TRANSCRIPTIONAL RECORDING BY CRISPR SPACER ACQUISITION FROM RNA

摘要

The present invention relates to a method for recording a transcriptome of a cell, the method comprising the steps of: providing a 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 a RT-Cas1-Cas2 complex formed by the expression products of said first transgene nucleic acid sequence and said second transgene nucleic acid sequence, 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 protospacers from RNA molecules and integrates spacers into said third transgene nucleic acid sequence yielding a modified third transgene nucleic acid sequence, isolating said modified third transgene nucleic acid sequence from said test cell yielding an isolated third transgene nucleic acid sequence, and sequencing said isolated modified third transgene nucleic acid sequence.