Multi-site editing in living cells
AssigneesThe Broad Institute, Inc., President and Fellows of Harvard College
InventorsVerena VOLF, George CHURCH, Fei CHEN
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Abstract
Methods and systems described herein enable robust and facile multi-site base editing through pooled editing, suitable for one-step encoding of information. gRNAs show robust editing in pools and demonstrate simultaneous multi-site editing across >100 genomic sites. Additionally, individual stem cells with more than two dozen edits can be obtained with minimal screening.
Claims22
A method of enriching for multi-site edited cells, comprising:
- a. delivering to a population of cells, a set of guide molecules and one or more programmable nucleases, wherein the set of guide molecules directs the one or more programmable nucleases to introduce a set of edits to a plurality of genomic loci to generate an edited population of cells;
- b. screening the population of cells for one or more enrichment markers;
- and
- c. isolating one or more cells from the population of cells that comprise the one or more enrichment markers.
The method of claim, wherein editing efficiency is determined by sequencing one or more genomic loci having a low overall editing efficiency in the plurality of genomic loci, wherein an edit above an editing efficiency threshold at the one or more genomic loci is an indicator that a cell comprises all edits in the set of edits.
The method of claim, wherein the one or more genomic loci having a low overall editing efficiency are associated with a functional phenotype or an encoded reporter gene, wherein the functional phenotype or encoded reported gene are expressed if the one or more genomic loci having the low overall editing efficiency are successfully edited.
The method of claim, wherein the functional phenotype is antibiotic resistance.
The method of claim, wherein the editing efficiency threshold is at least 0.1%, 0.2%, 0.5%, or 1%.
The method of claim, wherein the exogenous reporter is a fluorescent protein.
The method of claim, wherein one or more cells that express the exogenous reporter above an expression threshold are isolated.
The method of claim, wherein the expression threshold is in a top 20%, 15%, 10%, 5%, or 1% of the edited population of cells.
The method of claim, wherein the nucleotide deaminase is a cytidine deaminase or an adenosine deaminase.
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The engineered cell of claim, wherein the engineered cell is a primary cell or a stem cell.
The engineered cell of claim, wherein the engineered cell comprises one or more additional engineered modifications and wherein the cryptographically encoded information identifies the one or more additional engineered modifications.
Dependencies are inferred from the claim text.