IN SITU SEQUENCING OF RNA TRANSCRIPTS WITH NON-UNIFORM 5' ENDS
Disclosed herein are methods for performing in situ sequencing of RNA transcripts with non-uniform 5′ ends. During reverse transcription (RT) of RNA transcripts, RT enzyme is induced to “template-switch” to a separate oligonucleotide provided as the template for the upstream flanking region. This flanking region is grafted onto the beginning of the cDNA, enabling padlock probe detection, rolling circle amplification, and fluorescent in situ sequencing. Overall, the disclosed method for in situ sequencing can be applicable for analyzing exogenously introduced transcripts (e.g., identifying and determining impact of a perturbation including a CRISPR perturbation or shRNA/siRNA/ASO perturbation), analyzing naturally occurring transcripts (e.g., measuring gene expression, detecting splicing events), and analyzing modified, naturally occurring transcripts (e.g., detecting mutations or gene edits).
1 . A construct for performing rolling circle amplification, the construct comprising:
a RNA transcript; and
a cDNA strand hybridized with the RNA transcript,
wherein the cDNA strand comprises:
a template switching oligonucleotide (TSO) arm sequence;
a poly C sequence; and
a padlock arm sequence; and
a RNA template switching oligonucleotide (TSO) comprising a poly G sequence,
wherein the poly G sequence is hybridized to the poly C sequence of the cDNA strand, and wherein a portion of the RNA TSO is hybridized to the TSO arm sequence of the cDNA strand.
2 . The construct of claim 1 , wherein the cDNA strand further comprises a barcode sequence or a complement thereof, the barcode sequence encoding for a guide RNA (gRNA) that guides a CRISPR protein to a target site for imparting a perturbation at a particular location in genomic DNA of a cell.
3 . The construct of claim 1 wherein the poly C sequence comprises between 2 to 5 cytosine nucleobases.
4 . The construct of claim 1 , wherein the poly G sequence comprises between 2 to 5 guanine nucleobases.
5 . The construct of claim 1 , wherein the RNA TSO comprises one or more locked nucleic acids (LNAs).
6 . The construct of claim 5 , wherein the RNA TSO comprises one LNA at a 3′ end of the RNA TSO.
7 . The construct of claim 5 , wherein between 1% and 50% of the nucleotides of the RNA template switching oligonucleotide comprise locked nucleic acids.
8 . The construct of claim 1 , wherein the RNA transcript further comprises a reverse transcription priming site.
9 . The construct of claim 8 , wherein the cDNA strand further comprises a sequence complementary to the reverse transcription priming site.
10 . The construct of claim 1 , wherein the RNA transcript is a pol III—transcribed RNA transcript.
11 . The construct of claim 1 , wherein the RNA transcript further comprises a target sequence, and wherein the cDNA strand further comprises a reverse complement of the target sequence.
12 . The construct of claim 1 , wherein the TSO arm sequence is located at a 3′ end of the cDNA strand.
13 . A construct for performing rolling circle amplification, the construct comprising:
a cDNA strand reverse transcribed from a RNA transcript, wherein the cDNA strand comprises:
a template switching oligonucleotide (TSO) arm sequence;
a poly C sequence;
a padlock arm sequence;
a padlock probe, wherein a first arm of the padlock probe is hybridized with the TSO arm sequence and the poly C sequence, and wherein a second arm of the padlock probe hybridizes with the padlock arm sequence.
14 . The construct of claim 13 , wherein the cDNA strand further comprises a barcode sequence or a complement thereof, the barcode sequence encoding for a guide RNA (gRNA) that guides a CRISPR protein to a target site for imparting a perturbation at a particular location in genomic DNA of a cell.
15 . The construct of claim 13 , wherein the poly C sequence comprises between 2 to 5 cytosine nucleobases.
16 . The construct of claim 13 , wherein the cDNA strand further comprises a sequence complementary to a reverse transcription priming site of the RNA transcript.
17 . The construct of claim 13 , wherein the RNA transcript is a pol III—transcribed RNA transcript.
18 . The construct of claim 13 , wherein the RNA transcript further comprises a target sequence, and wherein the cDNA strand further comprises a reverse complement of the target sequence.
19 . The construct of claim 13 , wherein the first arm and the second arm of the padlock probe are located on two ends of the padlock probe.
20 . The construct of claim 13 , wherein the TSO arm sequence is located at a 3′ end of the cDNA strand.