Engineered multi-omic display constructs and display systems
AssigneesThe Broad Institute, Inc., Massachusetts Institute of Technology
InventorsAviv Regev, Evgenij Fiskin
Life of this patent
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Abstract
Described in several embodiments herein are engineered phagemids and bacteriophages containing the same. Also described in several embodiments herein are methods of using the engineered phagemids and bacteriophages containing the same. In some embodiments, the engineered phagemids and bacteriophages containing the same are capable of providing multi-omic information at the single-cell level.
Claims57
An engineered display construct comprising:
- optionally, a genetically encoded display molecule or a genetically encoded display molecule linker;
- a genetically encoded affinity molecule;
- and
- a genetically encoded sequencing molecule, wherein the genetically encoded sequencing molecule is fused to or operatively coupled to the genetically encoded affinity molecule and the genetically encoded display molecule.
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A method of multi-omic single cell or single nuclei analysis, comprising:
- specifically binding one or more individual cells, individual nuclei, or both with an engineered display system or plurality thereof of as in any one of the preceding claims;
- allowing each affinity molecule to specifically bind a target molecule present inside of and/or on the surface of the one or more individual cells and/or individual nuclei;
- fixing the specifically bound engineered display system(s) to the one or more individual cells and/or individual nuclei;
- accessing cellular polynucleotides within one or more individual specifically bound cells and/or individual specifically bound nuclei;
- accessing the engineered display construct(s) in the specifically bound engineered display construct(s);
- and
- characterizing one or more features of the one or more individual specifically bound cells and/or individual specifically bound nucleic based, at least in part, on sequencing, in whole or in part,
- (i) the accessed genetically encoded affinity molecule, genetically encoded sequencing molecule, or both present in the specifically bound engineered display construct and
- (ii) the one or more accessed cellular and/or nuclear polynucleotides, and optionally wherein sequencing comprises a single cell, single nucleus sequencing technique, or both.
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The method of 23, wherein sequencing the genetically encoded affinity molecule, the genetically encoded sequencing molecule, or both and sequencing the one or more cellular polynucleotides, one or more nuclear polynucleotides, or both occurs in situ.
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A method of generating a specific pool of engineered display constructs or engineered display systems having a desired target affinity, comprising:
- a. generating an input display construct or engineered display system library, wherein each display construct or display system present in the input library is as in any one of the preceding claims;
- b. removing from the input library via negative selection at least some of the engineered display constructs or engineered display systems in the input library that do not specifically bind or otherwise associate with a desired target;
- c. positively selecting engineered display constructs or engineered display systems form the pool formed after step (b) that specifically bind or otherwise associate with the desired target;
- and
- d. amplifying the positively selected engineered display constructs or engineered display systems;
- and
- e. optionally sequencing one or more regions of the positively selected engineered display constructs.
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A kit for performing multi-omic single cell analysis, comprising:
- an engineered display construct, an engineered display construct library, and/or an engineered display system or plurality thereof.
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Dependencies are inferred from the claim text.