Methods and Systems of Molecular Recording by Crispr-Cas System

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.

1 . 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 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.   
     
     
         2 - 3 . (canceled) 
     
     
         4 . The method of  claim 1  further comprising providing the cell with an oligo nucleotide sequence comprising a protospacer, wherein the oligo nucleotide sequence includes a modified AAG protospacer adjacent motif (PAM). 
     
     
         5 - 10 . (canceled) 
     
     
         11 . The method of  claim 1  wherein the eukaryotic cell is a yeast cell, plant cell or a mammalian cell. 
     
     
         12 . The method of  claim 1  wherein the nucleic acid sequence encoding the Cas1 protein and/or a Cas2 protein includes a first regulatory element operable in a eukaryotic cell. 
     
     
         13 . The method of  claim 1  wherein the nucleic acid sequence encoding the Cas1 protein and/or a Cas2 protein is codon optimized for expression of Cas1 and/or Cas2 in a eukaryotic cell. 
     
     
         14 . (canceled) 
     
     
         15 . 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. 
     
     
         16 - 17 . (canceled) 
     
     
         18 . 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. 
     
     
         19 . 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 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, 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.   
     
     
         20 - 21 . (canceled) 
     
     
         22 . The method of  claim 19  wherein the target DNA sequence includes a modified AAG protospacer adjacent motif (PAM). 
     
     
         23 - 26 . (canceled) 
     
     
         27 . The method of  claim 19 , wherein the cell is  E.   coli  BL21-AI. 
     
     
         28 - 29 . (canceled) 
     
     
         30 . The method of  claim 19  wherein the eukaryotic cell is a yeast cell, plant cell or a mammalian cell. 
     
     
         31 . The method of  claim 19  wherein the nucleic acid sequence encoding the Cas1 protein and/or a Cas2 protein includes a first regulatory element operable in a eukaryotic cell. 
     
     
         32 . The method of  claim 19  wherein the nucleic acid sequence encoding the Cas1 protein and/or a Cas2 protein is codon optimized for expression of Cas1 and/or Cas2 in a eukaryotic cell. 
     
     
         33 . 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.   
     
     
         34 - 59 . (canceled) 
     
     
         60 . 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.   
     
     
         61 . (canceled) 
     
     
         62 . The system of  claim 60  wherein the system records in single or multiple modalities, wherein the multiple modality recordation comprises altering Cas1 PAM recognition through directed evolution by specific cas1 or cas2 mutants. 
     
     
         63 . 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 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.   
     
     
         64 . The kit of  claim 63  wherein the recording is in single or multiple modalities. 
     
     
         65 . The kit of  claim 64  wherein the multiple modality recordation comprises altering Cas1 PAM recognition through directed evolution by specific cas1 or cas2 mutants. 
     
     
         66 - 92 . (canceled)