CRISPR/Cas-cytidine deaminase based compositions, systems, and methods for targeted nucleic acid editing
AssigneesThe Broad Institute, Inc., Massachusetts Institute of Technology, President and Fellows of Harvard College
InventorsFeng Zhang, Jonathan Gootenberg, David Benjamin Turitz Cox, Omar Abudayyeh, Soumya Kannan
Life of this patent
Estimating the term…
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 Cas protein, at least one guide molecule, and at least one cytidine deaminase protein or catalytic domain thereof.
Claims39
An engineered composition for site-directed base editing comprising a targeting domain and a cytidine deaminase protein or catalytic domain thereof, wherein the targeting domain is a Cas13 protein, or fragment thereof which retains RNA binding ability, and a guide molecule, and wherein the cytidine deaminase protein or catalytic domain thereof is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase and directly fused to or linked via a flexible linker to a C-terminus of the Cas13 protein or fragment thereof which retains RNA binding ability, and wherein the APOBEC family deaminase is an APOBEC1 deaminase comprising one or more mutations corresponding to W90A, W90Y, W90F, R118A, H121R, H122R, R126A, R126E, or R132E in rat APOBEC1, or an APOBEC3G deaminase comprising one or more mutations corresponding to W285A, W285Y, W285F, R313A, D316R, D317R, R320A, R320E, or R326E in human APOBEC3G.
An engineered composition for site-directed base editing of an RNA comprising:
- a Cas13 protein or fragment thereof, which retains RNA binding ability;
- a guide molecule;
- and
- a cytidine deaminase or a catalytic domain thereof, wherein the cytidine deaminase protein or the catalytic domain thereof is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase and directly fused to or linked via a flexible linker to a C-terminus of the Cas13 protein or fragment thereof which retains RNA binding ability, and wherein the APOBEC family deaminase is an APOBEC1 deaminase comprising one or more mutations corresponding to W90A, W90Y, W90F, R118A, H121R, H122R, R126A, R126E, or R132E in rat APOBEC1, or an APOBEC3G deaminase comprising one or more mutations corresponding to W285A, W285Y, W285F, R313A, D316R, D317R, R320A, R320E, or R326E in human APOBEC3G, and
- wherein the Cas13 protein or fragment thereof is or is derived from a Cas13 from a bacterial species selected from any one of
- Leptotrichia buccalis C-1013-b, Herbinix hemicellulosilytica, [Eubacterium] rectale, Eubacteriaceae bacterium CHKCI004, Blautia sp. Marseille-P2398, 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, Prevotella intermedia, Porphyromonas gingivalis, Bacteroides pyogenes, Riemerella anatipestifer, Prevotella aurantiaca, Prevotella saccharolytica, HMPREF9712_03108 [Myroides odoratimimus CCUG 10230], Flavobacterium branchiophilum, Myroides odoratimimus, Flavobacterium columnare, Porphyromonas sp. COT-052 OH4946, PIN17_0200 [Prevotella intermedia 17], HMPREF9144 1146 [Prevotella pallens ATCC 700821], HMPREF9714_02132 [Myroides odoratimimus CCUG 12901], HMPREF9711_00870 [Myroides odoratimimus CCUG 3837], 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 [Chryseobacterium ureilyticum], Prevotella pallens, Prevotella sp. MSX73, Psychroflexus torquis, Prevotella pleuritidis, Prevotella falsenii, Capnocytophaga cynodegmi, Prevotella sp. P5-119, Prevotella sp. P4-76, Prevotella sp. P5-60, Flavobacterium sp. 316, Chryseobacterium sp. YR477, Sinomicrobium oceani, Reichenbachiella agariperforans, Fusobacterium necrophorum subsp. funduliforme, Fusobacterium necrophorum, Fusobacterium necrophorum DJ-2, Fusobacterium necrophorum BFTR-1, Fusobacterium perfoetens, Fusobacterium ulcerans, Anaerosalibacter massiliensis, or Anaerosalibacter sp. ND11.
Dependencies are inferred from the claim text.