Systems methods, and compositions for targeted nucleic acid editing

Patent metadata

Field	Value
Publication number	US-12221636-B2
Application number	US-201816650480-A
Country	US
Kind code	B2
Filing date	Oct 4, 2018
Priority date	Oct 4, 2017
Publication date	Feb 11, 2025
Grant date	Feb 11, 2025

Abstract

The invention provides for systems, methods, and compositions for targeting and editing nucleic acids. In particular, the invention 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.

First claim

What is claimed is:

1. An engineered composition for site directed base editing comprising:
a) a targeting domain; and
b) an adenosine deaminase or catalytic domain thereof, wherein the adenosine deaminase is modified to convert activity to a cytidine deaminase, wherein the adenosine deaminase or catalytic domain thereof comprises one or more mutations selected from P462A, N597I, or both of a human ADAR2 or mutations corresponding thereto in a homologue, ortholog, or variant thereof.

2. The composition of claim 1 , wherein the adenosine deaminase further comprises one or more mutations
at one or more positions selected from E396, C451, V351, R455, T375, K376, S486, Q488, R510, K594, R348, G593, S397, H443, L444, Y445, F442, E438, T448, A353, V355, T339, P539, V525 and I520 of a human ADAR2 or in positions corresponding thereto in a homologue, ortholog, or variant thereof;
at one or more positions selected from E488, V351, S486, T375, and S370 of a human ADAR2 or in positions corresponding thereto in a homologue, ortholog, or variant thereof, or
selected from E488Q, V351G, S486A, T375S, and S370C of a human ADAR2 or mutations corresponding thereto in a homologue, ortholog, or variant thereof.

3. The composition of claim 1 , wherein the adenosine deaminase or catalytic domain thereof is a human, cephalopod, or Drosophila adenosine deaminase or catalytic domain thereof, or is covalently or non-covalently linked to the targeting domain.

4. The composition of claim 1 , wherein said adenosine deaminase or catalytic domain thereof has been modified to comprise a mutation at glutamic acid 488 of a hADAR2-D amino acid sequence, or a corresponding position in a homologous ADAR protein.

5. The composition of claim 4 , wherein said glutamic acid 488 or a corresponding position in a homologous ADAR protein is replaced by a glutamine residue (E488Q); or said adenosine deaminase or catalytic domain thereof is a mutated hADAR2d comprising mutation E488Q or a mutated hADAR1d comprising mutation E1008Q.

6. The composition of claim 1 , wherein the targeting domain is a catalytically inactive Cas13 protein, or a nucleotide sequence encoding said catalytically inactive Cas13 protein.

7. The composition of claim 6 , wherein the catalytically inactive Cas13 protein is catalytically inactive Cas13a, catalytically inactive Cas13b, or catalytically inactive Cas13c; is obtained from a Cas13 nuclease derived from a bacterial species selected from the group consisting of Leptotrichia shahii, L wadei, L wadei F0279, 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 , SAMN05216357_1045 [Porphyromonadaceae bacterium KH3CP3RA], Listeria riparia, Insolitispirillum peregrinum, Bergeyella zoohelcum, Prevotella intermedia, Prevotella buccae, Porphyromonas gingivalis, Alistipes sp. ZOR0009, Bacteroides pyogenes, Prevotella sp. MA2016 , Riemerella anatipestifer, Prevotella aurantiaca, Prevotella saccharolytica , HMPREF9712_03108 [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], HMPREF6485_0083 [ Prevotella buccae ATCC 33574], HMPREF9144_1146 [ Prevotella pallens ATCC 700821], HMPREF9714_02132 [Myroides odoratimimus CCUG 12901], HMPREF9711_00870 [Myroides odoratimimus CCUG 3837], HMPREF9699_02005 [Bergeyella zoohelcum ATCC 43767], HMPREF9151_01387 [ Prevotella saccharolytica F0055], A343_1752 [ Porphyromonas gingivalis JCVI SC001], HMPREF1981_03090 [ Bacteroides pyogenes F0041], HMPREF1553_02065 [ Porphyromonas gingivalis F0568], HMPREF1988_01768 [ Porphyromonas gingivalis F0185], HMPREF1990_01800 [ Porphyromonas gingivalis W4087], M573_117042 [ Prevotella intermedia ZT], A2033_10205 [Bacteroidetes bacterium GWA2_31_9], SAMN05421542_0666 [ Chryseobacterium jejuense ], SAMN05444360_11366 [ Chryseobacterium carnipullorum ], SAMN05421786_1011119, Prevotella pallens, [Chryseobacterium ureilyticum], Prevotella sp. MSX73 , Paludibacter propionicigenes, Psychroflexus torquis, Prevotella pleuritidis, Prevotella falsenii, Capnocytophaga cynodegmi, Prevotella sp. P5-119, Prevotella sp. P4-76, Prevotella sp. P5-60 , Phaeodactylibacter xiamenensis, Flavobacterium sp. 316 , Sinomicrobium oceani, Chryseobacterium sp. YR477 , Reichenbachiella agariperforans, Fusobacterium necrophorum, Fusobacterium necrophorum subsp. funduliforme, Fusobacterium necrophorum subsp. funduliforme ATCC 51357, Fusobacterium necrophorum DJ-2, Fusobacterium necrophorum BFTR-1, Fusobacterium necrophorum subsp. funduliforme 1_1_36S, Fusobacterium perfoetens ATCC 29250, Fusobacterium ulcerans ATCC 49185 , Anaerosalibacter sp. ND1, or Anaerosalibacter massiliensis ND1; or is a Cas13 protein having a sequence of any one of SEQ ID NO: 34-161.

8. An engineered, non-naturally occurring system for modifying an cytosine in a target locus of interest, comprising the composition of claim 6 and
c) a guide molecule which comprises a guide sequence linked to a direct repeat sequence, or a nucleotide sequence encoding said guide molecule;
wherein said adenosine deaminase or catalytic domain thereof is covalently or non-covalently linked to said catalytically inactive Cas13 protein or said guide molecule or is adapted to link thereto after delivery; and
wherein said guide sequence is capable of hybridizing with a target RNA sequence in a target RNA comprising an cytosine to form an RNA duplex, wherein said guide sequence comprises a non-pairing adenosine or uracil at a position corresponding to said cytosine resulting in an C-A/U mismatch in the RNA duplex formed.

9. The composition of claim 1 , further comprising a guide molecule which comprises a guide sequence linked to a direct repeat sequence, or a nucleotide sequence encoding said guide molecule.

10. An engineered, non-naturally occurring vector system for modifying a cytosine in a target locus of interest, comprising one or more polynucleotides comprising encoding sequences of a), and b) of claim 1 .

11. The engineered, non-naturally occurring vector system of claim 10 , comprising one or more vectors comprising:
i) a first regulatory element operably linked to a nucleotide sequence encoding a guide molecule which comprises a guide sequence,
ii) a second regulatory element operably linked to a nucleotide sequence encoding a catalytically inactive Cas13 protein, and
iii) a nucleotide sequence encoding the adenosine deaminase protein or catalytic domain thereof which is under control of said first regulatory element or said second regulatory element or operably linked to a third regulatory element;
wherein, if said nucleotide sequence encoding said adenosine deaminase protein or catalytic domain thereof is operably linked to a third regulatory element, said adenosine

Assignees

• Broad Inst Inc
• Massachusetts Inst Technology
• Harvard College
• President And Fellows Of Harvard Collage

Inventors

• Zhang Feng
• Gootenberg Jonathan
• Abudayyeh Omar

Classifications

• C07K2319/09

  containing a nuclear localisation signal · CPC title

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C12N15/113

  Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} · CPC title

• C12N9/226 Primary

  Class 2 CAS enzyme complex, e.g. single CAS protein · CPC title

• C12Y305/04005

  Cytidine deaminase (3.5.4.5) · CPC title