Systems, methods, and compositions for targeted nucleic acid editing
The present disclosure provides for systems, methods, and compositions for targeting and editing nucleic acids. In particular, the present disclosure provides non-naturally occurring or engineered RNA-targeting systems comprising a RNA-targeting Cas13 protein, at least one guide molecule, and at least one adenosine deaminase protein or catalytic domain thereof.
1 . An engineered, non-naturally occurring system suitable for modifying post-translational modification sites on a protein encoded by a target RNA, comprising:
(a) a catalytically inactive (dead) Cas13 protein, or a nucleotide sequence encoding said dead Cas13 protein;
(b) an adenosine deaminase acting on RNA (ADAR) protein or catalytic domain thereof comprising cytidine deaminase activity, a mutation of E488Q, and one or more mutations selected from L332I and K350 of an hADAR2-D amino acid sequence, or one or more corresponding mutations in a homologous ADAR protein;
(c) a guide molecule comprising a guide sequence designed to have a degree of complementarity with a target sequence in the target RNA at one or more codons that comprises an adenosine or cytosine and encodes an amino acid that is post-translationally modified;
wherein said adenosine deaminase protein or catalytic domain thereof is adapted to link thereto when contacted with the dead Cas13 protein or said guide molecule, and
wherein the post-translational modification sites are not phosphorylation sites.
2 . The system of claim 1 , wherein said guide sequence comprises a non-pairing nucleotide at a position corresponding to said adenosine or cytosine resulting in a mismatch in a RNA duplex formed between the target RNA and the guide molecule.
3 . The system of claim 1 , wherein the system is a particle delivery system comprising a ribonucleoprotein complex comprising (a), (b), and (c) encapsulated within or otherwise attached to a delivery particle, wherein the delivery particle further comprises a tissue-specific, cell-specific and/or cell compartment-specific targeting molecule.
4 . The system of claim 1 , where the system comprises a vector system comprising one or more vectors comprising:
i) a first regulatory element operably linked to a nucleotide sequence encoding said guide molecule which comprises said guide sequence;
ii) a second regulatory element operably linked to a nucleotide sequence encoding said dead Cas13 protein; and
iii) a nucleotide sequence encoding the adenosine deaminase protein or catalytic domain thereof which is under control of said first or second regulatory element or operably linked to a third regulatory element;
wherein if said nucleotide sequence encoding the adenosine deaminase protein or catalytic domain thereof is operably linked to the third regulatory element, said adenosine deaminase protein or catalytic domain thereof is adapted to link to said guide molecule or said dead Cas13 protein when contacted with said guide molecule or said dead Cas13 protein; and
wherein components (i), (ii) and (iii) are located on the same or different vectors of the system, wherein said first, second, and/or third regulatory element is an inducible promoter or wherein none of the first, second, and/or third regulatory element is an inducible promoter.
5 . The system of claim 1 , wherein the ADAR protein or catalytic domain thereof further comprises one or more mutations at amino acid position V351, S486, T375, S370, and N597 of an hADAR2-D amino acid sequence, or one or more corresponding mutations in a homologous ADAR protein;
or wherein the ADAR protein or catalytic domain thereof further comprises one or more mutations selected from V351G, S486A, T375S, S370C, P462A, N597I, and I398V in an hADAR2-D amino acid sequence, or one or more corresponding mutations in a homologous ADAR protein.
6 . The system of claim 1 , wherein the ADAR is a human (hu) ADAR or a catalytic domain thereof selected from (hu)ADAR1 or a catalytic domain thereof or (hu)ADAR2 or a catalytic domain thereof.
7 . The system of claim 1 , wherein said adenosine deaminase protein or catalytic domain thereof has been further modified to comprise one or more mutations selected from R348E, V351L, T375S, T375G, R455E, R455G, R455S, N473D, R474E, R477E, R481E, R481F, S486T, T490S, S495T, R510E, T490A, and K475Q, in a human ADAR2, or a corresponding mutation in an ADAR homologue or orthologue.
8 . The system of claim 1 , wherein said adenosine deaminase protein or catalytic domain thereof is delivered together with a uracil glycosylase inhibitor (UGI), where said UGI is covalently linked to said adenosine deaminase protein or catalytic domain thereof and/or said dead Cas13 protein.
9 . The system of claim 1 , wherein said dead Cas13 protein is a dead Cas13a, dead Cas13b, or dead Cas13c; or wherein said dead Cas13 originates from a Cas13 according to any one of SEQ ID NO: 34-161, or
originates from a bacterial species selected from Leptotrichia shahii, L wadei F0279 (Lw2), Listeria seeligeri , Lachnospiraceae bacterium MA2020, Lachnospiraceae bacterium NK4A179 , [Clostridium] aminophilum DSM 10710, Carnobacterium gallinarum DSM 4847, Paludibacter propionicigenes WB4 , Listeria weihenstephanensis FSL R9-0317, Listeriaceae bacterium FSL M6-0635 , Leptotrichia wadei F0279 , Rhodobacter capsulatus SB 1003 , Rhodobacter capsulatus R121 , Rhodobacter capsulatus DE442 , Leptotrichia buccalis C-1013-b, Herbinix hemicellulosilytica, [ Eubacterium ] rectale, Eubacteriaceae bacterium CHKCI004 , Blautia sp. Marseille-P2398 , Leptotrichia sp. oral taxon 879 str F0557, Lachnospiraceae bacterium NK4A144 , Chloroflexus aggregans, Demequina aurantiaca, Thalassospira sp. TSL5-1, SAMN04487830_13920 [Pseudobutyrivibrio sp. OR37], SAMN02910398_00008 [Butyrivibrio sp. YAB3001 ], Leptotrichia sp. Marseille-P3007 , Bacteroides ihuae, [Porphyromonadaceae bacterium KH3CP3RA], Listeria riparia, Insolitispirillum peregrinum, Bergeyella zoohelcum, Prevotella intermedia, Prevotella buccae, Porphyromonas gingivalis, Bacteroides pyogenes, Alistipes sp. ZOR0009 , Prevotella sp. MA2016 , Riemerella anatipestifer, Prevotella aurantiaca, Prevotella saccharolytica, [Myroides odoratimimus CCUG 10230 ], Capnocytophaga canimorsus, Porphyromonas gulae, Prevotella sp. P5-125 , Flavobacterium branchiophilum, Myroides odoratimimus, Flavobacterium columnare, Porphyromonas sp. COT-052 OH4946, PIN17_0200 [Prevotella intermedia 17 ], Prevotella buccae ATCC 33574 , Prevotella pallens ATCC 700821 , Myroides odoratimimus CCUG 12901 , Myroides odoratimimus CCUG 3837 , Bergeyella zoohelcum ATCC 43767 , Prevotella saccharolytica F0055 , Porphyromonas gingivalis JCVI SC001 , Bacteroides pyogenes F0041 , Porphyromonas gingivalis F0568 , Porphyromonas gingivalis F0185 , Porphyromonas gingivalis W4087 , Prevotella intermedia ZT, Bacteroidetes bacterium GWA2_31_9 , Chryseobacterium jejuense, Chryseobacterium carnipullorum, Chryseobacterium ureilyticum, Porphyromonas gingivalis, Prevotella pallens, Myroides odoratimimus, Prevotella sp. MSX73 , Paludibacter propionicigenes, Flavobacterium columnare, Psychroflexus torquis, Riemerella anatipestifer, Prevotella pleuritidis, Prevotella falsenii, Prevotella pleuritidis, Capnocytophaga cynodegmi, Prevotella sp. P5-119 , Prevotella sp. P4-76 , Prevotella sp. P5-60, WP_047431796 , Sinomicrobium oceani, Reichenbachiella agariperforans, Fusobacterium necrophorum subsp. funduliforme ATCC 51357 , Fusobacterium necrophorum DJ-2 , Fusobacterium necrophorum BFTR-1 , Fusobacterium necrophorum subsp. funduliforme 1_1_36S, Fusobacterium perfoetens, Fusobacterium ulcerans, Anaerosalibacter sp. ND1; or
wherein said dead Cas13 protein originates from a Prevotella sp. P5-125 Cas13b, Porphyromonas gulae Cas13b, or Riemerella anatipestifer Cas13b.
10 . The system of claim 1 , wherein said adenosine deaminase protein or catalytic domain thereof is directly fused to an N- or C-terminus of said dead Cas13 protein, or is fused to an N- or C-terminus of said dead Cas13 protein by a linker, wherein said linker is (GGGGS) 3-11 (SEQ ID NOS: 1-9), GSG 5 (SEQ ID NO: 10) or LEPGEKPYKCPECGKSFSQSGALTRHQRTHTR (SEQ ID NO: 11), or wherein said linker is an XTEN linker.
11 . The system of claim 1 , wherein said adenosine deaminase protein or catalytic domain thereof is linked to an adaptor protein and said guide molecule or said dead Cas13 protein comprises an aptamer sequence capable of binding to said adaptor protein, wherein said adaptor sequence is selected from MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, φCb5, φCb8r, φCb12r, #Cb23r, 7s and PRR1.
12 . The system of claim 1 , wherein said adenosine deaminase protein or catalytic domain thereof is inserted into an internal loop of said dead Cas13 protein.
13 . The system of claim 1 , wherein said dead Cas13 protein is a Cas13a protein and said Cas13a protein comprises one or more mutations in one or both of the two HEPN domains at positions R474 and R1046 of a Cas13a protein originating from Leptotrichia wadei or amino acid positions corresponding thereto of a Cas13a ortholog, or
wherein said dead Cas13 protein is a Cas13b protein and said Cas13b comprises a mutation in one or more of positions R116, H121, R1177, and H1182 of Cas13b protein originating from Bergeyella zoohelcum ATCC 43767 or amino acid positions corresponding thereto of a Cas13b ortholog, or
wherein said dead Cas13 protein is a Cas13b protein and said Cas13b comprises one or more mutations selected from R116A, H121A, R1177A, or any combination thereof of Cas13b protein originating from Bergeyella zoohelcum ATCC 43767 or amino acid positions corresponding thereto of a Cas13b ortholog, or
wherein said dead Cas13 protein is a Cas13b protein and said Cas13b protein comprises a mutation in one or more of positions R128, H133, R1053, and H1058of a Cas13b protein originating from Prevotella sp. P5-125 or amino acid positions corresponding thereto of a Cas13b ortholog, or
wherein said dead Cas13 protein is a Cas13b protein and said Cas13b protein comprises one or more mutations selected from H133A, H1058A, or both of a Cas13b protein originating from Prevotella sp. P5-125 or amino acid positions corresponding thereto of a Cas13b ortholog.
14 . The system of claim 1 , wherein said guide sequence has a length of from about 20 to about 53 nucleotides (nt), or from about 25 to about 53 nt, or from about 29 to about 53 nt, or from about 40 to about 50 nt capable of forming said RNA duplex with said target sequence.
15 . The system of claim 2 , wherein the distance between said non-pairing C and the 5′ end of said guide sequence is from about 20 to about 30 nucleotides.
16 . The system of claim 1 , wherein said dead Cas13 protein is a C-terminally truncated Cas13b functional variant of the corresponding wild type Cas13b wherein said C-terminally truncated Cas13b is encoded by nt 1-984 of Prevotella sp. P5-125 Cas13b or the corresponding nt of a Cas13b orthologue or homologue.
17 . The system of claim 1 , wherein the guide sequence comprises more than one mismatch corresponding to different adenosine or cytosine sites in the target sequence or wherein two guide molecules are used, each comprising a mismatch corresponding to a different adenosine or cytosine sites in the target RNA sequence.
18 . The system of claim 1 , wherein (a) said dead Cas13 protein, (b) said adenosine deaminase protein or catalytic domain thereof, or (c) both (a) and (b) comprise one or more heterologous nuclear export signal(s) (NES(s)) or nuclear localization signal(s) (NLS(s)), wherein the NES(s) are selected from an HIV Rev NES or MAPK NES, wherein the one or more NES(s), NLS(s), or both are at the C-terminus, N-terminus, or both of (a) the dead Cas13 protein, (b), the adenosine deaminase, or (c) both (a) and (b).
19 . The system of claim 1 , said target RNA is within a cell, or a eukaryotic cell, or a human cell, or a non-human animal cell, or a plant cell.
20 . The system of claim 1 , wherein the adenosine deaminase is linked to the dead Cas13 via one or more amino acids in amino acids 411-429, 114-124, 197-241, and 607-624 of a dead Prevotella buccae Cas13b or via one or more amino acids corresponding thereto in a dead Cas13 homologue or orthologue.
21 . A method of modifying post-translational modification sites on a protein encoded by a target RNA comprising:
contacting the target RNA with the system of claim 1 .
22 . The method of claim 21 , wherein (a), (b), and (c) are delivered or are to be delivered as one or more polynucleotide molecules, as a ribonucleoprotein complex, optionally via particles, vesicles, or one or more viral vectors.
23 . The method of claim 21 , wherein said target RNA is within a human or animal.
24 . The method of claim 6 , wherein the target RNA comprises a pathogenic G→A or C→T point mutation associated with a disease.
25 . The method of claim 24 , wherein said disease is selected from Meier-Gorlin syndrome, Seckel syndrome 4, Joubert syndrome 5, Leber congenital amaurosis 10; Charcot-Marie-Tooth disease, type 2; Charcot-Marie-Tooth disease, type 2; Usher syndrome, type 2C; Spinocerebellar ataxia 28; Spinocerebellar ataxia 28; Spinocerebellar ataxia 28; Long QT syndrome 2; Sjögren-Larsson syndrome; Hereditary fructosuria; Hereditary fructosuria; Neuroblastoma; Neuroblastoma; Kallmann syndrome 1; Kallmann syndrome 1; Kallmann syndrome 1; Metachromatic leukodystrophy, Rett syndrome, Amyotrophic lateral sclerosis type 10, Li-Fraumeni syndrome, Duchenne muscular dystrophy, Spinal muscular atrophy type II, Kugelberg-Welander disease, Cystic fibrosis, Hereditary pancreatitis, Parkinson disease 2, Joubert syndrome 3, Fanconi anemia complementation group C, Polycystic kidney disease adult type, Retinitis pigmentosa 64, Epilepsy focal with speech disorder and with or without mental retardation, Tuberous sclerosis 2, Tuberous sclerosis syndrome, Neurofibromatosis type 1, Tay-Sachs disease, Angelman syndrome, Niemann-Pick disease type A, Long QT syndrome, Primary familial hypertrophic cardiomyopathy, Familial adenomatous polyposis 1, Lynch syndrome, Nephrogenic diabetes insipidus X-linked, Primary pulmonary hypertension, Brachydactyly type B1, beta Thalassemia, Charcot-Marie-Tooth disease type 4C, Usher syndrome type 2A, Indifference to pain congenital autosomal recessive, Ehlers-Danlos syndrome type 4, Von Hippel-Lindau syndrome, Hypercholesterolemia autosomal recessive, Hereditary factor VIII deficiency disease, Severe combined immunodeficiency autosomal recessive T cell-negative B cell-positive NK cell-positive, Familial hypercholesterolemia, Danon disease, Niemann-Pick disease type C1, Primary ciliary dyskinesia, Leukocyte adhesion deficiency type III, Juvenile polyposis syndrome, X-linked severe combined immunodeficiency, Familial cancer of breast, Breast-ovarian cancer familial 1, Hereditary cancer-predisposing syndrome, Homocystinuria due to CBS deficiency, Breast-ovarian cancer familial 2, Wilson disease, or wherein said disease is a premature termination disease or cancer.
26 . The method of claim 21 , wherein said modification affects the fertility of an organism, wherein said modification affects splicing of said target RNA, wherein said modification introduces a mutation in a transcript introducing an amino acid change and causing expression of a new antigen in a cancer cell, wherein said deamination of an Adenine in said RNA causes a gain of function or a loss of function of a gene.
27 . The method of claim 2 , wherein the modification silences ubiquitination, methylation, acetylation, succinylation, glycosylation, O-GlcNAc, O-linked glycosylation, iodination, nitrosylation, sulfation, caboxyglutamation, or a combination thereof.
28 . The method of claim 21 , wherein the modification prevents pathological activated cell-signaling mediated by the post-translational modification.
29 . The method of claim 21 , wherein said modification increases or decreases a half-life of a target protein by modifying a degron encoded by the target RNA.