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 a rolling circle amplification reaction, the construct comprising:
an RNA transcript from a transfected cell, wherein the RNA transcript comprises a complementary sequence of a padlock arm sequence; and
a cDNA strand, wherein the cDNA strand is hybridized with the RNA transcript and is reverse transcribed from the RNA transcript,
wherein the cDNA strand comprises:
a template switching oligonucleotide (TSO) arm sequence;
a poly C sequence; and
a padlock arm sequence, wherein the transfected cell comprises the padlock arm sequence and is produced by inserting the padlock arm sequence into a genome of a cell using one of a construct of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), a cDNA construct, a Transcription Activator-like Effector Nuclease (TALEN), and a Zinc finger nuclease; and
an RNA template switching oligonucleotide (TSO) comprising a poly G sequence,
wherein the RNA transcript is transcribed from the genome of the transfected cell
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 which encodes for a guide RNA (gRNA) that guides a CRISPR protein to a target site in genomic DNA of a cell.
3 . The construct of claim 1 wherein the poly C sequence comprises 2 to 5 cytosine nucleobases.
4 . The construct of claim 1 , wherein the poly G sequence comprises 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 locked nucleic acid (LNA) at a 3′ end of the RNA TSO.
7 . The construct of claim 5 , wherein 1% to 50% of the nucleotides of the RNA TSO comprise LNAs.
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 an RNA polymerase 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 a rolling circle amplification reaction, the construct comprising:
an RNA transcript from a transfected cell, wherein the RNA transcript comprises a complementary sequence of a padlock arm sequence; and
a cDNA strand, wherein the cDNA strand is hybridized with the RNA transcript and is reverse transcribed from the RNA transcript,
wherein the cDNA strand comprises:
a template switching oligonucleotide (TSO) arm sequence;
a poly C sequence; and
a padlock arm sequence, wherein the transfected cell comprises the padlock arm sequence and is produced by inserting the padlock arm sequence into a genome of a cell such that the padlock arm sequence is located in a genome of the transfected cell at a position located less than 100 nucleobases downstream of an RNA polymerase II or RNA polymerase III promoter; and
an RNA template switching oligonucleotide (TSO) comprising a poly G sequence,
wherein the RNA transcript is transcribed from the genome of the transfected cell
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.
14 . The construct of claim 13 , wherein the cDNA strand further comprises a barcode sequence which encodes for a guide RNA (gRNA) that guides a CRISPR protein to a target site in genomic DNA of a cell.
15 . The construct of claim 13 , wherein the poly C sequence comprises 2 to 5 cytosine nucleobases or wherein the poly G sequence comprises 2 to 5 guanine nucleobases.
16 . The construct of claim 13 , wherein the RNA TSO comprises one or more LNAs.
17 . The construct of claim 16 , wherein the RNA TSO comprises one LNA at a 3′ end of the RNA TSO.
18 . The construct of claim 16 , wherein 1% to 50% of the nucleotides of the RNA TSO comprise LNAs.
19 . The construct of claim 13 , wherein the RNA transcript further comprises a reverse transcription priming site, and wherein the cDNA strand further comprises a sequence complementary to the reverse transcription priming site.
20 . 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.