Methods and compositions for nucleic acid-guided nuclease cell targeting screen
AssigneeSpotlight Therapeutics
InventorsAkshay Tambe, Hariharan Jayaram, Steven Strutt
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Abstract
Methods and compositions related to a nucleic acid-guided nuclease cell targeting screen are provided. The invention relates to compositions and methods for identifying cell targeting proteins that, when associated with a nucleic acid-guided nuclease (such as Cas9), enables at least the nucleic acid-guided nuclease to be targeted to the surface of a target cell or internalized by a target cell, i.e., a cell targeted by the cell targeting agent.
Claims56
A method of identifying a cell targeting agent, the method comprising:
- providing a plurality of ribonucleoproteins (RNPs) each comprising an RNA-guided nuclease fusion protein and a unique identifying RNA (uiRNA), wherein the RNA-guided nuclease fusion protein comprises an RNA-guided nuclease, or a functional fragment thereof, and a test protein;
- and wherein the uiRNA comprises a guide RNA (gRNA) and a sequence identifier;
- contacting the RNPs with a population of target cells;
- isolating RNA from the population of target cells, thereby obtaining isolated RNA;
- and
- testing the isolated RNA for the presence of the identifier sequence, wherein the presence of the identifier sequence indicates that the test protein is a cell targeting agent.
A method of identifying a cell targeting agent, the method comprising:
- providing a vector encoding an RNA-guided nuclease fusion protein comprising an RNA-guided nuclease, or a functional fragment thereof, and a test protein, and encoding a unique identifying RNA (uiRNA) comprising a guide RNA (gRNA) and a sequence identifier;
- transferring the vector to a host cell suitable to express the RNA-guided nuclease fusion protein and the uiRNA;
- expressing the RNA-guided nuclease fusion protein and the uiRNA in the host cell, such that ribonucleoproteins (RNPs) each comprising the RNA-guided nuclease fusion protein and the uiRNA are formed;
- isolating the RNPs from the host cell;
- contacting the RNPs with a population of target cells;
- isolating RNA from the population of target cells;
- and
- testing the isolated RNA for the presence of the identifier sequence, wherein the presence of the identifier sequence indicates that the test protein is a cell targeting agent.
A cell expression vector comprising:
- a nucleic acid encoding an RNA-guided nuclease operably linked to a cloning site for inserting a nucleic acid of a test protein, thereby forming an RNA-guided nuclease fusion protein comprising the RNA-guided nuclease and the test protein;
- and
- a nucleic acid encoding a unique identifying RNA (uiRNA), wherein the uiRNA comprises a guide RNA and a sequence identifier.
Dependencies are inferred from the claim text.