Systems, methods, and compositions 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
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Abstract
The disclosure 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.
Claims58
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An engineered, non-naturally occurring system comprising a catalytically inactive Cas13 effector protein (dCas13) or a nucleotide sequence encoding the catalytically inactive Cas13 effector protein, wherein the dCas13 protein is truncated at a C terminus, an N terminus, or both, and wherein the dCas13 is truncated by at least 20, at least 40, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 220, at least 240, at least 260, or at least 300 amino acids on the C terminus, wherein the Cas13 effector protein is Cas13a, and wherein the truncated form of the Cas13 effector protein has been truncated at C-terminal Δ984-1090.
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An engineered, non-naturally occurring system comprising a catalytically inactive Cas13 effector protein (dCas13) or a nucleotide sequence encoding the catalytically inactive Cas13 effector protein, wherein the dCas13 protein is truncated at a C terminus, an N terminus, or both, wherein the Cas13 effector protein is Cas13a, and wherein the truncated form of the Cas13 effector protein has been truncated at C-terminal Δ984-1090, wherein amino acid positions of the truncations correspond to amino acid positions of Prevotella sp. P5-125 Cas13b protein.
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An engineered, non-naturally occurring system comprising a catalytically inactive Cas13 effector protein (dCas13) or a nucleotide sequence encoding the catalytically inactive Cas13 effector protein, wherein the Cas13 effector protein is Cas13a, wherein the system further comprises a functional component or wherein the nucleotide sequence further encodes the functional component, wherein the functional component is a base editing component, wherein the base editing component comprises an adenosine deaminase or a catalytic domain thereof, and wherein the adenosine deaminase or the catalytic domain thereof, is inserted into an internal loop of the dCas13.
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A method for programmable and targeted base editing of a target sequence comprising delivering an engineered, non-naturally occurring system comprising a catalytically inactive Cas13 effector protein (dCas13) or a nucleotide sequence encoding the catalytically inactive Cas13 effector protein, wherein the Cas13 effector protein is Cas13a, wherein the Cas13 effector protein has been truncated at C-terminal Δ984-1090, to a cell, wherein the method further comprises determining the target sequence of interest and selecting an adenosine deaminase or catalytic domain thereof which most efficiently deaminates an adenine present in the target sequence, and wherein the deamination remedies a disease caused by transcripts containing a pathogenic G→A or C→T point mutation.
Dependencies are inferred from the claim text.