 83. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 83. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 83. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 83 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 83.

[0521] In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 83. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 83. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 83. In some embodiments, the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 83. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 83. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 83 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 83. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 83.

[0522] In some embodiments, the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 215-270 of SEQ ID NO: 83. In some embodiments, the HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 83. In some embodiments, the HVR2 region comprises residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of SEQ ID NO: 83. In some embodiments, the HVR2 region comprises Y, R, K, or D at a residue corresponding to residue 257 of SEQ ID NO: 83. In some embodiments, the HVR2 region comprises a residue corresponding to residue 241 of SEQ ID NO: 83. In some embodiments, the HVR2 region comprises a residue corresponding to residue 263 of SEQ ID NO: 83. In some embodiments, the HVR2 region comprises a residue corresponding to residue 264 of SEQ ID NO: 83. In some embodiments, the HVR2 region comprises residues 215-270 of SEQ ID NO: 83 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR2 region comprises residues 215- 270 of SEQ ID NO: 83.

[0523] In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 198-344 of SEQ ID NO: 83. In some embodiments, the second subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 196-354 of SEQ ID NO: 83. In some embodiments, the second subunit comprises G, S, or A at a residue corresponding to residue 210 of SEQ ID NO: 83. In some embodiments, the second subunit comprises E, Q, or K at a residue corresponding to residue 271 of SEQ ID NO: 83. In some embodiments, the second subunit comprises a residue corresponding to residue 271 of SEQ ID NO: 83. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 83 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the second subunit comprises residues 198-344 of SEQ ID NO: 83. In some embodiments, the second subunit comprises residues 196-354 of SEQ ID NO: 83.

[0524] In some embodiments, the engineered meganuclease is a single-chain meganuclease comprising a linker, wherein the linker covalently joins said first subunit and said second subunit. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 83. In some embodiments, the engineered meganuclease comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 2-354 of SEQ ID NO: 83. In some embodiments, the engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 83. In some embodiments, the engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 83. In some embodiments, the engineered meganuclease is encoded by a nucleic sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 128. In some embodiments, the engineered meganuclease is encoded by a nucleic acid sequence set forth in SEQ ID NO: 128.

[0525] CNR 21-22L.169 (SEQ ID NO: 84)

[0526] In some embodiments, the HVR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an amino acid sequence corresponding to residues 24-79 of SEQ ID NO: 84. In some embodiments, the HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 84. In some embodiments, the HVR1 region comprises residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of SEQ ID NO: 84. In some embodiments, the HVR1 region comprises Y, R, K, or D at a residue corresponding to residue 66 of SEQ ID NO: 84. In some embodiments, the HVR1 region comprises a residue corresponding to residue 48 of SEQ ID NO: 84. In some embodiments, the HVR1 region comprises a residue corresponding to residue 50 of SEQ ID NO: 84. In some embodiments, the HVR1 region comprises a residue corresponding to residue 72 of SEQ ID NO: 84. In some embodiments, the HVR1 region comprises a residue corresponding to residue 73 of SEQ ID NO: 84. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 84 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid substitutions. In some embodiments, the HVR1 region comprises residues 24-79 of SEQ ID NO: 84.

[0527] In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 7-153 of SEQ ID NO: 84. In some embodiments, the first subunit comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to residues 1-153 of SEQ ID NO: 84. In some embodiments, the first subunit comprises G, S, or A at a residue corresponding to residue 19 of SEQ ID NO: 84. In some embodiments, the first subunit comprises a residue corresponding to residue 19 of SEQ ID NO: 84. In some embodiments, the first subunit comprises E, Q, or K at a residue corresponding to residue 80 of SEQ ID NO: 84. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 84 with up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the first subunit comprises residues 7-153 of SEQ ID NO: 84. In some embodiments, the first subunit comprises residues 1-153 of SEQ ID NO: 84. In some embodiments, the HVR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or m...

Claims

CLAIMS1. An engineered meganuclease that binds and cleaves a recognition sequence comprising SEQ ID NO: 3 within a C90rf72 gene, wherein said engineered meganuclease comprises a first subunit and a second subunit, wherein said first subunit binds to a first recognition half-site of said recognition sequence and comprises a first hypervariable (HVR1) region, and wherein said second subunit binds to a second recognition half-site of said recognition sequence and comprises a second hypervariable (HVR2) region.

2. The engineered meganuclease of claim 1, wherein said HVR1 comprises an amino acid sequence having at least 80% sequence identity to residues 24-79 of any one of SEQ ID NOs: 7- 18.

3. The engineered meganuclease of claim 1 or claim 2, wherein said HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any one of SEQ ID NOs: 7-18.

4. The engineered meganuclease of any one of claims 1-3, wherein said HVR1 region comprises a residue corresponding to residue 29 of any one of SEQ ID NOs: 7-11.

5. The engineered meganuclease of any one of claims 1-4, wherein said HVR1 region comprises a residue corresponding to residue 41 of any one of SEQ ID NOs: 7-11.

6. The engineered meganuclease of any one of claims 1-5, wherein said HVR1 region comprises a residue corresponding to residue 48 of SEQ ID NO: 18.

7. The engineered meganuclease of any one of claims 1 -6, wherein said HVR1 region comprises a residue corresponding to residue 50 of any one of SEQ ID NOs: 7-18.

8. The engineered meganuclease of any one of claims 1-7, wherein said HVR1 region comprises a residue corresponding to residue 59 of any one of SEQ ID NOs: 10-18.

9. The engineered meganuclease of any one of claims 1-8, wherein said HVR1 region comprises a residue corresponding to residue 64 of any one of SEQ ID NOs: 7 or 9-17.

10. The engineered meganuclease of any one of claims 1-9, wherein said HVR1 region comprises a residue corresponding to residue 72 of any one of SEQ ID NOs: 7-18.

11. The engineered meganuclease of any one of claims 1-10, wherein said HVR1 region comprises a residue corresponding to residue 73 of any one of SEQ ID NOs: 7-18.

12. The engineered meganuclease of any one of claims 1-11, wherein said HVR1 region comprises residues 24-79 of any one of SEQ ID NOs: 7-18.

13. The engineered meganuclease of any one of claims 1-12, wherein said first subunit comprises an amino acid sequence having at least 80% sequence identity to residues 7-153 of any one of SEQ ID NOs: 7-18.

14. The engineered meganuclease of any one of claims 1-13, wherein said first subunit comprises an amino acid sequence having at least 80% sequence identity to residues 1-153 of any one of SEQ ID NOs: 7-18.

15. The engineered meganuclease of any one of claims 1-14, wherein said first subunit comprises a residue corresponding to residue 19 of any one of SEQ ID NOs: 7-18.

16. The engineered meganuclease of any one of claims 1-15, wherein said first subunit comprises a residue corresponding to residue 80 of any one of SEQ ID NOs: 8-12 or 14 or 18.

17. The engineered meganuclease of any one of claims 1-16, wherein said first subunit comprises a residue corresponding to residue 142 of SEQ ID NO: 9.

18. The engineered meganuclease of any one of claims 1-17, wherein said first subunit comprises residues 7-153 of any one of SEQ ID NOs: 7-18.

19. The engineered meganuclease of any one of claims 1-18, wherein said first subunit comprises residues 1-153 of any one of SEQ ID NOs: 7-18.

20. The engineered meganuclease of any one of claims 1-19, wherein said HVR2 region comprises an amino acid sequence having at least 80% sequence identity to residues 215-270 of any one of SEQ ID NOs: 7-18.

21. The engineered meganuclease of any one of claims 1-20, wherein said HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of any one of SEQ ID NOs: 7-18.

22. The engineered meganuclease of any one of claims 1-21, wherein said HVR2 region comprises a residue corresponding to residue 241 of any one of SEQ ID NOs: 7-18.

23. The engineered meganuclease of any one of claims 1-22, wherein said HVR2 region comprises a residue corresponding to residue 258 of SEQ ID NO: 8.

24. The engineered meganuclease of any one of claims 1-23, wherein said HVR2 region comprises a residue corresponding to residue 263 of any one of SEQ ID NOs: 7-18.

25. The engineered meganuclease of any one of claims 1-24, wherein said HVR2 region comprises residues 215-270 of any one of SEQ ID NOs: 7-18.

26. The engineered meganuclease of any one of claims 1-25, wherein said second subunit comprises an amino acid sequence having at least 80% sequence identity to residues 198-344 of any one of SEQ ID NOs: 7-18.

27. The engineered meganuclease of any one of claims 1 -26, wherein said second subunit comprises an amino acid sequence having at least 80% sequence identity to residues 196-354 of any one of SEQ ID NOs: 7-18.

28. The engineered meganuclease of any one of claims 1-27, wherein said second subunit comprises a residue corresponding to residue 271 of any one of SEQ ID NOs: 8, 10, or 12-14.

29. The engineered meganuclease of any one of claims 1 -28, wherein said second subunit comprises a residue corresponding to residue 330 of any one of SEQ ID NOs: 7, 10-12, 14, or 18.

30. The engineered meganuclease of any one of claims 1-29, wherein said second subunit comprises residues 198-344 of any one of SEQ ID NOs: 7-18.

31. The engineered meganuclease of any one of claims 1-30, wherein said second subunit comprises residues 196-354 of any one of SEQ ID NOs: 7-18.

32. The engineered meganuclease of any one of claims 1-31, wherein said engineered meganuclease is a single-chain meganuclease comprising a linker and wherein said linker covalently joins said first subunit and said second subunit.

33. The engineered meganuclease of any one of claims 1-32, wherein said engineered meganuclease comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 7-18.

34. The engineered meganuclease of any one of claims 1-33, wherein said engineered meganuclease comprises an amino acid sequence having at least 80% sequence identity to residues 2-354 of any one of SEQ ID NOs: 7-18.

35. The engineered meganuclease of any one of claims 1-34, wherein said engineered meganuclease comprises an amino acid sequence of any one of SEQ ID NOs: 7-18.

36. The engineered meganuclease of any one of claims 1-35, wherein said engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of any one of SEQ ID NOs: 7-18.

37. The engineered meganuclease of any one of claims 1-36, wherein said engineered meganuclease is encoded by a nucleic sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 67-78.

38. The engineered meganuclease of any one of claims 1-37, wherein said engineered meganuclease is encoded by a nucleic acid sequence of any one of SEQ ID NOs: 67-78.

39. The engineered meganuclease of any one of claims 1-32, wherein said engineered meganuclease comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 7.

40. The engineered meganuclease of any one of claims 1-32, wherein said engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 7.

41. The engineered meganuclease of any one of claims 1-32, wherein said engineered meganuclease comprises an amino acid sequence having at least 95% sequence identity to residues 2-354 of SEQ ID NO: 7.

42. The engineered meganuclease of any one of claims 1-32, wherein said engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 7.

43. An engineered meganuclease that binds and cleaves a recognition sequence comprising SEQ ID NO: 3 within a C90rf72 gene, wherein said engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 7.

44. An engineered meganuclease that binds and cleaves a recognition sequence comprising SEQ ID NO: 3 within a C90rf72 gene, wherein said engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 7.

45. An engineered meganuclease that binds and cleaves a recognition sequence comprising SEQ ID NO: 5 within a C90rf72 gene, wherein said engineered meganuclease comprises a first subunit and a second subunit, wherein said first subunit binds to a first recognition half-site of said recognition sequence and comprises a first hypervariable (HVR1) region, and wherein said second subunit binds to a second recognition half-site of said recognition sequence and comprises a second hypervariable (HVR2) region.

46. The engineered meganuclease of claim 45, wherein said HVR1 comprises an amino acid sequence having at least 80% sequence identity to residues 24-79 of any one of SEQ ID NOs: 79-87.

47. The engineered meganuclease of claim 45 or claim 46, wherein said HVR1 region comprises one or more residues corresponding to residues 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 68, 70, 75, and 77 of any one of SEQ ID NOs: 79-87.

48. The engineered meganuclease of any one of claims 45-47, wherein said HVR1 region comprises a residue corresponding to residue 48 of any one of SEQ ID NOs: 79-87.

49. The engineered meganuclease of any one of claims 45-48, wherein said HVR1 region comprises a residue corresponding to residue 50 of any one of SEQ ID NOs: 79-87.

50. The engineered meganuclease of any one of claims 45-49, wherein said HVR1 region comprises a residue corresponding to residue 59 of any one of SEQ ID NOs: 79-83 or 85.

51. The engineered meganuclease of any one of claims 45-50, wherein said HVR1 region comprises a residue corresponding to residue 72 of any one of SEQ ID NOs: 79-87.

52. The engineered meganuclease of any one of claims 45-51, wherein said HVR1 region comprises a residue corresponding to residue 73 of any one of SEQ ID NOs: 79-87.

53. The engineered meganuclease of any one of claims 45-52, wherein said HVR1 region comprises residues 24-79 of any one of SEQ ID NOs: 79-87.

54. The engineered meganuclease of any one of claims 45-53, wherein said first subunit comprises an amino acid sequence having at least 80% sequence identity to residues 7-153 of any one of SEQ ID NOs: 79-87.

55. The engineered meganuclease of any one of claims 45-54, wherein said first subunit comprises an amino acid sequence having at least 80% sequence identity to residues 1-153 of any one of SEQ ID NOs: 79-87.

56. The engineered meganuclease of any one of claims 45-55, wherein said first subunit comprises a residue corresponding to residue 19 of any one of SEQ ID NOs: 79-87.

57. The engineered meganuclease of any one of claims 45-56, wherein said first subunit comprises a residue corresponding to residue 80 of any one of SEQ ID NOs: 81, 82, 85, or 87.

58. The engineered meganuclease of any one of claims 45-57, wherein said first subunit comprises residues 7-153 of any one of SEQ ID NOs: 79-87.

59. The engineered meganuclease of any one of claims 45-58, wherein said first subunit comprises residues 1-153 of any one of SEQ ID NOs: 79-87.

60. The engineered meganuclease of any one of claims 45-59, wherein said HVR2 region comprises an amino acid sequence having at least 80% sequence identity to residues 215-270 of any one of SEQ ID NOs: 79-87.

61. The engineered meganuclease of any one of claims 45-60, wherein said HVR2 region comprises one or more residues corresponding to residues 215, 217, 219, 221, 223, 224, 229, 231, 233, 235, 237, 259, 261, 266, and 268 of any one of SEQ ID NOs: 79-87.

62. The engineered meganuclease of any one of claims 45-61, wherein said HVR2 region comprises a residue corresponding to residue 241 of any one of SEQ ID NOs: 79-87.

63. The engineered meganuclease of any one of claims 45-62, wherein said HVR2 region comprises a residue corresponding to residue 263 of any one of SEQ ID NOs: 79-87.

64. The engineered meganuclease of any one of claims 45-63, wherein said HVR2 region comprises a residue corresponding to residue 264 of any one of SEQ ID NOs: 79-87.

65. The engineered meganuclease of any one of claims 45-64, wherein said HVR2 region comprises residues 215-270 of any one of SEQ ID NOs: 79-87.

66. The engineered meganuclease of any one of claims 45-65, wherein said second subunit comprises an amino acid sequence having at least 80% sequence identity to residues 198-344 of any one of SEQ ID NOs: 79-87.

67. The engineered meganuclease of any one of claims 45-66, wherein said second subunit comprises an amino acid sequence having at least 80% sequence identity to residues 196-354 of any one of SEQ ID NOs: 79-87.

68. The engineered meganuclease of any one of claims 45-67, wherein said second subunit comprises a residue corresponding to residue 271 of any one of SEQ ID NOs: 79, 80, 81, 83, 84 or 87.

69. The engineered meganuclease of any one of claims 45-68, wherein said second subunit comprises a residue corresponding to residue 330 of SEQ ID NO: 81 or 86.

70. The engineered meganuclease of any one of claims 45-69, wherein said second subunit comprises residues 198-344 of any one of SEQ ID NOs: 79-87.

71. The engineered meganuclease of any one of claims 45-70, wherein said second subunit comprises residues 196-354 of any one of SEQ ID NOs: 79-87.

72. The engineered meganuclease of any one of claims 45-71, wherein said engineered meganuclease is a single-chain meganuclease comprising a linker and wherein said linker covalently joins said first subunit and said second subunit.

73. The engineered meganuclease of any one of claims 45-72, wherein said engineered meganuclease comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 79-87.

74. The engineered meganuclease of any one of claims 45-73, wherein said engineered meganuclease comprises an amino acid sequence having at least 80% sequence identity to residues 2-354 of any one of SEQ ID NOs: 79-87.

75. The engineered meganuclease of any one of claims 45-74, wherein said engineered meganuclease comprises an amino acid sequence of any one of SEQ ID NOs: 79-87.

76. The engineered meganuclease of any one of claims 45-75, wherein said engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of any one of SEQ ID NOs: 79-87.

77. The engineered meganuclease of any one of claims 45-76, wherein said engineered meganuclease is encoded by a nucleic sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 119-126.

78. The engineered meganuclease of any one of claims 45-77, wherein said engineered meganuclease is encoded by a nucleic acid sequence of any one of SEQ ID NOs: 124-132.

79. The engineered meganuclease of any one of claims 45-72, wherein said engineered meganuclease comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 80.

80. The engineered meganuclease of any one of claims 45-72, wherein said engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 80.

81. The engineered meganuclease of any one of claims 45-72, wherein said engineered meganuclease comprises an amino acid sequence having at least 95% sequence identity to residues 2-354 of SEQ ID NO: 80.

82. The engineered meganuclease of any one of claims 45-72, wherein said engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 80.

83. An engineered meganuclease that binds and cleaves a recognition sequence comprising SEQ ID NO: 5 within a C90rf72 gene, wherein said engineered meganuclease comprises an amino acid sequence of SEQ ID NO: 80.

84. An engineered meganuclease that binds and cleaves a recognition sequence comprising SEQ ID NO: 5 within a C90rf72 gene, wherein said engineered meganuclease comprises an amino acid sequence comprising residues 2-354 of SEQ ID NO: 80.

85. A polynucleotide comprising a nucleic acid sequence encoding said engineered meganuclease of any one of claims 1-84.

86. The polynucleotide of claim 85, wherein said polynucleotide comprises a promoter operably linked to said nucleic acid sequence encoding said engineered meganuclease.

87. The polynucleotide of claim 86, wherein said promoter is a CNS cell-specific promoter.

88. The polynucleotide of claim 85 or claim 86, wherein said promoter is active in cortex cells, upper motor neurons, spinal cells, lower motor neurons, neurons, neuron progenitor cells, astrocytes, excitatory neurons, inhibitory neurons, oligodendrocytes or motor neuron progenitor cells.

89. The polynucleotide of any one of claims 86-88, wherein said promoter is a CAG promoter or a human synapsin 1 (Syn-1) promoter.

90. A polynucleotide comprising a first nucleic acid sequence encoding a first engineered meganuclease and a second nucleic acid sequence encoding a second engineered meganuclease, wherein said first engineered meganuclease is said engineered meganuclease of any one of claims 1-44, and wherein said second engineered meganuclease is said engineered meganuclease of any one of claims 45-84.

91. The polynucleotide of claim 90, wherein said polynucleotide comprises, from 5' to 3', (i) said first nucleic acid sequence encoding said first engineered meganuclease; and (ii) said second nucleic acid sequence encoding said second engineered meganuclease.

92. The polynucleotide of claim 90, wherein said polynucleotide comprises, from 5' to 3', (i) said second nucleic acid sequence encoding said second engineered meganuclease; and (ii) said first nucleic acid sequence encoding said first engineered meganuclease.

93. The polynucleotide of any one of claims 90-92, wherein said polynucleotide comprises a promoter enhancer.

94. The polynucleotide of claim 93, wherein said promoter enhancer is a neuron-specific promoter enhancer.

95. The polynucleotide of any one of claims 90-94, wherein said first nucleic acid sequence and / or said second nucleic acid sequence is codon modified to reduce the percent sequence identity between said first nucleic acid sequence and said second nucleic acid sequence, whereinsaid codon modification does not alter the amino acid sequence of said first engineered meganuclease or said second engineered meganuclease.

96. The polynucleotide of claim 95, wherein said first nucleic acid sequence has no more than about 40% to about 80% sequence identity to said second nucleic acid sequence.

97. The polynucleotide of claim 95 or claim 96, wherein said first nucleic acid sequence has no more than about 60% sequence identity to said second nucleic acid sequence.

98. The polynucleotide of any one of claims 90-97, wherein said first engineered meganuclease comprises a nuclear localization sequence (NLS).

99. The polynucleotide of any one of claims 90-98, wherein said first engineered meganuclease comprises a first NLS attached at the N-terminus and a second NLS attached at the C-terminus.

100. The polynucleotide of claim 99, wherein said first NLS and said second NLS are not identical.

101. The polynucleotide of any one of claims 90-100, wherein said second engineered meganuclease comprises a third NLS attached at the N-terminus and a fourth NLS attached at the C-terminus.

102. The polynucleotide of claim 101, wherein said third NLS and said fourth NLS are not identical.

103. The polynucleotide of any one of claims 90-102, wherein said first nucleic acid sequence and said second nucleic acid sequence are separated by an internal ribosome entry site (IRES) element or a nucleic acid sequence encoding a 2A peptide.

104. The polynucleotide of claim 103, wherein said first nucleic acid sequence and said second nucleic acid sequence are separated by a nucleic acid sequence encoding a furin cleavage motif and a nucleic acid sequence encoding a 2A peptide.

105. The polynucleotide of claim 103 or claim 104, wherein said 2A peptide is a P2A peptide.

106. The polynucleotide of any one of claims 103-105, wherein said first nucleic acid sequence and said second nucleic acid sequence are separated by a nucleic acid sequence encoding a P2A / furin peptide comprising an amino acid sequence set forth in SEQ ID NO: 135.

107. The polynucleotide of any one of claims 90-106, wherein said first nucleic acid sequence and said second nucleic acid sequence are operably linked to a promoter.

108. The polynucleotide of claim 107, wherein said promoter is a CNS cell-specific promoter.

109. The polynucleotide of claim 107 or claim 108, wherein said promoter is active in in cortex cells, upper motor neurons, spinal cells, lower motor neurons, neurons, neuron progenitor cells, astrocytes, excitatory neurons, inhibitory neurons, oligodendrocytes or motor neuron progenitor cells.

110. The polynucleotide of any one of claims 107-109, wherein said promoter is a CAG promoter or a human Syn-1 promoter.

111. The polynucleotide of any one of claims 90-110, wherein said polynucleotide comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

112. The polynucleotide of any one of claims 90-111, wherein said polynucleotide comprises an intron sequence.

113. The polynucleotide of claim 112, wherein said intron sequence comprises a nucleic acid sequence set forth in SEQ ID NO: 136.

114. The polynucleotide of any one of claims 90-113, wherein said polynucleotide comprises a termination sequence.

115. The polynucleotide of claim 114, wherein said termination sequence is a poly A sequence.

116. The polynucleotide of claim 115, wherein said polyA sequence comprises a nucleic acid sequence set forth in SEQ ID NO: 137.

117. A polynucleotide comprising a first nucleic acid sequence encoding a first engineered meganuclease and a second nucleic acid sequence encoding a second engineered meganuclease, wherein:(a) said first engineered meganuclease binds and cleaves a recognition sequence comprising SEQ ID NO: 3 and comprises an amino acid sequence of SEQ ID NO: 7 or residues 2-354 of SEQ ID NO: 7; and(b) said second engineered meganuclease binds and cleaves a recognition sequence comprising SEQ ID NO: 5 and comprises an amino acid sequence of SEQ ID NO: 80 or residues 2-354 of SEQ ID NO: 80.

118. The polynucleotide of claim 85 or claim 90, wherein said polynucleotide is an mRNA.

119. A recombinant DNA construct comprising said polynucleotide of any one of claims 85- 117.

120. The recombinant DNA construct of claim 119, wherein said recombinant DNA construct is a plasmid DNA.

121. The recombinant DNA construct of claim 120 or claim 121, wherein said recombinant DNA construct encodes a recombinant virus comprising said polynucleotide.

122. The recombinant DNA construct of claim 120, wherein said recombinant virus is a recombinant adenovirus, a recombinant lentivirus, a recombinant retrovirus, or a recombinant adeno-associated virus (AAV).

123. The recombinant DNA construct of claim 121 or claim 122, wherein said recombinant virus is a recombinant AAV.

124. The recombinant DNA construct of claim 122 or claim 123, wherein said recombinant AAV has an AAV9 capsid.

125. The recombinant DNA construct of any one of claims 119-124, wherein said polynucleotide comprises a promoter operably linked to said nucleic acid sequence encoding said engineered meganuclease.

126. The recombinant DNA construct of claim 125, wherein said promoter is a CNS cellspecific promoter.

127. The recombinant DNA construct of claim 125 or claim 126, wherein said promoter is active in cortex cells, upper motor neurons, spinal cells, lower motor neurons, neurons, neuron progenitor cells, astrocytes, excitatory neurons, inhibitory neurons, oligodendrocytes or motor neuron progenitor cells.

128. The recombinant DNA construct of any one of claims 125-127, wherein said promoter is a CAG promoter or a human Syn-1 promoter.

129. A recombinant virus comprising a polynucleotide comprising said polynucleotide of any one of claims 85-117.

130. The recombinant virus of claim 129, wherein said recombinant virus is a recombinant adenovirus, a recombinant lentivirus, a recombinant retrovirus, or a recombinant AAV.

131. The recombinant virus of claim 129 or claim 130, wherein said recombinant virus is a recombinant AAV.

132. The recombinant virus of claim 130 or claim 131, wherein said recombinant AAV has an AAV9 capsid.

133. The recombinant virus of any one of claims 129-132, wherein said polynucleotide comprises a promoter operably linked to said nucleic acid sequence encoding said engineered meganuclease.

134. The recombinant virus of claim 133, wherein said promoter is a CNS cell-specific promoter.

135. The recombinant virus of claim 133 or claim 134, wherein said promoter is active in cortex cells, upper motor neurons, spinal cells, lower motor neurons, neurons, neuron progenitor cells, astrocytes, excitatory neurons, inhibitory neurons, oligodendrocytes or motor neuron progenitor cells.

136. The recombinant virus of any one of claims 133-135, wherein said promoter is a CAG promoter or a human Syn-1 promoter.

137. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and said engineered meganuclease of any one of claims 1-84.

138. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and said polynucleotide of any one of claims 85-118.

139. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and said recombinant DNA construct of any one of claims 119-128.

140. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and said recombinant virus of any one of claims 129-136.

141. A host cell comprising said polynucleotide of any one of claims 85-118.

142. A method for producing a genetically-modified eukaryotic cell comprising a modified C90rf72 gene, said method comprising: introducing into a eukaryotic cell one or more polynucleotides comprising a first nucleic acid sequence encoding a first engineered meganuclease and a second nucleic acid sequence encoding a second engineered meganuclease, wherein the first engineered meganuclease is said engineered meganuclease of any one of claims 1-44, and wherein said second engineered meganuclease is said engineered meganuclease of any one of claims 45-84, wherein said first engineered meganuclease and said second engineered meganuclease are expressed in said eukaryotic cell, wherein said first engineered meganuclease produces a first cleavage site in an endogenous C90rf72 gene at a recognition sequence comprising SEQ ID NO: 3, wherein said second engineered meganuclease produces a second cleavage site in said C90rf72 gene at a recognition sequence comprising SEQ ID NO: 5, wherein an intervening genomic DNA between said first cleavage site and said second cleavage site is excised from said C90rf72 gene, and wherein said C90rf72 gene is annealed to generate said modified C90rf72 gene.

143. The method of claim 142, wherein said modified C90rf72 gene comprises a reduced number of GGGGCC hexanucleotide repeats relative to said endogenous C90rf72 gene.

144. The method of claim 142 or claim 143, wherein said method comprises introducing into said eukaryotic cell a polynucleotide comprising a first nucleic acid encoding said first engineered meganuclease and a second nucleic acid sequence encoding said second engineered meganuclease.

145. The method of any one of claims 142-144, wherein said polynucleotide is said polynucleotide of any one of claims 85-118.

146. The method of any one of claims 142-145, wherein said polynucleotide is introduced into said eukaryotic cell by a recombinant virus.

147. The method of claim 146, wherein said recombinant virus is said recombinant virus of any one of claims 129-136.

148. The method of claim 146, wherein said recombinant virus is said recombinant AAV of any one of claims 130-136.

149. The method of any one of claims 142-145, wherein said polynucleotide is an mRNA.

150. The method of claim 149, wherein said mRNA is said mRNA of claim 118.

151. The method of any one of claims 142-150, wherein said eukaryotic cell is a mammalian cell.

152. The method of any one of claims 142-151, wherein said eukaryotic cell is a human cell.

153. The method of any one of claims 142-152, wherein said eukaryotic cell is a CNS cell.

154. The method of claim 153, wherein said CNS cell is a neuron or a motor neuron progenitor cell.

155. A method for modifying a C90rf72 gene in a target cell in a subject, said method comprising: delivering to said target cell one or more polynucleotides comprising a first nucleic acid sequence encoding a first engineered meganuclease and a second nucleic acid sequence encoding a second engineered meganuclease,wherein said first engineered meganuclease is said engineered meganuclease of any one of claims 1-44, wherein said second engineered meganuclease is said meganuclease of any one of claims 45-84, wherein said first engineered meganuclease and said second engineered meganuclease are expressed in said target cell, wherein said first engineered meganuclease produces a first cleavage site in said C90rf72 gene at a recognition sequence comprising SEQ ID NO: 3, wherein said second engineered meganuclease produces a second cleavage site in saidC90rf72 gene at a recognition sequence comprising SEQ ID NO: 5, wherein intervening genomic DNA between said first cleavage site and said second cleavage site is excised from said C90rf72 gene, and wherein said C90rf72 gene is annealed to generate a modified C90rf72 gene.

156. The method of claim 155, wherein said modified C90rf72 gene comprises a reduced number of GGGGCC hexanucleotide repeats relative to said endogenous C90rf72 gene.

157. The method of claim 155 or claim 156, wherein said method comprises introducing into said target cell a polynucleotide comprising a first nucleic acid encoding said first engineered meganuclease and a second nucleic acid sequence encoding said second engineered meganuclease.

158. The method of any one of claims 155-157, wherein said polynucleotide is said polynucleotide of any one of claims 85-118.

159. The method of any one of claims 155-158, wherein said polynucleotide is introduced into said eukaryotic cell by a recombinant virus.

160. The method of claim 159, wherein said recombinant virus is said recombinant virus of any one of claims 129-136.

161. The method of claim 159, wherein said recombinant virus is said recombinant AAV of any one of claims 130-136.

162. The method of any one of claims 155-158, wherein said polynucleotide is an mRNA.

163. The method of claim 162, wherein said mRNA is said mRNA of claim 118.

164. The method of any one of claims 155-163, wherein said eukaryotic cell is a mammalian cell.

165. The method of any one of claims 155-164, wherein said eukaryotic cell is a human cell.

166. The method of any one of claims 155-165, wherein said eukaryotic cell is a CNS cell.

167. The method of claim 166, wherein said CNS cell is a neuron or a motor neuron progenitor cell.

168. A method for treating a neurological disorder in a subject in need thereof, wherein said neurological disorder is characterized by a mutation in a C90rf72 gene that increases the number of GGGGCC hexanucleotide repeats relative to a full-length wild-type C90rf72 gene, said method comprising: administering to said subject an effective amount of one or more polynucleotides comprising a first nucleic acid sequence encoding a first engineered meganuclease and a second nucleic acid sequence encoding a second engineered meganuclease, wherein said first engineered meganuclease is said engineered meganuclease of any one of claims 1 -44, wherein said second engineered meganuclease is said engineered meganuclease of any one of claims 45-84, wherein said one or more polynucleotides are delivered to a target cell in said subject, wherein said first engineered meganuclease and said second engineered meganuclease are expressed in said target cell, wherein said first engineered meganuclease produces a first cleavage site in said C90rf72 gene at a recognition sequence comprising SEQ ID NO: 3, wherein said second engineeredmeganuclease produces a second cleavage site in said C90rf72 gene at a recognition sequence comprising SEQ ID NO: 5, wherein intervening genomic DNA between said first cleavage site and said second cleavage site is excised from said C90rf72 gene, and wherein said C90rf72 gene is annealed to generate a modified C90rf72 gene.

169. The method of claim 168, wherein said neurological disorder is Amyotrophic Lateral Sclerosis (ALS).

170. The method of claim 168 or claim 169, wherein said modified C90rf72 gene comprises a reduced number of GGGGCC hexanucleotide repeats relative to said endogenous C90rf72 gene.

171. The method of any one of claims 168-170, wherein said method comprises delivering to said target cell a polynucleotide comprising a first nucleic acid encoding said first engineered meganuclease and a second nucleic acid sequence encoding said second engineered meganuclease.

172. The method of any one of claims 168-171, wherein said polynucleotide is said polynucleotide of any one of claims 85-118.

173. The method of any one of claims 168-172, wherein said polynucleotide is introduced into said eukaryotic cell by a recombinant virus.

174. The method of claim 173, wherein said recombinant virus is said recombinant virus of any one of claims 129-136.

175. The method of claim 173, wherein said recombinant virus is said recombinant AAV of any one of claims 130-136.

176. The method of any one of claims 168-172, wherein said polynucleotide is an mRNA.

177. The method of claim 176, wherein said mRNA is said mRNA of claim 118.

178. The method of any one of claims 168-177, wherein said subject is a mammal.

179. The method of any one of claims 168-178, wherein said subject is a human.

180. The method of any one of claims 168-179, wherein said target cell is a CNS cell.

181. The method of claim 180, wherein said CNS cell is a neuron or a motor neuron progenitor cell.

182. The method of any one of claims 168-181, wherein the rate of motor function decline according to the ALS Functional Rating Scale is reduced compared to the decline prior to treatment or compared to an untreated subject having ALS.

183. The method of any one of claims 168-182, wherein neurofilament light chain levels in the cerebral spinal fluid are reduced.

184. The method of any one of claims 168-183, wherein said method reduces the number of GGGGCC dipeptides in the CSF and / or neurons of said subject compared to an untreated subject having ALS.

185. The method of any one of claims 168-184, wherein said method reduces the number of GGGGCC RNA foci in neurons of said subject compared to an untreated subject having ALS.

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