Transcriptional recording by CRISPR spacer acquisition from RNA

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.