# US 12,595,468 B2 · Transcriptional recording by CRISPR spacer acquisition from RNA > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,595,468 B2 - Document ID: US12595468B2 - Status: Granted - Kind: B2 - Application number: 17/274,443 - Filed: 2019-09-11 - Published: 2022-02-17 - Granted: 2026-04-07 - Earliest claimed: 2018-09-11 - Assignees: ETH Zurich - Applicants: Eth ZüRich - Inventors: Randall Jeffrey Platt; Florian Schmidt - Web page: https://patentgrep.com/patent/US12595468B2 ## CPC - C12N 9/222: {Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes} - C12N 15/102: {Mutagenizing nucleic acids} - C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} - C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} - C12N 15/62: DNA sequences coding for fusion proteins - C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) - C12Q 1/6869: Methods for sequencing - C12Q 1/6883: for diseases caused by alterations of genetic material - C07K 2319/00: Fusion polypeptide - C12N 2800/22: Vectors comprising a coding region that has been codon optimised for expression in a respective host - C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) ## Abstract The present invention relates to a method for recording a transcriptome of a cell by: providing a test cell that includes a first transgene nucleic acid sequence encoding a fusion protein that is a reverse transcriptase polypeptide and a Cas1 polypeptide and a second transgene nucleic acid sequence encoding a Cas2 polypeptide, wherein the first transgene nucleic acid sequence and the second transgene nucleic acid sequence are under transcriptional control of an inducible promoter sequence, and a third transgene nucleic acid sequence including a CRISPR direct repeat (DR) sequence; wherein the CRISPR direct repeat sequence is specifically recognizable by a RT-Cas1-Cas2 complex formed by the expression products of the first transgene nucleic acid sequence and the second transgene nucleic acid sequence. ## Claims ### Claim 1 A method for recording a transcript of a prokaryotic cell, the method comprising the steps of: providing a prokaryotic test cell comprising: a first transgene nucleic acid sequence encoding a fusion protein comprising a reverse transcriptase polypeptide and a Cas1 polypeptide and a second transgene nucleic acid sequence encoding a Cas2 polypeptide, wherein said first transgene nucleic acid sequence and said second transgene nucleic acid sequence are under transcriptional control of an inducible promoter sequence, and a third transgene nucleic acid sequence comprising a CRISPR direct repeat (DR) sequence; wherein said CRISPR direct repeat sequence is specifically recognizable by an RT-Cas1-Cas2 complex formed by the expression products of said first transgene nucleic acid sequence and said second transgene nucleic acid sequence, in an exposure step, exposing said test cell to conditions under which expression of said first transgene nucleic acid sequence and said second transgene nucleic acid sequence is induced, wherein said RT-Cas1-Cas2 complex formed by expression products of said first transgene nucleic acid sequence and said second transgene nucleic acid sequence acquires at least one protospacer from one or more RNA molecules, and integrates said at least one protospacer as spacer into said third transgene nucleic acid sequence yielding a modified third transgene nucleic acid sequence comprising at least one integrated spacer, isolating said modified third transgene nucleic acid sequence from said test cell yielding an isolated modified third transgene nucleic acid sequence, and sequencing said isolated modified third transgene nucleic acid sequence wherein said first transgene nucleic acid sequence is selected from SEQ ID NO 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, and 31, or a codon-optimized nucleic acid sequence thereof encoding an identical amino acid sequence. ### Claim 2 (depends on 1) The method according to claim 1, wherein said third transgene nucleic acid sequence further comprises a CRISPR leader sequence, wherein said CRISPR leader sequence is specifically recognizable by said RT-Cas1-Cas2 complex formed by the expression products of said first transgene nucleic acid sequence and said second transgene nucleic acid sequence. ### Claim 3 (depends on 1) The method according to claim 1, wherein said third transgene nucleic acid sequence does not comprise any further CRISPR direct repeat sequence. ### Claim 4 (depends on 1) The method according to claim 1, wherein said test cell additionally comprises a fourth transgene nucleic acid sequence encoding a sensor, wherein said sensor will be activated when contacted with an analyte molecule yielding an activated sensor, wherein said activated sensor will induce the expression of a record gene inside the cell; and wherein in said exposure step, if said analyte molecule is present, said activated sensor induces the expression of a record gene inside the cell and RNA obtained from said record gene is acquired as a spacer. ### Claim 5 (depends on 1) The method according to claim 1, wherein said CRISPR leader sequence and/or said CRISPR direct repeat sequence are specifically recognizable by an RT-Cas1-Cas2 complex of F. saccharivorans, Candidatus accumlibacter, Eubacterium saburreum, Bacteroides fragiles, Campylobacter fetus, Teredinibacter turnerae, Woodsholea maritima, Desulfaculus baarsii, Azospirillum lipoferum, Cellulomonospora bogoriensis, Micromonospora rosaria, Tolypothirx camplyonemoides, Oscillatoriales cyanobacterium, or Rivularia sp, or an RT-Cas1-Cas2 complex originating thereof. ### Claim 6 (depends on 1) The method according to claim 1, wherein said third transgene nucleic acid sequence comprises an endonuclease recognition site sequence downstream of or within said CRISPR direct repeat, and said endonuclease recognition site sequence is specifically recognizable by a site-specific endonuclease and said isolated modified third transgene nucleic acid sequence is contacted with said site-specific endonuclease before said sequencing, wherein said (full length CRISPR direct repeat (adjacent to said endonuclease recognition site sequence is cleaved into a truncated CRISPR direct repeat sequence. ### Claim 7 (depends on 6) The method according to claim 6, wherein said sequencing comprises the use of a PCR primer, wherein said PCR primer comprises a nucleic acid sequence that binds to part of a full length CRISPR direct repeat sequence, but does not bind to said truncated CRISPR direct repeat sequences resulting from said endonuclease recognition site sequence being cleaved, within said modified third nucleic acid sequence, wherein said full length CRISPR direct repeat sequence results from or is formed by at least one spacer acquisition event. ### Claim 8 (depends on 1) The method according to claim 1, wherein said first transgene nucleic acid sequence is selected from SEQ ID NO 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, and 31 or selected from a codon-optimized nucleic acid sequence encoding a fusion protein comprising a reverse transcriptase polypeptide and a Cas1 polypeptide with identical amino acid sequence, and/or said second transgene nucleic acid sequence is selected from SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, and 32 or selected from a codon-optimized nucleic acid sequence encoding a Cas2 polypeptide with identical amino acid sequence, and/or said first transgene nucleic acid sequence and said second transgene nucleic acid sequence together comprise or essentially consist of a sequence of SEQ ID NO 34, and/or said third transgene nucleic acid sequence comprising a CRISPR direct repeat (DR) sequence comprises or essentially consists of a sequence selected from SEQ ID NO 35 to 103.