In situ sequencing of RNA transcripts with non-uniform 5 prime ends
Inventors
Hao, Cynthia • Salick, Max R. • Chu, Ci
Assignees
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Publication Number
US-12163190-B2
Publication Date
2024-12-10
Expiration Date
Abstract
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).
Core Innovation
The invention provides a method for determining a barcode sequence in a cell by inserting a barcode sequence and a padlock arm sequence into a genome of cells, wherein transcription of the inserted barcode sequence and the padlock arm sequence is controlled by RNA polymerase III. The barcode sequence encodes a guide RNA sequence that guides a CRISPR protein to its target site for imparting a perturbation at a particular location in genomic DNA of the cell.
The method forms a fixed cell by fixing and permeabilizing the cell, and generates cDNA strands from a plurality of RNA transcripts of the fixed cell. The RNA transcripts comprise non-uniform 5′ ends, and reverse transcribing the RNA transcripts generates nucleic acid strands comprising an untemplated poly C sequence on their 3′ ends.
A RNA template switching oligonucleotide comprising a poly G sequence is incorporated by hybridizing the poly G sequence to the untemplated poly C sequence, and the cDNA strands are further extended to incorporate a TSO arm sequence complementary to the RNA TSO. A padlock probe hybridizes to the cDNA strands to generate a circular template, rolling circle amplification generates one or more amplicons comprising the barcode sequence, and in situ sequencing within the fixed cell determines the barcode sequence in the cell.
Claims Coverage
The independent claim covers 5 main inventive features: genome-encoded barcode and padlock arm transcription under RNA polymerase III, CRISPR guide RNA-based perturbation, fixed-cell cDNA generation with polyC/polyG template switching to graft a TSO arm, padlock-probe hybridization to form a circular template, rolling circle amplification to generate barcode-containing amplicons, and in situ sequencing within the fixed cell to determine the barcode sequence.
Genome-encoded barcode and padlock arm transcription for CRISPR perturbation
A method for determining a barcode sequence in a cell by inserting the barcode sequence and a padlock arm sequence into a genome of cells, wherein transcription of the inserted barcode sequence and the padlock arm sequence is controlled by RNA polymerase III, and wherein the barcode sequence encodes a guide RNA sequence that guides a CRISPR protein to its target site for imparting a perturbation at a particular location in genomic DNA of the cell.
Fixed-cell cDNA generation from non-uniform 5′ ends
forming a fixed cell by fixing and permeabilizing the cell; generating cDNA strands from a plurality of RNA transcripts of the fixed cell, the plurality of RNA transcripts comprising non-uniform 5′ ends having the barcode sequence and the padlock arm sequence located 3′ to the barcode sequence; wherein said generating the cDNA strands comprises reverse transcribing the plurality of RNA transcripts using a reverse transcriptase and generating nucleic acid strands comprising an untemplated poly C sequence on their 3′ ends.
PolyC/polyG RNA TSO template switching to graft a TSO arm
incorporating a RNA template switching oligonucleotide comprising a poly G sequence in its 3′ end to the nucleic acid strands by hybridizing the poly G sequence of the RNA TSO to the untemplated poly C sequence of the nucleic acid strands such that the RNA TSO is incorporated at 5′ ends of the plurality of RNA transcripts; generating the cDNA strands by further extending the nucleic acid strands and incorporating a TSO arm sequence complementary to the RNA TSO to the nucleic acid strands; wherein the cDNA strands comprise the TSO arm sequence at their 3′ ends, the poly C sequence, a complement of the barcode sequence, and a complement of the padlock arm sequence.
Padlock-probe circular template formation and RCA barcode amplification
contacting one of the cDNA strands in the fixed cell with a padlock probe, wherein a first arm of the padlock probe hybridizes with the TSO arm sequence and/or the poly C sequence, and wherein a second arm of the padlock probe hybridizes with the complement of the padlock arm sequence, the first arm and the second arm are located on two ends of the padlock probe; generating a circular template comprising the padlock probe hybridized to the one of the cDNA strands in the fixed cell; generate one or more amplicons comprising the barcode sequence in the fixed cell by performing a rolling circle amplification using the circular template as a template.
In situ sequencing of barcode-containing amplicons to determine the barcode sequence
within the fixed cell, in situ sequencing the barcode sequence of the one or more amplicons, thereby determining the barcode sequence in the cell.
Across the independent claim, barcode determination is achieved by combining a genome-inserted barcode/CRISPR guide design under RNA polymerase III with fixed-cell polyC/polyG RNA TSO template switching that grafts a TSO arm onto cDNA, padlock-probe formation of a circular template, rolling circle amplification to produce barcode-containing amplicons, and in situ sequencing within the fixed cell to determine the barcode sequence.
Stated Advantages
higher detected amplicon counts
~20–30× increased sensitivity for pol III transcripts
improving barcode sequencing confidence
enabling spatial, single-cell readout
Documented Applications
Sequencing of exogenous barcode-bearing transcripts for pooled CRISPR (gRNA barcodes) and shRNA/siRNA/ASO/label readouts.
Sequencing of naturally occurring transcripts for gene expression, transcription start site (TSS) mapping, and splicing detection.
Mutation/edit detection for modified/transcribed-edited RNAs near TSS.
Spatial, single-cell readout using improved barcode sequencing sensitivity and confidence.
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