Methods and systems of molecular recording by crispr-cas system
AssigneePresident and Fellows of Harvard College
InventorsGeorge M. Church, Seth Lawler Shipman, Jeffrey D. Manklis, Jeffrey Matthew Nivala
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Abstract
This invention provides methods of altering a cell including providing the cell with a nucleic acid sequence encoding a Cas1 protein and/or a Cas2 protein of a CRISPR adaptation system, providing the cell with a CRISPR array nucleic acid sequence including a leader sequence and at least one repeat sequence, wherein the cell expresses the Cas1 protein and/or the Cas2 protein and wherein the CRISPR array nucleic acid sequence is within genomic DNA of the cell or on a plasmid. Also provided are methods and systems for nucleic acid storage and in vivo molecular recordings of events into a cell.
Claims72
A method of altering a cell comprising
- providing the cell with a nucleic acid sequence encoding a Cas1 protein and/or a Cas2 protein of a CRISPR adaptation system,
- providing the cell with a CRISPR array nucleic acid sequence including a leader sequence and at least one repeat sequence,
- wherein the cell expresses the Cas 1 protein and/or the Cas2 protein and wherein the CRISPR array nucleic acid sequence is within genomic DNA of the cell or on a plasmid.
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The method of claim 6 wherein the cell is a yeast cell, plant cell or a mammalian cell.
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An engineered, non-naturally occurring cell including a nucleic acid sequence encoding a Cas1 protein and/or a Cas2 protein of a CRISPR adaptation system wherein the cell expresses the Cas1 protein and/or the Cas 2 protein.
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An engineered, non-naturally occurring cell including a nucleic acid sequence encoding a Cas1 protein and/or a Cas2 protein of a CRISPR adaptation system and a CRISPR array nucleic acid sequence including a leader sequence and at least one repeat sequence,
- wherein the cell expresses the Cas1 protein and/or the Cas 2 protein, and wherein the CRISPR array nucleic acid sequence is inserted within genomic DNA of the cell or on a plasmid.
A method of inserting a target DNA sequence within genomic DNA of a cell comprising
- administering the target DNA sequence to the cell including a nucleic acid sequence encoding a Cas 1 protein and/or a Cas2 protein of a CRISPR adaptation system and a CRISPR array nucleic acid sequence including a leader sequence and at least one repeat sequence,
- wherein the cell expresses the Cas 1 protein and/or the Cas2 protein and wherein the CRISPR array nucleic acid sequence is within genomic DNA of the cell or on a plasmid, and
- wherein the target DNA sequence is administered under conditions within the cell wherein the Cas1 protein and/or the Cas2 protein processes the target DNA and the target DNA is inserted into the CRISPR array nucleic acid sequence adjacent a corresponding repeat sequence.
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A nucleic acid storage system comprising an engineered, non-naturally occurring cell including a nucleic acid sequence encoding a Cas1 protein and/or a Cas2 protein of a CRISPR adaptation system and a CRISPR array nucleic acid sequence including a leader sequence and at least one repeat sequence,
- wherein the cell expresses the Cas1 protein and/or the Cas2 protein and wherein the CRISPR array nucleic acid sequence is within genomic DNA of the cell or on a plasmid.
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A method of recording molecular events into a cell comprising
- administering a DNA sequence containing information of the molecular events to the cell including a nucleic acid sequence encoding a Cas 1 protein and/or a Cas2 protein of a CRISPR adaptation system and a CRISPR array nucleic acid sequence including a leader sequence and at least one repeat sequence,
- wherein the cell expresses the Cas 1 protein and/or the Cas2 protein and wherein the CRISPR array nucleic acid sequence is within genomic DNA of the cell or on a plasmid, and
- wherein the DNA sequence is administered under conditions within the cell wherein the Cas1 protein and/or the Cas2 protein processes the DNA and the DNA is inserted into the CRISPR array nucleic acid sequence adjacent a corresponding repeat sequence.
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-56. (canceled)
A system for in vivo molecular recording comprising an engineered, non-naturally occurring cell including a nucleic acid sequence encoding a cas1 protein and/or a cas2 protein of a CRISPR adaptation system and a CRISPR array nucleic acid sequence including a leader sequence and at least one repeat sequence,
- wherein the cell expresses the cas1 protein and/or the cas 2 protein and wherein the CRISPR array nucleic acid sequence is within genomic DNA of the cell or on a plasmid.
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A kit of directed recording of molecular events into a cell comprising an engineered, non-naturally occurring cell including a nucleic acid sequence encoding a cas 1 protein and/or a cas2 protein of a CRISPR adaptation system and a CRISPR array nucleic acid sequence including a leader sequence and at least one repeat sequence,
- wherein the cell expresses the cas 1 protein and/or the cas 2 protein and wherein the CRISPR array nucleic acid sequence is within genomic DNA of the cell or on a plasmid.
A method of recording information into a cell comprising
- converting a format of information into a plurality of bit sequences with the plurality of bit sequences comprising in series a bit stream representative of the format of information,
- converting the plurality of bit sequences to a set of corresponding oligonucleotide protospacer sequences,
- synthesizing the set of corresponding oligonucleotide protospacer sequences,
- introducing the set of corresponding oligonucleotide protospacer sequences into an engineered, non-naturally occurring cell including a nucleic acid sequence encoding a Cas1 protein and/or a Cas2 protein of a CRISPR adaptation system and a CRISPR array nucleic acid sequence including a leader sequence and at least one repeat sequence, wherein the CRISPR array nucleic acid sequence is inserted within genomic DNA of the cell or on a plasmid,
- wherein the cell expresses the Cas1 protein and/or the Cas 2 protein, and
- wherein the Cas1 protein and/or the Cas2 protein processes and inserts each member of the set of corresponding oligonucleotide protospacer sequences into the CRISPR array nucleic acid sequence adjacent a corresponding repeat sequence.
A method of recording an image into a cell comprising
- converting an image into a two dimensional array of rows and columns of pixels, wherein each pixel corresponds to a color of the image, wherein each color is represented by one or more nucleotides, and wherein each nucleotide is represented by one or more binary digits,
- identifying portions of the array of pixels as unique pixel sets and providing each pixel set with a unique pixel set identifier,
- converting each unique pixel set to a protospacer sequence to generate a set of protospacer sequences representative of the image,
- synthesizing the set of protospacer sequences,
- introducing the set of protospacer sequences into an engineered, non-naturally occurring cell including a nucleic acid sequence encoding a Cas 1 protein and/or a Cas2 protein of a CRISPR adaptation system and a CRISPR array nucleic acid sequence including a leader sequence and at least one repeat sequence, wherein the CRISPR array nucleic acid sequence is inserted within genomic DNA of the cell or on a plasmid,
- wherein the cell expresses the Cas1 protein and/or the Cas 2 protein, and
- wherein the Cas1 protein and/or the Cas2 protein processes and inserts each member of the set of corresponding oligonucleotide protospacer sequences into the CRISPR array nucleic acid sequence adjacent a corresponding repeat sequence.
A method of recording multiple images into a cell in a series relationship comprising
- creating a set of protospacer sequences for each image wherein each set of protospacers sequences are separately and in series introduced into an engineered, non-naturally occurring cell including a nucleic acid sequence encoding a Cas 1 protein and/or a Cas2 protein of a CRISPR adaptation system and a CRISPR array nucleic acid sequence including a leader sequence and at least one repeat sequence, wherein the CRISPR array nucleic acid sequence is inserted within genomic DNA of the cell or on a plasmid,
- wherein the cell expresses the Cas1 protein and/or the Cas 2 protein, and
- wherein the Cas1 protein and/or the Cas2 protein processes and inserts each member of the set of protospacer sequences into the CRISPR array nucleic acid sequence adjacent a corresponding repeat sequence, resulting in a CRISPR array nucleic acid sequence representing the order in which each set of protospacer sequences were introduced into the cell.
Dependencies are inferred from the claim text.