Methods of crispr guide RNA sequencing in single cell workflows

Abstract

Provided herein are methods and compositions for sequencing gRNAs. The methods and compositions are compatible with single-cell sequencing workflows, including in combination with analysis of additional non-gRNA analytes such as cellular transcripts. The methods provided herein can facilitate high-resolution phenotypic analysis in large-scale CRISPR / Cas-based screens.

Description

Attorney Docket No.43487-1046601 METHODS OF CRISPR GUIDE RNA SEQUENCING IN SINGLE CELL WORKFLOWS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 549,400,filed February 2, 2024, U.S. Provisional Application No.63 / 700,430, filed September 27, 2024, US. Provisional Application No.63 / 740,126, filed December 30, 2024, and U.S. Provisional Application No.63 / 740,918, filed December 31, 2024, the contents of which are incorporated by reference in their entirety. FIELD

[0002] The present disclosure relates to methods and compositions for guide RNA (gRNA)sequencing in single-cell sequencing workflows. BACKGROUND

[0003] A sample may be processed for various purposes, such as identification of a type ofmoiety within the sample. The sample may be a biological sample. Biological samples may be processed, such as for detection of a disease (e.g., cancer) or identification of a particular species. There are various approaches for processing samples, such as polymerase chain reaction (PCR) and sequencing. Biological samples may be processed within various reaction environments, such as partitions. Partitions may be wells or droplets. Droplets or wells may be employed to process biological samples in a manner that enables the biological samples to be partitioned and processed separately. For example, such droplets may be fluidically isolated from other droplets, enabling accurate control of respective environments in the droplets. Biological samples in partitions may be subjected to various processes, such as chemical processes or physical processes. Samples in partitions may be subjected to heating or cooling, or chemical reactions, such as to yield species that may be qualitatively or quantitatively processed. Biological molecules, such as nucleic acids and proteins, within biological samples may be probed and / or processed for quantitative or qualitative assessment. Improved methods are needed for detecting and sequencing analytes in a biological sample. SUMMARY

[0004] In some aspects, the present disclosure provides methods and compositions forsequencing gRNAs, such as from CRISPR / Cas systems. The methods can be performed in single-cell sequencing workflows. The methods can be performed alone and are also compatible with sequencing and / or detection of additional analytes, such as transcripts. In some aspects, theAttorney Docket No.43487-1046601 methods comprise single-cell gRNA and transcript (e.g. transcriptome) expression analysis. In some aspects, the methods facilitate analysis of gRNA-expressing cells, such as in large-scale CRISPR / Cas screens.

[0005] In some aspects, provided herein is a method comprising: providing a gRNA-expressing cell comprising a gRNA having a spacer sequence and a constant region comprising a scaffold sequence; contacting the gRNA-expressing cell with a gRNA-targeting probe that hybridizes to the constant region of the gRNA; contacting the gRNA-expressing cell with a ligatable probe pair comprising a first ligatable probe and a second ligatable probe that hybridize to a target nucleic acid in the gRNA-expressing cell; ligating the first ligatable probe to the second ligatable probe using the target nucleic acid as template to generate a ligated probe pair; generating a partition comprising 1) the gRNA-expressing cell, and 2) a plurality of barcoded oligonucleotides comprising a partition-specific barcode; extending the 3’ end of the gRNA- targeting probe to generate an extended gRNA-targeting probe comprising a sequence complementary to the spacer sequence; using the extended gRNA-targeting probe and a first barcoded oligonucleotide of the plurality of barcoded oligonucleotides to generate a barcoded spacer oligonucleotide comprising the spacer sequence or complement thereof, and the partition- specific barcode or complement thereof; and using the ligated probe pair and a second barcoded oligonucleotide of the plurality of barcoded oligonucleotides to generate a barcoded analyte oligonucleotide comprising a sequence of the ligated probe pair or complement thereof, and the partition-specific barcode or a complement thereof. In some embodiments, the method further comprises sequencing the barcoded spacer oligonucleotide or a derivative thereof and the barcoded analyte oligonucleotide or a derivative thereof. In some embodiments, the method comprises analyzing the results of the sequencing to determine the sequence of the spacer sequence. In some embodiments, the method comprises analyzing the results of the sequencing to determine the presence and / or abundance of the gRNA and the target nucleic acid in the gRNA-expressing cell. In some embodiments, the method comprises hybridizing the extended gRNA-targeting probe to the first barcoded oligonucleotide, and extending the 3’ end of the extended gRNA-targeting probe and / or extending the first barcoded oligonucleotide to generate the barcoded spacer oligonucleotide. In some embodiments, the extending the 3’ end of the gRNA-targeting probe comprises extending the 3’ end of the gRNA-targeting probe using a reverse transcriptase having terminal deoxynucleotidyl transferase (TdT) activity to incorporate a sequence complementary to the spacer sequence and a non-templated 3’ terminal sequence. In some embodiments, the method comprises hybridizing the 3’ terminal sequence to the first barcoded oligonucleotide, and extending the 3’ end of the extended gRNA-targeting probe and / or extending the first barcoded oligonucleotide to generate the barcoded spacerAttorney Docket No.43487-1046601 oligonucleotide. In some embodiments, the gRNA-targeting probe comprises a 5’ overhang. In some embodiments, the 5’ overhang of the gRNA-targeting probe comprises a barcode sequence, optionally wherein the barcode sequence is a sample-specific barcode sequence. In some embodiments, the 5’ overhang of the gRNA-targeting probe comprises one or more functional sequences, optionally wherein the one or more functional sequences of the 5’ overhang of the gRNA-targeting probe comprise a primer hybridization sequence, a sequencing primer binding site, or complement thereof. In some embodiments, the gRNA-targeting probe hybridizes to a sequence in the gRNA that is at least 10bp, at least 20bp, at least 30bp, or at least 40bp away from the spacer sequence. In some embodiments, the gRNA-targeting probe hybridizes to a sequence in the constant region of the gRNA that is non-structured and / or that does not form a secondary structure of the scaffold sequence via base-pairing. In some embodiments, the first ligatable probe comprises a 3’ overhang and a 5’ hybridizing region that hybridizes to the target nucleic acid, and the second ligatable probe comprises a 5’ overhang and a 3’ hybridizing region that hybridizes to the target nucleic acid. In some embodiments, the ligated probe pair comprises a sequence that is complementary to and / or indicative of the target nucleic acid. In some embodiments, the barcoded analyte oligonucleotide comprises a sequence that is complementary to and / or indicative of the target nucleic acid. In some embodiments, the method comprises hybridizing a sequence of the 3’ overhang of the ligated probe pair to the second barcoded oligonucleotide, and extending the 3’ end of the ligated probe pair and / or extending the 3’ end of the second barcoded oligonucleotide to generate the barcoded analyte oligonucleotide. In some embodiments, the target nucleic acid is an mRNA. In some embodiments, the target nucleic acid is not a gRNA. In some embodiments, the method comprises removing unhybridized probes from the gRNA-expressing cell. In some embodiments, the method comprises performing one or more wash steps to remove the unhybridized probes. In some embodiments, the wash steps are performed prior to generating the partition. In some embodiments, the method further comprises: contacting the gRNA-expressing cell with a plurality of ligatable probe pairs that hybridize to a plurality of different target nucleic acids in the cell; ligating the plurality of ligatable probe pairs using the plurality of different target nucleic acids as templates to generate a plurality of ligated probe pairs; and using the plurality of ligated probe pairs and the plurality of barcoded oligonucleotides to generate a plurality of barcoded analyte oligonucleotides; wherein a barcoded analyte oligonucleotide of the plurality of barcoded analyte oligonucleotides comprises a sequence of a ligated probe pair of the plurality of ligated probe pairs or a complement thereof and a sequence of the partition-specific barcode or complement thereof. In some embodiments, a barcoded analyte oligonucleotide of the plurality of barcoded analyte oligonucleotides comprises a sequence of a target nucleic acid of the plurality of different target nucleic acids or aAttorney Docket No.43487-1046601 complement thereof and a sequence of the partition-specific barcode or complement thereof. In some embodiments, the method further comprises sequencing the plurality of barcoded analyte oligonucleotides or derivatives thereof. In some embodiments, the method further comprises analyzing the results of the sequencing to determine the presence and / or abundance of the different target nucleic acids in the gRNA-expressing cell.

[0006] In some aspects, provided herein is a method for analyzing a gRNA-expressing cell.In some embodiments, provided herein is a method comprising: providing a gRNA-expressing cell comprising a gRNA having a spacer sequence and a constant region comprising a scaffold sequence; contacting the gRNA-expressing cell with a gRNA-targeting probe that hybridizes to the constant region of the gRNA; generating a partition comprising 1) the gRNA-expressing cell, and 2) a plurality of barcoded oligonucleotides comprising a partition-specific barcode and a capture sequence; extending the 3’ end of the gRNA-targeting probe using a reverse transcriptase having terminal deoxynucleotidyl transferase (TdT) activity to incorporate a sequence complementary to the spacer sequence and a non-templated 3’ terminal sequence; hybridizing the 3’ terminal sequence to the capture sequence of a barcoded oligonucleotide of the plurality of barcoded oligonucleotides; and further extending the 3’ end of the gRNA-targeting probe using the barcoded oligonucleotide as template and / or extending the barcoded oligonucleotide using the extended gRNA-targeting probe as template, thereby generating a barcoded spacer oligonucleotide comprising the spacer sequence or complement thereof, and the partition- specific barcode or complement thereof. In some embodiments, the gRNA-targeting probe hybridizes to a sequence in the gRNA that is at least 10bp, at least 20bp, at least 30bp, or at least 40bp away from the spacer sequence. In some embodiments, the gRNA-targeting probe hybridizes to a sequence in the constant region of the gRNA that is non-structured and / or that does not form a secondary structure of the scaffold sequence via base-pairing. In some embodiments, the method further comprises sequencing the barcoded spacer oligonucleotide to determine the sequence of the spacer sequence and the partition-specific barcode, and associating the spacer sequence with the partition-specific barcode. In some embodiments, the gRNA- targeting probe comprises a 5’ overhang. In some embodiments, the 5’ overhang of the gRNA- targeting probe comprises a barcode sequence. In some embodiments, the 5’ overhang of the gRNA-targeting probe comprises a sample-specific barcode sequence. In some embodiments, the 5’ overhang of the gRNA-targeting probe comprises one or more functional sequences. In some embodiments, the one or more functional sequences of the 5’ overhang of the gRNA-targeting probe comprise a primer hybridization sequence, a sequencing primer binding site, or complement thereof. In some embodiments, the partition comprises the gRNA-expressing cell and no other cells.Attorney Docket No.43487-1046601

[0007] In some aspects, provided herein is a method for analyzing a gRNA-expressing cell.In some embodiments, provided herein is a method comprising: providing a gRNA-expressing cell comprising a gRNA having a spacer sequence and a constant region comprising a scaffold sequence; contacting the gRNA-expressing cell with a gRNA-targeting probe that hybridizes to the constant region of the gRNA; extending the 3’ end of the gRNA-targeting probe using a reverse transcriptase having terminal deoxynucleotidyl transferase (TdT) activity to incorporate a sequence complementary to the spacer sequence and a non-templated 3’ terminal sequence; hybridizing the 3’ terminal sequence to a template-switching oligonucleotide (TSO) and further extending the 3’ end of the gRNA-targeting probe to incorporate a sequence complementary to the TSO, thereby generating a TSO-tagged probe; generating a partition comprising 1) the gRNA-expressing cell, and 2) a plurality of barcoded oligonucleotides comprising a partition- specific barcode and a capture sequence; hybridizing the TSO-tagged probe to the capture sequence of a barcoded oligonucleotide of the plurality of barcoded oligonucleotides; and extending the TSO-tagged probe using the barcoded oligonucleotide as template and / or extending the barcoded oligonucleotide using the TSO-tagged probe as template, thereby generating a barcoded spacer oligonucleotide comprising the spacer sequence or complement thereof, and the partition-specific barcode or complement thereof. In some embodiments, the method further comprises sequencing the barcoded spacer oligonucleotide to determine the sequence of the spacer sequence and the partition-specific barcode, and associating the spacer sequence with the partition-specific barcode. In some embodiments, the TSO comprises a barcode sequence. In some embodiments, the TSO comprises a sample-specific barcode sequence. In some embodiments, the TSO comprises a capturing sequence, and the TSO-tagged probe comprises a complement of the capturing sequence. In some embodiments, the complement of the capturing sequence in the TSO-tagged probe hybridizes to the capture sequence of the barcoded oligonucleotide. In some embodiments, all or a portion of the TSO is dehybridized from the TSO-tagged probe. In some embodiments, all or a portion of the TSO is dehybridized from the TSO-tagged probe prior to hybridizing the TSO-tagged probe to the capture sequence of the barcoded oligonucleotide. In some embodiments, dehybridizing all or a portion of the TSO from the TSO-tagged probe comprises degrading the TSO. In some embodiments, degrading the TSO comprises contacting the TSO with an enzyme. In some embodiments, the TSO comprises ribonucleotides and dehybridizing all or a portion of the TSO from the TSO-tagged probe comprises contacting the TSO with Ribonuclease H (RNAse H) to digest the TSO. In some embodiments, the TSO comprises uracil residues and dehybridizing all or a portion of the TSO from the TSO-tagged probe comprises contacting the TSO with an enzyme to remove the uracil residues. In some embodiments, the enzyme is a Uracil-DNAAttorney Docket No.43487-1046601 Glycosylase (UDG) enzyme. In some embodiments, the enzyme is a uracil-specific excision reagent (USER) enzyme. In some embodiments, the TSO hybridized to the TSO-tagged probe is displaced by hybridization of the capture sequence of the barcoded oligonucleotide to the TSO- tagged probe. In some embodiments, the gRNA-targeting probe comprises a 5’ overhang. In some embodiments, the 5’ overhang of the gRNA-targeting probe comprises a barcode sequence. In some embodiments, the 5’ overhang of the gRNA-targeting probe comprises a sample-specific barcode sequence. In some embodiments, the 5’ overhang of the gRNA-targeting probe comprises one or more functional sequences. In some embodiments, the one or more functional sequences of the 5’ overhang of the gRNA-targeting probe comprise a primer hybridization sequence, a sequencing primer binding site, or complement thereof. In some embodiments, the gRNA-targeting probe hybridizes to a sequence in the gRNA that is at least 10bp, at least 20bp, at least 30bp, or at least 40bp away from the spacer sequence. In some embodiments, the gRNA- targeting probe hybridizes to a sequence in the constant region of the gRNA that is non- structured and / or that does not form a secondary structure of the scaffold sequence via base- pairing. In some embodiments, the partition comprises the gRNA-expressing cell and no other cells.

[0008] In some embodiments, provided herein is a method for analyzing a gRNA-expressingcell. In some embodiments, provided herein is a method comprising: providing a gRNA- expressing cell comprising a gRNA having a spacer sequence and a constant region comprising a scaffold sequence, wherein the gRNA comprises a 5’ monophosphate; contacting the gRNA- expressing cell with a gRNA ligation adapter comprising a functional region and a 3’ ligation end; ligating the 3’ ligation end of the gRNA ligation adapter to the gRNA, thereby generating a tagged gRNA comprising the functional region; generating a partition comprising 1) the gRNA- expressing cell, and 2) a plurality of barcoded oligonucleotides comprising a partition-specific barcode and a capture sequence; hybridizing the constant region of the tagged gRNA to the capture sequence of a barcoded oligonucleotide of the plurality of barcoded oligonucleotides; and extending the barcoded oligonucleotide using the tagged gRNA as template, thereby generating a barcoded spacer oligonucleotide comprising the partition-specific barcode, a sequence complementary to the spacer sequence, and a sequence complementary to the functional region. In some embodiments, the constant region of the gRNA comprises a capturing sequence, and wherein the constant region of the tagged gRNA is hybridized via the capturing sequence to the capture sequence of the barcoded oligonucleotide. In some embodiments, the capturing sequence is at the 3’ end of the constant region of the gRNA. In some embodiments, the capturing sequence is within and / or flanked by the scaffold sequence of the gRNA. In some embodiments, the capturing sequence is complementary to the capture sequence. In someAttorney Docket No.43487-1046601 embodiments, the method further comprises sequencing the barcoded spacer oligonucleotide to determine the sequence of the spacer sequence and the partition-specific barcode, and associating the spacer sequence with the partition-specific barcode.

[0009] In some embodiments, provided herein is a method for analyzing a gRNA-expressingcell. In some embodiments, provided herein is a method comprising: providing a gRNA- expressing cell comprising a gRNA having a spacer sequence and a constant region comprising a scaffold sequence, wherein the gRNA comprises a 5’ monophosphate; contacting the gRNA- expressing cell with a gRNA ligation adapter comprising a 3’ ligation end, and a functional region comprising a capturing sequence; ligating the 3’ end of the gRNA ligation adapter to the gRNA, thereby generating a tagged gRNA; contacting the tagged gRNA with a primer that hybridizes to the constant region of the gRNA, and extending the primer using the tagged gRNA as template, thereby generating a tagged gRNA complement that comprises a sequence complementary to the spacer sequence and a complement of the capturing sequence; generating a partition comprising 1) the gRNA-expressing cell, and 2) a plurality of barcoded oligonucleotides comprising a partition-specific barcode and a capture sequence; hybridizing the complement of the capturing sequence in the tagged gRNA complement to the capture sequence of a barcoded oligonucleotide of the plurality of barcoded oligonucleotides; and extending the barcoded oligonucleotide using the tagged gRNA complement as template and / or extending the tagged gRNA complement using the barcoded oligonucleotide as template, thereby generating a barcoded spacer oligonucleotide comprising the partition-specific barcode or a complement thereof, and the sequence of the spacer sequence or a complement thereof. In some embodiments, the primer that hybridizes to the constant region of the gRNA comprises a 5’ overhang. In some embodiments, the 5’ overhang of the primer that hybridizes to the constant region of the gRNA comprises a barcode sequence. In some embodiments, the 5’ overhang of the primer that hybridizes to the constant region of the gRNA comprises a sample-specific barcode sequence. In some embodiments, the 5’ overhang of the primer that hybridizes to the constant region of the gRNA comprises one or more functional sequences. In some embodiments, the one or more functional sequences of the 5’ overhang of the primer that hybridizes to the constant region of the gRNA comprise a primer hybridization sequence, a sequencing primer binding site, or complement thereof. In some embodiments, the method further comprises sequencing the barcoded spacer oligonucleotide to determine the sequence of the spacer sequence and the partition-specific barcode, and associating the spacer sequence with the partition-specific barcode. In some embodiments, the partition comprises the gRNA-expressing cell and no other cells.Attorney Docket No.43487-1046601

[0010] In some embodiments, the gRNA ligation adapter comprises the functional region; a5’ hybridizing region that hybridizes to the gRNA; and a self-hybridizing region, wherein the self-hybridizing region comprises a first sequence and second sequence that hybridize to one another, wherein the second sequence of the self-hybridizing region comprises the 3’ ligation end, and wherein the 3’ ligation end is configured to be ligated to the 5’ end of the gRNA upon hybridization of the 5’ hybridizing region to the gRNA.

[0011] In some embodiments, the gRNA ligation adapter comprises a first gRNA ligationadapter nucleic acid molecule and a second gRNA ligation adapter nucleic acid molecule. In some embodiments, the first gRNA ligation adapter nucleic acid molecule comprises the 5’ hybridizing region that hybridizes to the gRNA, and the first sequence of the self-hybridizing region; and the second gRNA ligation adapter nucleic acid molecule comprises the functional region and the second sequence of the self-hybridizing region comprising the 3’ ligation end.

[0012] In some embodiments, the gRNA ligation adapter is a single molecule gRNA ligationadapter. In some embodiments, the single molecule gRNA ligation adapter comprises in the 5’ to 3’ direction: the 5’ hybridizing region, the first sequence of the self-hybridizing region, the functional region, and the second sequence of the self-hybridizing region comprising the 3’ ligation end that is configured to be ligated to the 5’ end of the gRNA upon hybridization of the 5’ hybridizing region to the gRNA. In some embodiments, the single molecule gRNA ligation adapter has a stem-loop structure. In some embodiments, the functional region is in the loop of the stem-loop structure.

[0013] In some embodiments, the functional region comprises a barcode sequence. In someembodiments, the functional region comprises a sample-specific barcode sequence. In some embodiments, the functional region comprises one or more functional sequences. In some embodiments, the one or more functional sequences of the functional region comprise a primer hybridization sequence, a sequencing primer binding site, or complement thereof.

[0014] In some embodiments, the gRNA ligation adapter comprises a polymerase block sitethat is configured to terminate 3’ extension of a polynucleotide by a polymerase using the gRNA ligation adapter as template. In some embodiments, the polymerase block site is 5’ of the functional region and / or 3’ of the first sequence of the self-hybridizing region. In some embodiments, the polymerase block site comprises an abasic site. In some embodiments, the polymerase block site comprises uracil, and the uracil is removed to generate the abasic site. In some embodiments, the uracil is removed by contacting the uracil with a Uracil-DNA Glycosylase (UDG) enzyme or a Uracil-Specific Excision Reagent (USER) enzyme. In some embodiments, the polymerase block site terminates extension of the barcoded oligonucleotide using the tagged gRNA as template. In some embodiments, the polymerase block site is 5’ of theAttorney Docket No.43487-1046601 capturing sequence in the gRNA ligation adapter. In some embodiments, the polymerase block site terminates extension of the primer that hybridizes to the constant region of the gRNA during the generation of the tagged gRNA complement.

[0015] In some embodiments, the method comprises modifying a pre-modified gRNA togenerate the gRNA comprising the 5’ monophosphate. In some embodiments, the pre-modified gRNA comprises a 5’ triphosphate, and the method comprises modifying the 5’ triphosphate to generate the 5’ monophosphate. In some embodiments, the method comprises contacting the pre- modified gRNA with an enzyme to generate gRNA comprising the 5’ monophosphate. In some embodiments, the enzyme is RNA 5’ Pyrophosphohydrolase (RppH).

[0016] In some embodiments, the 5’ hybridizing region hybridizes to the spacer sequence ofthe gRNA. In some embodiments, the 5’ hybridizing region hybridizes to the constant region of the gRNA. In some embodiments, the 5’ hybridizing region hybridizes to the spacer sequence of the gRNA and the constant region of the gRNA. In some embodiments, the 5’ hybridizing region comprises a non-specific hybridization region. In some embodiments, the non-specific hybridization region comprises a sequence of residues capable of hybridizing to different spacer sequences. In some embodiments, the non-specific hybridization region comprises inosine residues. In some embodiments, the non-specific hybridization region comprises a sequence of inosine residues capable of hybridizing to different spacer sequences. In some embodiments, the 5’ hybridizing region comprises a sequence that is complementary to a portion of the constant region of the gRNA. In some embodiments, the sequence that is complementary to a portion of the constant region of the gRNA is at the 5’ end of the 5’ hybridizing region. In some embodiments, the 5’ hybridizing region comprises a non-hybridizing portion and a hybridizing portion. In some embodiments, the non-hybridizing portion comprises a carbon spacer. In some embodiments, the hybridizing portion hybridizes to at least a portion of the gRNA spacer and / or at least a portion of the constant region of the gRNA.

[0017] In some aspects, provided herein is a method for analyzing a gRNA-expressing cell.In some aspects, provided herein is a method comprising: providing a gRNA-expressing cell comprising a gRNA having a spacer sequence and a constant region comprising a scaffold sequence; contacting the gRNA-expressing cell with a gRNA ligation adapter comprising a capturing sequence and a 5’ ligation end; ligating the 5’ ligation end of the gRNA ligation adapter to the gRNA, thereby generating a tagged gRNA comprising the capturing sequence; generating a partition comprising 1) the gRNA-expressing cell, and 2) a plurality of barcoded oligonucleotides comprising a partition-specific barcode and a capture sequence; hybridizing the capturing sequence to the capture sequence of a barcoded oligonucleotide of the plurality of barcoded oligonucleotides; and using the barcoded oligonucleotide and the tagged gRNA toAttorney Docket No.43487-1046601 generate a barcoded spacer oligonucleotide comprising 1) the partition-specific barcode or a complement thereof, and 2) a sequence of the spacer or a complement thereof. In some embodiments, the method comprises extending the barcoded oligonucleotide using the tagged gRNA as template, thereby generating a barcoded spacer oligonucleotide comprising the partition-specific barcode and a sequence complementary to the spacer sequence. In some embodiments, the 5’ ligation end of the gRNA ligation adapter is ligated to the gRNA prior to generating the partition. In some embodiments, the 5’ ligation end of the gRNA ligation adapter is ligated to the gRNA after generating the partition. In some embodiments, the gRNA ligation adapter comprises: the capturing sequence; a 3’ hybridizing region that hybridizes to the gRNA; and a self-hybridizing region, wherein the self-hybridizing region comprises a first sequence and second sequence that hybridize to one another, wherein the second sequence of the self- hybridizing region comprises the 5’ ligation end, and wherein the 5’ ligation end is configured to be ligated to the 3’ end of the gRNA upon hybridization of the 3’ hybridizing region to the gRNA. In some embodiments, the gRNA ligation adapter comprises a first gRNA ligation adapter nucleic acid molecule and a second gRNA ligation adapter nucleic acid molecule. In some embodiments, the first gRNA ligation adapter nucleic acid molecule comprises the 3’ hybridizing region that hybridizes to the gRNA and the first sequence of the self-hybridizing region; and the second gRNA ligation adapter nucleic acid molecule comprises the capturing sequence and the second sequence of the self-hybridizing region comprising the 5’ ligation end. In some embodiments, the gRNA ligation adapter is a single molecule gRNA ligation adapter. In some embodiments, the single molecule gRNA ligation adapter comprises in the 3’ to 5’ direction: the 3’ hybridizing region, the first sequence of the self-hybridizing region, the capturing sequence, and the second sequence of the self-hybridizing region comprising the 5’ ligation end that is configured to be ligated to the 3’ end of the gRNA upon hybridization of the 3’ hybridizing region to the gRNA. In some embodiments, the single molecule gRNA ligation adapter has a stem-loop structure. In some embodiments, the capturing sequence is in the loop of the stem-loop structure. In some embodiments, the 5’ ligation end of the gRNA ligation adapter comprises a 5’ monophosphate. In some embodiments, the gRNA ligation adapter further comprises a sample-specific barcode sequence, and wherein the barcoded spacer oligonucleotide further comprises the sample-specific barcode sequence or a complement thereof. In some embodiments, the constant region of the gRNA further comprises a functional sequence. In some embodiments, the functional sequence is at the 5’ end of the constant region of the gRNA. In some embodiments, the functional sequence is within and / or flanked by the scaffold sequence of the gRNA. In some embodiments, the functional sequence comprises a primer hybridization sequence, a sequencing primer binding site, or a complement thereof. In some embodiments, theAttorney Docket No.43487-1046601 3’ hybridizing region hybridizes to the spacer sequence of the gRNA. In some embodiments, the 3’ hybridizing region hybridizes to the constant region of the gRNA. In some embodiments, the 3’ hybridizing region hybridizes to the spacer sequence of the gRNA and the constant region of the gRNA. In some embodiments, the 3’ hybridizing region comprises a non-specific hybridization region. In some embodiments, the non-specific hybridization region comprises a sequence of residues capable of hybridizing to different spacer sequences. In some embodiments, the non-specific hybridization region comprises inosine residues. In some embodiments, the non- specific hybridization region comprises a sequence of inosine residues capable of hybridizing to different spacer sequences. In some embodiments, the 3’ hybridizing region comprises a sequence that is complementary to a portion of the constant region of the gRNA. In some embodiments, the sequence that is complementary to a portion of the constant region of the gRNA is at the 3’ end of the 3’ hybridizing region. In some embodiments, the 3’ hybridizing region comprises a non-hybridizing portion and a hybridizing portion. In some embodiments, the non-hybridizing portion comprises a carbon spacer. In some embodiments, the hybridizing portion hybridizes to at least a portion of the gRNA spacer and / or at least a portion of the constant region of the gRNA. In some embodiments, the partition comprises the gRNA- expressing cell and no other cells.

[0018] In some embodiments, the method further comprises sequencing the barcoded spaceroligonucleotide or a derivative thereof. In some embodiments, the method comprises analyzing the results of the sequencing to determine the sequence of the spacer sequence. In some embodiments, the method comprises analyzing the results of the sequencing to determine the presence and / or abundance of the gRNA in the gRNA-expressing cell. In some embodiments, the partition comprises the gRNA-expressing cell and no other cells. In some embodiments, the method comprises removing unhybridized probes from the gRNA-expressing cell. In some embodiments, the method comprises performing one or more wash steps to remove unhybridized probes. In some embodiments, the method comprises performing one or more wash steps prior to generating the partition.

[0019] In some embodiments, the method further comprises: contacting the gRNA-expressing cell with a ligatable probe pair comprising a first ligatable probe and a second ligatable probe that hybridize to a target nucleic acid in the gRNA-expressing cell; ligating the first ligatable probe to the second ligatable probe using the target nucleic acid as template to generate a ligated probe pair; and using the ligated probe pair and a second barcoded oligonucleotide of the plurality of barcoded oligonucleotides to generate a barcoded analyte oligonucleotide comprising a sequence of the ligated probe pair or complement thereof, and the partition-specific barcode or a complement thereof. In some embodiments, the method comprisesAttorney Docket No.43487-1046601 sequencing the barcoded spacer oligonucleotide or a derivative thereof and the barcoded analyte oligonucleotide or a derivative thereof. In some embodiments, the method comprises analyzing the results of the sequencing to determine the sequence of the spacer sequence. In some embodiments, the method comprises analyzing the results of the sequencing to determine the presence and / or abundance of the gRNA and / or the target nucleic acid in the gRNA-expressing cell. In some embodiments, the first ligatable probe comprises a 3’ overhang and a 5’ hybridizing region that hybridizes to the target nucleic acid, and the second ligatable probe comprises a 5’ overhang and a 3’ hybridizing region that hybridizes to the target nucleic acid. In some embodiments, the ligated probe pair comprises a sequence that is complementary to and / or indicative of the target nucleic acid. In some embodiments, the barcoded analyte oligonucleotide comprises a sequence that is complementary to and / or indicative of the target nucleic acid. In some embodiments, the method comprises hybridizing a sequence of the 3’ overhang of the ligated probe pair to the second barcoded oligonucleotide, and extending the 3’ end of the ligated probe pair and / or extending the 3’ end of the barcoded oligonucleotide to generate the barcoded analyte oligonucleotide. In some embodiments, the target nucleic acid is not a gRNA. In some embodiments, the target nucleic acid is an mRNA.

[0020] In some embodiments, the method further comprises: contacting the gRNA-expressing cell with a plurality of ligatable probe pairs that hybridize to a plurality of different target nucleic acids in the cell; ligating the plurality of ligatable probe pairs using the plurality of different target nucleic acids as templates to generate a plurality of ligated probe pairs; and using the plurality of ligated probe pairs and the plurality of barcoded oligonucleotides to generate a plurality of barcoded analyte oligonucleotides; wherein a barcoded analyte oligonucleotide of the plurality of barcoded analyte oligonucleotides comprises a sequence of a ligated probe pair of the plurality of ligated probe pairs or a complement thereof and a sequence of the partition-specific barcode or complement thereof. In some embodiments, a barcoded analyte oligonucleotide of the plurality of barcoded analyte oligonucleotides comprises a sequence of a target nucleic acid of the plurality of different target nucleic acids or a complement thereof and a sequence of the partition-specific barcode or complement thereof. In some embodiments, the method further comprises sequencing the plurality of barcoded analyte oligonucleotides or derivatives thereof. In some embodiments, the method further comprises analyzing the results of the sequencing to determine the presence and / or abundance of the different target nucleic acids in the gRNA- expressing cell.

[0021] In some embodiments, the method is performed in parallel for a plurality of gRNA-expressing cells, wherein different partitions are generated for different gRNA-expressing cells of the plurality of gRNA-expressing cells, and wherein barcoded spacer oligonucleotidesAttorney Docket No.43487-1046601 comprising partition-specific barcodes are generated from the different gRNA-expressing cells. In some embodiments, barcoded analyte oligonucleotides are generated from the different gRNA-expressing cells. In some embodiments, the method comprises sequencing the barcoded spacer oligonucleotides or derivatives thereof and / or the barcoded analyte oligonucleotides or derivatives thereof. In some embodiments, the method comprises analyzing the results of the sequencing to determine the presence and / or abundance of one or more gRNAs and one or more target nucleic acids in the different gRNA-expressing cells of the plurality of gRNA-expressing cells.

[0022] In some embodiments, the method further comprises: contacting the gRNA-expressing cell with a ligatable probe pair comprising 1) a first ligatable probe having a 3’ overhang, and a 5’ hybridizing region that hybridizes to a target nucleic acid in the cell, and 2) a second ligatable probe having a 3’ hybridizing region that hybridizes to the target nucleic acid in the cell, and a 5’ overhang; ligating the 5’ hybridizing region of the first ligatable probe to the 3’ hybridizing region of the second ligatable probe using the target nucleic acid as template, thereby generating a ligated probe pair comprising a sequence complementary to and / or indicative of the target nucleic acid; hybridizing a sequence of the 3’ overhang to the capture sequence of a barcoded oligonucleotide of the plurality of barcoded oligonucleotides in the partition; extending the 3’ end of the ligated probe pair to incorporate a sequence complementary to the barcoded oligonucleotide and / or extending the 3’ end of the barcoded oligonucleotide to incorporate a sequence complementary to the ligated probe pair, thereby generating a barcoded analyte oligonucleotide comprising: the sequence of the ligated probe pair or complement thereof, and the sequence of the barcoded capture oligonucleotide or complement thereof. In some embodiments, the method further comprises sequencing the barcoded analyte oligonucleotide to determine the sequence complementary to and / or indicative of the target nucleic acid and the sequence of the partition-specific barcode, and associating the target nucleic acid with the partition-specific barcode. In some embodiments, the 3’ overhang of the first ligatable probe and / or the 5’ overhang of the second ligatable probe comprise a barcode sequence. In some embodiments, the 3’ overhang of the first ligatable probe and / or the 5’ overhang of the second ligatable probe comprise a sample-specific barcode sequence. In some embodiments, the 3’ overhang of the first ligatable probe and / or the 5’ overhang of the second ligatable probe comprise one or more functional sequences. In some embodiments, the one or more functional sequences of the 3’ overhang of the first ligatable probe and / or the 5’ overhang of the second ligatable probe comprise a primer hybridization sequence, a sequencing primer binding site, or complement thereof. In some embodiments, the first ligatable probe is ligated to the second ligatable probe in the partition. In some embodiments, the first ligatable probe is ligated to theAttorney Docket No.43487-1046601 second ligatable probe prior to generating the partition. In some embodiments, the plurality of barcoded oligonucleotides comprise one or more functional sequences.

[0023] In some embodiments, the one or more functional sequences of the plurality ofbarcoded oligonucleotides comprise a primer hybridization sequence, a sequencing primer binding site, or complement thereof. In some embodiments, each barcoded oligonucleotide of the plurality of barcoded oligonucleotides comprises a unique molecular identifier (UMI) sequence.

[0024] In some embodiments, the method comprises sequencing the barcoded analyteoligonucleotide and the barcoded spacer oligonucleotide, thereby determining the presence of the target analyte and the presence of the gRNA having the spacer sequence in the same cell. In some embodiments, the barcoded spacer oligonucleotide and barcoded analyte oligonucleotide are amplified and / or sequenced outside of the partition.

[0025] In some embodiments, the method is performed in parallel for a plurality of gRNA-expressing cells, wherein different partitions are generated for different gRNA-expressing cells of the plurality of gRNA-expressing cells, and wherein barcoded spacer oligonucleotides comprising partition-specific barcodes are generated from the different gRNA-expressing cells. In some embodiments, barcoded analyte oligonucleotides comprising partition-specific barcodes are generated from the different gRNA-expressing cells. In some embodiments, the method comprises sequencing the one or more barcoded spacer oligonucleotides and / or the one or more barcoded analyte oligonucleotides from the different gRNA-expressing cells. In some embodiments, for the gRNA expressing cells, the presence and / or abundance of one or more gRNA spacer sequences is determined. In some embodiments, for the gRNA expressing cells, the presence and / or abundance of one or more target nucleic acids is determined.

[0026] The present disclosure provides methods for use in sample processing and analysis.The methods provided herein may involve hybridizing a probe to a molecule of interest (e.g., target protein, target nucleic acid molecule) and processing the probe-molecule complex. Such processing can include barcoding the probe, the probe-molecule complex, or the molecule, and / or performing a nucleic acid reaction. The probe may comprise a nucleic acid molecule, and further processing can include extension, denaturation, and amplification processes to provide nucleic acid molecules comprising a sequence the same or substantially the same as or complementary to that of a target region of a nucleic acid molecule of interest (e.g., target nucleic acid molecule). A method may comprise hybridizing a first probe and a second probe to first and second target regions of the nucleic acid molecule, linking the first and second probes to provide a probe-linked nucleic acid molecule, and barcoding the probe-linked nucleic acid molecule. A method may comprise hybridizing a first probe to a first target region of a nucleic acid molecule, barcoding the probe, and hybridizing a second probe to a second target region ofAttorney Docket No.43487-1046601 the nucleic acid molecule to generate a barcoded, probe-linked nucleic acid molecule. In some aspects, the method may comprise hybridizing a probe to a nucleic acid molecule attached to a feature-binding moiety to provide a probe-binding moiety complex and barcoding the probe. One or more processes of the methods provided herein may be performed within a partition such as a droplet or well. The methods of the present disclosure be useful, for example, in controlled analysis and processing of analytes such as biological particles, nucleic acids, and proteins. One or more of the methods described herein may allow for genomic, transcriptomic, or exomic profiling with high sensitivity, for example in comparison to certain other methods. The methods of the present disclosure may be useful in detecting variants and characterizing nucleic acid molecules, e.g., for assessment of single nucleotide polymorphisms (SNPs), alternative splice junctions, insertions, deletions, V(D)J rearrangements, etc. The methods of the present disclosure may be useful for multiplexed analysis of nucleic acids and proteins while minimizing reagent usage, e.g., by decreasing the number of unoccupied partitions for analysis.

[0027] Another aspect of the present disclosure provides a non-transitory computer readablemedium comprising machine executable code that, upon execution by one or more computer processors, implements any of the methods above or elsewhere herein.

[0028] Another aspect of the present disclosure provides a system comprising one or morecomputer processors and computer memory coupled thereto. The computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods above or elsewhere herein.

[0029] Additional aspects and advantages of the present disclosure will become readilyapparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive. INCORPORATION BY REFERENCE

[0030] All publications, patents, and patent applications mentioned in this specification areherein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.Attorney Docket No.43487-1046601 BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The drawings illustrate certain features and advantages of this disclosure. Theseembodiments are not intended to limit the scope of the appended claims in any manner. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which principles of the disclosure are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0032] FIG. 1 shows an example of a microfluidic channel structure for partitioningindividual biological particles.

[0033] FIG. 2 shows an example of a microfluidic channel structure for the controlledpartitioning of beads into discrete droplets.

[0034] FIG. 3 illustrates an example of a barcode carrying bead.

[0035] FIG. 4 illustrates another example of a barcode carrying bead.

[0036] FIG. 5 schematically illustrates an example microwell array.

[0037] FIG. 6 schematically illustrates an example workflow for processing nucleic acidmolecules.

[0038] FIG. 7 schematically illustrates another example workflow for processing nucleicacid molecules.

[0039] FIG. 8 schematically illustrates another example workflow for processing nucleicacid molecules.

[0040] FIG. 9 schematically illustrates another example workflow for processing nucleicacid molecules.

[0041] FIG. 10 schematically illustrates an example workflow for analyzing cells, nuclei orcell beads.

[0042] FIG. 11 schematically illustrates example labelling agents with nucleic acidmolecules attached thereto.

[0043] FIG. 12A schematically shows an example of labelling agents. FIG. 12Bschematically shows another example workflow for processing nucleic acid molecules. FIG. 12C schematically shows another example workflow for processing nucleic acid molecules.

[0044] FIG. 13 schematically shows another example of a barcode-carrying bead.

[0045] FIG. 14 shows a computer system that is programmed or otherwise configured toimplement methods provided herein.

[0046] FIG. 15 shows an example processed nucleic acid molecule described herein.

[0047] FIG. 16A shows an example workflow for processing multiple analytes in a partition.Attorney Docket No.43487-1046601

[0048] FIG. 16B shows another example workflow for processing multiple analytes in apartition.

[0049] FIG. 17 schematically shows a feature-binding group described herein.

[0050] FIG. 18 shows example data from a workflow described herein.

[0051] FIG. 19 shows additional example data from a workflow described herein.

[0052] FIG. 20 shows additional example data from a workflow described herein.

[0053] FIG. 21A shows example data comparing fixed cells and unfixed cells. FIG. 21Bshows additional example data comparing fixed cells and unfixed cells. FIG.21C shows additional example data comparing fixed cells and unfixed cells.

[0054] FIG. 22 schematically shows an example workflow for assaying two different analytetypes.

[0055] FIG. 23 shows example data of a barcoding approach described herein.

[0056] FIG. 24 shows example data of different analyte types using the barcodingapproaches described herein.

[0057] FIG. 25 schematically shows an example method for processing nucleic acidmolecules.

[0058] FIG. 26 shows another example method for processing nucleic acid molecules.

[0059] FIG. 27 shows an example workflow for generating probe-linked nucleic acidmolecules.

[0060] FIG. 28 shows another example workflow for generating probe-linked nucleic acidmolecules.

[0061] FIG. 29 shows an example workflow for processing cells according to the methodsdescribed herein.

[0062] FIG. 30A shows example protein expression data resulting from barcoding ofmultiple analytes using different sample preparation parameters. FIG.30B shows additional protein expression data resulting from barcoding of multiple analytes using different sample preparation parameters.

[0063] FIG. 31 shows example gene expression data resulting from barcoding of multipleanalytes using different sample preparation parameters.

[0064] FIGs. 32A-C shows example data of multiple analyte probing for a negative controlgroup. FIG.32A shows example data showing different immune cell clusters. FIG.32B shows example data of gene expression of GZMB gene. FIG.32C shows example data of protein expression resulting from antibody staining.

[0065] FIGs. 33A-C shows example data of multiple analyte probing for an experimentalgroup. FIG.33A shows example data showing different immune cell clusters. FIG.33B showsAttorney Docket No.43487-1046601 example data of gene expression of GZMB gene. FIG.33C shows example data of protein expression resulting from antibody staining.

[0066] FIGs. 34A-C shows example data of multiple analyte probing for an experimentalgroup. FIG.34A shows example data showing different immune cell clusters. FIG.34B shows example data of gene expression of GZMB gene. FIG.34C shows example data of protein expression resulting from antibody staining.

[0067] FIGs. 35A-C shows example data of multiple analyte probing for an experimentalgroup. FIG.35A shows example data showing different immune cell clusters. FIG.35B shows example data of gene expression of GZMB gene. FIG.35C shows example data of protein expression resulting from antibody staining.

[0068] FIGs. 36A-C shows example data of multiple analyte probing for an experimentalgroup. FIG.35A shows example data showing different immune cell clusters. FIG.36B shows example data of gene expression of GZMB gene. FIG.36C shows example data of protein expression resulting from antibody staining.

[0069] FIGs. 37A-C shows example data of multiple analyte probing for an experimentalgroup. FIG.37A shows example data showing different immune cell clusters. FIG.37B shows example data of gene expression of GZMB gene. FIG.37C shows example data of protein expression resulting from antibody staining.

[0070] FIG. 38 shows another example workflow for assaying two different analyte types.

[0071] FIG. 39 shows an exemplary workflow for sequencing gRNAs and analytes (e.g.cellular transcripts) from the same single cells.

[0072] FIG. 40 shows an exemplary analyte sequencing workflow that is compatible and canbe performed in parallel with gRNA sequencing workflows described herein to achieve transcript and gRNA sequencing in the same single cells.

[0073] FIG. 41 shows an exemplary workflow for gRNA sequencing using a gRNA-targeting probe.

[0074] FIG. 42 shows an exemplary workflow for gRNA sequencing using a gRNA-targeting probe with template-switching.

[0075] FIGS. 43A-C show exemplary workflows for gRNA sequencing using a gRNAligation adapter. FIG.43A shows an exemplary embodiment of the workflow in which the gRNA includes a capturing sequence. FIG.43B shows an exemplary embodiment in which the gRNA ligation adapter includes a capturing sequence. FIG.43C shows an exemplary embodiment of a gRNA ligation adapter for sequencing a gRNA having a 3’ spacer.Attorney Docket No.43487-1046601

[0076] FIG. 44 shows exemplary data of transcriptome sequencing library and the gRNAsequencing library yields resulting from a combined gRNA and transcriptome single-cell sequencing workflow as described in Example 2.

[0077] FIG. 45 shows exemplary data showing key metrics of transcriptome sequencingresults from a combined gRNA and transcriptome single-cell sequencing workflow as described in Example 2.

[0078] FIG. 46 shows exemplary data showing key metrics of gRNA sequencing resultsfrom a combined gRNA and transcriptome single-cell sequencing workflow as described in Example 2.

[0079] FIG. 47 shows exemplary data showing gRNA sequencing efficiency resulting fromuse of two different gRNA-targeting probes in a combined gRNA and transcriptome single-cell sequencing workflow as described in Example 2. DETAILED DESCRIPTION

[0080] While various embodiments of the invention have been shown and described herein,it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed. OVERVIEW

[0081] Large-scale screens using CRISPR / Cas systems to identify how specific genetic andepigenetic perturbations affect cellular phenotypes such as gene expression have the potential to provide transformative insights for biology and disease. However, the ability to gain insight from such screens is limited by the quality and resolution of data that is acquired.

[0082] In certain CRISPR / Cas screening strategies, cells are transduced with a library ofgRNAs that complex with a Cas protein to target different loci and mediate a genetic or epigenetic effect. Subsequently, cells are selected or enriched in bulk for a particular phenotype (e.g. using flow cytometry for expression of a marker), and the cells with the particular phenotype are sequenced to identify gRNAs that are enriched, thereby revealing specific targets and / or perturbations that affect the particular phenotype. These and other methods for leveraging CRISPR / Cas screening strategies are inherently limited in their ability to facilitate discovery of new or unexpected phenotypes, and to probe CRISPR / Cas-mediated perturbations in an unbiased manner.

[0083] A potentially much more powerful approach for generating biological insights can beachieved with the ability to analyze a large number of cells at the single cell level in order toAttorney Docket No.43487-1046601 associate the expression of specific gRNAs with specific phenotypes, such as gene expression, in an unbiased manner. For example, the ability to perform both transcriptome sequencing and gRNA sequencing at the single cell level, and in the same single cells, in the context of a large- scale CRISPR / Cas screen can allow insight into how various different genes or pathways affected by different gRNAs contribute to any number of different phenotypes, all within a single experiment. However, there is a paucity of available methods available that facilitate such an approach. The methods provided herein facilitate such approaches, and address these and other challenges.

[0084] In some aspects, provided herein are methods for sequencing gRNAs. In someaspects, the methods for sequencing gRNAs are compatible and can be performed in parallel with single-cell analysis and / or single-cell sequencing assays (e.g. single-cell transcriptome sequencing assays), such as any described herein, thereby facilitating powerful insights, for example as described above in combination with CRISPR / Cas perturbations and screens. In some aspects, the methods can be performed in a variety of tissue types. In some aspects, the methods can be performed in fixed cells, thereby providing increased power to analyze a large number of samples collected at any number of time points. The methods may also be performed in suitable alternatives to cells, such as cell nuclei (e.g. for analysis of a gRNA-expressing nucleus, which may be a nucleus of a gRNA-expressing cell).

[0085] In some aspects, the methods provided herein are scalable approaches for sequencinga large number of gRNAs having different spacer sequences (e.g. from a gRNA library). The methods are scalable because they facilitate detection of any number of different gRNA spacer sequences present in a sample without any required change in the workflow or the number of provided gRNA-targeting probes. For example, the methods can facilitate sequencing a large number (e.g. a plurality) of different gRNAs using a single gRNA-targeting probe that hybridizes to a constant region of the gRNAs, such as a scaffold sequence (e.g. as described in detail herein and as illustrated in FIG.41 and FIG.42). The methods can also facilitate sequencing a large number (e.g. a plurality) of different gRNAs using a single gRNA ligation adapter capable of hybridizing to gRNAs having different spacer sequences (e.g. as described in detail herein and as illustrated in FIGS.43A-C). In some aspects, this inherent scalability is in contrast to certain other methods of gRNA sequencing in which an increase in the number of different gRNAs having different spacer sequences requires a corresponding increase in the number of gRNA- targeting probes (e.g. gRNA-targeting probes that hybridize to specific gRNA spacer sequences). Thus, the scalability of the methods provided herein can provide increased flexibility to assay various customized gRNA libraries, and can reduce costs in comparison to methods that require generating new reagents and probes for screening different gRNA libraries.Attorney Docket No.43487-1046601

[0086] In some aspects, provided herein are methods for analyzing gRNA-expressing cells.In some aspects, a method described herein can be described with reference to a single gRNA in a single cell (e.g. a single gRNA-expressing cell). However, it is to be understood that for all such described methods, it is envisioned that the method can be performed in parallel for a plurality of gRNAs expressed in a plurality of single cells. For example, for each individual cell of a plurality of cells, the methods can be employed to sequence a plurality of gRNAs therein. Similarly, the methods can be employed to sequence a plurality of gRNAs and a plurality of analytes (e.g. cellular transcripts) in each of a plurality of cells, at the single-cell level. In some embodiments, the methods can be employed to sequence a plurality of gRNAs in a plurality of single cells. Similarly, the methods can be employed to sequence a plurality of gRNAs and a plurality of analytes (e.g. cellular transcripts, such as mRNAs) in a plurality of cells, at the single-cell level.

[0087] Provided herein are methods for sample processing and / or analysis. A method of thepresent disclosure may comprise barcoding one or more types of biomolecules (e.g., a nucleic acid molecule, a protein, a lipid, a carbohydrate, or a combination thereof). The biomolecule may be, for instance, a nucleic acid molecule (e.g., a ribonucleic acid (RNA) molecule) or a protein. Such a method may involve attaching one or more probes (e.g., nucleic acid probes) to the biomolecules and subsequently attaching a nucleic acid barcode molecule comprising a barcode sequence to the one or more probes. For example, the nucleic acid barcode molecule may attach to an overhanging sequence of a probe or to the end of a probe. Extension from an end of the probe to an end of the nucleic acid barcode molecule may form an extended nucleic acid molecule comprising both a sequence complementary to the barcode sequence and a sequence complementary to a target region of the nucleic acid molecule. The extended nucleic acid molecule may then be denatured from the nucleic acid barcode molecule and the nucleic acid molecule may be duplicated. One or more processes of the method may be carried out within a partition such as a droplet or well.

[0088] The present disclosure also provides a method of processing a sample (e.g., a cellsample or a tissue sample) that provides a barcoded nucleic acid molecule having linked probe molecules attached thereto. The method may comprise providing a sample comprising a nucleic acid molecule (e.g., an RNA molecule) having a first and second target region; a first probe having a (i) first probe sequence that is complementary to the first target region and (ii) an additional probe sequence; and a second probe having a second probe sequence that is complementary to the second target region. In some instances, the first target region and the second target region are adjacent. The first and second probe sequences may also comprise first and second reactive moieties, respectively. Upon hybridization of the first probe sequence of theAttorney Docket No.43487-1046601 first probe to the first target region of the nucleic acid molecule, and hybridization of the second probe sequence of the second probe to the second target region of the nucleic acid molecule, the reactive moieties may be adjacent to one another. Subsequent reaction between the adjacent reactive moieties under sufficient conditions may link the first and second probes to yield a probe-linked nucleic acid molecule. The probe-linked nucleic acid molecule may also be referred to as a probe-ligated nucleic acid molecule. In other instances, the first target region and the second target region are not adjacent, and a nucleic acid reaction (e.g., a nucleic acid extension reaction, a gap-filling reaction) may be performed to yield a probe-linked nucleic acid molecule.

[0089] The probe-linked nucleic acid molecule may be barcoded with a barcode sequence ofa nucleic acid barcode molecule to provide a barcoded probe-linked nucleic acid molecule. Barcoding may be achieved by hybridizing a binding sequence of the nucleic acid barcode molecule to the additional probe sequence of the first probe of the probe-linked nucleic acid molecule. The barcoded probe linked-nucleic acid molecule may be subjected to amplification reactions to yield an amplified product comprising the first and second target regions and the barcode sequence or sequences complementary to these sequences. Accordingly, the method may provide amplified products without the use of reverse transcription. One or more processes may be performed within a partition such as a droplet or well.

[0090] The present disclosure also provides a method of generating barcoded, probe-linkednucleic acid molecules. The method may comprise providing a sample comprising a nucleic acid molecule (e.g., an RNA molecule) having a first target region and a second target region; a first probe having a first probe sequence that is complementary to the first target region and optionally an additional probe sequence; and a second probe having a second probe sequence that is complementary to the second target region. The additional probe sequence of the first probe may comprise a probe capture sequence. Alternatively or in addition to, the second probe may comprise a probe capture sequence. The first probe sequence of the first probe may hybridize to the first target region of the nucleic acid molecule, generating a probe-associated nucleic acid molecule, and a nucleic acid reaction (e.g., a nucleic acid extension reaction using a polymerase or reverse transcriptase) may be performed to generate an extended nucleic acid molecule comprising a sequence complementary to the second target region. Prior to, during, or subsequent to the nucleic acid extension reaction, the second probe may hybridize to the nucleic acid molecule (or extended nucleic acid molecule, or complement thereof), and optionally, a nucleic acid extension reaction may be performed. The extended nucleic acid molecule may be barcoded, such as by (a) hybridization of a barcode binding sequence of the nucleic acid barcode molecule to the first probe (e.g., the additional probe sequence of the first probe) or the secondAttorney Docket No.43487-1046601 probe (e.g., a probe capture sequence of the second probe), or (b) via a probe binding molecule (also referred to herein as a “splint molecule” or “splint oligonucleotide”), in which the probe binding molecule comprises (i) a probe binding sequence complementary to the additional probe sequence of the first probe (which may comprise the probe capture sequence) and / or a capture sequence of the second probe and a (ii) barcode binding sequence complementary to a sequence (e.g., a common sequence) of the barcode molecule. In some instances, the barcoding may be performed prior to hybridization of the second probe to the second target region. In such cases, the barcoded nucleic acid molecule may be subjected to conditions sufficient for hybridization of the second probe sequence of the second probe to the second target region of the nucleic acid molecule (or barcoded nucleic acid molecule). A nucleic acid reaction (e.g., nucleic acid extension) may be performed, thereby generating a barcoded, probe-linked nucleic acid molecule.

[0091] Another aspect of the present disclosure provides a method of barcoding multipleanalytes, such as the probe-linked nucleic acid molecules described herein, as well as other types of biomolecules (e.g., proteins). The method may comprise providing (i) a sample comprising a nucleic acid molecule (e.g., an RNA molecule) having first and second target regions and (ii) a feature-binding moiety comprising a reporter oligonucleotide comprising a capture sequence; (iii) a first probe having a first probe sequence that is complementary to the first target region and an additional probe sequence; (iv) a second probe having a second probe sequence that is complementary to the second target region; and (v) a third probe having a third probe sequence that is complementary to a sequence of the reporter oligonucleotide. The first probe and the second probe may be subjected to conditions sufficient to hybridize to the first target region and the second target region, respectively, and to generate a probe-linked nucleic acid molecule. The third probe sequence of the third probe may be subjected to conditions sufficient to hybridize to the capture sequence of the reporter oligonucleotide, generating a probe-binding moiety complex. The probe-linked nucleic acid molecule and the probe-binding moiety complex may be subjected to conditions sufficient for barcoding, thereby generating a barcoded probe-linked nucleic acid molecule and a barcoded probe-binding moiety complex. The barcoded probe- linked molecule may be subjected to amplification reactions to yield an amplified product comprising the first and second target regions and the barcode sequence or sequences complementary to these sequences. The barcoded probe-binding moiety complex may similarly be subjected to amplification reactions to yield an amplified product comprising the fourth probe sequence and the barcode sequence. One or more processes may be performed within a cell bead and / or a partition, such as a droplet or well. Beneficially, the methods described herein may be useful in indexing cells, nuclei, or cell beads to partitions; such indexing may be useful inAttorney Docket No.43487-1046601 partitions occupied by more than one cell and identifying the cell, nucleus, cell bead or partition from which an analyte was derived. TERMINOLOGY

[0092] Where values are described as ranges, it will be understood that such disclosureincludes the disclosure of all possible sub-ranges within such ranges, as well as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated.

[0093] The terms “a,” “an,” and “the,” as used herein, generally refers to singular and pluralreferences unless the context clearly dictates otherwise.

[0094] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedesthe first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0095] Whenever the term “no more than,” “less than,” or “less than or equal to” precedesthe first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0096] The term “barcode,” as used herein, generally refers to a label, or identifier, thatconveys or is capable of conveying information about an analyte. A barcode can be part of an analyte. A barcode can be independent of an analyte. A barcode can be a tag attached to an analyte (e.g., nucleic acid molecule) or a combination of the tag in addition to an endogenous characteristic of the analyte (e.g., size of the analyte or end sequence(s)). A barcode may be unique. Barcodes can have a variety of different formats. For example, barcodes can include: polynucleotide barcodes; random nucleic acid and / or amino acid sequences; and synthetic nucleic acid and / or amino acid sequences. A barcode can be attached to an analyte in a reversible or irreversible manner. A barcode can be added to, for example, a fragment of a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before, during, and / or after sequencing of the sample. Barcodes can allow for identification and / or quantification of individual sequencing-reads.

[0097] The term “real time,” as used herein, can refer to a response time of less than about 1second, a tenth of a second, a hundredth of a second, a millisecond, or less. The response timeAttorney Docket No.43487-1046601 may be greater than 1 second. In some instances, real time can refer to simultaneous or substantially simultaneous processing, detection or identification.

[0098] The term “subject,” as used herein, generally refers to an animal, such as a mammal(e.g., human, mouse, rat) or avian (e.g., bird), or other organism, such as a plant. For example, the subject can be a vertebrate, such as a mammal, a rodent (e.g., a mouse), a primate, a simian or a human. Animals may include, but are not limited to, farm animals, sport animals, and pets. A subject can be a healthy or asymptomatic individual, an individual that has or is suspected of having a disease (e.g., cancer) or a pre-disposition to the disease, and / or an individual that is in need of therapy or suspected of needing therapy. A subject can be a patient. A subject can be a microorganism or microbe (e.g., bacteria, fungi, archaea, viruses).

[0099] The term “genome,” as used herein, generally refers to genomic information from asubject, which may be, for example, at least a portion or an entirety of a subject’s hereditary information. A genome can be encoded either in DNA or in RNA. A genome can comprise coding regions (e.g., that code for proteins) as well as non-coding regions. A genome can include the sequence of all chromosomes together in an organism. For example, the human genome ordinarily has a total of 46 chromosomes. The sequence of all of these together may constitute a human genome.

[0100] The terms “adaptor(s)”, “adapter(s)” and “tag(s)” may be used synonymously. Anadaptor or tag can be coupled to a polynucleotide sequence to be “tagged” by any approach, including ligation, hybridization, or other approaches.

[0101] The term “sequencing,” as used herein, generally refers to methods and technologiesfor determining the sequence of nucleotide bases in one or more polynucleotides. The polynucleotides can be, for example, nucleic acid molecules such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), including variants or derivatives thereof (e.g., single stranded DNA). Sequencing can be performed by various systems currently available, such as, without limitation, a sequencing system by Illumina®, Pacific Biosciences (PacBio®), Oxford Nanopore®, or Life Technologies (Ion Torrent®). Alternatively or in addition, sequencing may be performed using nucleic acid amplification, polymerase chain reaction (PCR) (e.g., digital PCR, quantitative PCR, or real time PCR), or isothermal amplification. Such systems may provide a plurality of raw genetic data corresponding to the genetic information of a subject (e.g., human), as generated by the systems from a sample provided by the subject. In some examples, such systems provide sequencing reads (also “reads” herein). A read may include a string of nucleic acid bases corresponding to a sequence of a nucleic acid molecule that has been sequenced. In some situations, systems and methods provided herein may be used with proteomic information.Attorney Docket No.43487-1046601

[0102] The term “bead,” as used herein, generally refers to a particle. The bead may be asolid or semi-solid particle. The bead may be a gel bead. The gel bead may include a polymer matrix (e.g., matrix formed by polymerization or cross-linking). The polymer matrix may include one or more polymers (e.g., polymers having different functional groups or repeat units). Polymers in the polymer matrix may be randomly arranged, such as in random copolymers, and / or have ordered structures, such as in block copolymers. Cross-linking can be via covalent, ionic, or inductive, interactions, or physical entanglement. The bead may be a macromolecule. The bead may be formed of nucleic acid molecules bound together. The bead may be formed via covalent or non-covalent assembly of molecules (e.g., macromolecules), such as monomers or polymers. Such polymers or monomers may be natural or synthetic. Such polymers or monomers may be or include, for example, nucleic acid molecules (e.g., DNA or RNA). The bead may be formed of a polymeric material. The bead may be magnetic or non-magnetic. The bead may be rigid. The bead may be flexible and / or compressible. The bead may be disruptable or dissolvable. The bead may be a solid particle (e.g., a metal-based particle including but not limited to iron oxide, gold or silver) covered with a coating comprising one or more polymers. Such coating may be disruptable or dissolvable.

[0103] As used herein, the term “barcoded nucleic acid molecule” generally refers to anucleic acid molecule that results from, for example, the processing of a nucleic acid barcode molecule with a nucleic acid sequence (e.g., nucleic acid sequence complementary to a nucleic acid primer sequence encompassed by the nucleic acid barcode molecule). The nucleic acid sequence may be a targeted sequence or a non-targeted sequence. For example, in the methods and systems described herein, hybridization and reverse transcription of a nucleic acid molecule (e.g., a messenger RNA (mRNA) molecule) of a cell or nucleus with a nucleic acid barcode molecule (e.g., a nucleic acid barcode molecule containing a barcode sequence and a nucleic acid primer sequence complementary to a nucleic acid sequence of the mRNA molecule) results in a barcoded nucleic acid molecule that has a sequence corresponding to the nucleic acid sequence of the mRNA and the barcode sequence (or a reverse complement thereof). A barcoded nucleic acid molecule may serve as a template, such as a template polynucleotide, that can be further processed (e.g., amplified) and sequenced to obtain the target nucleic acid sequence. For example, in the methods and systems described herein, a barcoded nucleic acid molecule may be further processed (e.g., amplified) and sequenced to obtain the nucleic acid sequence of the mRNA. In some embodiments, a barcoded nucleic acid molecule is a barcoded spacer oligonucleotide, such as any described herein. In some embodiments, a barcoded nucleic acid molecule is a barcoded analyte oligonucleotide, such as any described herein. In someAttorney Docket No.43487-1046601 embodiments, a nucleic acid barcode molecule is a barcoded oligonucleotide, such as any described herein.

[0104] The term “sample,” as used herein, generally refers to a biological sample of asubject. The biological sample may comprise any number of macromolecules, for example, cellular macromolecules. The sample may be a cell sample. The sample may be a cell line or cell culture sample. The sample can include one or more cells or nuclei. The sample can include one or more microbes. The biological sample may be a nucleic acid sample or protein sample. The biological sample may also be a carbohydrate sample or a lipid sample. The biological sample may be derived from another sample. The sample may be a tissue sample, such as a biopsy, core biopsy, needle aspirate, or fine needle aspirate. The tissue sample may be a fresh tissue sample, a frozen tissue sample (e.g., flash frozen, lyophilized, cryo-sectioned, etc.), or a fixed tissue sample (e.g., a formalin-fixed and paraffin-embedded tissue sample). The sample may be a fluid sample, such as a blood sample, urine sample, or saliva sample. The sample may be a skin sample. The sample may be a cheek swab. The sample may be a plasma or serum sample. The sample may be a cell-free or cell free sample. A cell-free sample may include extracellular polynucleotides. Extracellular polynucleotides may be isolated from a bodily sample that may be selected from the group consisting of blood, plasma, serum, urine, saliva, mucosal excretions, sputum, stool and tears.

[0105] The term “biological particle,” as used herein, generally refers to a discrete biologicalsystem derived from a biological sample. The biological particle may be a macromolecule. The biological particle may be a small molecule. The biological particle may be a virus. The biological particle may be a cell or derivative of a cell. The biological particle may be an organelle. Examples of an organelle from a cell include, without limitation, a nucleus, a ribosome, a Golgi apparatus, an endoplasmic reticulum, a chloroplast, an endocytic vesicle, an exocytic vesicle, a vacuole, and a lysosome. The biological particle may be a rare cell from a population of cells. The biological particle may be any type of cell, including without limitation prokaryotic cells, eukaryotic cells, bacterial, fungal, plant, mammalian, or other animal cell type, mycoplasmas, normal tissue cells, tumor cells, or any other cell type, whether derived from single cell or multicellular organisms. The biological particle may be a constituent of a cell. The biological particle may be or may include DNA, RNA, organelles, proteins, or any combination thereof. The biological particle may be or may include a matrix (e.g., a gel or polymer matrix) comprising a cell or one or more constituents from a cell (e.g., cell bead), such as DNA, RNA, organelles, proteins, or any combination thereof, from the cell. The biological particle may be obtained from a tissue of a subject (e.g., a human, a mouse, a rat, or other mammal). The biological particle may be a hardened cell. Such hardened cell may or may not include a cellAttorney Docket No.43487-1046601 wall or cell membrane. The biological particle may include one or more constituents of a cell, but may not include other constituents of the cell. An example of such constituents is a nucleus or an organelle. A cell may be a live cell. The live cell may be capable of being cultured, for example, being cultured when enclosed in a gel or polymer matrix, or cultured when comprising a gel or polymer matrix.

[0106] The term “macromolecular constituent,” as used herein, generally refers to amacromolecule contained within or from a biological particle. The macromolecular constituent may comprise a nucleic acid. In some cases, the biological particle may be a macromolecule. The macromolecular constituent may comprise DNA. The macromolecular constituent may comprise RNA. The RNA may be coding or non-coding. The RNA may be messenger RNA (mRNA), ribosomal RNA (rRNA) or transfer RNA (tRNA), for example. The RNA may be a transcript. The RNA may be small RNA that are less than 200 nucleic acid bases in length, or large RNA that are greater than 200 nucleic acid bases in length. Small RNAs may include 5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA), microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNAs), Piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA) and small rDNA-derived RNA (srRNA). The RNA may be double-stranded RNA or single-stranded RNA. The RNA may be circular RNA. The macromolecular constituent may comprise a protein. The macromolecular constituent may comprise a peptide. The macromolecular constituent may comprise a polypeptide.

[0107] The term “molecular tag,” as used herein, generally refers to a molecule capable ofbinding to a macromolecular constituent. The molecular tag may bind to the macromolecular constituent with high affinity. The molecular tag may bind to the macromolecular constituent with high specificity. The molecular tag may comprise a nucleotide sequence. The molecular tag may comprise a nucleic acid sequence. The nucleic acid sequence may be at least a portion or an entirety of the molecular tag. The molecular tag may be a nucleic acid molecule or may be part of a nucleic acid molecule. The molecular tag may be an oligonucleotide or a polypeptide. The molecular tag may comprise a DNA aptamer. The molecular tag may be or comprise a primer. The molecular tag may be, or comprise, a protein. The molecular tag may comprise a polypeptide. The molecular tag may be a barcode.

[0108] The term “partition,” as used herein, generally, refers to a space or volume that maybe suitable to contain one or more species or conduct one or more reactions. A partition may be a physical compartment, such as a droplet or well. The partition may isolate space or volume from another space or volume. The droplet may be a first phase (e.g., aqueous phase) in a second phase (e.g., oil) immiscible with the first phase. The droplet may be a first phase in a second phase that does not phase separate from the first phase, such as, for example, a capsule orAttorney Docket No.43487-1046601 liposome in an aqueous phase. A partition may comprise one or more other (inner) partitions. In some cases, a partition may be a virtual compartment that can be defined and identified by an index (e.g., indexed libraries) across multiple and / or remote physical compartments. For example, a physical compartment may comprise a plurality of virtual compartments. gRNAs AND CRISPR / CAS SYSTEMS

[0109] Clustered regularly interspaced short palindromic repeats (CRISPR) / Cas (CRISPR-associated proteins) systems are a component of prokaryotic adaptive immune systems represented in archaea and bacteria. Various naturally occurring CRISPR / Cas systems from different species have been engineered to allow sequence-specific targeting for genetic and epigenetic perturbations in a wide variety of contexts. CRISPR / Cas systems are composed of a Cas protein component and a guide RNA (gRNA) component. When co-expressed, a Cas protein and gRNA form a Cas / gRNA complex in which the Cas protein binds to a structured region of the gRNA known as the scaffold. The gRNA further includes a spacer, which provides specificity by hybridizing to a specific target site (e.g. genomic locus), at which the complexed Cas protein mediates a genetic or epigenetic effect. gRNAs from different CRISPR / Cas systems include different scaffolds that allow them to complex with a particular Cas protein. In general, for a particular CRISPR / Cas system, the sequence of the scaffold remains constant, whereas the spacer sequence varies according to the target site. Naturally occurring Cas proteins can mediate DNA cleavage (such as a double-stranded break) at the target site. Engineered Cas proteins and CRISPR / Cas systems can be used to mediate a variety of different effects at the target site, including double-stranded breaks and single-stranded breaks. In addition, certain engineered Cas proteins, commonly referred to as dCas proteins, have been engineered to lack nuclease activity. dCas proteins (e.g. dCas9) can be recruited to a locus alone, for example to repress gene expression, or can be fused to epigenetic effectors, for example to repress (e.g. dCas-KRAB) or activate (e.g. dCas-VP64) gene expression. A variety of engineered CRISPR / Cas systems can be leveraged for large-scale CRISPR / Cas perturbation screens. Naturally occurring and engineered CRISPR / Cas systems, and methods of their use, are described in detail elsewhere, for example in Bock et al., “High-content CRISPR screening” Nat. Rev. Methods Primers, 2022, 2(1):9; and in Liu et al., “The CRISPR-Cas toolbox and gene editing technologies” Mol. Cell, 2022, 82(2):333- 347; each of which is incorporated by reference herein in its entirety.

[0110] The methods provided herein can be readily applied for sequencing gRNAs from anysuitable CRISPR / Cas system, including in single-cell workflows, as described herein. In addition, one of skill in the art would readily be able to apply the methods provided herein for sequencing gRNAs from any suitable CRISPR / Cas system, including within the context of aAttorney Docket No.43487-1046601 large-scale CRISPR / Cas screen involving the generation of a plurality of gRNA-expressing cells expressing different gRNAs (e.g. from a gRNA library).

[0111] In some aspects, the methods provided herein facilitate sequencing gRNAs, forexample in single-cell sequencing workflows. A gRNA provided herein can be any suitable gRNA to be sequenced in accordance with the provided methods. gRNAs can from any suitable CRISPR / Cas system, and / or can be compatible with (e.g. capable of complexing with) any suitable Cas protein. For example, in some embodiments, the gRNA is capable of complexing with Cas9 (e.g. a Cas9-compatible gRNA). In some embodiments, the gRNA is capable of complexing with a Cas12 (e.g. Cas12a or Cpf1). In some embodiments, the gRNA is capable of complexing with Cas12a). A gRNA provided herein can be from any other CRISPR / Cas system, such as those described in detail elsewhere.

[0112] In some aspects, a gRNA can be composed of more than one RNA molecule (e.g. asin naturally occurring CRISPR / Cas systems). For example, Cas9 system gRNAs include a crRNA that includes a spacer sequence, and a tracrRNA that hybridizes to the crRNA and facilitates complexing with the Cas9 via a scaffold. In contrast, the gRNAs of many engineered CRISPR / Cas systems have been engineered to comprise a single gRNA (which may be referred to as a “sgRNA” or simply “gRNA”) that includes both the spacer and a full scaffold in a single molecule. The methods provided herein can be readily adapted to sequence any suitable gRNA, including those composed of a single RNA molecule or those composed of more than one RNA molecule (e.g. a gRNA consisting of a crRNA / tracrRNA duplex).

[0113] In some aspects, the gRNA comprises a spacer. In some embodiments, the spacer ofthe gRNA is at the 5’ end of the gRNA (e.g. as in Cas9-compatible gRNAs). In some embodiments, the spacer of the gRNA is at the 5’ end of the gRNA (e.g. as in gRNAs from Cas9 CRISPR / Cas systems). In some embodiments, the gRNA is from a Cas9 CRISPR / Cas system, e.g. is Cas9-compatible. In some embodiments, the spacer of the gRNA is at the 3’ end of the gRNA (e.g. as in gRNAs from Cas12a (Cpf1) CRISPR / Cas systems). In some embodiments, the gRNA is from a Cas12 CRISPR / Cas system, e.g. is Cas12-compatible. In some embodiments, the gRNA is from a Cas12a (i.e. Cpf1) CRISPR / Cas system, e.g. is Cas12a-compatible. In some embodiments, the spacer is flanked by non-spacer sequences.

[0114] In some embodiments, the gRNA comprises a constant region. In some embodiments,the gRNA comprises a scaffold sequence. In some embodiments, the gRNA comprises a scaffold region. In some embodiments, the gRNA comprises a scaffold. In some embodiments, the constant region is a scaffold sequence. In some embodiments, the constant region comprises a scaffold sequence. In some embodiments, the constant region comprises a scaffold sequence and an additional sequence. In some embodiments, the constant region comprises a scaffold sequenceAttorney Docket No.43487-1046601 and a functional sequence. In some embodiments, the functional sequence is a primer hybridization sequence, a sequencing primer binding site, or complement thereof. In some embodiments, the constant region comprises a capturing sequence. The constant region of the gRNA can be designed to include any suitable additional sequence in accordance with the methods described herein. For example, the constant region can include a capturing sequence that facilitates downstream capture of the gRNA or a product thereof (e.g. a tagged gRNA), for example by hybridization to a barcoded oligonucleotide comprising a partition-specific barcode. gRNA SEQUENCING WORKFLOWS

[0115] In some aspects, provided herein are methods for analyzing a gRNA-expressing cell.In some aspects, provided herein are methods for analyzing a plurality of gRNA-expressing cells. In some aspects, provided herein are methods for gRNA sequencing in a plurality of cells. In some aspects, the gRNA sequencing is performed at the single-cell level (e.g. single-cell gRNA sequencing). For example, in some aspects, the methods for analyzing a gRNA- expressing cell can be performed in parallel for a plurality of gRNA-expressing cells. In some embodiments, the methods can be performed in a single-cell sequencing workflow (e.g. single- cell gRNA and / or analyte sequencing workflow). In some embodiments, different cells (e.g. gRNA-expressing cells) are partitioned into different partitions to facilitate single-cell analysis. The partitions can be any suitable partition, such as a droplet or a well. The different partitions can comprise barcoded oligonucleotides having partition-specific barcodes.

[0116] The barcoded oligonucleotides having partition-specific barcodes and gRNAs (orproducts generated therefrom) can be used to generate single nucleic acids (e.g. barcoded spacer oligonucleotides as described herein) comprising both a gRNA spacer sequence (or complement thereof) and the partition-specific barcode (or complement thereof). Sequencing the barcoded spacer oligonucleotides can thus reveal the sequence of a gRNA spacer sequence and the partition (e.g. single-cell) that the gRNA spacer sequence was present in. This can be readily performed (e.g. in parallel) for a plurality of gRNAs from a plurality of single cells. In some aspects, the methods for analyzing a gRNA-expressing cell are compatible with detecting and / or sequencing additional analytes, such as target nucleic acids, in the same single cells, as described herein. In some aspects, provided herein are workflows for combined gRNA sequencing and analyte (e.g. cellular transcript) sequencing in the same single cells.

[0117] In some embodiments, the barcoded oligonucleotides are nucleic acid barcodemolecules, such as any of the nucleic acid barcode molecules described herein. Accordingly, the barcoded oligonucleotides described herein can be used to generate barcoded nucleic acid molecules as described herein, for example in combination with gRNAs (e.g. barcoded spacerAttorney Docket No.43487-1046601 oligonucleotides) and / or other target nucleic acids (e.g. barcoded analyte oligonucleotides). In some embodiments, the barcoded oligonucleotides are nucleic acid barcode molecules. In some embodiments, the barcoded oligonucleotide is a nucleic acid barcode molecule. In some embodiments, the barcoded analyte oligonucleotide is a barcoded nucleic acid molecule. In some embodiments, the barcoded spacer oligonucleotide is a barcoded nucleic acid molecule.

[0118] In some aspects, the methods herein are described for analysis of a gRNA-expressingcell. In some embodiments, the gRNA-expressing cell is a cell comprising a gRNA. The gRNA- expressing cell can be any cell comprising a gRNA, regardless of how the gRNA was generated (e.g. transcribed within the cell or directly transduced into the cell). In some embodiments, the gRNA is transcribed in the cell (e.g. from an expression construct). In some embodiments, the gRNA is not transcribed in the cell. For example, the gRNA can be transduced directly into the cell without needing to be transcribed within the cell. The gRNA-expressing cell can be any suitable cell or derivative or product thereof. In some embodiments, the gRNA-expressing cell is a fixed cell, such as any fixed cell described herein or any cell fixed or prepared according to the methods provided herein. In some embodiments, the methods can be applied to cell derivatives or components thereof. For example, in some embodiments, any of the methods provided herein can be performed to analyze a gRNA-expressing nucleus.

[0119] In any of the embodiments provided herein, such as any of the gRNA sequencingworkflows described above, the method can comprise sequencing the barcoded spacer oligonucleotide or a derivative thereof. In some embodiments, the method comprises analyzing the results of the sequencing to determine the sequence of the spacer sequence. In some embodiments, the method comprises analyzing the results of the sequencing to determine the presence and / or abundance of the gRNA in the gRNA-expressing cell.

[0120] In some embodiments, the partition comprises the gRNA-expressing cell. In someembodiments, the partition comprises the gRNA-expressing cell and no other cells. In some embodiments, one or more steps (e.g. wash steps) can be performed to remove unhybridized probes, such as unhybridized gRNA-targeting probes or probes of a ligatable probe pair. In some embodiments, the method comprises removing unhybridized probes from the gRNA-expressing cell. In some embodiments, the method comprises performing one or more wash steps to remove unhybridized probes. In some embodiments, the method comprises performing one or more wash steps prior to generating the partition.Attorney Docket No.43487-1046601 gRNA sequencing using gRNA-targeting probe extension

[0121] In some aspects, provided herein is a method for gRNA sequencing involving gRNA-targeting probe extension, for example as exemplified by Example 11B and as illustrated in FIG. 41.

[0122] In some aspects, provided herein is a method for analyzing a gRNA-expressing cell.In some embodiments, the method comprises providing a gRNA-expressing cell comprising a gRNA having a spacer sequence and a constant region comprising a scaffold sequence. In some embodiments, the method comprises contacting the gRNA-expressing cell with a gRNA- targeting probe that hybridizes to the constant region of the gRNA. In some embodiments, the method comprises generating a partition comprising 1) the gRNA-expressing cell, and 2) a plurality of barcoded oligonucleotides comprising a partition-specific barcode and a capture sequence. In some embodiments, the partition comprises the gRNA-expressing cell and no other cells. In some embodiments, each barcoded oligonucleotide of the plurality of barcoded oligonucleotides comprises the partition-specific barcode and the capture sequence. In some embodiments, the method comprises extending the 3’ end of the gRNA-targeting probe using a reverse transcriptase having terminal deoxynucleotidyl transferase (TdT) activity to incorporate a sequence complementary to the spacer sequence and a non-templated 3’ terminal sequence. In some embodiments, the method comprises hybridizing the 3’ terminal sequence to the capture sequence of a barcoded oligonucleotide of the plurality of barcoded oligonucleotides. In some embodiments, the method comprises further extending the 3’ end of the gRNA-targeting probe using the barcoded oligonucleotide as template and / or extending the barcoded oligonucleotide using the extended gRNA-targeting probe as template, thereby generating a barcoded spacer oligonucleotide comprising the spacer sequence or complement thereof, and the partition- specific barcode or complement thereof. In some embodiments, the method comprises sequencing the barcoded spacer oligonucleotide to determine the sequence of the spacer sequence and the partition-specific barcode, and associating the spacer sequence with the partition-specific barcode.

[0123] In some embodiments, the gRNA-targeting probe comprises a 5’ overhang. In someaspects, the 5’ overhang can be used for downstream processing and / or sequencing purposes. For example, in some embodiments, the 5’ overhang of the gRNA-targeting probe comprises a barcode sequence. In some embodiments, the 5’ overhang of the gRNA-targeting probe comprises a sample-specific barcode sequence. In some aspects, a sample-specific barcode sequence (such as the sample-specific barcode sequence described in the current section or any of the gRNA sequencing workflows described herein) can be used as an indicator (e.g. during sequencing analysis) of which sample the method was performed in. Thus, in some aspects, aAttorney Docket No.43487-1046601 sample-specific barcode sequence facilitates multiplexed analysis of the method having been performed in different reactions, which can be subsequently combined and sequenced together while preserving information regarding the sample of origin. In some embodiments, the 5’ overhang of the gRNA-targeting probe comprises one or more functional sequences. In some aspects, functional sequences (such as the functional sequences described in the current section or any of the gRNA sequencing workflows described herein) can be used for any suitable downstream processing and / or sequencing purposes. In some embodiments, the one or more functional sequences of the 5’ overhang of the gRNA-targeting probe comprise a primer hybridization sequence, a sequencing primer binding site, or complement thereof.

[0124] In some embodiments, the gRNA-targeting probe hybridizes to a sequence in thegRNA that is at least 10bp, at least 20bp, at least 30bp, or at least 40bp away from the spacer sequence. In some embodiments, the gRNA-targeting probe hybridizes to a sequence in the gRNA that is at least 20bp away from the spacer sequence. In some embodiments, the gRNA- targeting probe hybridizes to a sequence in the gRNA that is at least 30bp away from the spacer sequence. In some embodiments, the gRNA-targeting probe hybridizes to a sequence in the gRNA that is at least 40bp away from the spacer sequence. In some embodiments, the gRNA- targeting probe hybridizes to a sequence in the gRNA that is at least 50bp away from the spacer sequence. In some embodiments, the gRNA-targeting probe hybridizes to a sequence in the constant region of the gRNA that is non-structured and / or that does not form a secondary structure of the scaffold sequence via base-pairing. As shown in the Examples, gRNA-targeting probes that are not hybridized immediately upstream of the spacer (e.g. that hybridize at least 10bp, at least 20bp, at least 30bp, or at least 40bp away from the spacer) can facilitate increased gRNA sequencing efficiency. As shown in the Examples, gRNA-targeting probes that hybridize to a sequence in the constant region of the gRNA that is non-structured and / or that does not form a secondary structure of the scaffold sequence via base-pairing can facilitate increased gRNA sequencing efficiency.

[0125] In some aspects, any of the methods described above for gRNA sequencing can beperformed in combination with methods for detecting one or more other target nucleic acids. Such methods can facilitate single-cell analysis of gRNA-expressing cells, for example to allow gRNA detection and transcriptome analysis in the same single cells. These methods can facilitate powerful large-scale CRISPR perturbation screens, for example as described herein.

[0126] Accordingly, in some aspects, provided herein is a method for analyzing a cellcomprising a gRNA and a target nucleic acid. In some embodiments, the method comprises providing a gRNA-expressing cell comprising a gRNA having a spacer sequence and a constant region comprising a scaffold sequence. In some embodiments, the method comprises contactingAttorney Docket No.43487-1046601 the gRNA-expressing cell with a gRNA-targeting probe that hybridizes to the constant region of the gRNA. In some embodiments, the method comprises contacting the gRNA-expressing cell with a ligatable probe pair comprising a first ligatable probe and a second ligatable probe that hybridize to a target nucleic acid in the gRNA-expressing cell. In some embodiments, the method comprises ligating the first ligatable probe to the second ligatable probe using the target nucleic acid as template to generate a ligated probe pair. In some embodiments, the method comprises generating a partition comprising 1) the gRNA-expressing cell, and 2) a plurality of barcoded oligonucleotides comprising a partition-specific barcode. In some embodiments, the method comprises extending the 3’ end of the gRNA-targeting probe to generate an extended gRNA-targeting probe comprising a sequence complementary to the spacer sequence. In some embodiments, the method comprises using the extended gRNA-targeting probe and a first barcoded oligonucleotide of the plurality of barcoded oligonucleotides to generate a barcoded spacer oligonucleotide comprising the spacer sequence or complement thereof, and the partition- specific barcode or complement thereof. In some embodiments, the method comprises using the ligated probe pair and a second barcoded oligonucleotide of the plurality of barcoded oligonucleotides to generate a barcoded analyte oligonucleotide comprising a sequence of the ligated probe pair or complement thereof, and the partition-specific barcode or a complement thereof.

[0127] In some embodiments, the method further comprises sequencing the barcoded spaceroligonucleotide or a derivative thereof and the barcoded analyte oligonucleotide or a derivative thereof. In some embodiments, the method comprises analyzing the results of the sequencing to determine the sequence of the spacer sequence. In some embodiments, the method comprises analyzing the results of the sequencing to determine the presence and / or abundance of the gRNA and the target nucleic acid in the gRNA-expressing cell. In some embodiments, the method comprises hybridizing the extended gRNA-targeting probe to the first barcoded oligonucleotide, and extending the 3’ end of the extended gRNA-targeting probe and / or extending the first barcoded oligonucleotide to generate the barcoded spacer oligonucleotide. In some embodiments, the extending the 3’ end of the gRNA-targeting probe comprises extending the 3’ end of the gRNA-targeting probe using a reverse transcriptase having terminal deoxynucleotidyl transferase (TdT) activity to incorporate a sequence complementary to the spacer sequence and a non-templated 3’ terminal sequence. In some embodiments, the method comprises hybridizing the 3’ terminal sequence to the first barcoded oligonucleotide, and extending the 3’ end of the extended gRNA-targeting probe and / or extending the first barcoded oligonucleotide to generate the barcoded spacer oligonucleotide. In some embodiments, the gRNA-targeting probe comprises a 5’ overhang. In some embodiments, the 5’ overhang of the gRNA-targeting probeAttorney Docket No.43487-1046601 comprises a barcode sequence. In some embodiments, the barcode sequence is a sample-specific barcode sequence. In some embodiments, the 5’ overhang of the gRNA-targeting probe comprises one or more functional sequences. In some embodiments, the one or more functional sequences of the 5’ overhang of the gRNA-targeting probe comprise a primer hybridization sequence, a sequencing primer binding site, or complement thereof. In some embodiments, the gRNA-targeting probe hybridizes to a sequence in the gRNA that is at least 10bp, at least 20bp, at least 30bp, or at least 40bp away from the spacer sequence. In some embodiments, the gRNA- targeting probe hybridizes to a sequence in the constant region of the gRNA that is non- structured and / or that does not form a secondary structure of the scaffold sequence via base- pairing. In some embodiments, the first ligatable probe comprises a 3’ overhang and a 5’ hybridizing region that hybridizes to the target nucleic acid, and the second ligatable probe comprises a 5’ overhang and a 3’ hybridizing region that hybridizes to the target nucleic acid. In some embodiments, the ligated probe pair comprises a sequence that is complementary to and / or indicative of the target nucleic acid. In some embodiments, the barcoded analyte oligonucleotide comprises a sequence that is complementary to and / or indicative of the target nucleic acid. In some embodiments, the method comprises hybridizing a sequence of the 3’ overhang of the ligated probe pair to the second barcoded oligonucleotide, and extending the 3’ end of the ligated probe pair and / or extending the 3’ end of the second barcoded oligonucleotide to generate the barcoded analyte oligonucleotide.

[0128] The target nucleic acid can be any suitable nucleic acid. In some embodiments, thetarget nucleic acid is an mRNA. In some embodiments, the target nucleic acid is not a gRNA. In some embodiments, the method comprises removing unhybridized probes from the gRNA- expressing cell. In some embodiments, the method comprises performing one or more wash steps to remove the unhybridized probes. In some embodiments, the wash steps are performed prior to generating the partition. The method can be performed to analyze a plurality of target nucleic acids in the cell. For example, in some embodiments, the method can comprise both gRNA sequencing and transcriptome sequencing. In some embodiments, the method further comprises contacting the gRNA-expressing cell with a plurality of ligatable probe pairs that hybridize to a plurality of different target nucleic acids in the cell. In some embodiments, the method comprises ligating the plurality of ligatable probe pairs using the plurality of different target nucleic acids as templates to generate a plurality of ligated probe pairs. In some embodiments, the method comprises using the plurality of ligated probe pairs and the plurality of barcoded oligonucleotides to generate a plurality of barcoded analyte oligonucleotides. In some embodiments, a barcoded analyte oligonucleotide of the plurality of barcoded analyte oligonucleotides comprises a sequence of a ligated probe pair of the plurality of ligated probeAttorney Docket No.43487-1046601 pairs or a complement thereof and a sequence of the partition-specific barcode or complement thereof. In some embodiments, a barcoded analyte oligonucleotide of the plurality of barcoded analyte oligonucleotides comprises a sequence of a target nucleic acid of the plurality of different target nucleic acids or a complement thereof and a sequence of the partition-specific barcode or complement thereof. In some embodiments, the method further comprises sequencing the plurality of barcoded analyte oligonucleotides or derivatives thereof. In some embodiments, the method further comprises analyzing the results of the sequencing to determine the presence and / or abundance of the different target nucleic acids in the gRNA-expressing cell. gRNA sequencing using gRNA-targeting probe extension and template switching

[0129] In some aspects, provided herein is a method for gRNA sequencing involving gRNA-targeting probe extension and template switching, for example as exemplified by Example 11C and as illustrated in FIG.42.

[0130] In some aspects, provided herein is a method for analyzing a gRNA-expressing cell.In some embodiments, the method comprises providing a gRNA-expressing cell comprising a gRNA having a spacer sequence and a constant region comprising a scaffold sequence. In some embodiments, the method comprises contacting the gRNA-expressing cell with a gRNA- targeting probe that hybridizes to the constant region of the gRNA. In some embodiments, the method comprises extending the 3’ end of the gRNA-targeting probe using a reverse transcriptase having terminal deoxynucleotidyl transferase (TdT) activity to incorporate a sequence complementary to the spacer sequence and a non-templated 3’ terminal sequence. In some embodiments, the method comprises hybridizing the 3’ terminal sequence to a template- switching oligonucleotide (TSO) and further extending the 3’ end of the gRNA-targeting probe to incorporate a sequence complementary to the TSO, thereby generating a TSO-tagged probe. In some embodiments, the method comprises generating a partition comprising 1) the gRNA- expressing cell, and 2) a plurality of barcoded oligonucleotides comprising a partition-specific barcode and a capture sequence. In some embodiments, the partition comprises the gRNA- expressing cell and no other cells. In some embodiments, each barcoded oligonucleotide of the plurality of barcoded oligonucleotides comprises the partition-specific barcode and the capture sequence. In some embodiments, the method further comprises sequencing the barcoded spacer oligonucleotide to determine the sequence of the spacer sequence and the partition-specific barcode, and associating the spacer sequence with the partition-specific barcode. In some embodiments, the method comprises hybridizing the TSO-tagged probe to the capture sequence of a barcoded oligonucleotide of the plurality of barcoded oligonucleotides. In some embodiments, the method comprises extending the TSO-tagged probe using the barcodedAttorney Docket No.43487-1046601 oligonucleotide as template and / or extending the barcoded oligonucleotide using the TSO-tagged probe as template, thereby generating a barcoded spacer oligonucleotide comprising the spacer sequence or complement thereof, and the partition-specific barcode or complement thereof.

[0131] In some embodiments, the TSO comprises a barcode sequence. In someembodiments, the TSO comprises a sample-specific barcode sequence. In some embodiments, the TSO comprises a capturing sequence, and the TSO-tagged probe comprises a complement of the capturing sequence. In some embodiments, the complement of the capturing sequence in the TSO-tagged probe hybridizes to the capture sequence of the barcoded oligonucleotide.

[0132] In some embodiments, all or a portion of the TSO is dehybridized from the TSO-tagged probe. In some aspects, dehybridizing the TSO from the TSO-tagged probe can allow the TSO-tagged probe to more efficiently hybridize to the barcoded oligonucleotide, and can thus increase the efficiency of generating the barcoded spacer oligonucleotide, and ultimately the efficiency of gRNA sequencing. In some embodiments, all or a portion of the TSO is dehybridized from the TSO-tagged probe prior to hybridizing the TSO-tagged probe to the capture sequence of the barcoded oligonucleotide. In some embodiments, dehybridizing all or a portion of the TSO from the TSO-tagged probe comprises degrading the TSO. In some embodiments, dehybridizing all or a portion of the TSO from the TSO-tagged probe comprises contacting the TSO with an enzyme, such as any enzyme capable of degrading (e.g. digesting, cleaving, etc.) or otherwise contributing to dehybridizing the TSO. In some embodiments, degrading the TSO comprises contacting the TSO with an enzyme, such as any enzyme capable of degrading (e.g. digesting, cleaving, etc.) the TSO. In some embodiments, the TSO comprises ribonucleotides and dehybridizing all or a portion of the TSO from the TSO-tagged probe comprises contacting the TSO with Ribonuclease H (RNAse H) to digest the TSO. In some embodiments, the TSO comprises uracil residues and dehybridizing all or a portion of the TSO from the TSO-tagged probe comprises contacting the TSO with an enzyme to remove the uracil residues. In some embodiments, the enzyme is a Uracil-DNA Glycosylase (UDG) enzyme. In some embodiments, the enzyme is a uracil-specific excision reagent (USER) enzyme. In some embodiments, the TSO hybridized to the TSO-tagged probe is displaced by hybridization of the capture sequence of the barcoded oligonucleotide to the TSO-tagged probe.

[0133] In some embodiments, the gRNA-targeting probe comprises a 5’ overhang. In someembodiments, the 5’ overhang of the gRNA-targeting probe comprises a barcode sequence. In some embodiments, the 5’ overhang of the gRNA-targeting probe comprises a sample-specific barcode sequence. In some embodiments, the 5’ overhang of the gRNA-targeting probe comprises one or more functional sequences. In some embodiments, the one or more functionalAttorney Docket No.43487-1046601 sequences of the 5’ overhang of the gRNA-targeting probe comprise a primer hybridization sequence, a sequencing primer binding site, or complement thereof.

[0134] In some embodiments, the gRNA-targeting probe hybridizes to a sequence in thegRNA that is at least 10bp, at least 20bp, at least 30bp, or at least 40bp away from the spacer sequence. In some embodiments, the gRNA-targeting probe hybridizes to a sequence in the gRNA that is at least 20bp away from the spacer sequence. In some embodiments, the gRNA- targeting probe hybridizes to a sequence in the gRNA that is at least 30bp away from the spacer sequence. In some embodiments, the gRNA-targeting probe hybridizes to a sequence in the gRNA that is at least 40bp away from the spacer sequence. In some embodiments, the gRNA- targeting probe hybridizes to a sequence in the gRNA that is at least 50bp away from the spacer sequence. In some embodiments, the gRNA-targeting probe hybridizes to a sequence in the constant region of the gRNA that is non-structured and / or that does not form a secondary structure of the scaffold sequence via base-pairing. As shown in the Examples, gRNA-targeting probes that are not hybridized immediately upstream of the spacer (e.g. that hybridize at least 10bp, at least 20bp, at least 30bp, or at least 40bp away from the spacer) can facilitate increased gRNA sequencing efficiency. As shown in the Examples, gRNA-targeting probes that hybridize to a sequence in the constant region of the gRNA that is non-structured and / or that does not form a secondary structure of the scaffold sequence via base-pairing can facilitate increased gRNA sequencing efficiency. gRNA sequencing using a gRNA ligation adapter

[0135] In some aspects, provided herein is a method for gRNA sequencing involving ligationof a gRNA ligation adapter, for example as exemplified in Example 11D and as illustrated in FIGS.43A-C.

[0136] In some aspects, the method comprises ligating a gRNA ligation adapter to a gRNAto facilitate sequencing. In some embodiments, a capturing sequence is included in either the gRNA or the gRNA ligation adapter.

[0137] In some aspects, the method can be employed in different configurations dependingon the location of a capturing sequence, which can be included in either the gRNA or the gRNA ligation adapter. In one configuration (e.g. as shown in FIG.43A), the capturing sequence is included in the gRNA (e.g. as part of the constant region such as the scaffold), and the capturing sequence hybridizes to a barcoded oligonucleotide in a partition. In another configuration (e.g. as shown in FIG.43B), the capturing sequence is included in the gRNA ligation adapter, and a product of the capturing sequence (e.g. complement of the capturing sequence in a tagged gRNA resulting from ligation of the gRNA ligation adapter and gRNA) hybridizes to the barcodedAttorney Docket No.43487-1046601 oligonucleotide in the partition. In another configuration (e.g. as shown in FIG.43C), the capturing sequence is included in the gRNA ligation adapter, and the capturing sequence hybridizes to a barcoded oligonucleotide in a partition.

[0138] In some aspects, provided herein is a method for analyzing a gRNA-expressing cell,such as illustrated in FIG.43A and Example 11D. In some embodiments, the method comprises providing a gRNA-expressing cell comprising a gRNA having a spacer sequence and a constant region comprising a scaffold sequence, wherein the gRNA comprises a 5’ monophosphate. In some embodiments, the method comprises contacting the gRNA-expressing cell with a gRNA ligation adapter comprising a functional region and a 3’ ligation end. In some embodiments, the method comprises ligating the 3’ ligation end of the gRNA ligation adapter to the gRNA, thereby generating a tagged gRNA comprising the functional region. In some embodiments, the method comprises generating a partition comprising 1) the gRNA-expressing cell, and 2) a plurality of barcoded oligonucleotides comprising a partition-specific barcode and a capture sequence. In some embodiments, the partition comprises the gRNA-expressing cell and no other cells. In some embodiments, each barcoded oligonucleotide of the plurality of barcoded oligonucleotides comprises the partition-specific barcode and the capture sequence. In some embodiments, the method comprises hybridizing the constant region of the tagged gRNA to the capture sequence of a barcoded oligonucleotide of the plurality of barcoded oligonucleotides. In some embodiments, the method comprises extending the barcoded oligonucleotide using the tagged gRNA as template, thereby generating a barcoded spacer oligonucleotide comprising the partition-specific barcode, a sequence complementary to the spacer sequence, and a sequence complementary to the functional region. In some embodiments, the constant region of the gRNA comprises a capturing sequence. In some embodiments, the constant region of the tagged gRNA is hybridized via the capturing sequence to the capture sequence of the barcoded oligonucleotide. In some embodiments, the capturing sequence is at the 3’ end of the constant region of the gRNA. In some embodiments, the capturing sequence is within and / or flanked by the scaffold sequence of the gRNA. In some embodiments, the capturing sequence is complementary to the capture sequence. In some embodiments, the method further comprises sequencing the barcoded spacer oligonucleotide to determine the sequence of the spacer sequence and the partition- specific barcode, and associating the spacer sequence with the partition-specific barcode.

[0139] In some aspects, provided herein is a method for analyzing a gRNA-expressing cell,such as illustrated in FIG.43B and Example 11D. In some aspects, the method comprises providing a gRNA-expressing cell comprising a gRNA having a spacer sequence and a constant region comprising a scaffold sequence, wherein the gRNA comprises a 5’ monophosphate. In some aspects, the method comprises contacting the gRNA-expressing cell with a gRNA ligationAttorney Docket No.43487-1046601 adapter comprising a 3’ ligation end, and a functional region comprising a capturing sequence. In some aspects, the method comprises ligating the 3’ end of the gRNA ligation adapter to the gRNA, thereby generating a tagged gRNA. In some aspects, the method comprises contacting the tagged gRNA with a primer that hybridizes to the constant region of the gRNA, and extending the primer using the tagged gRNA as template, thereby generating a tagged gRNA complement that comprises a sequence complementary to the spacer sequence and a complement of the capturing sequence. In some aspects, the method comprises generating a partition comprising 1) the gRNA-expressing cell, and 2) a plurality of barcoded oligonucleotides comprising a partition-specific barcode and a capture sequence. In some embodiments, the partition comprises the gRNA-expressing cell and no other cells. In some embodiments, each barcoded oligonucleotide of the plurality of barcoded oligonucleotides comprises the partition- specific barcode and the capture sequence. In some aspects, the method comprises hybridizing the complement of the capturing sequence in the tagged gRNA complement to the capture sequence of a barcoded oligonucleotide of the plurality of barcoded oligonucleotides. In some aspects, the method comprises extending the barcoded oligonucleotide using the tagged gRNA complement as template and / or extending the tagged gRNA complement using the barcoded oligonucleotide as template, thereby generating a barcoded spacer oligonucleotide comprising the partition-specific barcode or a complement thereof, and the sequence of the spacer sequence or a complement thereof. In some embodiments, the primer that hybridizes to the constant region of the gRNA comprises a 5’ overhang. In some embodiments, the 5’ overhang of the primer that hybridizes to the constant region of the gRNA comprises a barcode sequence. In some embodiments, the 5’ overhang of the primer that hybridizes to the constant region of the gRNA comprises a sample-specific barcode sequence. In some embodiments, the 5’ overhang of the primer that hybridizes to the constant region of the gRNA comprises one or more functional sequences. In some embodiments, the one or more functional sequences of the 5’ overhang of the primer that hybridizes to the constant region of the gRNA comprise a primer hybridization sequence, a sequencing primer binding site, or complement thereof. In some embodiments, the method further comprises sequencing the barcoded spacer oligonucleotide to determine the sequence of the spacer sequence and the partition-specific barcode, and associating the spacer sequence with the partition-specific barcode.

[0140] In some aspects, provided herein is a method for analyzing a gRNA-expressing cell,such as illustrated in FIG.43C. In some embodiments, the method comprises providing a gRNA- expressing cell comprising a gRNA having a spacer sequence and a constant region comprising a scaffold sequence. In some embodiments, the method comprises contacting the gRNA- expressing cell with a gRNA ligation adapter comprising a capturing sequence and a 5’ ligationAttorney Docket No.43487-1046601 end. In some embodiments, the method comprises ligating the 5’ ligation end of the gRNA ligation adapter to the gRNA, thereby generating a tagged gRNA comprising the capturing sequence. In some embodiments, the method comprises generating a partition comprising 1) the gRNA-expressing cell, and 2) a plurality of barcoded oligonucleotides comprising a partition- specific barcode and a capture sequence. In some embodiments, the partition comprises the gRNA-expressing cell and no other cells. In some embodiments, each barcoded oligonucleotide of the plurality of barcoded oligonucleotides comprises the partition-specific barcode and the capture sequence. In some embodiments, the method comprises hybridizing the capturing sequence to the capture sequence of a barcoded oligonucleotide of the plurality of barcoded oligonucleotides. In some embodiments, the method comprises using the barcoded oligonucleotide and the tagged gRNA to generate a barcoded spacer oligonucleotide. In some embodiments, the barcoded spacer oligonucleotide comprises 1) the partition-specific barcode or a complement thereof, and 2) a sequence of the spacer or a complement thereof. In some embodiments, the method comprises extending the barcoded oligonucleotide using the tagged gRNA as template, thereby generating a barcoded spacer oligonucleotide comprising the partition-specific barcode and a sequence complementary to the spacer sequence. In some embodiments, the 5’ ligation end of the gRNA ligation adapter is ligated to the gRNA prior to generating the partition. In some embodiments, the 5’ ligation end of the gRNA ligation adapter is ligated to the gRNA after generating the partition. In some embodiments, the method further comprises sequencing the barcoded spacer oligonucleotide. In some embodiments, the method comprises analyzing the results of the sequencing to determine the sequence of the spacer sequence and the partition-specific barcode, and associating the spacer sequence with the partition-specific barcode.

[0141] In various embodiments of any of the methods provided herein involving use of agRNA ligation adapter, the gRNA ligation adapter can be a single molecule (e.g. one nucleic acid) or more than one molecule (e.g. two nucleic acids). In some embodiments, the gRNA ligation adapter is configured to provide efficient ligation to the gRNA. In some aspects, hybridization of the gRNA ligation adapter to the gRNA brings the ligation adapter and 3’ ligation end thereof into proximity with the 5’ end of the gRNA (e.g. as in FIGS.43A-B). In some embodiments, hybridization of the gRNA ligation adapter to the gRNA brings the ligation adapter and 5’ ligation end thereof into proximity with the 3’ end of the gRNA (e.g. as shown in FIG.43C). In some aspects, the ligation adapter can further serve as a template for the ligation. For example, in some embodiments, the 5’ end of the gRNA and the 3’ ligation end of the gRNA ligation adapter hybridize to adjacent sequences on the gRNA ligation adapter, and are ligated using the gRNA ligation adapter as template. Alternatively, the 3’ end of the gRNA and the 5’Attorney Docket No.43487-1046601 ligation end of the gRNA ligation adapter hybridize to adjacent sequences on the gRNA ligation adapter, and are ligated using the gRNA ligation adapter as template. In some embodiments, the gRNA ligation adapter does not need to hybridize to the gRNA, and ligation can still be achieved (e.g. by using an increased concentration of gRNA ligation adapter and / or enzyme facilitating ligation). However, in some aspects, hybridization of the gRNA ligation adapter to the gRNA and to itself (e.g. within a self-hybridizing region) as described herein provide the advantage of increasing the efficiency and specificity of ligation.

[0142] In some aspects, the gRNA ligation adapter facilitates gRNA sequencing of gRNAscomprising a spacer sequence at a 5’ end (i.e. a 5’ spacer), such as Cas9-compatible gRNAs. In some embodiments, provided herein are gRNA ligation adapters for sequencing gRNAs having a spacer at a 5’ end of the gRNA. In some embodiments, provided herein are gRNA ligation adapters for sequencing gRNAs having a 5’ spacer. In some aspects, provided herein is a method for sequencing a gRNA having a 5’ spacer using a gRNA ligation adapter, such as any gRNA ligation adapter described herein. Exemplary gRNA ligation adapters for sequencing gRNAs with 5’ spacers are illustrated in FIGS.43A-B.

[0143] In various embodiments of any of the methods provided herein involving use of agRNA ligation adapter, the gRNA ligation adapter comprises the functional region; a 5’ hybridizing region that hybridizes to the gRNA; and a self-hybridizing region, wherein the self- hybridizing region comprises a first sequence and second sequence that hybridize to one another, wherein the second sequence of the self-hybridizing region comprises the 3’ ligation end, and wherein the 3’ ligation end is configured to be ligated to the 5’ end of the gRNA upon hybridization of the 5’ hybridizing region to the gRNA.

[0144] In some embodiments, the gRNA ligation adapter comprises a first gRNA ligationadapter nucleic acid molecule and a second gRNA ligation adapter nucleic acid molecule. In some embodiments, the first gRNA ligation adapter nucleic acid molecule comprises the 5’ hybridizing region that hybridizes to the gRNA, and the first sequence of the self-hybridizing region; and the second gRNA ligation adapter nucleic acid molecule comprises the functional region and the second sequence of the self-hybridizing region comprising the 3’ ligation end.

[0145] In some embodiments, the gRNA ligation adapter is a single molecule gRNA ligationadapter. In some embodiments, the single molecule gRNA ligation adapter comprises in the 5’ to 3’ direction: the 5’ hybridizing region, the first sequence of the self-hybridizing region, the functional region, and the second sequence of the self-hybridizing region comprising the 3’ ligation end that is configured to be ligated to the 5’ end of the gRNA upon hybridization of the 5’ hybridizing region to the gRNA. In some embodiments, the single molecule gRNA ligation adapter has a stem-loop structure. In some embodiments, the functional region is in the loop ofAttorney Docket No.43487-1046601 the stem-loop structure. In some embodiments, the functional region comprises a barcode sequence.

[0146] In some embodiments, the functional region comprises a sample-specific barcodesequence. In some embodiments, the functional region comprises one or more functional sequences. In some embodiments, the one or more functional sequences of the functional region comprise a primer hybridization sequence, a sequencing primer binding site, or complement thereof.

[0147] In some embodiments, the gRNA ligation adapter comprises a polymerase block site.In some embodiments, the polymerase block site is configured to terminate 3’ extension of a polynucleotide by a polymerase using the gRNA ligation adapter as template. In some aspects, the polymerase block site allows for a polymerization reaction in the workflow to terminate without incorporating unwanted and / or unnecessary sequences in a product which may interfere, for example, in downstream processing steps. For example, a complement of the first sequence of the self-hybridizing region can be excluded from an extension product by termination prior to the polymerase reaching the first sequence of the self-hybridizing region, such that the extension product does not self-hybridize. In some embodiments, the polymerase block site is 5’ of the functional region. In some embodiments, the polymerase block site is 5’ of the capturing sequence in the gRNA ligation adapter. In some embodiments, the polymerase block site is 3’ of the first sequence of the self-hybridizing region. In some embodiments, the polymerase block site comprises an abasic site. In some embodiments, the polymerase block site comprises uracil, and the uracil is removed to generate the abasic site. In some embodiments, the uracil is removed by contacting the uracil with a Uracil-DNA Glycosylase (UDG) enzyme or a Uracil-Specific Excision Reagent (USER) enzyme. In some embodiments, the polymerase block site terminates extension of the barcoded oligonucleotide using the tagged gRNA as template. In some embodiments, the polymerase block site is 5’ of the capturing sequence in the gRNA ligation adapter. In some embodiments, the polymerase block site terminates extension of the primer that hybridizes to the constant region of the gRNA during the generation of the tagged gRNA complement.

[0148] In some aspects, gRNAs transcribed in cells from an expression vector (e.g. from aPol III promoter such as a U6 promoter) do not comprise a 5’ monophosphate. For example, pre- modified gRNAs typically include a 5’ triphosphate. Thus, in some embodiments, the method comprises modifying a pre-modified gRNA to generate the gRNA comprising the 5’ monophosphate. The modification can be performed by any suitable means and chemistry available to one having skill in the art. In some embodiments, the pre-modified gRNA comprises a 5’ triphosphate, and the method comprises modifying the 5’ triphosphate to generate the 5’Attorney Docket No.43487-1046601 monophosphate. In some embodiments, the method comprises contacting the pre-modified gRNA with an enzyme to generate gRNA comprising the 5’ monophosphate. In some embodiments, the enzyme is RNA 5’ Pyrophosphohydrolase (RppH). In some embodiments, gRNAs comprising a 5’ monophosphate can be directly introduced into cells, such that no modification is necessary.

[0149] The hybridization region of the gRNA ligation adapter can be provided in anysuitable configuration to allow hybridization to the gRNA. In some embodiments, the 5’ hybridizing region hybridizes to the spacer sequence of the gRNA. In some embodiments, the 5’ hybridizing region hybridizes to the constant region of the gRNA. In some embodiments, the 5’ hybridizing region hybridizes to the spacer sequence of the gRNA and the constant region of the gRNA. In some embodiments, the 5’ hybridizing region hybridizes only to the spacer sequence of the gRNA and not to the constant region of the gRNA. In some embodiments, the 5’ hybridizing region hybridizes only to the constant region of the gRNA and not to the spacer sequence of the gRNA.

[0150] In some embodiments, the 5’ hybridizing region comprises a non-specifichybridization region. In some embodiments, the non-specific hybridization region comprises a sequence of residues capable of hybridizing to different spacer sequences. In some embodiments, the non-specific hybridization region comprises inosine residues. In some embodiments, the non- specific hybridization region comprises a sequence of inosine residues capable of hybridizing to different spacer sequences. In some embodiments, the 5’ hybridizing region comprises a sequence that is complementary to a portion of the constant region of the gRNA. In some embodiments, the sequence that is complementary to a portion of the constant region of the gRNA is at the 5’ end of the 5’ hybridizing region. In some aspects, the non-specific hybridization region can allow the same gRNA ligation adapter to be used for a wide range of different gRNA molecules having different spacer sequences. In some embodiments, the 5’ hybridizing region can comprise a non-specific hybridization region (e.g. inosine residues for non-specifically hybridizing to gRNA spacers), as well as a sequence that hybridizes to a constant region sequence adjacent to the gRNA spacer, thus allowing both non-specific spacer hybridization while providing specificity for gRNA molecules in general (e.g. versus non-gRNA molecules in the cell).

[0151] In some embodiments, the 5’ hybridizing region comprises a non-hybridizing portionand a hybridizing portion. In some embodiments, the non-hybridizing portion comprises a carbon spacer. In some embodiments, the hybridizing portion hybridizes to at least a portion of the gRNA spacer and / or at least a portion of the constant region of the gRNA. In some embodiments, hybridizing portion provides specificity for hybridizing to the gRNA, whereas theAttorney Docket No.43487-1046601 non-hybridizing portion allows the gRNA ligation adapter to not be limited to hybridizing to gRNA molecules with specific gRNA spacers.

[0152] In some embodiments, the gRNA ligation adapter facilitates gRNA sequencing ofgRNAs comprising a spacer sequence at a 3’ end (i.e. a 3’ spacer), such as Cas12a-compatible gRNAs. In some embodiments, provided herein are gRNA ligation adapters for sequencing gRNAs having a spacer at a 3’ end of the gRNA. In some embodiments, provided herein are gRNA ligation adapters for sequencing gRNAs having a 3’ spacer. In some aspects, provided herein is a method for sequencing a gRNA having a 3’ spacer using a gRNA ligation adapter, such as any gRNA ligation adapter described herein. An exemplary gRNA ligation adapter for sequencing a gRNA having a 3’ spacer is illustrated in FIG.43C.

[0153] In some embodiments, the gRNA ligation adapter comprises a capturing sequence. Insome aspects, the capturing sequence facilitates hybridization of the tagged gRNA to the barcoded oligonucleotide to allow generation of the barcoded spacer oligonucleotide, e.g. by a nucleic acid extension reaction. In some embodiments, the gRNA ligation adapter comprises a 5’ ligation end. In some embodiments, the gRNA ligation adapter is configured to promote ligation of the 5’ ligation end to the 3’ end of the gRNA, such as via hybridization, as described below. In some embodiments, the gRNA ligation adapter comprises a 5’ monophosphate. In some aspects, for methods involving gRNA ligation adapters for sequencing a gRNA having a 3’ spacer, the gRNA does not need to be modified to generate a 5’ monophosphate on the gRNA, since the 5’ end of the gRNA is not included in the ligation reaction to generate the tagged gRNA.

[0154] In some embodiments, the gRNA ligation adapter comprises a 3’ hybridizing regionthat hybridizes to the gRNA. In some embodiments, the gRNA ligataion adapter comprises a self-hybridizing region. In some embodiments, the self-hybridizing region comprises a first sequence and second sequence that hybridize to one another. In some embodiments, the second sequence of the self-hybridizing region comprises the 5’ ligation end. In some embodiments, the 5’ ligation end is configured to be ligated to the 3’ end of the gRNA upon hybridization of the 3’ hybridizing region to the gRNA. In some embodiments, the gRNA ligation adapter comprises: the capturing sequence; a 3’ hybridizing region that hybridizes to the gRNA; and a self- hybridizing region, wherein the self-hybridizing region comprises a first sequence and second sequence that hybridize to one another, wherein the second sequence of the self-hybridizing region comprises the 5’ ligation end, and wherein the 5’ ligation end is configured to be ligated to the 3’ end of the gRNA upon hybridization of the 3’ hybridizing region to the gRNA.

[0155] The gRNA ligation adapter may consist of one or more molecules. In someembodiments, the gRNA ligation adapter comprises a first gRNA ligation adapter nucleic acidAttorney Docket No.43487-1046601 molecule and a second gRNA ligation adapter nucleic acid molecule. In some embodiments, the first gRNA ligation adapter nucleic acid molecule comprises the 3’ hybridizing region that hybridizes to the gRNA and the first sequence of the self-hybridizing region. In some embodiments, the second gRNA ligation adapter nucleic acid molecule comprises the capturing sequence and the second sequence of the self-hybridizing region comprising the 5’ ligation end.

[0156] In some embodiments, the gRNA ligation adapter is a single molecule gRNA ligationadapter. In some embodiments, the single molecule gRNA ligation adapter comprises in the 3’ to 5’ direction: the 3’ hybridizing region, the first sequence of the self-hybridizing region, the capturing sequence, and the second sequence of the self-hybridizing region comprising the 5’ ligation end. In some embodiments, the 5’ ligation end is configured to be ligated to the 3’ end of the gRNA upon hybridization of the 3’ hybridizing region to the gRNA. In some embodiments, the single molecule gRNA ligation adapter has a stem-loop structure. In some embodiments, the capturing sequence is in the loop of the stem-loop structure. In some embodiments, the 5’ ligation end of the gRNA ligation adapter comprises a 5’ monophosphate.

[0157] The gRNA ligation adapter can comprise one or more additional sequences, such as afunctional sequence and / or a barcode. In some embodiments, the gRNA ligation adapter further comprises a sample-specific barcode sequence, and wherein the barcoded spacer oligonucleotide further comprises the sample-specific barcode sequence or a complement thereof.

[0158] In some embodiments, the constant region of the gRNA further comprises afunctional sequence. In some embodiments, the functional sequence is at the 5’ end of the constant region of the gRNA. In some embodiments, the functional sequence is within and / or flanked by the scaffold sequence of the gRNA. In some embodiments, the functional sequence comprises a primer hybridization sequence, a sequencing primer binding site, or a complement thereof.

[0159] The hybridization region of the gRNA ligation adapter can be provided in anysuitable configuration to allow hybridization to the gRNA, and / or to configure the 5’ ligation end to be ligated to the 3’ end of the gRNA. In some embodiments, the 3’ hybridizing region hybridizes to the spacer sequence of the gRNA. In some embodiments, 3’ hybridizing region hybridizes to the constant region of the gRNA. In some embodiments, the 3’ hybridizing region hybridizes to the spacer sequence of the gRNA and the constant region of the gRNA. In some embodiments, the 3’ hybridizing region comprises a non-specific hybridization region. In some embodiments, the non-specific hybridization region comprises a sequence of residues capable of hybridizing to different spacer sequences. In some embodiments, the non-specific hybridization region comprises inosine residues. In some embodiments, the non-specific hybridization region comprises a sequence of inosine residues capable of hybridizing to different spacer sequences. InAttorney Docket No.43487-1046601 some embodiments, the 3’ hybridizing region comprises a sequence that is complementary to a portion of the constant region of the gRNA. In some embodiments, the sequence that is complementary to a portion of the constant region of the gRNA is at the 3’ end of the 3’ hybridizing region. In some aspects, the non-specific hybridization region can allow the same gRNA ligation adapter to be used for a wide range of different gRNA molecules having different spacer sequences. In some embodiments, the 3’ hybridizing region can comprise a non-specific hybridization region (e.g. inosine residues for non-specifically hybridizing to gRNA spacers), as well as a sequence that hybridizes to a constant region sequence adjacent to the gRNA spacer, thus allowing both non-specific spacer hybridization while providing specificity for gRNA molecules in general (e.g. versus non-gRNA molecules in the cell).

[0160] In some embodiments, the 3’ hybridizing region comprises a non-hybridizing portionand a hybridizing portion. In some embodiments, the non-hybridizing portion comprises a carbon spacer. In some embodiments, the hybridizing portion hybridizes to at least a portion of the gRNA spacer and / or at least a portion of the constant region of the gRNA. In some embodiments, the hybridizing portion provides specificity for hybridizing to the gRNA, whereas the non-hybridizing portion allows the gRNA ligation adapter to not be limited to hybridizing to gRNA molecules with specific gRNA spacers. gRNA sequencing, gRNA sequencing in a plurality of single cells, and gRNA sequencing in combination with additional analyte sequencing

[0161] In some aspects, any of the workflows for analyzing and / or sequencing gRNAs canbe performed in combination with analysis of additional analytes. In some embodiments, the additional analytes are target nucleic acids. For example, FIG.39 shows an exemplary workflow in which gRNA-expressing cells expressing gRNAs and other analytes (e.g. cellular transcripts) are analyzed. Barcoded spacer oligonucleotides and barcoded analyte oligonucleotides can be generated from the same single cells, for example as described herein. In some embodiments, the method comprises sequencing the barcoded spacer oligonucleotides or derivatives thereof and barcoded analyte oligonucleotides or derivatives thereof. In some embodiments, the method comprises analyzing the results of the sequencing. For example, the barcoded spacer oligonucleotides and barcoded analyte oligonucleotides can be amplified and sequenced to determine the presence and / or abundance of gRNAs and analytes at the single-cell level in a plurality of single cells.

[0162] In some aspects, a workflow for detecting and / or sequencing an analyte in parallelwith gRNA sequencing as described herein is shown in FIG.40. Ligatable probe pairs specific for any number of analytes (e.g. cellular transcripts) can be ligated and used to generate barcodedAttorney Docket No.43487-1046601 analyte oligonucleotides, for example as described in Example 11A and in various sections of the specification. While specific aspects of how sequencing an analyte can be performed are described in this section, any suitable alternative method that can be employed in parallel with the gRNA sequencing workflows described herein can be used.

[0163] In some aspects, any of the methods described above for gRNA sequencing can beperformed in combination with methods for detecting one or more other target nucleic acids. Such methods can facilitate single-cell analysis of gRNA-expressing cells, for example to allow gRNA detection and transcriptome analysis in the same single cells. These methods can facilitate powerful large-scale CRISPR perturbation screens, for example as described herein. For example, in some embodiments, the method further comprises contacting the gRNA-expressing cell with a ligatable probe pair comprising a first ligatable probe and a second ligatable probe that hybridize to a target nucleic acid in the gRNA-expressing cell. In some embodiments, the method comprises ligating the first ligatable probe to the second ligatable probe using the target nucleic acid as template to generate a ligated probe pair. In some embodiments, the method comprises using the ligated probe pair and a second barcoded oligonucleotide of the plurality of barcoded oligonucleotides to generate a barcoded analyte oligonucleotide comprising a sequence of the ligated probe pair or complement thereof, and the partition-specific barcode or a complement thereof.

[0164] In some embodiments, the method comprises sequencing the barcoded spaceroligonucleotide or a derivative thereof and the barcoded analyte oligonucleotide or a derivative thereof. In some embodiments, the method comprises analyzing the results of the sequencing to determine the sequence of the spacer sequence. In some embodiments, the method comprises analyzing the results of the sequencing to determine the presence and / or abundance of the gRNA and / or the target nucleic acid in the gRNA-expressing cell. In some embodiments, the first ligatable probe comprises a 3’ overhang and a 5’ hybridizing region that hybridizes to the target nucleic acid, and the second ligatable probe comprises a 5’ overhang and a 3’ hybridizing region that hybridizes to the target nucleic acid. In some embodiments, the ligated probe pair comprises a sequence that is complementary to and / or indicative of the target nucleic acid. In some embodiments, the barcoded analyte oligonucleotide comprises a sequence that is complementary to and / or indicative of the target nucleic acid. In some embodiments, the method comprises hybridizing a sequence of the 3’ overhang of the ligated probe pair to the second barcoded oligonucleotide, and extending the 3’ end of the ligated probe pair and / or extending the 3’ end of the barcoded oligonucleotide to generate the barcoded analyte oligonucleotide. In some embodiments, the target nucleic acid can be any suitable nucleic acid for analysis described herein. The target nucleic acid can be an endogenous analyte. The target nucleic acid can be aAttorney Docket No.43487-1046601 nucleic acid associated with an analyte to be detected in the cell. In some embodiments, the target nucleic acid is not a gRNA. In some embodiments, the target nucleic acid comprises DNA. In some embodiments, the target nucleic acid comprises RNA. In some embodiments, the target nucleic acid is an RNA molecule. In some embodiments, the target nucleic acid is an mRNA.

[0165] In some embodiments, a plurality of target nucleic acids can be analyzed in additionto the gRNA. For example, in some embodiments, the method further comprises contacting the gRNA-expressing cell with a plurality of ligatable probe pairs that hybridize to a plurality of different target nucleic acids in the cell. In some embodiments, the method comprises ligating the plurality of ligatable probe pairs using the plurality of different target nucleic acids as templates to generate a plurality of ligated probe pairs. In some embodiments, the method comprises using the plurality of ligated probe pairs and the plurality of barcoded oligonucleotides to generate a plurality of barcoded analyte oligonucleotides. In some embodiments, a barcoded analyte oligonucleotide of the plurality of barcoded analyte oligonucleotides comprises a sequence of a ligated probe pair of the plurality of ligated probe pairs or a complement thereof and a sequence of the partition-specific barcode or complement thereof. In some embodiments, a barcoded analyte oligonucleotide of the plurality of barcoded analyte oligonucleotides comprises a sequence of a target nucleic acid of the plurality of different target nucleic acids or a complement thereof and a sequence of the partition-specific barcode or complement thereof. In some embodiments, the method further comprises sequencing the plurality of barcoded analyte oligonucleotides or derivatives thereof. In some embodiments, the method further comprises analyzing the results of the sequencing to determine the presence and / or abundance of the different target nucleic acids in the gRNA-expressing cell. In some embodiments, the method is performed in parallel for a plurality of gRNA-expressing cells. In some embodiments, different partitions are generated for different gRNA-expressing cells of the plurality of gRNA-expressing cells. In some embodiments, barcoded spacer oligonucleotides comprising partition-specific barcodes are generated from the different gRNA-expressing cells. In some embodiments, barcoded analyte oligonucleotides are generated from the different gRNA-expressing cells. In some embodiments, the method comprises sequencing the barcoded spacer oligonucleotides or derivatives thereof. In some embodiments, the method comprises sequencing the barcoded analyte oligonucleotides or derivatives thereof. In some embodiments, the method comprises analyzing the results of the sequencing to determine the presence and / or abundance of one or more gRNAs and one or more target nucleic acids in the different gRNA- expressing cells of the plurality of gRNA-expressing cells.

[0166] In some embodiments, the method comprises contacting the gRNA-expressing cellwith a ligatable probe pair comprising 1) a first ligatable probe having a 3’ overhang, and a 5’Attorney Docket No.43487-1046601 hybridizing region that hybridizes to a target nucleic acid in the cell, and 2) a second ligatable probe having a 3’ hybridizing region that hybridizes to the target nucleic acid in the cell, and a 5’ overhang. In some embodiments, the method comprises ligating the 5’ hybridizing region of the first ligatable probe to the 3’ hybridizing region of the second ligatable probe using the target nucleic acid as template, thereby generating a ligated probe pair comprising a sequence complementary to and / or indicative of the target nucleic acid. In some embodiments, the method comprises hybridizing a sequence of the 3’ overhang to the capture sequence of a barcoded oligonucleotide of the plurality of barcoded oligonucleotides in the partition, In some embodiments, the method comprises extending the 3’ end of the ligated probe pair to incorporate a sequence complementary to the barcoded oligonucleotide and / or extending the 3’ end of the barcoded oligonucleotide to incorporate a sequence complementary to the ligated probe pair, thereby generating a barcoded analyte oligonucleotide comprising: the sequence of the ligated probe pair or complement thereof, and the sequence of the barcoded capture oligonucleotide or complement thereof. In some embodiments, the method further comprises sequencing the barcoded analyte oligonucleotide to determine the sequence complementary to and / or indicative of the target nucleic acid and the sequence of the partition-specific barcode, and associating the target nucleic acid with the partition-specific barcode. In some embodiments, the 3’ overhang of the first ligatable probe and / or the 5’ overhang of the second ligatable probe comprise a barcode sequence. In some embodiments, the 3’ overhang of the first ligatable probe and / or the 5’ overhang of the second ligatable probe comprise a sample-specific barcode sequence. In some embodiments, the 3’ overhang of the first ligatable probe and / or the 5’ overhang of the second ligatable probe comprise one or more functional sequences. In some embodiments, the one or more functional sequences of the 3’ overhang of the first ligatable probe and / or the 5’ overhang of the second ligatable probe comprise a primer hybridization sequence, a sequencing primer binding site, or complement thereof. In some embodiments, the first ligatable probe is ligated to the second ligatable probe in the partition. In some embodiments, the first ligatable probe is ligated to the second ligatable probe prior to generating the partition. In some embodiments, the plurality of barcoded oligonucleotides comprise one or more functional sequences.

[0167] In some embodiments, the one or more functional sequences of the plurality ofbarcoded oligonucleotides comprise a primer hybridization sequence, a sequencing primer binding site, or complement thereof. In some embodiments, each barcoded oligonucleotide of the plurality of barcoded oligonucleotides comprises a unique molecular identifier (UMI) sequence. In some embodiments, the method comprises sequencing the barcoded analyte oligonucleotide and the barcoded spacer oligonucleotide, thereby determining the presence of the target analyte and the presence of the gRNA having the spacer sequence in the same cell. In someAttorney Docket No.43487-1046601 embodiments, the barcoded spacer oligonucleotide and barcoded analyte oligonucleotide are amplified and / or sequenced outside of the partition. In some embodiments, the method is performed in parallel for a plurality of gRNA-expressing cells, such that a different partition is generated for each gRNA-expressing cell of the plurality of gRNA-expressing cells, and wherein one or more barcoded spacer oligonucleotides are generated from each gRNA-expressing cell. In some embodiments, one or more barcoded analyte oligonucleotides are generated from each gRNA-expressing cell. In some embodiments, the method comprises sequencing the one or more barcoded spacer oligonucleotides and / or the one or more barcoded analyte oligonucleotides from each gRNA-expressing cell. In some embodiments, for each gRNA expressing cell, the presence and / or abundance of one or more gRNA spacer sequences is determined. In some embodiments, for each gRNA expressing cell, the presence and / or abundance of one or more target nucleic acids is determined.

[0168] In some embodiments, provided herein is a composition or kit. In some embodiments,the composition or kit comprises any of the probes and / or other nucleic acids provided in connection with the methods herein for sequencing gRNAs, and / or sequencing or detecting one or more non-gRNA analytes (e.g. target nucleic acids). In some embodiments, the composition or kit comprises a gRNA-targeting probe, such as any described in connection with the methods provided herein. In some embodiments, the composition or kit comprises a gRNA ligation adapter, such as any described in connection with the methods provided herein. In some embodiments, the composition or kit comprises one or a plurality of ligatable probe pairs, such as any described in connection with the methods provided herein. In some embodiments, the composition or kit comprises the gRNA-targeting probe and one or a plurality of ligatable probe pairs. In some embodiments, the composition or kit comprises the gRNA ligation adapter and one or a plurality of ligatable probe pairs. In some embodiments, the composition or kit comprises a template switch oligonucleotide (TSO), such as any described in connection with the methods provided herein. In some embodiments, the composition or kit comprises a plurality of barcoded oligonucleotides, such as any described in connection with the methods provided herein. In some embodiments, the composition or kit comprises one or more enzymes, such as any described in connection with the methods provided herein, including a ligase, RppH, RNAse H, a USER enzyme, a UDG enzyme, and / or a ligase.

[0169] In some embodiments, provided herein are systems for analyzing gRNA-expressingcells according to any of the methods provided herein. In some embodiments, the systems comprise any of the compositions or kits provided herein. In some embodiments, the system further comprises one or more components for performing the methods. In some embodiments, the system comprises a partition or a plurality of partitions. In some embodiments, the systemAttorney Docket No.43487-1046601 comprises a device for generating partitions, such as wells or droplets. In some embodiments, the system comprises wells for the partitioning. In some embodiments, the system comprises means for sequencing the barcoded spacer oligonucleotides and / or the barcoded analyte oligonucleotides. In some embodiments, the system comprises a sequencer. In some embodiments, the system comprises one or more devices, processors, and / or computers for analyzing the results of the sequencing. SAMPLES, COMPOSITIONS, SYSTEMS, AND ANALYSIS Fixed Samples

[0170] A sample may be a fixed sample. For example, a sample may comprise a plurality offixed samples, such as a plurality of fixed cells or fixed nuclei. Alternatively or in addition, a sample may comprise a fixed tissue. Fixation of cell or cellular constituent, or a tissue comprising a plurality of cells or nuclei, may comprise application of a chemical species or chemical stimulus. The term “fixed” as used herein with regard to biological samples generally refers to the state of being preserved from decay and / or degradation. “Fixation” generally refers to a process that results in a fixed sample, and in some instances can include contacting the biomolecules within a biological sample with a fixative (or fixation reagent) for some amount of time, whereby the fixative results in covalent bonding interactions such as crosslinks between biomolecules in the sample. A “fixed biological sample” may generally refer to a biological sample that has been contacted with a fixation reagent or fixative. For example, a formaldehyde- fixed biological sample has been contacted with the fixation reagent formaldehyde. “Fixed cells”, “fixed nuclei” or “fixed tissues” refer to cells / nuclei or tissues that have been in contact with a fixative under conditions sufficient to allow or result in the formation of intra- and inter- molecular covalent crosslinks between biomolecules in the biological sample. Generally, contact of biological sample (e.g., a cell or nucleus) with a fixation reagent (e.g., paraformaldehyde or PFA) results in the formation of intra- and inter-molecular covalent crosslinks between biomolecules in the biological sample. In some cases, the fixation reagent, formaldehyde, may result in covalent aminal crosslinks within RNA, DNA, and / or protein molecules. For example, the widely used fixative reagent, paraformaldehyde or PFA, fixes tissue samples by catalyzing crosslink formation between basic amino acids in proteins, such as lysine and glutamine. Both intra-molecular and inter-molecular crosslinks can form in the protein. These crosslinks can preserve protein secondary structure and also eliminate enzymatic activity in the preserved tissue sample. Examples of fixation reagents include but are not limited to aldehyde fixatives (e.g., formaldehyde, also commonly referred to as “paraformaldehyde,” “PFA,” and “formalin”; glutaraldehyde; etc.), imidoesters, NHS (N-Hydroxysuccinimide) esters, and the like.Attorney Docket No.43487-1046601

[0171] In some embodiments, the fixative or fixation reagent useful for fixing samples isformaldehyde. The term “formaldehyde” when used in the context of a fixative may also refer to “paraformaldehyde” (or “PFA”) and “formalin”, both of which are terms with specific meanings related to the formaldehyde composition (e.g., formalin is a mixture of formaldehyde and methanol). Thus, a formaldehyde-fixed biological sample may also be referred to as formalin- fixed or PFA-fixed. Protocols and methods for the use of formaldehyde as a fixation reagent to prepare fixed biological samples are well known in the art and can be used in the methods and compositions of the present disclosure. For example, suitable ranges of formaldehyde concentrations for use in preparing a fixed biological sample is 0.1 to 10%, 1-8%, 1-4%, 1-2%, 3-5%, or 3.5-4.5%. In some embodiments of the present disclosure the biological sample is fixed using a final concentration of 1% formaldehyde, 4% formaldehyde, or 10% formaldehyde. Typically, the formaldehyde is diluted from a more concentrated stock solution – e.g., a 35%, 25%, 15%, 10%, 5% PFA stock solution.

[0172] Other examples of fixatives include, for example, organic solvents such as alcohols(e.g., methanol or ethanol), ketones (e.g., acetone), and aldehydes (e.g., paraformaldehyde, formaldehyde (e.g., formalin), or glutaraldehyde). As described herein, cross-linking agents may also be used for fixation including, without limitation, disuccinimidyl suberate (DSS), dimethylsuberimidate (DMS), formalin, and dimethyladipimidate (DMA), dithio-bis(- succinimidyl propionate) (DSP), disuccinimidyl tartrate (DST), and ethylene glycol bis(succinimidyl succinate) (EGS). In some cases, a cross-linking agent may be a cleavable cross-linking agent (e.g., thermally cleavable, photocleavable, etc.).

[0173] In some cases, more than one fixation reagent can be used in combination whenpreparing a fixed biological sample. For example, a first fixation agent, such as an organic solvent, may be used in combination with a second fixation agent, such as a cross-linking agent. The organic solvent may be an alcohol (e.g., ethanol or methanol), ketone (e.g., acetone), or aldehyde (e.g., paraformaldehyde, formaldehyde, or glutaraldehyde). The cross-linking agent may be selected from the group consisting of disuccinimidyl suberate (DSS), dimethylsuberimidate (DMS), formalin, and dimethyladipimidate (DMA), dithio-bis(- succinimidyl propionate) (DSP), disuccinimidyl tartrate (DST), and ethylene glycol bis(succinimidyl succinate) (EGS). In some cases, a first fixation agent may be provided to or brought into contact with the cell or nucleus to bring about a change in a first characteristic or set of characteristics of the cell / nucleus, and a fixation agent may be provided to or brought into contact with the cell or nucleus to bring about a change in a second characteristic or set of characteristics of the cell or nucleus. For example, a first fixation agent may be provided to or brought into contact with a cell or nucleus to bring about a change in a dimension of the cellAttorney Docket No.43487-1046601 (e.g., a reduction in cross-sectional diameter, see, e.g., U.S. Pat. Pub. No.2020 / 0033237, which is incorporated herein by reference in its entirety), and a second fixation agent may be provided to or brought into contact with a cell or nucleus to bring about a change in a second characteristic or set of characteristics of the cell (e.g., forming crosslinks within and / or surrounding the cell or nucleus). The first and second fixation agents may be provided to or brought into contact with the cell or nucleus at the same or different times. Other suitable fixing agents include those disclosed in, e.g., International PCT App. No. PCT / US2020 / 066705, which is incorporated herein by reference in its entirety.

[0174] In an example, a first fixation agent that is an organic solvent may be provided to acell to change a first characteristic (e.g., cell size) and a second fixation agent that is a cross- linking agent may be provided to a cell to change a second characteristic (e.g., cell fluidity or rigidity). The first fixation agent may be provided to the cell before the second fixation agent.

[0175] In another embodiment, biomolecules (e.g., biological samples such as tissuespecimens) are contacted with a fixation reagent containing both formaldehyde and glutaraldehyde, and thus the contacted biomolecules can include fixation crosslinks resulting both from formaldehyde induced fixation and glutaraldehyde induced fixation. Typically, a suitable concentration of glutaraldehyde for use as a fixation reagent can be 0.1 to 1%. Fixation and wash reagents may also include commercially available products, e.g., BioLegend® Fixation Buffer (420801) and Permeabilization Wash Buffer (421002).

[0176] Changes to a characteristic or a set of characteristics of a cell or cellular constituents(e.g., incurred upon interaction with one or more fixation agents) may be at least partially reversible (e.g., via rehydration or de-crosslinking). Alternatively, changes to a characteristic or set of characteristics of a cell or cellular constituents (e.g., incurred upon interaction with one or more fixation agents) may be substantially irreversible.

[0177] A sample (e.g., a cell sample) may be subjected to a fixation process at any usefulpoint in time. For example, cells, nuclei and / or cellular / nuclear constituents of a sample may be subjected to a fixation process involving one or more fixation agents (e.g., as described herein) prior to commencement of any subsequent processing, such as for storage. Cells, nuclei and / or cellular / nuclear constituents, such as cells, nuclei and / or cellular / nuclear constituents of a tissue sample, subjected to a fixation process prior to storage, may be stored in an aqueous solution, optionally in combination with one or more preserving agents configured to preserve morphology, size, or other features of the cells and / or cellular components. Fixed cells, nuclei and / or cellular / nuclear constituents may be stored below room temperature, such as in a freezer. Alternatively, cells, nuclei and / or cellular / nuclear constituents of a sample may be subjected to a fixation process involving one or more fixation agents subsequent to one or more otherAttorney Docket No.43487-1046601 processes, such as filtration, centrifugation, agitation, selective precipitation, purification, permeabilization, isolation, heating, etc. For example, cells, nuclei, and / or cellular / nuclear constituents of a given type from a sample may be subjected to a fixation process following a separation and / or enrichment procedure (e.g., as described herein). In an example, a sample comprising a plurality of cells including a plurality of cells of a given type may be subjected to a positive separation process to provide a sample enriched in the plurality of cells of the given type. The enriched sample may then be subjected to a fixation process involving one or more fixation agents (e.g., as described herein) to provide an enriched sample comprising a plurality of fixed cells. A fixation process may be performed in a bulk solution. In some cases, fixed samples (e.g., fixed cells, fixed nuclei, and / or cellular / nuclear constituents) may be partitioned amongst a plurality of partitions (e.g., droplets or wells) and subjected to processing as described elsewhere herein. In some cases, fixed samples may undergo additional processing, such as partial or complete reversal of a fixation process by, for example, rehydration or de-crosslinking, prior to partitioning and any subsequent processing. In some cases, fixed samples may undergo partial or complete reversal of a fixation process within a plurality of partitions (e.g., prior to or concurrent with additional processing described elsewhere herein).

[0178] In some cases, a tissue specimen comprising a plurality of cells, nuclei and / orcellular / nuclear constituents may be processed to provide formalin-fixed paraffin-embedded (FFPE) tissue. A tissue specimen may be contacted (e.g., saturated) with formalin and then embedded in paraffin wax. FFPE processing may facilitate preservation of a tissue sample (e.g., prior to subsequent processing and analysis). A tissue sample, including an FFPE tissue sample, may additionally or alternatively be subjected to storage in a low-temperature freezer. Cells, nuclei and / or cellular / nuclear constituents may be dissociated from a tissue sample (e.g., FFPE tissue sample) prior to undergoing subsequent processing. In some cases, individual cells, nuclei and / or cellular / nuclear constituents of a tissue sample such as an FFPE tissue sample may be optically detected, labeled, or otherwise processed prior to any such dissociation. Such detection, labeling, or other processing may be performed according to a 2- or 3-dimensional array and optionally according to a pre-determined pattern. Methods of Nucleic Acid Analysis

[0179] In an aspect, the present disclosure provides a method for barcoding nucleic acidmolecules. The method may generally comprise contacting a nucleic acid molecule with a pair of probes and a barcode molecule to generate a barcoded molecule (e.g., a barcoded probe-linked molecule). The nucleic acid molecule may comprise a sequence corresponding to a target sequence or a template sequence. One or more nucleic acid reactions (e.g., a ligation, a nucleicAttorney Docket No.43487-1046601 acid extension reaction, amplification, etc.) may be performed to generate the barcoded molecule. In some aspects, the method comprises: contacting a nucleic acid molecule with a first probe to generate a probe-associated nucleic acid molecule, wherein the nucleic acid molecule comprises a first target region and a second target region, wherein the first probe comprises a first probe sequence complementary to the first target region; performing a nucleic acid reaction (e.g., a nucleic acid extension reaction, e.g., by using a polymerase or reverse transcriptase, etc.) to generate an extended probe molecule comprising a sequence complementary to the second target region; providing (i) a second probe comprising a second probe sequence corresponding to or complementary to the second target region and (ii) a nucleic acid barcode molecule; and subjecting the extended probe molecule or derivative thereof to conditions sufficient to generate a barcoded molecule. The first target region and the second target region may be disposed adjacent to one another or may be separate from one another (e.g., disposed on opposite ends of a gap region). In some instances, barcoding may be facilitated by providing a probe binding molecule (also referred to herein as a “splint molecule” or in some instances, a “splint oligonucleotide”). For example, the first probe and / or the second probe may comprise a probe capture sequence, and the probe-binding molecule may comprise a probe-binding sequence complementary to the probe capture sequence. In addition to or alternatively, the nucleic acid barcode molecule may comprise a barcode sequence and a barcode capture sequence, and the probe-binding molecule may comprise a barcode binding sequence complementary to the barcode capture sequence. In some instances, the probe-binding molecule may be pre-annealed to the nucleic acid barcode molecule. Barcoding may comprise hybridization of the probe binding molecule to the probe capture sequence (or complement thereof) of the first probe and / or second probe and to the barcode capture sequence of the nucleic acid barcode molecule. Accordingly, the barcoded molecule may comprise a sequence corresponding to the first target region, a sequence corresponding to the second target region, a sequence corresponding to the probe capture sequence, and a sequence corresponding to the barcode sequence. One or more operations may be performed within a partition (e.g., droplet or well).

[0180] The methods described herein may facilitate gene expression profiling with single-cell, single-nucleus or single-cell bead resolution using, for example, nucleic acid extension reactions, probe hybridization, chemical or enzymatic ligation, barcoding, amplification, and sequencing. The methods described herein may allow for gene expression analysis while avoiding the use of specialized imaging equipment and, in certain instances, reverse transcription, which may be highly error prone and inefficient. In some instances, the methods may be used to analyze a pre-determined panel of target genes in a population of single cells, nuclei, or cell beads in a sensitive and accurate manner. The methods described herein may alsoAttorney Docket No.43487-1046601 be useful in detecting or characterizing genetic variants, for example, in instances where the sequence of a region disposed between the target regions (e.g., a gap region) is not known. In some cases, the methods described herein may be useful in analyzing a single nucleotide polymorphism (SNP), an alternative-spliced junction, an insertion, a mutation, a deletion, a gene rearrangement (e.g., V(D)J rearrangements), a transposon, or other genetic element or variants. In some cases, the nucleic acid molecule analyzed by the methods described herein may comprise a fusion gene (e.g., a hybrid gene generated via translocation, interstitial deletion, or chromosomal inversion). In some cases, the methods described herein may be useful in analyzing genomic, transcriptomic, exomic and / or proteomic elements in cells, nuclei, cell beads, tissue samples, spatial arrays of cells, nuclei or tissues, etc.

[0181] The nucleic acid molecule analyzed by the methods described herein may be a single-stranded or a double-stranded nucleic acid molecule. A double-stranded nucleic acid molecule may be completely or partially denatured to provide access to a target region (e.g., a target sequence) of a strand of the nucleic acid molecule. Denaturation may be achieved by, for example, adjusting the temperature or pH of a solution comprising the nucleic acid molecule; using a chemical agent such as formamide, guanidine, sodium salicylate, dimethyl sulfoxide, propylene glycol, urea, or an alkaline agent (e.g., NaOH); or using mechanical agitation (e.g., centrifuging or vortexing a solution including the nucleic acid molecule).

[0182] The nucleic acid molecule may be a target nucleic acid molecule. The target nucleicacid molecule may be an RNA molecule. The RNA molecule may be, for example, a transfer RNA (tRNA) molecule, ribosomal RNA (rRNA) molecule, mitochondrial RNA (mtRNA) molecule, messenger RNA (mRNA) molecule, non-coding RNA molecule, synthetic RNA molecule, or another type of RNA molecule. For example, the RNA molecule may be an mRNA molecule. In some cases, the nucleic acid molecule may be a viral or pathogenic RNA. In some cases, the nucleic acid molecule may be a synthetic nucleic acid molecule previously introduced into or onto a cell. For example, the nucleic acid molecule may comprise a plurality of barcode sequences, and two or more barcode sequences may be target regions of the nucleic acid molecule. In some instances, the nucleic acid molecule is a guide RNA (gRNA), which may be exogenously introduced in a cell or cell bead. In some instances, the nucleic acid molecule is an RNA molecule derived from an exogenously introduced nucleic acid molecule, e.g., an RNA derived from a plasmid, an integrated DNA sequence (e.g. using viral transduction in a cell), a gRNA from a CRISPR genetic element, etc. See also US20240002901.

[0183] The nucleic acid molecule (e.g., RNA molecule) may comprise one or more featuresselected from the group consisting of a 5’ cap structure, an untranslated region (UTR), a 5’ triphosphate moiety, a 5’ hydroxyl moiety, a Kozak sequence, a Shine-Dalgarno sequence, aAttorney Docket No.43487-1046601 coding sequence, a codon, an intron, an exon, an open reading frame, a regulatory sequence, an enhancer sequence, a silencer sequence, a promoter sequence, and a poly(A) sequence (e.g., a poly(A) tail). For example, the nucleic acid molecule may comprise one or more features selected from the group consisting of a 5’ cap structure, an untranslated region (UTR), a Kozak sequence, a Shine-Dalgarno sequence, a coding sequence, and a poly(A) sequence (e.g., a poly(A) tail).

[0184] Features of the nucleic acid molecule may have any useful characteristics. A 5’ capstructure may comprise one or more nucleoside moieties joined by a linker such as a triphosphate (ppp) linker. A 5’ cap structure may comprise naturally occurring nucleoside and / or non- naturally occurring (e.g., modified) nucleosides. For example, a 5’ cap structure may comprise a guanine moiety or a modified (e.g., alkylated, reduced, or oxidized) guanine moiety such as a 7- methylguanylate (m7G) cap. Examples of 5’ cap structures include, but are not limited to, m7GpppG, m7Gpppm7G, m7GpppA, m7GpppC, GpppG, m2,7GpppG, m2,2,7GpppG, and anti- reverse cap analogs such as m7,2’OmeGpppG, m7,2’dGpppG, m7,3’OmeGpppG, and m7,3’dGpppG. An untranslated region (UTR) may be a 5’ UTR or a 3’ UTR. A UTR may include any number of nucleotides. For example, a UTR may comprise at least 3, 5, 7, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more nucleotides. In some cases, a UTR may comprise fewer than 20 nucleotides. In other cases, a UTR may comprise at least 100 nucleotides, such as more than 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides. Similarly, a coding sequence may include any number of nucleotides, such as at least 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more nucleotides. A UTR, coding sequence, or other sequence of a nucleic acid molecule may have any nucleotide or base content or arrangement. For example, a sequence of a nucleic acid molecule may comprise any number or concentration of guanine, cytosine, uracil, and adenine bases. A nucleic acid molecule may also include non-naturally occurring (e.g., modified) nucleosides. A modified nucleoside may comprise one or more modifications (e.g., alkylations, hydroxylation, oxidation, or other modification) in its nucleobase and / or sugar moieties.

[0185] The nucleic acid molecule may comprise one or more target regions. In some cases, atarget region may correspond to a gene or a portion thereof. Each region may have the same or different sequences. For example, the nucleic acid molecule may comprise two target regions having the same sequence located at different positions along a strand of the nucleic acid molecule. Alternatively, the nucleic acid molecule may comprise two or more target regions having different sequences. Different target regions may be interrogated by different probes. Target regions may be located adjacent to one another or may be spatially separated along a strand of the nucleic acid molecule. The target regions may be located on the same strand or different strands. As used herein with regard to two entities, “adjacent,” may mean that theAttorney Docket No.43487-1046601 entities directly next to one other (e.g., contiguous) or in proximity to one another. For example, a first target region may be directly next to a second target region (e.g., having no other entity disposed between the first and second target regions) or in proximity to a second target region (e.g., having an intervening sequence or molecule between the first and second target regions). In some cases, a double-stranded nucleic acid molecule may comprise a target region in each strand that may be the same or different. For a nucleic acid molecule comprising multiple target regions, the methods described herein may be performed for one or more target regions at a time. For example, a single target region of the multiple target regions may be analyzed (e.g., as described herein) or two or more target regions may be analyzed at the same time. Analyzing two or more target regions may involve providing two or more probes, where a first probe has a sequence that is complementary to the first target region, a second probe has a sequence that is complementary to the second target region, etc.

[0186] Each probe (e.g., the first probe and the second probe) may further comprise one ormore additional sequences (e.g., additional probe sequences, unique molecular identifiers (UMIs), a barcode sequence, a primer sequence, a capture sequence, or other functional sequence). For example, in some instances, the first probe and / or the second probe may comprise the same or different barcode sequences. In some examples, the first probe and the second probe may be configured to hybridize to one or more nucleic acid barcode molecules. For example, the first probe and / or the second probe may comprise a probe capture sequence, which may be configured to hybridize to a nucleic acid barcode molecule or to a probe binding molecule (e.g., a splint oligonucleotide) that is configured to hybridize to a nucleic acid barcode molecule (e.g., via a barcode binding sequence that is complementary to a capture sequence of the nucleic acid barcode molecule). The probe capture sequence may be any useful length; for example, the probe capture sequence may be about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100 or more nucleotides in length. The probe capture sequence may be at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100 or more nucleotides in length. The probe capture sequence may be at most 100, at most 90, at most 80, at most 70, at most 60, at most 50, at most 40, at most 30, at most 20, at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 nucleotide in length. A range of lengths of the probe capture sequence, such as from about 8 to about 50 nucleotides in length, etc. In some instances, the probe capture sequence length may be varied based on any useful application and properties, e.g., melting temperature, annealing temperature, annealing strength (e.g., GC content), hybridization stringency, etc.Attorney Docket No.43487-1046601

[0187] Similarly, the probe binding molecule and nucleic acid barcode molecule may furthercomprise one or more additional sequences (e.g., unique molecular identifiers (UMIs), a barcode sequence, a primer sequence, a capture sequence, or other functional sequence). For example, in some instances, the probe binding molecule or barcode molecule may comprise a functional sequence, a primer sequence (e.g., sequencing primer sequence or partial sequencing primer sequence), a UMI, etc. The probe binding molecule and the nucleic acid barcode molecule may be any useful length; for example, either or both may be about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100 or more nucleotides in length. The probe binding molecule or the barcode molecule may be at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100 or more nucleotides in length. The probe capture binding molecule or the barcode molecule may be at most 100, at most 90, at most 80, at most 70, at most 60, at most 50, at most 40, at most 30, at most 20, at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 nucleotide in length. A range of lengths of the probe binding molecule or barcode molecule may be used, such as from about 16 to about 100 nucleotides in length, etc. In some instances, the probe binding molecule or barcode molecule length may be varied based on any useful application and properties, e.g., melting temperature, annealing temperature, etc. In some instances, the first target region and the second target region of the nucleic acid molecule are not adjacent. For instance, the first target region and the second target region may be separated by one or more gap regions disposed between the first target region and the second target region. The gap region may comprise, for example, at least one nucleotide base, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, or more bases. The gap region may comprise at most about 1000, at most about 500, at most about 400, at most about 300, at most about 200, at most about 100, at most about 90, at most about 80, at most about 70, at most about 60, at most about 50, at most about 40, at most about 30, at most about 20, at most about 10, or at most about 5 bases. The gap region may comprise a range of number of bases, such as between about 1 and 30 bases.

[0188] A target region of the nucleic acid molecule may have one or more usefulcharacteristics. For example, a target region may have any useful length, base content, sequence, melting point, or other characteristic. A target region may comprise, for example, at least 10Attorney Docket No.43487-1046601 bases, such as at least about 20, 25, 30, 35, 40, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, or more bases. A target region may have any useful base content and any useful sequence and combination of bases. For example, a target region may comprise one or more adenine, thymine, uracil, cytosine, and / or guanine bases (e.g., natural or canonical bases). A target region may also comprise one or more derivatives or modified versions of a natural or canonical base, such as an oxidized, alkylated (e.g., methylated), hydroxylated, or otherwise modified base. Similarly, a target region may comprise ribose or deoxyribose moieties and phosphate moieties or derivatives or modified versions thereof.

[0189] A target region of the nucleic acid molecule may comprise one or more sequences orfeatures, or portions thereof, of the nucleic acid molecule. For example, a target region may comprise all or a portion of a UTR (e.g., a 3’ UTR or a 5’ UTR), a Kozak sequence, a Shine- Dalgarno sequence, a coding sequence, a polyA sequence, a cap structure, an intron, an exon, or any other sequence or feature of the nucleic acid molecule.

[0190] The nucleic acid molecule (e.g., RNA molecule, such as an mRNA molecule) of asample may be included within a cell, nucleus or cell bead. For example, the sample may comprise a cell or nucleus comprising the nucleic acid molecule. The cell, nucleus, or cell bead may comprise additional nucleic acid molecules that may be the same as or different from the nucleic acid molecule of interest. In some cases, the sample may comprise a plurality of cells, and each cell may contain one or more nucleic acid molecules. The cell may be, for example, a human cell, an animal cell, or a plant cell. In some cases, the cell may be derived from a tissue or fluid, as described herein. The cell may be a prokaryotic cell or a eukaryotic cell. The cell may be a lymphocyte such as a B cell or T cell. The cell may be comprised within a bead, such as those disclosed in U.S. Pat. No.10,428,326, which is incorporated by reference herein in its entirety. In some instances, the cell is comprised within a tissue sample and may be fixed to a substrate. For example, the cell may be a cell of a formalin-fixed, paraffin-embedded (FFPE) sample, as described above. In such instances, the method may comprise additional operations for preparing the cell or nucleic acid molecule comprised therein, e.g., deparaffinization, staining (e.g., using immunological agents) or destaining, decrosslinking, washing, enzymatic treatment, etc. Additional examples of treating FFPE samples prior to and following hybridization of probes are included in PCT / US2020 / 066720, which is included by reference herein in its entirety.

[0191] Access to a nucleic acid molecule included in a cell, nucleus or cell bead may beprovided by lysing or permeabilizing the cell or nucleus. Lysing the cell, nucleus or cell bead may release the nucleic acid molecule contained therein from the cell, nucleus or cell bead. AAttorney Docket No.43487-1046601 cell or nucleus may be lysed using a lysis agent such as a bioactive agent. A bioactive agent useful for lysing a cell or nucleus may be, for example, an enzyme (e.g., as described herein). An enzyme used to lyse a cell or nucleus may or may not be capable of carrying out additional functions such as degrading, extending, reverse transcribing, or otherwise altering a nucleic acid molecule. Alternatively, an ionic or non-ionic surfactant such as TritonX-100, Tween 20, sarcosyl, or sodium dodecyl sulfate may be used to lyse a cell or nucleus. Cell / nucleus lysis may also be achieved using a cellular disruption method such as an electroporation or a thermal, acoustic, or mechanical disruption method. Alternatively, a cell or nucleus may be permeabilized to provide access to a nucleic acid molecule included therein. Permeabilization may involve partially or completely dissolving or disrupting a cell / nuclear membrane or a portion thereof. Permeabilization may be achieved by, for example, contacting a cell membrane with an organic solvent (e.g., methanol) or a detergent such as Triton X-100 or NP-40. The cell, nucleus or cell bead may be fixed, as described elsewhere herein.

[0192] In some cases, the cell may be lysed within the cell bead, and a subset of theintracellular contents may associate with the bead. In some cases, the cell bead may comprise thioacrydite-modified nucleic acid molecules that can hybridize with nucleic acids from the cell. For example, a poly-T nucleic acid sequence may be thioacrydite-modified and bound to the cell bead matrix. Upon cell or nucleus lysis, the cellular nucleic acids (e.g., mRNA) may hybridize with the poly-T sequence. The retained intracellular / intranuclear contents may be released, for example, by addition of a reducing agent, e.g., DTT, TCEP, etc. The release may occur at any convenient step, such as before or after partitioning.

[0193] The nucleic acid molecule or probe-associated nucleic acid molecule may besubjected to conditions sufficient to generate a probe-linked molecule. For instance, the first target region may be adjacent to the second target region, and the first probe and the second probe may hybridize to the first target region and the second target region, respectively. The first probe may comprise a first reactive moiety, and the second probe may comprise a second reactive moiety. In some instances, the first reactive moiety of the first probe is adjacent to the second reactive moiety of the second probe. The reactive moieties may then be subjected to conditions sufficient to cause them to react to yield a probe-linked nucleic acid molecule comprising the first probe linked to the second probe. For example, the reactive moieties may be joined together via click chemistry or enzymatic ligation, such as those disclosed in in U.S. Pat. Pub. No.2020 / 0239874, International Pub. No. WO 2019 / 165318, and International Pat. Pub. No. WO2021 / 237087, each of which is incorporated by reference herein in its entirety. In some examples, the first probe or the second probe may comprise an adenylated oligonucleotide or moiety (e.g., an adenylated phosphate group), which may be useful in reducing non-specificAttorney Docket No.43487-1046601 ligation reactions. In some instances, the linking of the probes (e.g., via ligation) may be performed in substantially ATP-free conditions, optionally using an enzyme (e.g., ligase) that does not require ATP (e.g., truncated T4 RNA ligase) or that is pre-activated (e.g., a preactivated T4 DNA ligase). Additional examples of such ligation schemes can be found in PCT / US2020 / 066720 and International Pat. App. No. PCT / US2021 / 33649, filed May 21, 2021, which is incorporated by reference herein in its entirety.

[0194] In some instances, the first target region of the nucleic acid molecule (e.g., RNAmolecule) may not be adjacent to the second target region. In such cases, the nucleic acid molecule may be subjected to conditions sufficient for hybridization of the first probe sequence of the first probe to the first target region to generate a probe-associated nucleic acid molecule. The probe-associated nucleic acid molecule may be subjected to a nucleic acid reaction (e.g., a nucleic acid extension reaction, reverse transcription, etc.) to generate an extended probe molecule comprising a sequence complementary to the second target region. A second probe comprising a second probe sequence may hybridize to the extended probe molecule (or complement thereof) and subjected to conditions sufficient (e.g., nucleic acid extension, amplification, hybridization of additional probe molecules, ligation, etc.) to generate a probe- linked molecule comprising a sequence corresponding to the first target region and a sequence corresponding to the second target region. Alternatively or in addition to, the first probe and the second probe may be provided simultaneously, and following hybridization of the first probe sequence and the second probe sequence to the first target region and the second target region, respectively, to generate a dual-probe-associated nucleic acid molecule, the gap (e.g., the region disposed between the first target region and the second region) may be filled (e.g., via a nucleic acid extension or gap-fill reaction and / or hybridization of additional probe molecules that hybridize to at least a portion of the gap region). In some instances, one or both probes may comprise an overhang or flap sequence (e.g., at a 5’ end) that is recognizable or cleavable by an enzyme (e.g., an endonuclease such as FEN1 endonuclease). For example, the second probe may comprise a 5’ flap sequence that is cleaved by FEN1 endonuclease if at least a specific portion of the second probe hybridizes to the nucleic acid molecule (e.g., target molecule). Subsequent to hybridization of the second probe to the second target sequence of the nucleic acid molecule, an endonuclease (e.g., FEN1) may be used to cleave the flap sequence and leave a ligatable end (e.g., a phosphorylated end) of the second probe. In instances in which the first target region is not adjacent to the second target region, the gap region may be filled, followed by cleavage of the flap sequence. In some instances, the first probe or the second probe and the gap-filled region may be ligated, e.g., chemically or enzymatically. Additional examples of systems and methods for generating probe-linked nucleic acid molecules and gap-filling reactions can beAttorney Docket No.43487-1046601 found, for example in U.S. Pat. Pub. No.2020 / 0239874, International Pub. No. WO 2019 / 165318, and International Pat. Pub. No. WO2021 / 237087, each of which is incorporated by reference herein in its entirety.

[0195] The probe-linked nucleic acid molecule may be barcoded to provide a barcodedprobe-linked nucleic acid molecule, or barcoding may occur prior to generation of the probe- linked nucleic acid molecule. Barcoding may be performed using a variety of techniques. For example, the first probe or the second probe may comprise a probe capture sequence. The nucleic acid barcode molecule may comprise a barcode capture sequence capable of hybridizing to the probe capture sequence. Alternatively, barcoding may be mediated by a probe binding molecule (e.g., a splint oligonucleotide) comprising (i) a probe binding sequence, which may be complementary to the probe capture sequence of the first probe or the second probe, and (ii) a barcode binding sequence, which may be complementary to the barcode capture sequence of the nucleic acid barcode molecule. In some instances, the barcoding may be followed by ligation, e.g., chemically or enzyme-mediated, to covalently link the nucleic acid barcode molecule to the probe (or to the probe binding sequence, and the probe binding sequence may be ligated to the probe). Examples of chemical ligation of nucleic acid molecules may include “click chemistry” approaches, e.g., reaction of azide and alkyne moieties, as described in U.S. Pat. Pub. No. 2020 / 0239874, which is incorporated by reference herein in its entirety.

[0196] By way of example, the first probe may comprise a first probe sequence and a probecapture sequence, and the first probe may be subjected to conditions sufficient to hybridize the first probe sequence to the first target region, thereby generating a probe-associated nucleic acid molecule. In some instances, the probe-associated nucleic acid molecule may be subjected to washing or other conditions to remove unannealed probes from a mixture. The probe-associated nucleic acid molecule may be extended from an end of the first probe towards an end of the nucleic acid molecule to which it is hybridized (towards the end which is proximal to the second target region) to provide an extended nucleic acid molecule. The extended nucleic acid barcode molecule may comprise the first probe sequence and a complement to the second target region. In some instances, the extended nucleic acid molecule may be barcoded, e.g., by hybridizing the barcode capture sequence of the nucleic acid barcode molecule to the probe capture sequence, or by hybridizing (i) a probe-binding molecule comprising a probe binding sequence and a barcode binding sequence to the probe capture sequence and (ii) the barcode capture sequence of the nucleic acid barcode molecule to the barcode binding sequence of the probe binding molecule. In some instances, the probe-binding molecule may be provided pre-annealed to the nucleic acid barcode molecule. Subsequently, a second probe comprising a second probe sequence may be provided. The barcoded, extended nucleic acid molecule may be subjected to conditionsAttorney Docket No.43487-1046601 sufficient to hybridize the second probe sequence to the second target region or complement thereof. A nucleic acid extension reaction may be performed, thereby generating a barcoded molecule (e.g., barcoded probe-linked molecule) comprising a sequence corresponding to the first target region, a sequence corresponding to the second target region, a sequence corresponding to the probe capture sequence, and a sequence corresponding to the barcode sequence.

[0197] FIG. 7 schematically shows a method for generating a barcoded nucleic acidmolecule, as described herein. A nucleic acid molecule (e.g., RNA molecule) 700 comprising a first target region 702 and a second target region 704 may be provided. The nucleic acid molecule 700 may be contacted with a first probe 706 comprising a first probe sequence 708 and, optionally, a functional sequence 710, thereby generating a probe-associated nucleic acid molecule. The first probe sequence 708 may be complementary to the first target region 702. The functional sequence 710 may comprise, for instance, a probe capture sequence used for downstream barcoding, or it may comprise a different functional sequence, such as a primer sequence, a partial primer sequence, a barcode sequence, a sequencing primer sequence, etc.

[0198] In operation 701, the probe-associated nucleic acid molecule may be subjected toconditions sufficient to extend the first probe 706, thereby generating an extended probe molecule 712 comprising a sequence complementary to the second target region 704. In some instances, the extended probe molecule 712 may be released from the nucleic acid molecule 700, e.g., via denaturing and / or degrading the nucleic acid molecule 700 (e.g., using an RNAse, increased temperature or heat cycling, pH, etc.). In operation 703, a nucleic acid barcode molecule may be provided. In some instances, the nucleic acid barcode molecule may be partially double-stranded and may comprise a first strand 720 comprising a barcode sequence, and a second strand 722 comprising a sequence 724 at least partially complementary to the barcode sequence and a probe binding sequence 726, which may be at least partially complementary to the functional sequence (e.g., probe capture sequence) 710 of the first probe 706. In some instances, the nucleic acid barcode molecule is single-stranded and comprises only first strand 720 comprising the barcode sequence and a barcode capture sequence. A probe binding molecule (e.g., a splint oligonucleotide) 722 may be provided, comprising barcode- binding sequence 724, which is at least partially complementary to the barcode capture sequence, and the probe binding sequence 726. In some instances, the probe binding molecule and the nucleic acid barcode molecule may be provided as a pre-annealed complex. The nucleic acid barcode molecule (or the pre-annealed complex) may be coupled to a bead, such as a gel bead, as described herein, and may comprise additional functional sequences, including, but not limitedAttorney Docket No.43487-1046601 to, a unique molecular identifier (UMI), a capture sequence, a primer sequence (e.g., a R1 / R2 sequence).

[0199] In operation 705, the extended probe molecule may be barcoded by hybridizing theprobe binding sequence 726 to the functional sequence (e.g., probe capture sequence 710). In some instances, the nucleic acid barcode molecule may be covalently linked to the extended probe molecule (e.g., via the probe capture sequence), e.g., enzymatically (e.g., using a ligase) or chemically (e.g., using click chemistry). In operation 707, a second probe molecule 716 may be provided. In some instances, operation 707 may also include a denaturation of the double- stranded molecule. The second probe molecule 716 may comprise a second probe sequence 714 corresponding to the second target region 704 and optionally a functional sequence 718, which may comprise a probe capture sequence, a barcode sequence, a primer sequence, a sequencing primer sequence, etc. In operation 709, a nucleic acid extension reaction may be performed, e.g., using a polymerase, to extend the second probe 716 along the extended probe molecule, thereby generating a barcoded molecule comprising a sequence corresponding to the first target region 702, the second target region 704, a sequence corresponding to the probe capture sequence 710, and a sequence corresponding to the barcode sequence 720.

[0200] In another example, the first probe and the second probe may be linked (e.g., bychemical ligation or enzymatic extension and / or ligation) prior to barcoding. In such an example, the first probe may be hybridized to the nucleic acid molecule (e.g., via hybridization of the first probe sequence to the first target region) to generate a probe-associated nucleic acid molecule. The probe-associated nucleic acid molecule may be extended from an end of the first probe to an end of the nucleic acid molecule to which it is hybridized, to provide an extended nucleic acid molecule. The extended molecule may be subjected to conditions sufficient to hybridize the second probe to the second target region or complement thereof (e.g., via hybridization of the second probe sequence to the second target region or complement thereof). An additional nucleic acid extension reaction may be performed, to generate an extended, and the resultant extension product may be barcoded, generating a barcoded molecule. The barcoded molecule may comprise a sequence corresponding to the first target region, a sequence corresponding to the second target region, a sequence corresponding to the probe capture sequence, and a sequence corresponding to the barcode sequence. In some instances, the nucleic acid barcode molecule (or the probe binding molecule) may be chemically linked to the first probe or the second probe, such as by ligation or click chemistry. For example, the nucleic acid barcode molecule may comprise a first reactive moiety, and the first or the second probe may comprise a second reactive moiety; the first reactive moiety may be configured to react with the second reactive moiety to generate a covalent linkage. Barcoded nucleic acid molecules or derivatives thereofAttorney Docket No.43487-1046601 may then be optionally further processed and analyzed by any suitable technique, including nucleic acid sequencing (e.g., Illumina sequencing).

[0201] FIG. 8 schematically shows another method for generating a barcoded nucleic acidmolecule, as described herein. A nucleic acid molecule (e.g., RNA molecule) 800 comprising a first target region 802 and a second target region 804 may be provided. The nucleic acid molecule 800 may be contacted with a first probe 806 comprising a first probe sequence 808 and, optionally, a functional sequence 810, thereby generating a probe-associated nucleic acid molecule. The first probe sequence 808 may be complementary to the first target region 802. The functional sequence 810 may comprise, for instance, a probe capture sequence used for downstream barcoding, or it may comprise a different functional sequence, such as a primer sequence, a partial primer sequence, a barcode sequence, a sequencing primer sequence, etc.

[0202] In operation 801, the probe-associated nucleic acid molecule may be subjected toconditions sufficient to extend the first probe 806, thereby generating an extended probe molecule 812 comprising a sequence complementary to the second target region 804. In some instances, the extended probe molecule 812 may be released from the nucleic acid molecule 800, e.g., via denaturing and / or degrading the nucleic acid molecule 800 (e.g., using an RNAse, increased temperature or heat cycling, pH, etc.). In operation 803, a nucleic acid barcode molecule and a second probe 816 may be provided. The second probe 816 may comprise a second probe sequence 814 corresponding to the second target region 804 and optionally a functional sequence 818, which may comprise a probe capture sequence. In some instances, the nucleic acid barcode molecule may be partially double-stranded and may comprise a first strand 820 comprising a barcode sequence, and a second strand 822 comprising a sequence 824 complementary to the barcode sequence and a probe binding sequence 826, which may be complementary to the functional sequence (e.g., probe capture sequence) 818 of the second probe 816. In some instances, the nucleic acid barcode molecule is single-stranded and comprises only first strand 820 comprising the barcode sequence and a barcode capture sequence. A probe binding molecule (e.g., a splint oligonucleotide) 822 may be provided, comprising barcode-binding sequence 824 that is complementary to the barcode capture sequence, and the probe binding sequence 826. In some instances, the probe binding molecule and the nucleic acid barcode molecule may be provided as a pre-annealed complex. The nucleic acid barcode molecule (or the pre-annealed complex) may be coupled to a bead, such as a gel bead, as described herein, and may comprise additional functional sequences, including, but not limited to, a unique molecular identifier (UMI), a capture sequence, a primer sequence (e.g., a R1 / R2 sequence). In operation 803, the second probe 816 may hybridize to the extended probe molecule 812 (e.g., via hybridization of the second probe sequence 814 to the second targetAttorney Docket No.43487-1046601 region 804 or complement thereof), and the nucleic acid barcode molecule may be attached or coupled to the second probe 816, e.g., via hybridization of the probe binding sequence 826 to the probe capture sequence 818. In some instances, the nucleic acid barcode molecule or the probe binding molecule may be ligated to the second probe 816, e.g., using a ligase or via chemical linkage, such as click chemistry.

[0203] In operation 805, a nucleic acid extension reaction may be performed, e.g., using apolymerase (e.g., DNA polymerase, Hot Start polymerase, etc.), to extend the nucleic acid barcode molecule and the second probe 816 along the extended probe molecule, thereby generating a barcoded molecule comprising a sequence corresponding to the first target region 802, the second target region 804, a sequence corresponding to the probe capture sequence 818, and a sequence corresponding to the barcode sequence 820. Barcoded nucleic acid molecules or derivatives thereof may then be optionally further processed and analyzed by any suitable technique, including nucleic acid sequencing (e.g., Illumina sequencing).

[0204] FIG. 9 schematically shows another method for generating a barcoded nucleic acidmolecule, similar to that shown in FIG.8. A nucleic acid molecule (e.g., RNA molecule) 900 comprising a first target region 902 and a second target region 904 may be provided. The nucleic acid molecule 900 may be contacted with a first probe 906 comprising a first probe sequence 908 and, optionally, a functional sequence 910, thereby generating a probe-associated nucleic acid molecule. The first probe sequence 908 may be complementary to the first target region 902. The functional sequence 910 may comprise, for instance, a probe capture sequence, or it may comprise a different functional sequence, such as a primer sequence, a partial primer sequence, a barcode sequence, a sequencing primer sequence, etc.

[0205] In operation 901, the probe-associated nucleic acid molecule may be subjected toconditions sufficient to extend the first probe 906, thereby generating an extended probe molecule 912 comprising a sequence complementary to the second target region 906. In some instances, the extended probe molecule 912 may be released from the nucleic acid molecule 900, e.g., via denaturing and / or degrading the nucleic acid molecule 900 (e.g., using an RNAse, increased temperature or heat cycling, pH, etc.). In operation 903, a second probe 916 may be provided. The second probe 916 may comprise a second probe sequence 914 corresponding to the second target region 904 and optionally a functional sequence 918, which may comprise a probe capture sequence. In operation 905, a nucleic acid extension reaction may be performed, e.g., using a polymerase, to extend the nucleic acid barcode molecule and the second probe 916 along the extended probe molecule, thereby generating a probe-linked molecule comprising a sequence corresponding to the first target region 902 and the second target region 904.Attorney Docket No.43487-1046601

[0206] In operation 905, a nucleic acid barcode molecule may also be provided with thesecond probe. In some instances, the nucleic acid barcode molecule may be partially double- stranded and may comprise a first strand 920 comprising a barcode sequence, and a second strand 922 comprising a sequence 924 complementary to the barcode sequence and a probe binding sequence 926, which may be complementary to the functional sequence (e.g., probe capture sequence) 918 of the second probe 916. In some instances, the nucleic acid barcode molecule is single-stranded and comprises only first strand 920 comprising the barcode sequence and a barcode capture sequence. A probe binding molecule (e.g., a splint oligonucleotide) 922 may be provided, comprising barcode-binding sequence 924 that is complementary to the barcode capture sequence, and the probe binding sequence 926. In some instances, the probe binding molecule and the nucleic acid barcode molecule may be provided as a pre-annealed complex. The nucleic acid barcode molecule (or the pre-annealed complex) may be coupled to a bead, such as a gel bead, as described herein, and may comprise additional functional sequences, including, but not limited to, a unique molecular identifier (UMI), a capture sequence, a primer sequence (e.g., a R1 / R2 sequence). In operation 907, the nucleic acid barcode molecule may be attached or coupled to the second probe 916, e.g., via hybridization of the probe binding sequence 926 to the probe capture sequence 918. The resultant barcoded product may comprise a sequence corresponding to the first target region 902, the second target region 904, a sequence corresponding to the probe capture sequence 918, and a sequence corresponding to the barcode sequence 920. In some instances, the nucleic acid barcode molecule may be covalently linked to the extended probe molecule (e.g., via the probe capture sequence 918), e.g., enzymatically (e.g., using a ligase) or chemically (e.g., using click chemistry). Barcoded nucleic acid molecules or derivatives thereof may then be optionally further processed and analyzed by any suitable technique, including nucleic acid sequencing (e.g., Illumina sequencing).

[0207] In additional examples, the methods of the present disclosure may comprisegenerating probe-associated nucleic acid molecules, and barcoding the probe-associated nucleic acid molecules, optionally with a linking operation (e.g., prior to or subsequent to barcoding of the probe-associated nucleic acid molecules). For example, a nucleic acid molecule (e.g., RNA molecule) comprising a first target region and a second target region may be provided. The nucleic acid molecule may be contacted with (i) a first probe comprising a first probe sequence complementary to the first target region and (ii) a second probe comprising a second probe sequence complementary to the second target region, thereby generating a probe-associated nucleic acid molecule. In some instances, the probe-associated nucleic acid molecule may be subjected to conditions sufficient to link the first probe to the second probe (e.g., enzymatically, such as with a polymerase, reverse transcriptase, and / or ligase, or chemically), therebyAttorney Docket No.43487-1046601 generating a probe-linked nucleic acid molecule. The probe-associated nucleic acid molecule or the probe-linked molecule may subsequently be barcoded (e.g., in a partition) to generate a barcoded nucleic acid molecule.

[0208] For example, FIG. 25 schematically shows an example method for generating aprobe-linked nucleic acid molecule, which may subsequently be barcoded, e.g., in a partition, to generate a barcoded nucleic acid molecule. A nucleic acid molecule (e.g., RNA molecule) 2500 comprising a first target region 2502 and a second target region 2504 may be provided. In some instances, the first target region is adjacent to the second target region. The nucleic acid molecule 2500 may be contacted, in operation 2501, with a first probe 2506 comprising a first probe sequence 2508 complementary to the first target region 2502 and a second probe 2516 comprising a second probe sequence 2514 complementary to the second target region 2504, thereby generating a probe-associated nucleic acid molecule. The first probe 2506 and / or the second probe 2516 may comprise a functional sequence, e.g., a probe capture sequence, a primer sequence, a partial primer sequence, a barcode sequence, a sequencing primer sequence, etc.

[0209] In some instances, one of the probes (e.g., the second probe 2516) comprises a flap oroverhang sequence 2530, which may be recognized by an endonuclease (e.g., FEN1) upon annealing of the second probe sequence 2514 to the second target region 2504. For example, the second probe 2516 may comprise a 5’ flap sequence 2530, and subsequent to annealing of the first probe 2506 and the second probe 2516 to the nucleic acid molecule 2500, the flap sequence may be adjacent to an end of the first probe (e.g., a 3’ end) as well as an end of the second probe (e.g., a 5’ end). In operation 2503, an endonuclease, e.g., FEN1 may be used to remove the flapsequence 2530 ļeaving a ligatable end (e.g., 5’phosphorylated end) of the second probe 2516. Inoperation 2507, a ligation reaction may be performed (e.g., using a ligase) to link the first probe to the second probe, thereby generating a probe-linked nucleic acid molecule. The probe-linked nucleic acid molecule may subsequently be barcoded, e.g., in partitions, as is described elsewhere herein. In some instances, the probe-associated nucleic acid molecules may be barcoded and linked (e.g., in partitions).

[0210] FIG. 26 shows another example workflow, similar to that shown in FIG. 25, inwhich the target regions of the nucleic acid molecule are not adjacent. Such a workflow may comprise an additional gap-fill reaction to generate the probe-associated molecule. In one such example, the first target region 2602 of nucleic acid molecule 2600 may not be adjacent to the second target region 2604. For example, the a gap region may be disposed between the first target region and the second target region. In operation 2601, the first probe 2606 may anneal to the first target region 2602 and the second probe 2616 may anneal to the second target region 2604. In operation 2603, an extension reaction (e.g., using a polymerase, reverse transcriptase,Attorney Docket No.43487-1046601 etc.) may be performed to fill in the gap region between the first probe 2606 and the second probe 2616, yielding a gap-filled nucleic acid molecule. In some instances, the second probe2616 comprises a flap sequence 2630. In such instances, in operation 2605, an endonuclease,e.g., FEN1 may be used to remove the flap sequence 2630 ̧leaving a ligatable end (e.g.,5’phosphorylated end) of the second probe 2616. In operation 2607, a ligation reaction may be performed (e.g., using a ligase) to link the first probe to the second probe, thereby generating a probe-linked nucleic acid molecule. The probe-linked nucleic acid molecule, or alternatively, the un-linked molecule, may be barcoded, e.g., in a partition.

[0211] FIG. 27 shows an additional scheme of generating a probe-linked nucleic acidmolecule by performing a gap-filling reaction using a third probe. In FIG.27 Panel A, a first probe 2706 and a second probe 2716 anneal (e.g., via a first probe sequence and a second probe sequence, respectively) to a first target region 2702 and a second target region 2704 of nucleic acid molecule 2700 to generate a probe-associated nucleic acid molecule. A gap sequence may be disposed between the first target region 2702 and the second target region 2704. Third probe molecules 2770 may be provided (illustrated as two different probe molecules, which may be used for SNP detection), which may anneal to the gap sequence (FIG.27 Panel B). In FIG.27 Panel C, the first probe, the third probe, and the second probe may be ligated (e.g., using a ligase) to generate a probe-linked nucleic acid molecule. The probe-linked nucleic acid molecule, or alternatively, the probe-associated nucleic acid molecule, may be barcoded, e.g., in a partition.

[0212] FIG. 28 shows an example of a ligation scheme used to generate probe-linked nucleicacid molecules. In such an example, the probe molecules may hybridize to the nucleic acid molecule. The first probe may be ligated to the second probe, optionally with a gap-fill operation, as described above, using an enzyme. In some instances, the enzyme may be a pre- activated enzyme, e.g., a preactivated T4 DNA ligase, and the ligation may occur under ATP- reduced or ATP-removed conditions, e.g. using Apyrase.

[0213] Additional examples of methods and systems for generating probe-associated nucleicacid molecules, and barcoding the probe-associated nucleic acid molecules, can be found in, for example U.S. Pat. Pub. No.2020 / 0239874, International Pub. No. WO 2019 / 165318, International App. No. PCT / US2020 / 066720, and International Pat. App. No. PCT / US2021 / 33649, filed May 21, 2021, each of which is incorporated by reference herein in its entirety.

[0214] It will be appreciated that, e.g., referring to FIGs. 7-9 and FIGs. 25-28, the nucleicacid barcode molecule may be attached (e.g., via hybridization) to either the first probe and / or the second probe (e.g., via a probe capture sequence comprised in the first probe or the secondAttorney Docket No.43487-1046601 probe). Similarly, the first probe and the second probe may comprise any useful functional sequences, such as primer sequences, barcode sequences, unique molecular identifier (UMI) sequences, flow cell attachment sequences, primer-binding sequences, capture sequences, etc. The first probe may hybridize to the left-hand side (e.g., a 3’ end) of a nucleic acid molecule (e.g., 700, 800, or 900) or to the right-hand side (e.g., a 5’ end). Similarly, the second probe may hybridize to the left-hand side or to the right-hand side of the nucleic acid molecule.

[0215] As described herein, one or more extension reactions may be performed on the probe-hybridized nucleic acid molecules. For example, the probe may be extended from an end of the probe to an end of the nucleic acid barcode molecule, or a second probe may be extended from an end of the second probe to an end of the first probe of a probe-associated nucleic acid molecule. Extension may comprise the use of an enzyme (e.g., a polymerase, reverse transcriptase) to add one or more nucleotides to the end of the probe. Extension may provide an extended nucleic acid molecule comprising sequences complementary to the target region of the nucleic acid molecule of interest, the barcode sequence, and optionally, one or more additional sequences of the nucleic acid barcode molecule such as one or more binding sequences. In some instances, appropriate conditions and or chemical agents (e.g., as described herein) may be applied to denature the extended nucleic acid molecule from the nucleic acid barcode molecule and the target nucleic acid molecule. In some cases, one or more processes may involve the use of thermosensitive agents. For example, in some cases, probes may be annealed or hybridized under one set of temperature conditions, and extension may occur under a different set of temperature conditions. In some cases, a Warm or Hot Start polymerase may be used. In some cases, hybridization of the nucleic acid barcode molecule to one or more of the probes (e.g., directly hybridizing or via a probe binding molecule such as a splint oligonucleotide) may precede hybridization of the probe to the target region of the nucleic acid molecule. Following barcoding, the barcoded nucleic acid molecule may be duplicated or amplified by, for example, one or more amplification reactions. The amplification reactions may comprise polymerase chain reactions (PCR) and may involve the use of one or more primers or polymerases. The extension, denaturation, and / or amplification processes may take place within a partition, or in bulk. In some cases, the extended nucleic acid molecule or derivatives thereof (e.g., the barcoded molecule) may be duplicated or amplified within a partition to provide an amplified product. The barcoded product, or a complement thereof (e.g., an amplified product), may be detected via sequencing (e.g., as described herein).

[0216] The nucleic acid molecule or a derivative thereof (e.g., a probe-linked nucleic acidmolecule, a nucleic acid molecule having one or more probes hybridized thereto, a barcoded probe-linked nucleic acid molecule, or an extended nucleic acid molecule or complementAttorney Docket No.43487-1046601 thereof) or a cell or cell bead comprising the nucleic acid molecule or a derivative thereof may be provided within a partition such as a well or droplet, e.g., as described herein. One or more reagents may be co-partitioned with a nucleic acid molecule or a derivative thereof or a cell comprising the nucleic acid molecule or a derivative thereof. For example, a nucleic acid molecule or a derivative thereof or a cell comprising the nucleic acid molecule or a derivative thereof may be co-partitioned with one or more reagents selected from the group consisting of lysis agents or buffers, permeabilizing agents, enzymes (e.g., enzymes capable of digesting one or more RNA molecules, extending one or more nucleic acid molecules, reverse transcribing an RNA molecule, permeabilizing or lysing a cell, or carrying out other actions), fluorophores, oligonucleotides, primers, probes, barcodes, nucleic acid barcode molecules (e.g., nucleic acid barcode molecules comprising one or more barcode sequences), buffers, deoxynucleotide triphosphates, detergents, reducing agents, chelating agents, oxidizing agents, nanoparticles, beads, and antibodies. In some cases, a nucleic acid molecule or a derivative thereof, or a cell comprising the nucleic acid molecule or a derivative thereof (e.g., a cell bead), may be co- partitioned with one or more reagents selected from the group consisting of temperature- sensitive enzymes, pH-sensitive enzymes, light-sensitive enzymes, reverse transcriptases, proteases, ligase, polymerases, restriction enzymes, nucleases, protease inhibitors, exonucleases, and nuclease inhibitors. For example, a nucleic acid molecule or a derivative thereof or a cell comprising the nucleic acid molecule or a derivative thereof may be co-partitioned with a polymerase and nucleotide molecules. Partitioning a nucleic acid molecule or a derivative thereof or a cell comprising the nucleic acid molecule or a derivative thereof and one or more reagents may comprise flowing a first phase comprising an aqueous fluid, the cell, and the one or more reagents and a second phase comprising a fluid that is immiscible with the aqueous fluid toward a junction. Upon interaction of the first and second phases, a discrete droplet of the first phase comprising the nucleic acid molecule or a derivative thereof or a cell comprising the nucleic acid molecule or a derivative thereof (e.g., a cell bead) and the one or more reagents may be formed. In some cases, the partition may comprise a single cell. The cell may be lysed or permeabilized within the partition (e.g., droplet) to provide access to the nucleic acid molecule of the cell.

[0217] One or more processes may be carried out within a partition (e.g., droplet, well, etc.).For instance, the nucleic acid molecule, or a cell or cell bead comprising the nucleic acid molecule, may be co-partitioned with one or more reagents (e.g., as described herein) at any useful stage of the method. For example, the probe-associated nucleic acid molecule (e.g., the nucleic acid molecule with the first probe hybridized thereto) may be generated in bulk (e.g., in a population of cells, which may be alive or fixed and / or permeabilized, in a tissue sample, etc.)Attorney Docket No.43487-1046601 and subjected to conditions sufficient for generating for generating an extended probe molecule. The extended probe molecule may be subsequently partitioned in a partition among a plurality of partitions. The partition may comprise the second probe and a nucleic acid barcode molecule and optionally, a probe binding molecule. As described herein, the second probe may hybridize (e.g., via the second probe sequence) to the second target region or complement thereof of the probe- associated molecule. The partition may comprise additional reagents for performing a nucleic acid reaction (e.g., digestion, ligation, extension, amplification). For instance, the probe- associated nucleic acid molecule may comprise or be hybridized to the nucleic acid molecule, and the partition may comprise a degrading enzyme (e.g., RNAse), which may be useful in digesting or removing the template strand (e.g., the nucleic acid molecule, such as an RNA molecule) from the extended probe molecule. The partition may comprise a polymerase, which may be used to extend the second probe hybridized to the extended probe molecule. In some instances, the partition comprises a linking enzyme (e.g., ligase), which may be used to ligate the nucleic acid barcode molecule to the first probe or the second probe (e.g., via a probe capture sequence). The ligase may in some instances be used to ligate the probe binding molecule to the probe capture sequence of the first probe or the second probe. In some instances, the probe binding molecule, the probe capture sequence, and / or the barcode capture sequence comprises one or more reactive moieties, which may be used to chemically or enzymatically link the nucleic acid barcode molecule to the probe capture sequence, or complement thereof. The resultant barcoded product may comprise a sequence corresponding to the first target region, a sequence corresponding to the second target region, a sequence corresponding to the probe capture sequence, and a sequence corresponding to the barcode sequence.

[0218] For example, referring again to FIG. 7, operation 701 may be performed in bulk (e.g.,outside a partition), while operations 703, 705 may be performed in a partition. Operations 707 and 709 may be performed in bulk or within the partition. Similarly, referring to FIG.8, operation 801 may be performed in bulk, while operation 803 may be performed in a partition. Operation 805 may be performed in bulk or in a partition. Referring to FIG.9, operation 901 may be performed in bulk, while operations 903, 905, and 907 may be performed in a partition. It will be appreciated that any of the operations may be performed in bulk or in partitions at any convenient step and that the order of the operations may be changed for a suitable or useful purpose.

[0219] Similarly, the nucleic acid molecule or the cell or cell bead comprising the nucleicacid molecule, or derivatives thereof (e.g., the probe-associated molecule, the extended molecule, the barcoded molecule, etc.) may be released from a partition at any useful stage of the method. For example, the extended probe molecule may be hybridized to the second probe andAttorney Docket No.43487-1046601 released from the partition subsequent to hybridization of the barcode capture sequence of the nucleic acid barcode molecule to the first probe, the second probe, or the probe binding molecule. Alternatively, the extended probe molecule may be released from the partition subsequent to (i) hybridization of the second probe and nucleic acid barcode molecule and (ii) extension of the second probe to generate the barcoded molecule comprising a sequence corresponding to the first target region, a sequence corresponding to the second target region, a sequence corresponding to the probe capture sequence, and a sequence corresponding to the barcode sequence. Duplication and / or amplification of the extended nucleic acid molecule may be carried out within the partition or in bulk, e.g., within a solution. In some cases, the solution may comprise additional extended nucleic acid molecules generated through the same process carried out in different partitions. Each extended nucleic acid molecule may comprise a different barcode sequence, and the barcode sequence may be useful in identifying the partition or cell from whence the extended nucleic acid molecules originated. In such cases, the solution may comprise a pooled mixture comprising the contents of two or more partitions (e.g., droplets).

[0220] Additional processes or operations may be performed within a partition, including,but not limited to: lysis, permeabilization, denaturation, hybridization, extension, duplication, and amplification of one or more components of a sample. In some cases, multiple processes are carried out within a partition.

[0221] Hybridization of the probe sequences to the target regions of the nucleic acidmolecule may be performed within or outside of a partition. In some cases, hybridization may be preceded by denaturation of a double-stranded nucleic acid molecule to provide a single- stranded nucleic acid molecule or by lysis or permeabilization of a cell. In some cases, the hybridization may occur in a cell bead comprising a cell. The sequence of the probe that is complementary to the target region may be situated at an end of the probe. Alternatively, this sequence may be disposed between other sequences such that when the probe sequence is hybridized to the target region, additional probe sequences extend beyond the hybridized sequence in one or more directions. The probe sequence that hybridizes to the target region of the nucleic acid molecule may be of the same or different length as the target region. For example, the probe sequence may be shorter than the target region and may only hybridize to a portion of the target region. Alternatively, the probe sequence may be longer than the target region and may hybridize to the entirety of the target region and extend beyond the target region in one or more directions. In addition to a probe sequence complementary to a target region of the nucleic acid molecule, the probe may comprise one or more additional probe sequences. For example, the probe may comprise the probe sequence complementary to the target region and aAttorney Docket No.43487-1046601 second probe sequence. The second probe sequence may have any useful length and other characteristics.

[0222] The probe (e.g., the first probe or the second probe) may comprise one or moreadditional sequences or moieties, such as one or more barcode sequences or unique molecule identifier (UMI) sequences, adapter sequences, functional sequences (e.g., primer sequences, sequencing primer sequences, etc.). In some cases, one or more probe sequences of the probe may comprise a detectable moiety such as a fluorophore or a fluorescent moiety. In some instances, the first probe or the second probe may comprise a reactive moiety, as described elsewhere herein. For example, the first probe or the second probe may comprise an azide moiety, an alkyne moiety, a phosphorothioate moiety, an iodide moiety, an amine moiety, a phosphate moiety, or a combination thereof. The first probe may comprise a first reactive moiety and the second probe may comprise a second reactive moiety, and reaction of the first reactive moiety and the second reactive moiety may be sufficient to yield a probe-linked molecule comprising the first probe linked to the second probe. In some instances, the first reactive moiety and the second reactive moiety is linked via ligation. Accordingly, the first probe or the second probe may comprise one or more moieties or modified nucleotides to facilitate ligation, e.g., one or more ribonucleotides or dideoxynucleotides (ddNTPs), which may be ligated to a phosphorylated end of the second probe using a ligase (e.g., T4 DNA ligase, SplintR ligase). In some instances, the probe (e.g., the first probe or the second probe) may comprise an overhang or flap sequence which is recognizable or cleavable by an endonuclease (e.g., FEN1 endonuclease). Other suitable enzymes, e.g., ligases, may be used, for example, the enzymes and ligases disclosed in U.S. Provisional App. No.63 / 171,031, filed April 5, 2021, which is incorporated herein by reference in its entirety.

[0223] As described herein, a probe sequence of the probe may be capable of hybridizingwith a sequence of a nucleic acid barcode molecule or a probe binding molecule (e.g., splint oligonucleotide). A nucleic acid barcode molecule may comprise a first binding sequence (e.g., a barcode capture sequence) that is complementary to a probe sequence of the probe (e.g., a probe capture sequence). The nucleic acid barcode molecule may comprise one or more additional functional sequences , e.g., primer sequences, primer annealing sequences, and immobilization sequences. The binding sequences may have any useful length and other characteristics. In some cases, the binding sequence (e.g., barcode capture sequence) that is complementary to a probe sequence of the probe may be the same length as the probe sequence. Alternatively, the binding sequence may be a different length of the probe sequence. For example, the binding sequence may be shorter than the probe sequence and may only hybridize to a portion of the probe sequence. Alternatively, the binding sequence may be longer than theAttorney Docket No.43487-1046601 probe sequence and may hybridize to the entirety of the probe sequence and extend beyond the probe sequence in one or more directions. Similarly, in instances when a probe-binding molecule is used, the binding sequence (e.g., barcode capture sequence) of the nucleic acid barcode molecule may be the same length as the barcode binding sequence of the probe-binding molecule, or the binding sequence may be longer or shorter than the barcode binding sequence.

[0001] One or more processes described herein may be performed in a cell, nucleus or cellbead. For example, in some embodiments, a plurality of cells, nuclei or cell beads may comprise a plurality of nucleic acid molecules. The cells, nuclei or cell beads may be alive or fixed and / or permeabilized. In some instances, the first probes may be provided to the cells, nuclei or cell beads, such as in a bulk solution. Optionally, the cells, nuclei or cell beads may be washed to remove unbound first probes, and the nucleic acid extension reaction, as described herein, may be performed. Subsequently, the cells, nuclei or cell beads comprising the plurality of nucleic acid molecules (or the extended, probe nucleic acid molecules) may be partitioned into a plurality of separate partitions, where at least a subset of the plurality of separate partitions comprises a single cell, single nucleus, or single cell bead. Access to a target nucleic acid molecule contained within a cell, nucleus or cell bead in a partition may be provided by lysing or permeabilizing the nucleus or cell (e.g., as described herein), which may be performed prior to or during partitioning. Additional probe hybridization (e.g., providing of the second probe) and / or barcoding may be performed within the separate partitions. Barcoding, as described herein, may comprise using a nucleic acid barcode molecule to attach or hybridize to the target nucleic acid molecule or derivative thereof (e.g., the extended probe molecule, or complement thereof). Nucleic acid barcode molecules provided within each partition of the plurality of separate partitions may be provided attached to beads. In some instances, as described elsewhere herein, the nucleic acid barcode molecule may be releasably attached to a bead (e.g., via a labile bond). Each partition (or a subset of partitions) of the plurality of separate partitions may comprise a bead comprising a plurality of nucleic acid barcode molecules attached thereto (e.g., as described herein). The plurality of nucleic acid barcode molecules attached to each bead may comprise a unique barcode sequence, such that each partition of the plurality of separate partitions comprises a different barcode sequence. Upon release of components from the plurality of different partitions of the plurality of separate partitions (e.g., following barcoding), the barcoded molecules arising from a single cell, single nucleus, or single cell bead may have a same barcode sequence (e.g., a common barcode sequence), such that each barcoded nucleic acid molecule can be traced to a given partition and / or, in some instances, a given cell, nucleus or cell bead.

[0224] The methods described herein may comprise additional barcoding operations, whichmay be useful, for example, in indexing nucleic acid molecules to a cell, nucleus, cell bead, aAttorney Docket No.43487-1046601 sample, a partition, or a plurality of partitions. Such indexing may be useful in situations when a single partition is occupied by multiple cells, nuclei, or cell beads. In some instances, it may be beneficial to overload partitions such that a partition comprises more than a single cell, single nucleus, or single cell bead; for example, it may be useful in certain situations to overload partitions, e.g., to overcome Poisson loading statistics in partitions and / or to prevent reagent waste (e.g., from unoccupied partitions). Accordingly, such indexing may be useful in attributing cells, nuclei or nucleic acid molecules in multiply-occupied partitions to the originating cell, nucleus, cell bead, partition, sample, etc.

[0225] In an example, a barcoded molecule, such as the barcoded molecules generated usingthe methods described herein (e.g., in FIGs.7-9, FIGs.25-28, as well as barcoded, probe-linked nucleic acid molecules described in U.S. Pat. Pub. No.2020 / 0239874 and International Pub. No. WO 2019 / 165318, each of which is incorporated by reference herein) may be provided. The barcoded molecule may comprise, as described herein, a sequence corresponding to the first target region, a sequence corresponding to the second target region, a sequence corresponding to the probe capture sequence (which may be disposed on the first probe or the second probe), and a sequence corresponding to the barcode sequence of the nucleic acid barcode molecule. Such a barcode sequence may be specific to the partition and may differ from other barcode sequences of other partitions and thus may be used to identify a partition from which a nucleic acid molecule (or derivative thereof) originated. In some instances, some of the partitions may comprise a single cell, single nucleus, or single cell bead and thus the nucleic acid barcode molecule or barcode sequence may be used to identify a cell, nucleus, or cell bead from which a nucleic acid molecule (or derivative thereof) originated.

[0226] In some instances, the barcoded molecule may be subjected to an additionalbarcoding operation, e.g., in partitions or in bulk. For example, the barcoded molecule may be re-partitioned in a partition among a plurality of partitions comprising a plurality of additional nucleic acid barcode molecules. The plurality of additional nucleic acid barcode molecules may comprise additional barcode sequences that differ across the partitions. The barcoded molecules may be subjected to conditions sufficient to barcode the barcoded molecules to generate a combinatorially barcoded molecule comprising two barcode sequences. As each barcode sequence pertains to a unique partition, the combination of barcodes may be useful in generating a greater diversity of barcoded molecules, as well as for identifying the originating partitions of the combinatorially barcoded molecule.

[0227] In some cases, combinatorial assembly of barcode segments may be performed using,e.g., a split-pool approach. For example, in some embodiments, the probe-linked nucleic acidmolecules may be combinatorially barcoded using a split pool approach. In one such example, aAttorney Docket No.43487-1046601 plurality of permeabilized cells (or permeabilized nuclei or cell beads) comprising, e.g., probe- linked nucleic acid molecule, which may optionally be barcoded (e.g., the product following operation 709 of FIG.7, 805 of FIG.8, or 905 or 907 of FIG.9) may be partitioned into a plurality of partitions (e.g., a plurality of wells), wherein each partition of the plurality of partitions comprises a different (i.e., unique) barcode sequence segment. Alternatively, the plurality of permeabilized cells (or permeabilized nuclei or cell beads) may be partitioned, and then the different barcode sequence segments delivered to the respective partitions containing the cells, nuclei, and / or cell beads. After addition of the barcode sequence segment, cells (or nuclei or cell beads) can be collected from the plurality of partitions, pooled, and partitioned into an additional plurality of partitions (e.g., a plurality of wells) wherein each partition of the additional plurality of partitions comprises a different (i.e., unique) second barcode sequence segment. Repeating this split-pool process allows the generation of barcodes or barcoded molecules comprising any suitable amount of barcode sequence segments. Combinatorial barcoding as described herein may comprise at least 1, 2, 3, 4, 5, 6, 7, 8 or more operations (e.g., split-pool cycles). Combinatorial barcoding comprising multiple operations may be useful, for example, in generation of greater barcode diversity and to synthesize a unique barcode sequence on nucleic acid molecules derived from each single cell, nucleus, or cell bead of a plurality of cells, nuclei, cell beads. For example, combinatorial barcoding comprising three operations, each comprising attachment of a unique nucleic acid sequence in each of 96 partitions, will yield up to 884,736 unique barcode combinations. Generally, where there are M partitions, and N number of split-pool iterations are performed, up to MNunique barcode combinations may be generated. Cells or nuclei or cell beads may be partitioned such that at least one cell (or nuclei or cell bead) is present in each partition of a plurality of partitions. Cells, nuclei, or cell beads may be partitioned such that at least 1; 2; 3; 4; 5; 10; 20; 50; 100; 500; 1,000; 5,000; 10,000; 100,000; 1,000,000; or more cells, nuclei, or cell beads are present in a single partition. Cells, nuclei, or cell beads may be partitioned such that at most 1,000,000; 100,000; 10,000; 5,000; 1,000; 500; 100; 50; 20; 10; 5; 4; 3; 2; or 1 cell (or nucleus or cell bead) is present in a single partition. Cells, nuclei, and / or cell beads may be partitioned in a random configuration.

[0228] In some instances, the additional barcoding operations may be performed prior tosome of the operations described herein. For example, it may be beneficial to combinatorially barcode the first probe in a bulk solution, e.g., prior to or following generation of the extended probe molecule or probe-linked molecule. In such cases, the nucleic acid molecule may be contacted, e.g., in bulk, with a first probe to generate a probe-associated molecule. The probe- associated molecule may optionally be extended, e.g., using the methods described herein, to generate an extended probe molecule. The probe-associated molecule or the extended probeAttorney Docket No.43487-1046601 molecule may then be subjected to combinatorial barcoding, e.g., in partitions, as described above, to generate a combinatorially barcoded molecule. The combinatorially barcoded molecule may then be partitioned with a second probe and a nucleic acid barcode molecule, which, as described herein, may attach to either the first probe (or combinatorially barcoded probe), the second probe, or both probes. As each partition of the combinatorial barcoding process comprises a different barcode sequence segment, a plurality of the combinatorially barcoded molecules may be traced back to the individual partitions from which they originated. Moreover, the combinatorial barcoding may be useful in generating greater probe diversity.

[0229] Beneficially, the combinatorial barcoding of the first probe may be particularly usefulwhen combined with the second probe and nucleic acid barcode molecule, which may comprise a barcode sequence that is specific to the partition. For example, the presence of the probe- specific barcode(s) and the partition-specific barcode sequence may allow for indexing of individual cells (or nuclei or cell beads) within a partition. For instance, partitions comprising cell / nucleus / cell bead multiplets (e.g., cell doublets, triplets, etc.) can be computationally deconvolved into single cells / nuclei / cell beads. Thus, in some instances, cells, nuclei, or cell beads may be “overloaded” into partitions using conditions such that a higher probability of cell / nucleus / cell bead multiplets (2,3,4,5+ cells, nuclei, or cell beads per partition) are formed, wherein target libraries of these cell multiplets may be computationally deconvolved into single cells, nuclei, or cell beads.

[0230] FIG. 10 schematically shows an example workflow of barcoding nucleic acidmolecules in partitions comprising cell / nucleus / cell bead multiplets. In operation 1010, one or more populations of cells / nuclei / cell beads (or nucleic acid molecules contained therein) may be subjected to barcoding, as described herein (e.g., using processes shown and described in FIGs. 7-9 and FIGs.15-16). For example, a first population of cells (or nuclei or cell beads) 1002 (comprising a first plurality of nucleic acid molecules) may be subjected to barcoding in a first subset of a first plurality of partitions, generating a first plurality of barcoded nucleic acid molecules comprising a first barcode sequence. A second population of cells (or nuclei or cell beads) 1004 may be barcoded in a second subset of the first plurality of partitions, generating a second plurality of barcoded nucleic acid molecules comprising a second barcode sequence. The first barcode sequence may be different than the second barcode sequence. In operation 1020, the first population of cells (or nuclei or cell beads) 1002 may be pooled together with the second population of cells (or nuclei or cell beads) 1004 to generate a mixture of cells. In operation 1030, the mixture of cells (or nuclei or cell beads) may be partitioned into a second plurality of partitions. In some instances, the mixture of cells / nuclei / cell beads may be partitioned into the second plurality of partitions such that some partitions of the second plurality of partitionsAttorney Docket No.43487-1046601 comprises more than one cell (e.g., a cell multiplet partition). For example, a partition 1035 of the second plurality of partitions may comprise a cell, nucleus, or cell bead (“Cell A”) from the first population of cells 1002 and a cell, nucleus, or cell bead (“Cell B”) from the second population of cells 1004. The partition 1035 may comprise an additional barcode sequence, which may be unique to the partition. The cells / nuclei / cell beads in each partition may be subjected to an additional barcoding operation to append the additional barcode sequence on the barcoded nucleic acid molecules. In operation 1040, the barcoded nucleic acid molecules may be deconvolved, using the different barcode sequences (e.g., the first barcode sequence, the second barcode sequence, and the additional barcode sequences), to identify the originating cell / nucleus / cell bead. For instance, a barcoded nucleic acid molecule comprising the additional barcode sequence from partition 1035 and the first barcode sequence from the first population of cells (or nuclei or cell beads) 1002 may be used to identify that barcoded nucleic acid molecule as originating from Cell A. Similarly, a barcoded nucleic acid molecule comprising the additional barcode sequence from partition 1035 and the second barcode sequence from the second populations of cells (or nuclei or cell beads) 1004 may be used to identify that barcoded nucleic acid molecule as originating from Cell B.

[0231] Following partition-based barcoding, the contents of the partitions may be pooled andthe barcoded molecules (e.g., barcoded probe-linked nucleic acid molecules) may be duplicatedor amplified by, for example, one or more amplification reactions, which may in some instances be isothermal. The amplification reactions may comprise polymerase chain reactions (PCR) and may involve the use of one or more primers or polymerases. The one or more primers may comprise one or more functional sequences (e.g., a primer sequence / primer binding sequence, a sequencing primer sequence (e.g., R1 or R2), a partial sequencing primer sequence (e.g., partial R1 or partial R2), a sequence configured to attach to the flow cell of a sequencer (e.g., P5 or P7, or partial sequences thereof), etc.) and may facilitate addition of said one or more functional sequences to the extended nucleic acid molecule. The barcoded molecules, or derivatives thereof, may be detected via nucleic acid sequencing (e.g., as described herein).

[0232] In some aspects, provided herein are systems useful for barcoding nucleic acidmolecules. The systems may comprise any of the components described herein, e.g., a plurality of partitions (e.g., droplets, wells), which may be provided in any useful format, e.g., a microfluidic device, a multi-well array or plate, etc. The systems may include nucleic acid barcode molecules, optionally coupled to supports (e.g., particles, beads, gel beads, etc.). In some instances, the systems may comprise any of the probes described herein, such as a first probe or plurality of first probes, a second probe or plurality of second probes, and any useful reaction components (e.g., for performing a nucleic acid reaction, e.g., extension, ligation,Attorney Docket No.43487-1046601 amplification, etc.). Such useful reaction components can include, in non-limiting examples, enzymes (e.g., ligases, polymerases, reverse transciptases, restriction enzymes, etc.), nucleotides bases, etc.

[0233] Also provided herein are compositions useful for systems and methods for barcodingnucleic acid molecules. A composition may comprise any of the probes described herein. For example, a composition may comprise a plurality of first probes, a plurality of second probes, and / or a plurality of first probes and a plurality of second probes. A probe or a set of probes may be designed to target a specific sequence or a set of specific sequences. Such probes may be designed to have the same or different sequences within different partitions. For example, a first composition may comprise a first probe and a second probe designed to target two regions of a first gene, and a second composition may comprise a first probe and a second probe designed to target two regions of a second gene, which second gene is different than the first gene. A composition may comprise nucleic acid barcode molecules, and / or probe binding molecules, which may optionally be provided coupled to a support (e.g., particle, bead). A composition may be a part of or comprise a reaction mixture, which can include reaction components or reagents, e.g., enzymes, nucleotide bases, catalysts, buffers etc. Multiplexed analysis of nucleic acids and proteins

[0234] In another aspect, the present disclosure provides methods for performingmultiplexed assays. Such a multiplexed assay may comprise assaying or analyzing one or more biomolecules (e.g., nucleic acid molecules, proteins, lipids, carbohydrates, etc.). A method may comprise using one or more probes and a nucleic acid barcode molecule to barcode a nucleic acid molecule of a cell / nucleus / cell bead, thereby generating a first barcoded nucleic acid molecule; attaching or coupling a feature-binding group to a feature of the cell / nucleus / cell bead, wherein the feature-binding group comprises a reporter oligonucleotide comprising a reporter sequence that identifies the feature-binding group; using an additional nucleic acid barcode molecule, and optionally, an additional probe, to barcode the reporter sequence to generate a second barcoded nucleic acid molecule; and optionally barcoding the first barcoded nucleic acid molecule and the second barcoded nucleic acid molecule to generate a third barcoded nucleic acid molecule and a fourth barcoded nucleic acid molecule. One or more operations may be performed within a partition (e.g., droplet or well).

[0235] The methods described herein may facilitate profiling of one or more biomoleculeswith single-cell / single nucleus / single cell bead resolution, using, for example, probe hybridization, feature binding groups (e.g., antibodies, antibody fragments, epitope-binding groups, etc.), barcoding, amplification, and sequencing. The methods may be useful in providingAttorney Docket No.43487-1046601 genomic, transcriptomic, proteomic, exomic, or other “-omic” information from a single cell / nucleus / cell bead. As described herein, the methods may be used to analyze a pre- determined panel of target genes and a pre-determined panel of target features (e.g., proteins, peptides, or other biomolecules) in a sensitive and accurate manner. Alternatively or in addition to, the methods may be used to analyze whole genomic, whole transcriptomic, whole exomic, etc. characteristics of a cell.

[0236] In some aspects, the methods comprise contacting a cell / nucleus / cell bead with a firstprobe, a second probe, and a third probe under conditions sufficient to generate a first probe- associated molecule and a second probe-associated molecule. The cell / nucleus / cell bead may comprise (i) a nucleic acid molecule (e.g., a target nucleic acid molecule such as RNA or DNA) comprising a first target region and a second target region and (ii) a feature (e.g., protein, peptide, or other biomolecule) coupled to a feature-binding group. The feature binding group may comprise or be coupled to (i) a reporter oligonucleotide comprising a reporter sequence, which may be associated with the feature or may be used to identify the feature, and (ii) a feature probe-binding sequence. The first probe may comprise a first probe sequence complementary to the first target region of the nucleic acid molecule and, optionally, an additional probe sequence, such as a probe capture sequence or other functional sequence. The second probe may comprise a second probe sequence complementary to the second target region and, optionally, a probe capture sequence or functional sequence. The third probe may comprise (i) a third probe sequence complementary to the feature probe-binding sequence and (ii) a probe capture sequence or functional sequence, which may be the same sequence as the probe capture sequence of the first probe and / or second probe.

[0237] In some instances, the first probe-associated molecule may comprise the nucleic acidmolecule, the first probe, the second probe, or combinations or complements thereof. The second probe-associated molecule may comprise the reporter oligonucleotide (which comprises the reporter sequence) and the third probe, or complements thereof.

[0238] In some aspects, the method comprises providing the first probe-associated moleculeand the second probe-associated molecule, and barcoding the first probe-associated molecule and the second probe-associated molecules. Such barcoding operations may occur in a first set of partitions (e.g., droplets or wells). Such an example method may comprise contacting the first probe-associated molecule and the second-probe-associated molecule with probe binding molecules (e.g., a splint oligonucleotide) and barcode molecules (e.g., nucleic acid barcode molecules) under conditions sufficient to generate a first barcoded nucleic acid molecule and a second barcoded nucleic acid molecule. The barcode molecules may comprise (i) a barcode capture sequence, e.g., a common sequence that is common to a plurality of barcode moleculesAttorney Docket No.43487-1046601 and (ii) a first barcode sequence. In instances where partitions are used, the first barcode sequence may be unique to a first partition of a first set of partitions, and the barcode molecules within the first partition may share the same first barcode sequence. The probe-binding molecule may comprise (i) a probe-binding sequence complementary to the probe capture sequence (of the first probe, the second probe, and / or the third probe) and (ii) a barcode binding sequence complementary to the barcode capture sequence (e.g., common sequence) of the plurality of barcode molecules. As such, barcoding of the first probe-associated molecule and the second probe-associated molecule may comprise hybridization of the probe binding molecule to (i) the probe capture sequence (or complement thereof) of the first probe, the second probe, and / or the third probe, and (ii) the barcode capture sequence (or common sequence) of the nucleic acid barcode molecule. In some examples, the first barcoded nucleic acid molecule comprises a sequence corresponding to the first probe sequence, a sequence corresponding to the second probe sequence, and a sequence corresponding to the first barcode sequence. Similarly, the second barcoded nucleic acid molecule may comprise a sequence corresponding to the reporter sequence, a sequence corresponding to the third probe sequence, and a sequence corresponding to the first barcode sequence.

[0239] The method may further comprise providing a second set of partitions, and in asecond partition of the second set of partitions, (i) contacting the first barcoded nucleic acid molecule, or derivative thereof (e.g., complements, amplicons, extension products thereof), to a first capture molecule of a plurality of capture molecules under conditions sufficient to generate a third barcoded nucleic acid molecule, and (ii) contacting the second barcoded nucleic acid molecule, or derivative thereof, to a second capture molecule of the plurality of capture molecules under conditions sufficient to generate a fourth barcoded nucleic acid molecule. The plurality of capture molecules may each comprise a second barcode sequence, which may be the same or different than the first barcode sequence from the first set of partitions. The second barcode sequence may be unique to the partition (i.e., differ across partitions). The third barcoded nucleic acid molecule and the fourth barcoded molecule may each comprise a sequence corresponding to the first barcode sequence and a sequence corresponding to the second barcode sequence. For example, the third barcoded nucleic acid molecule may comprise a sequence corresponding to the first target region, a sequence corresponding to the second target region, a sequence corresponding to a probe capture sequence, the first barcode sequence and the second barcode sequence. The fourth barcoded nucleic acid molecule may comprise a sequence corresponding to the reporter sequence, a sequence corresponding to the feature probe binding sequence, a sequence corresponding to the third probe, the first barcode sequence and the second barcode sequence.Attorney Docket No.43487-1046601

[0240] The feature binding group may comprise a labelling agent, as described elsewhereherein. Accordingly, the feature binding group may comprise, in some examples, an antibody or antibody fragment, an epitope binding moiety, a protein, a peptide, a lipophilic moiety (such as cholesterol), a cell surface receptor binding molecule, a receptor ligand, a small molecule, a bi- specific antibody, a bi-specific T-cell engager, a T-cell receptor engager, a B-cell receptor engager, a pro-body, an aptamer, a monobody, an affimer, a darpin, and a protein scaffold, or any combination thereof.

[0241] The probe capture sequence of the first probe (or the second probe) may be commonto a plurality of first probes (or second probes), a plurality of partitions, and / or a plurality of cells / nuclei / cell beads. For instance, the first set of partitions may comprise one or more additional partitions that comprise additional probe-associated nucleic acid molecules. The additional probe-associated nucleic acid molecules may comprise identical sequences (e.g., first probe sequence, second probe sequence) to the probe-associated nucleic acid molecule of the first partition, or the additional probe-associated nucleic acid molecules of the additional partitions may comprise different sequences (e.g., different probe sequences) than the probe- associated nucleic acid molecule of the first partition. In some instances, each of the one or more additional probe-associated nucleic acid molecules comprises a probe capture sequence, which may be identical or different across the first set of partitions.

[0242] The probe-associated molecules may be a probe-linked molecule. For example, theprobe-associated molecules may be the probe-associated molecules or barcoded molecules described herein (e.g., in FIGS.7-9), or a probe-linked molecule, such as those described in U.S. Pat. Pub. No.2020 / 0239874 and International Pub. No. WO 2019 / 165318, each of which is incorporated by reference herein in its entirety. In some examples, two sets of probe-associated molecules may be generated, in which: (i) a first probe-associated molecule comprises the nucleic acid molecule, with the first probe and the second probe hybridized thereto (e.g., via hybridization of the first probe sequence to the first target region and the second probe sequence to the second target region) and (ii) a second probe-associated molecule comprises the reporter oligonucleotide (which comprises the reporter sequence), with the third probe hybridized thereto.

[0243] The first probe, the second probe, and / or the third probe may comprise a probecapture sequence. The probe capture sequence on the first probe may be the same or different than the probe capture sequence of the second probe or the third probe. Similarly, the probe capture sequence of the second probe may be the same or different than the probe capture sequence of the third probe. Accordingly, the barcoding operations described herein may occur on the first probe, the second probe, the third probe, or any combination thereof. For example, for a probe-associated molecule comprising a nucleic acid molecule and the first probe (“probeAttorney Docket No.43487-1046601 1”) and second probe (“probe 2”) hybridized thereto, a first barcode molecule comprising the first barcode sequence (“BC1”) may hybridize (e.g., directly or via a probe-binding molecule) to the first probe to generate a first barcoded nucleic acid molecule, and subsequently, a capture molecule comprising a second barcode sequence (“BC2”) may be annealed to a region of the first barcode molecule, thereby generating a molecule comprising a sequence, or complementary sequences, of BC2-BC1-probe 1-probe 2. Alternatively or in addition to, the first barcode molecule comprising the first barcode sequence (“BC1”) may hybridize (e.g., directly or via a probe-binding molecule) to the second probe to generate a first barcoded nucleic acid molecule, and subsequently, a capture molecule comprising the second barcode sequence (“BC2”) may be annealed to a region of the first barcode molecule, thereby generating a molecule comprising a sequence of probe 1-probe 2-BC1-BC2. Alternatively or in addition to, the barcode molecules and the capture molecules may be annealed to different probes. For example, the first barcode molecule comprising the first barcode sequence (“BC1”) may hybridize (e.g., directly or via a probe-binding molecule) to the first probe to generate a first barcoded nucleic acid molecule, and subsequently, a capture molecule comprising the second barcode sequence (“BC2”) may be annealed to the second probe, thereby generating a molecule comprising a sequence of BC1- probe 1-probe 2-BC2. Alternatively or in addition to, the first barcode molecule comprising the first barcode sequence (“BC1”) may hybridize (e.g., directly or via a probe-binding molecule) to the second probe to generate a first barcoded nucleic acid molecule, and subsequently, a capture molecule comprising the second barcode sequence (“BC2”) may be annealed to the first probe, thereby generating a molecule comprising a sequence of BC2-probe 1-probe 2-BC1. It will be appreciated that while several examples of barcoding schemes are described herein, additional combinations and positioning of barcode sequences are possible; for example, combinatorial barcoding may be used to generate greater barcode diversity, as described herein, and such barcoding may occur on any of the probe molecules (or already barcoded molecules).

[0244] In some instances, the barcode molecules may comprise a capture-binding sequencecomplementary to a capture sequence of the plurality of capture molecules. For example, the first probe may comprise a probe capture sequence which may hybridize to a probe binding molecule, which may mediate hybridization of the barcode molecule (e.g., via hybridization of the barcode binding sequence of the probe binding molecule to the barcode capture sequence (e.g., common sequence) of the barcode molecule). The barcode molecule may additionally comprise the capture-binding sequence, which may allow for hybridization of the capture sequence of the capture molecules to the barcode molecule.

[0245] FIG. 15 schematically illustrates an example barcoded nucleic acid molecule asdescribed herein. Referring to Panel A, a nucleic acid molecule (e.g., RNA molecule) 1500Attorney Docket No.43487-1046601 comprising a first target region 1502 and a second target region 1504 may be provided. The nucleic acid molecule 1500 may be contacted with a first probe 1506 comprising a first probe sequence 1508 and, optionally, a first probe capture sequence 1510. The first probe sequence 1508 may be complementary to the first target region 1502. The first probe capture sequence 1510 may additionally, in some instances, comprise a functional sequence, such as a primer sequence, a partial primer sequence, a barcode sequence, a sequencing primer sequence, etc. The nucleic acid molecule 1500 may also be contacted with a second probe 1516 comprising a second probe sequence 1514 and, optionally, a second probe capture sequence 1518. The second probe sequence 1514 may be complementary to the second target region 1504. The second probe capture sequence 1518 may additionally comprise a functional sequence. Hybridization of the first probe 1506 and the second probe 1516 to the nucleic acid molecule 1500 may generate a probe-associated molecule.

[0246] As described herein, the probe-associated molecule may be subjected to one or morebarcoding operations. Such a barcoding operation may occur in one or more partitions (e.g., a first set of partitions) and may include hybridizing a probe binding molecule 1517 and a barcode molecule 1519 comprising a barcode capture sequence (e.g., a common sequence), to the probe- associated molecule. In some instances, the probe binding molecule 1517 and the barcode molecule 1519 may be provided as a pre-annealed complex, or they may be provided as separate molecules. The barcode capture sequence (e.g., common sequence) may be a sequence that is common to the plurality of barcode molecules in the first set of partitions, or the common sequence may be unique to the barcode molecules in only a single first partition (i.e., the common sequence differs across partitions of the first set of partitions). The probe binding molecule 1517 may comprise a probe binding sequence complementary to the probe capture sequence 1518 of the second probe 1516, as well as a barcode binding sequence complementary to a sequence of the barcode molecule 1519. The probe-associated molecule may be subjected to conditions sufficient to generate a first barcoded nucleic acid molecule, which can include annealing of the probe-binding molecule 1517 to (i) the probe capture sequence 1518 and (ii) the barcode capture sequence (e.g., common sequence) of the barcode molecule 1519. The barcoding process may comprise additional operations, such as ligation, which may be performed chemically or enzymatically, as described elsewhere herein.

[0247] The first barcoded nucleic acid molecule or derivatives thereof (e.g., a complement,an amplicon, an extension product, a combinatorially barcoded nucleic acid molecule, as described elsewhere herein), may be subjected to a second barcoding operation. Such a second barcoding operation may occur in a second set of partitions. For example, the first barcoded nucleic acid molecule may be removed from the first set of partitions, pooled (e.g., with otherAttorney Docket No.43487-1046601 barcoded nucleic acid molecules from other first partitions of the first set of partitions), and partitioned in a second partition of a second set of partitions. The second partition may comprise a capture molecule 1520. The capture molecule 1520 may comprise a second barcode sequence and a sequence complementary to the probe capture sequence 1510 of the first probe 1506. The second barcode sequence may be a sequence that is common to the plurality of capture molecules in the second set of partitions, or the barcode sequence may be unique to the capture molecules in only the second partition (i.e., differ across partitions). The capture molecule 1520 may hybridize to the probe capture sequence 1510 to generate an additional barcoded molecule (also referred to herein as a “third barcoded nucleic acid molecule”). The additional barcoded molecule may comprise a sequence corresponding to the first barcode sequence (of the barcode molecule 1519), and a sequence corresponding to the second barcode sequence (of the capture molecule 1520).

[0248] Panel B of FIG. 15 schematically illustrates another example barcoded molecule inwhich the capture molecule 1520 is hybridized to the barcode molecule 1519. Similar to Panel A, in Panel B, the nucleic acid molecule (e.g., RNA molecule) 1500 comprising a first target region 1502 and a second target region 1504 may be provided. The nucleic acid molecule 1500 may be contacted with a first probe 1506 comprising a first probe sequence 1508 and a probe capture sequence 1510. The first probe sequence 1508 may be complementary to the first target region 1502. The probe capture sequence 1510 may additionally comprise a functional sequence, such as a primer sequence, a partial primer sequence, a barcode sequence, a sequencing primer sequence, etc. The nucleic acid molecule 1500 may also be contacted with a second probe 1516 comprising a second probe sequence 1514 and, optionally, an additional sequence 1518. The second probe sequence 1514 may be complementary to the second target region 1504. The additional sequence 1518 may comprise, for instance, a probe capture sequence, or a functional sequence (e.g., primer, primer binding site, sequencing primer sequence, etc.). Hybridization of the first probe 1506 and the second probe 1516 to the nucleic acid molecule 1500 may generate a probe-associated molecule.

[0249] The probe-associated molecule may be contacted with one or more barcodemolecules. Such barcoding operations, as described herein, may occur in a plurality of partitions (e.g., a first partition of a first set of partitions and / or a second partition of a second set of partitions). The probe-associated molecule may be contacted with a probe binding molecule 1517 and a barcode molecule 1519, which may comprise a first barcode capture sequence (e.g., a common sequence) and a second barcode capture sequence 1521 (also referred to herein as “capture binding sequence”). In some instances, the probe binding molecule 1517 and the barcode molecule 1519 may be provided as a pre-annealed complex or as separate molecules.Attorney Docket No.43487-1046601 The first barcode capture sequence (e.g., common sequence) may be a sequence that is common to the plurality of barcode molecules in the first set of partitions, or the common sequence may be unique to the barcode molecules in only the first partition (i.e., differ across partitions). The probe binding molecule 1517 may comprise a probe binding sequence complementary to the probe capture sequence 1510 as well as a barcode binding sequence complementary to the first barcode capture sequence (e.g., common sequence) of the barcode molecule 1519. The probe- associated molecule may be subjected to conditions sufficient to generate a first barcoded nucleic acid molecule, which can include annealing of the probe-binding molecule 1517 to (i) the probe capture sequence 1510 and (ii) the first barcode capture sequence (e.g., common sequence) of the barcode molecule 1519. The barcoding process may comprise additional operations, such as ligation, which may be performed chemically or enzymatically, as described elsewhere herein.

[0250] The first barcoded nucleic acid molecule or derivatives thereof, may be subjected to asecond barcoding operation. Such a second barcoding operation may occur in a second set of partitions. For example, the first barcoded nucleic acid molecule may be removed from the first partition and partitioned in a second partition of a second set of partitions (e.g., droplets). The second partition may comprise a capture molecule 1520. The capture molecule 1520 may comprise a second barcode sequence and a sequence complementary to the second barcode capture sequence 1521 of the barcode molecule 1519. The second barcode sequence may be a sequence that is common to the plurality of capture molecules in the second set of partitions, or the barcode sequence may be unique to the capture molecules in only the second partition (i.e., differ across partitions). The capture molecule may hybridize to the second barcode capture sequence 1521 to generate an additional barcoded molecule (also referred to herein as a “third barcoded nucleic acid molecule”). The additional barcoded molecule may comprise a sequence corresponding to the first barcode sequence (of the barcode molecule 1519), and a sequence corresponding to the second barcode sequence (of the capture molecule 1520).

[0251] Panel C of FIG. 15 illustrates another example barcoded nucleic acid molecule. Anucleic acid molecule (e.g., RNA molecule) 1500 comprising a first target region 1502 and a second target region 1504 may be provided. The nucleic acid molecule 1500 may be contacted with a first probe 1506 comprising a first probe sequence 1508 and, optionally, a first probe capture sequence 1510. The first probe sequence 1508 may be complementary to the first target region 1502. The first probe or first probe capture sequence 1510 may additionally, in some instances, comprise a functional sequence, such as a primer sequence, a partial primer sequence, a barcode sequence, a sequencing primer sequence, etc. The nucleic acid molecule 1500 may also be contacted with a second probe 1516 comprising a second probe sequence 1514 and, optionally, a second probe capture sequence 1518. The second probe sequence 1514 may beAttorney Docket No.43487-1046601 complementary to the second target region 1504. The second probe capture sequence 1518 may additionally comprise a functional sequence. Hybridization of the first probe 1506 and the second probe 1516 to the nucleic acid molecule 1500 may generate a probe-associated molecule or complex.

[0252] As described herein, the probe-associated molecule may be subjected to one or morebarcoding operations. Such a barcoding operation may occur in one or more partitions (e.g., a first set of partitions) and may include hybridizing a probe binding molecule 1517 and a barcode molecule 1519 comprising a barcode capture sequence (e.g., a common sequence), to the probe- associated molecule or complex. In some instances, the probe binding molecule 1517 and the barcode molecule 1519 are provided as a pre-annealed complex (e.g., a partially double-stranded molecule comprising the probe binding molecule 1517 and the barcode molecule 1519), or they may be provided as separate molecules, which may separately anneal to the probe-associated molecule or complex (e.g., the probe binding molecule 1517 may hybridize to the probe- associated molecule or complex, e.g., via the second probe capture sequence 1518, and the barcode molecule 1519 may hybridize to the probe binding molecule 1517). The barcode capture sequence (e.g., common sequence) may be a sequence that is common to the plurality of barcode molecules in the first set of partitions, or the common sequence may be unique to the barcode molecules in only a single first partition (i.e., the common sequence differs across partitions of the first set of partitions). The probe binding molecule 1517 may comprise a probe binding sequence complementary to the probe capture sequence 1518 of the second probe 1516, as well as a barcode binding sequence complementary to a sequence of the barcode molecule 1519. In some instances, the probe binding molecule 1517 and / or the barcode molecule 1519 comprise an additional sequence, e.g., an adapter sequence, a primer sequence (e.g., sequencing primer sequence or partial sequencing primer sequence), a UMI, a sample index sequence, etc. In some instances, the probe binding molecule 1517 comprises the entire sequence of the barcode molecule 1519, such that no overhang remains. In some instances, the probe binding molecule 1517 and barcode molecule 1519 comprise a sample index sequence, which may be useful in identifying the partition, cell, nucleus, or cell bead from which the target nucleic acid molecule 1500 originates. The probe-associated molecule may be subjected to conditions sufficient to generate a first barcoded nucleic acid molecule, which can include annealing of the probe- binding molecule 1517 to (i) the probe capture sequence 1518 and (ii) the barcode capture sequence (e.g., common sequence) of the barcode molecule 1519. The barcoding process may comprise additional operations, such as ligation (e.g., ligation of the barcode molecule 1519 to the probe capture sequence 1518), which may be performed chemically or enzymatically, as described elsewhere herein.Attorney Docket No.43487-1046601

[0253] The first barcoded nucleic acid molecule or derivatives thereof (e.g., a complement,an amplicon, an extension product, a combinatorially barcoded nucleic acid molecule, as described elsewhere herein), may be subjected to a second barcoding operation. Such a second barcoding operation may occur in a second set of partitions. For example, the first barcoded nucleic acid molecule may be removed from the first set of partitions, pooled (e.g., with other barcoded nucleic acid molecules from other first partitions of the first set of partitions), and partitioned in a second partition of a second set of partitions. The second partition may comprise a capture molecule 1520. The capture molecule 1520 may comprise a second barcode sequence and a sequence complementary to the probe capture sequence 1510 of the first probe 1506 (and / or the second probe 1516). The second barcode sequence may be a sequence that is common to the plurality of capture molecules in the second set of partitions, or the barcode sequence may be unique to the capture molecules in only the second partition (i.e., differ across partitions). The capture molecule 1520 may hybridize to the probe capture sequence 1510 to generate an additional barcoded molecule (also referred to herein as a “third barcoded nucleic acid molecule”). The additional barcoded molecule may comprise a sequence corresponding to the first barcode sequence (of the barcode molecule 1519), and a sequence corresponding to the second barcode sequence (of the capture molecule 1520).

[0254] In addition to barcoding of nucleic acid molecules, the present disclosure provides formethods of multiplexed analysis, e.g., processing of additional biomolecule types, such as proteins and peptides. The method may comprise providing a feature-binding group (e.g., antibody, protein, binding moiety, etc.), which may couple to or bind to a feature (e.g., protein, peptide) of a cell, nucleus or cell bead. Such a method may comprise providing a cell, nucleus or cell bead having a feature of interest (e.g., protein) and contacting the cell, nucleus or cell bead with the feature-binding group. The feature-binding group may couple to the feature of interest. The feature-binding group may comprise a reporter oligonucleotide comprising a reporter sequence coupled thereto, which may be specific for a particular feature and thus be used to identify the feature. For example, the feature-binding group may be an antibody and the reporter oligonucleotide may comprise a reporter sequence that identifies the antigen or binding moiety (e.g., epitope, epitope fragment) to which the antibody couples or binds. Alternatively or in addition to, the feature binding group may comprise a feature probe binding sequence, which may be used for downstream probe-binding and / or barcoding. Following the contacting of the cell (nucleus or cell bead) with the feature binding group, the cell / nucleus / cell bead may comprise the feature coupled to the feature binding group.

[0255] In some instances, the methods described herein may additionally comprise:providing a cell, nucleus or cell bead comprising (i) the nucleic acid molecule comprising theAttorney Docket No.43487-1046601 first target region and the second target region and (ii) the feature coupled to the feature binding group and contacting the cell, nucleus or cell bead with a plurality of probes. The cell / nucleus / cell bead may be contacted (e.g., in a first partition) with a first probe, a second probe, and a third probe. As described herein, the first probe and the second probe may associate with the first target region and the second target region of the nucleic acid molecule, thereby generating a first probe-associated molecule. Similarly, the third probe may associate with (e.g., via hybridization) with the feature binding group, thereby generating a second probe-associated molecule. In some instances, the third probe may comprise a third probe sequence that is complementary to the feature probe binding sequence, and in some instances, the third probe may additionally comprise a probe capture sequence. The first probe and / or the second comprise may also comprise a probe capture sequence, which may be the same or different than the probe capture sequence of the third probe.

[0256] In the first set of partitions, the first probe-associated molecule (e.g., the nucleic acidmolecule with the first probe and the second probe associated therewith) and the second-probe- associated molecule (e.g., the feature binding group with the third probe associated therewith) may be barcoded. Such a barcoding operation may comprise, for example, providing barcode molecules comprising a first barcode sequence and a barcode-capture sequence such as a common sequence, which may hybridize directly with the first probe-associated molecule and the second probe-associated molecule, e.g., via the probe capture sequences. Alternatively or in addition to, the barcode molecules may be provided with probe-binding molecules which comprise (i) a probe binding sequence complementary to the probe capture sequence of the first probe, the second probe, and / or the third probe and (ii) a barcode binding sequence, which may be complementary to the common sequence of the barcode molecules. In some instances, the probe binding molecules and the barcode molecules may be provided as a pre-annealed complex. Barcoding of the first probe-associated molecule and the second probe-associated molecule may include hybridization of the barcode molecules (e.g., the barcode capture sequence such as a common sequence) to a portion (e.g., the probe capture sequence) of the first probe-associated molecule and the second probe-associated molecule, or the barcoding may include hybridization of the barcode molecules to the probe binding molecule and hybridization of the probe binding molecule to the first probe-associated molecule or the second probe-associated molecule. Additional operations such as ligation (e.g., enzymatic or chemical ligation) may be performed to generate the first barcoded molecule and the second barcoded molecule.

[0257] The first barcoded molecule and the second barcoded molecule may be subjected toadditional barcoding operations, e.g., in a second set of partitions. Such additional barcoding operations may include: contacting the first barcoded nucleic acid molecule or derivative thereofAttorney Docket No.43487-1046601 to a first capture molecule of a plurality of capture molecules to generate a third barcoded nucleic acid molecule and contacting the second barcoded nucleic acid molecule or derivative thereof to a second capture molecule of the plurality of capture molecules to generate a fourth barcoded nucleic acid molecule. The capture molecules within a partition may each comprise a second barcode sequence, which may be unique to the partition (i.e., differ across partitions). Accordingly, both the third barcoded nucleic acid molecule and the fourth barcoded nucleic acid molecule may comprise a first barcode sequence (or complement thereof) and a second barcode sequence (or complement thereof).

[0258] FIG. 16A schematically illustrates an example workflow for barcoding multipleanalytes of a cell, nucleus or cell bead. The cell, nucleus or cell bead 1600 may comprise a nucleic acid molecule (e.g., RNA molecule or other target nucleic acid molecule) 1601 comprising a first target region 1602 and a second target region 1604. The cell, nucleus or cell bead may additionally comprise a feature (e.g., a protein, such as a cell surface receptor (or nuclear membrane protein) or an intracellular / intranuclear protein) 1650. In some instances, the cell, nucleus or cell bead 1600 may be processed, e.g., fixed, permeabilized, treated with a treatment, etc. In some instances, such processing may include providing one or more feature binding groups (e.g., antibodies, antibody fragments, etc.) 1652, which may couple to the feature 1650. The feature binding group 1652 may comprise or be coupled to a reporter oligonucleotide 1657, which may comprise a reporter sequence 1654. The reporter sequence 1654 may be indicative of the feature binding group 1652 or feature 1650. For instance, the reporter sequence 1654 may be pre-indexed or assigned to a particular antibody or other feature binding group, such that presence of the reporter sequence 1654 indicates presence of the particular feature 1650 in a sample. The feature binding group 1652 or the reporter oligonucleotide 1657 may also comprise or be coupled to feature probe binding sequence 1656. In some instances, the cell, nucleus or cell bead 1600 may be contacted with the feature binding group 1652 and fixed, e.g., either in addition to or alternatively to a fixation and permeabilization operation before the contacting.

[0259] In some cases, the analysis of both intracellular and / or intranuclear proteins andmembrane proteins of a cell (or nucleus) can be performed. In one embodiment, a permeabilized (and optionally fixed) cell (or nucleus) may be contacted with (i) one or more feature binding groups (or labeling agents) that are configured to couple to intracellular proteins (or intranuclear proteins) and / or (ii) one or more feature binding groups (or labeling agents) that are configured to couple to cell membrane proteins (or nuclear membrane proteins). As further described herein, permeabilization may involve partially or completely dissolving or disrupting a cell membrane (or nuclear membrane) or a portion thereof. Permeabilization may be achieved by,Attorney Docket No.43487-1046601 for example, contacting a cell membrane (or a nuclear membrane) with an organic solvent (e.g., methanol) or a detergent such as Triton X-100 or NP-40. The cell, nucleus, or cell bead may be fixed, as described elsewhere herein.

[0260] Referring again to FIG. 16A, a second feature binding group (or labeling agent)similar to 1652 (not shown) can be used to couple to an intracellular feature, such as an intracellular protein, and comprise or be coupled to a second reporter oligonucleotide, which may comprise a second reporter sequence. The second reporter sequence may be indicative ofthe second feature binding group or the intracellular feature. For instance, the second reportersequence may be pre-indexed or assigned to a particular antibody or other feature binding group, such that presence of the second reporter sequence indicates presence of the particular intracellular feature in a sample. The second feature binding group or the second reporter oligonucleotide may also comprise or be coupled to a second feature probe binding sequence, similar to that of 1656.

[0261] The cell, nucleus or cell bead 1600 may be contacted with a first probe 1606, asecond probe 1616, and a third probe 1658, under conditions sufficient to generate a first probe- associated molecule (or probe-associated complex) 1630 and a second probe-associated molecule (or probe-associated complex) 1665. The first probe-associated molecule 1630 may be or comprise a probe-linked molecule, as described elsewhere herein. For example, the first probe-associated molecule 1630 (or probe-linked molecule) may be any of the probe-associated molecules or probe-linked molecules described herein (e.g., generated from an extended probe, a barcoded extended probe, etc.). The first probe 1606 may comprise a first probe sequence 1608 and, optionally, a probe capture sequence 1610. The first probe sequence 1608 may be complementary to the first target region 1602. The second probe 1616 may comprise a second probe sequence 1615 and, optionally, a probe capture sequence 1618. The second probe sequence 1615 may be complementary to the second target region 1604. The third probe 1658 may comprise a third probe sequence 1660 and a probe capture sequence 1662. The third probe sequence 1660 may be complementary to the feature probe binding sequence 1656. In some instances, the probe capture sequence 1662 is the same probe capture sequence as the probe capture sequences 1610, 1618 of the first probe and / or the second probe, respectively.

[0262] In one embodiment, the cell, cell bead or nucleus 1600 may be further contacted withadditional probes under conditions to generate additional probe-associated molecules or probe-associated complexes. The additional probe-associated molecule(s) may be or comprise a probe-linked molecule, as described elsewhere herein. For example, the additional probe-associatedmolecule(s) or probe-linked molecule(s) may be any of the probe-associated molecules or probe-linked molecules described herein (e.g., generated from an extended probe, a barcoded extendedAttorney Docket No.43487-1046601 probe, etc.). In one embodiment, the cell (or cell bead or nucleus) 1600 may be further contacted with a fourth probe (not shown) similar to 1658 which comprises (i) a fourth probe sequence similar to 1660 and (ii) a fourth probe capture sequence similar to 1662. The fourth probe sequence may be complementary to the second feature probe binding sequence, as further described herein. In some instances, the fourth probe capture sequence is the same probe capture sequence as the probe capture sequences 1610, 1618 of the first probe and / or the second probe, respectively.

[0263] In one embodiment, the cell, nucleus or cell bead 1600 may be partitioned into a firstpartition of a first set of partitions prior to any processing operations described above including, without limitation, fixing, permeabilizing, contacting with probes, and generating probe- associated or probe-linked molecules. In another embodiment, the cell, nucleus or cell bead 1600 may be fixed and optionally permeabilized prior to partitioning in the first partition and then subsequently processed in the first partition, e.g., contacting with probes and generating molecules.

[0264] In operation 1670, the cell, nucleus or cell bead 1600 comprising the first probe-associated molecule 1630 and the second probe-associated molecule 1665 may be partitioned into a first partition of a first set of partitions or further processed in the first partition. In another embodiment, the cell, cell bead or nucleus 1600 may further comprise additional probe- associated molecules or complexes. For instance, referring to FIG.16A, 1600 may comprise a third probe-associated complex (not shown) that is similar to 1665 but comprises (i) a fourth probe comprising a fourth probe sequence complementary to the second feature probe binding sequence and (ii) a reporter oligonucleotide (similar to 1657) as further described herein. The reporter oligonucleotide may be provided as part of or coupled to the second feature binding group, e.g., a feature binding group configured to couple to an intracellular protein. In some instances, the cell, nucleus or cell bead 1600 may be subjected to processing within the partition, such as lysis, to release the cellular / nuclear components (e.g., the first probe-associated molecule and the second probe-associated molecule) within the partition. Alternatively, the cell, nucleus or cell bead 1600 may remain intact. In one embodiment, the cell bead is processed to release cellular components while keeping the cell bead intact. Within the first partition, a probe binding molecule 1617 and a barcode molecule 1619 may be provided. The first probe- associated molecule 1630 and the second probe-associated molecule 1665 may be contacted with one or more probe binding molecules 1617 and barcode molecules 1619. In some examples, the first partition further comprises one or more additional probe-associated molecules or complexes similar to 1665 (not shown). The additional probe-associated complex may comprise the third probe-associated complex described above, which comprises a fourth probe and a reporterAttorney Docket No.43487-1046601 oligonucleotide for a second feature binding group, e.g., a feature binding group configured to couple to an intracellular protein. Additional probe-associated complexes, such as the third probe-associated complex, may be contacted with one or more probe binding molecules 1617 and barcode molecules 1619. In one embodiment, the contacting of a cell, nucleus or cell bead 1600 in the first partition with one or more probe binding molecules may be simultaneously as the contacting with the probes (e.g., 1606, 1616, 1658 and optionally the fourth probe) as described above. The barcode molecules 1619 may comprise a barcode capture sequence or a common sequence common to a plurality of barcode molecules and a first barcode sequence common to the first partition of the first set of partitions. The nucleic acid barcode molecule may, in some instances, be coupled to a bead, such as a gel bead, or other support, as described herein, and can comprise additional functional sequences, including, but not limited to, a unique molecular identifier (UMI), a capture sequence, a primer sequence (e.g., a R1 / R2 sequence), additional barcode sequence segments, etc.. The probe binding molecules 1617 may comprise a probe binding sequence complementary to any or a combination of the probe capture sequences1610, 1618, 1662, a fourth probe capture sequence, and a barcode binding sequencecomplementary to the common sequence of the barcode molecule 1619. In some instances, the probe binding molecules 1617 and the barcode molecules 1619 may be provided as a pre- annealed complex. The probe binding molecules 1617 and the barcode molecules 1619 may hybridize to the first probe-associated molecule 1630 and the second probe-associated molecule 1665 and / or an additional probe-associated complex, such as the third probe-associated complex (e.g., via hybridization of the probe binding molecules 1617 to the probe capture sequences 1610, 1618, 1662, and the fourth probe capture sequence), thereby generating a first barcoded nucleic acid molecule and a second barcoded nucleic acid molecule, and optionally additional barcoded nucleic acid molecules. Additional processing may occur within the first partition, e.g., ligation of the barcode molecules 1619 to the probes (1606, 1616, 1658 or the fourth prob...

Claims

Attorney Docket No.43487-1046601 CLAIMS WHAT IS CLAIMED IS:

1. A method comprising: providing a gRNA-expressing cell comprising a gRNA having a spacer sequence and a constant region comprising a scaffold sequence; contacting the gRNA-expressing cell with a gRNA-targeting probe that hybridizes to the constant region of the gRNA; contacting the gRNA-expressing cell with a ligatable probe pair comprising a first ligatable probe and a second ligatable probe that hybridize to a target nucleic acid in the gRNA- expressing cell; ligating the first ligatable probe to the second ligatable probe using the target nucleic acid as template to generate a ligated probe pair; generating a partition comprising 1) the gRNA-expressing cell, and 2) a plurality of barcoded oligonucleotides comprising a partition-specific barcode; extending the 3’ end of the gRNA-targeting probe to generate an extended gRNA- targeting probe comprising a sequence complementary to the spacer sequence; using the extended gRNA-targeting probe and a first barcoded oligonucleotide of the plurality of barcoded oligonucleotides to generate a barcoded spacer oligonucleotide comprising the spacer sequence or complement thereof, and the partition-specific barcode or complement thereof; and using the ligated probe pair and a second barcoded oligonucleotide of the plurality of barcoded oligonucleotides to generate a barcoded analyte oligonucleotide comprising a sequence of the ligated probe pair or complement thereof, and the partition-specific barcode or a complement thereof.

2. The method of claim 1, wherein the method further comprises sequencing the barcoded spacer oligonucleotide or a derivative thereof and the barcoded analyte oligonucleotide or a derivative thereof.

3. The method of claim 2, wherein the method comprises analyzing the results of the sequencing to determine the sequence of the spacer sequence.

4. The method of claim 2 or 3, wherein the method comprises analyzing the results of the sequencing to determine the presence and / or abundance of the gRNA and the target nucleic acid in the gRNA-expressing cell.Attorney Docket No.43487-1046601 5. The method of any of claims 1-4, wherein the method comprises hybridizing the extended gRNA-targeting probe to the first barcoded oligonucleotide, and extending the 3’ end of the extended gRNA-targeting probe and / or extending the first barcoded oligonucleotide to generate the barcoded spacer oligonucleotide.

6. The method of any of claims 1-5, wherein the extending the 3’ end of the gRNA- targeting probe comprises extending the 3’ end of the gRNA-targeting probe using a reverse transcriptase having terminal deoxynucleotidyl transferase (TdT) activity to incorporate a sequence complementary to the spacer sequence and a non-templated 3’ terminal sequence.

7. The method of claim 6, wherein the method comprises hybridizing the 3’ terminal sequence to the first barcoded oligonucleotide, and extending the 3’ end of the extended gRNA- targeting probe and / or extending the first barcoded oligonucleotide to generate the barcoded spacer oligonucleotide.

8. The method of any of claims 1-7, wherein the gRNA-targeting probe comprises a 5’ overhang.

9. The method of claim 8, wherein the 5’ overhang of the gRNA-targeting probe comprises a barcode sequence, optionally wherein the barcode sequence is a sample-specific barcode sequence.

10. The method of claim 8 or 9, wherein the 5’ overhang of the gRNA-targeting probe comprises one or more functional sequences, optionally wherein the one or more functional sequences of the 5’ overhang of the gRNA-targeting probe comprise a primer hybridization sequence, a sequencing primer binding site, or complement thereof.

11. The method of any of claims 1-10, wherein the gRNA-targeting probe hybridizes to a sequence in the gRNA that is at least 10bp, at least 20bp, at least 30bp, or at least 40bp away from the spacer sequence.

12. The method of any of claims 1-11, wherein the gRNA-targeting probe hybridizes to a sequence in the constant region of the gRNA that is non-structured and / or that does not form a secondary structure of the scaffold sequence via base-pairing.

13. The method of any of claims 1-12, wherein the first ligatable probe comprises a 3’ overhang and a 5’ hybridizing region that hybridizes to the target nucleic acid, and the secondAttorney Docket No.43487-1046601 ligatable probe comprises a 5’ overhang and a 3’ hybridizing region that hybridizes to the target nucleic acid.

14. The method of any of claims 1-13, wherein the ligated probe pair comprises a sequence that is complementary to and / or indicative of the target nucleic acid.

15. The method of any of claims 1-14, wherein the barcoded analyte oligonucleotide comprises a sequence that is complementary to and / or indicative of the target nucleic acid.

16. The method of any of claims 13-15, wherein the method comprises hybridizing a sequence of the 3’ overhang of the ligated probe pair to the second barcoded oligonucleotide, and extending the 3’ end of the ligated probe pair and / or extending the 3’ end of the second barcoded oligonucleotide to generate the barcoded analyte oligonucleotide.

17. The method of any of claims 1-16, wherein the target nucleic acid is an mRNA.

18. The method of any of claims 1-17, wherein the target nucleic acid is not a gRNA.

19. The method of any of claims 1-18, wherein the method comprises removing unhybridized probes from the gRNA-expressing cell.

20. The method of claim 19, wherein the method comprises performing one or more wash steps to remove the unhybridized probes.

21. The method of claim 20, wherein the wash steps are performed prior to generating the partition.

22. The method of any of claims 1-21, wherein the method further comprises: contacting the gRNA-expressing cell with a plurality of ligatable probe pairs that hybridize to a plurality of different target nucleic acids in the cell; ligating the plurality of ligatable probe pairs using the plurality of different target nucleic acids as templates to generate a plurality of ligated probe pairs; and using the plurality of ligated probe pairs and the plurality of barcoded oligonucleotides to generate a plurality of barcoded analyte oligonucleotides; wherein a barcoded analyte oligonucleotide of the plurality of barcoded analyte oligonucleotides comprises a sequence of a ligated probe pair of the plurality of ligated probe pairs or a complement thereof and a sequence of the partition-specific barcode or complement thereof.Attorney Docket No.43487-1046601 23. The method of claim 22, wherein a barcoded analyte oligonucleotide of the plurality of barcoded analyte oligonucleotides comprises a sequence of a target nucleic acid of the plurality of different target nucleic acids or a complement thereof and a sequence of the partition-specific barcode or complement thereof.

24. The method of claim 22 or 23, wherein the method further comprises sequencing the plurality of barcoded analyte oligonucleotides or derivatives thereof.

25. The method of claim 24, wherein the method further comprises analyzing the results of the sequencing to determine the presence and / or abundance of the different target nucleic acids in the gRNA-expressing cell.

26. A method comprising: providing a gRNA-expressing cell comprising a gRNA having a spacer sequence and a constant region comprising a scaffold sequence; contacting the gRNA-expressing cell with a gRNA-targeting probe that hybridizes to the constant region of the gRNA; generating a partition comprising 1) the gRNA-expressing cell, and 2) a plurality of barcoded oligonucleotides comprising a partition-specific barcode and a capture sequence; extending the 3’ end of the gRNA-targeting probe using a reverse transcriptase having terminal deoxynucleotidyl transferase (TdT) activity to incorporate a sequence complementary to the spacer sequence and a non-templated 3’ terminal sequence; hybridizing the 3’ terminal sequence to the capture sequence of a barcoded oligonucleotide of the plurality of barcoded oligonucleotides; and further extending the 3’ end of the gRNA-targeting probe using the barcoded oligonucleotide as template and / or extending the barcoded oligonucleotide using the extended gRNA-targeting probe as template, thereby generating a barcoded spacer oligonucleotide comprising the spacer sequence or complement thereof, and the partition-specific barcode or complement thereof.

27. The method of claim 26, wherein the method further comprises sequencing the barcoded spacer oligonucleotide to determine the sequence of the spacer sequence and the partition-specific barcode, and associating the spacer sequence with the partition-specific barcode.

28. The method of claim 26 or 27, wherein the gRNA-targeting probe comprises a 5’ overhang.Attorney Docket No.43487-1046601 29. The method of claim 28, wherein the 5’ overhang of the gRNA-targeting probe comprises a barcode sequence, optionally wherein the barcode sequence is a sample-specific barcode sequence.

30. The method of claim 28 or 29, wherein the 5’ overhang of the gRNA-targeting probe comprises one or more functional sequences, optionally wherein the one or more functional sequences of the 5’ overhang of the gRNA-targeting probe comprise a primer hybridization sequence, a sequencing primer binding site, or complement thereof.

31. The method of any of claims 26-30, wherein the gRNA-targeting probe hybridizes to a sequence in the gRNA that is at least 10bp, at least 20bp, at least 30bp, or at least 40bp away from the spacer sequence.

32. The method of any of claims 26-31, wherein the gRNA-targeting probe hybridizes to a sequence in the constant region of the gRNA that is non-structured and / or that does not form a secondary structure of the scaffold sequence via base-pairing.

33. A method comprising: providing a gRNA-expressing cell comprising a gRNA having a spacer sequence and a constant region comprising a scaffold sequence; contacting the gRNA-expressing cell with a gRNA-targeting probe that hybridizes to the constant region of the gRNA; extending the 3’ end of the gRNA-targeting probe using a reverse transcriptase having terminal deoxynucleotidyl transferase (TdT) activity to incorporate a sequence complementary to the spacer sequence and a non-templated 3’ terminal sequence; hybridizing the 3’ terminal sequence to a template-switching oligonucleotide (TSO) and further extending the 3’ end of the gRNA-targeting probe to incorporate a sequence complementary to the TSO, thereby generating a TSO-tagged probe; generating a partition comprising 1) the gRNA-expressing cell, and 2) a plurality of barcoded oligonucleotides comprising a partition-specific barcode and a capture sequence; hybridizing the TSO-tagged probe to the capture sequence of a barcoded oligonucleotide of the plurality of barcoded oligonucleotides; and extending the TSO-tagged probe using the barcoded oligonucleotide as template and / or extending the barcoded oligonucleotide using the TSO-tagged probe as template, thereby generating a barcoded spacer oligonucleotide comprising the spacer sequence or complement thereof, and the partition-specific barcode or complement thereof.Attorney Docket No.43487-1046601 34. The method of claim 33, wherein the method further comprises sequencing the barcoded spacer oligonucleotide to determine the sequence of the spacer sequence and the partition-specific barcode, and associating the spacer sequence with the partition-specific barcode.

35. The method of claim 33 or 34, wherein the TSO comprises a barcode sequence, optionally wherein the TSO comprises a sample-specific barcode sequence.

36. The method of any of claims 33-35, wherein the TSO comprises a capturing sequence, and the TSO-tagged probe comprises a complement of the capturing sequence.

37. The method of claim 36, wherein the complement of the capturing sequence in the TSO-tagged probe hybridizes to the capture sequence of the barcoded oligonucleotide.

38. The method of any of claims 33-37, wherein all or a portion of the TSO is dehybridized from the TSO-tagged probe.

39. The method of any of claims 33-38, wherein all or a portion of the TSO is dehybridized from the TSO-tagged probe prior to hybridizing the TSO-tagged probe to the capture sequence of the barcoded oligonucleotide.

40. The method of claim 38 or 39, wherein dehybridizing all or a portion of the TSO from the TSO-tagged probe comprises degrading the TSO.

41. The method of claim 40, wherein degrading the TSO comprises contacting the TSO with an enzyme.

42. The method of any of claims 38-41, wherein the TSO comprises ribonucleotides and dehybridizing all or a portion of the TSO from the TSO-tagged probe comprises contacting the TSO with Ribonuclease H (RNAse H) to digest the TSO.

43. The method of any of claims 38-42, wherein the TSO comprises uracil residues and dehybridizing all or a portion of the TSO from the TSO-tagged probe comprises contacting the TSO with an enzyme to remove the uracil residues.

44. The method of claim 43, wherein the enzyme is a Uracil-DNA Glycosylase (UDG) enzyme.

45. The method of claim 43, wherein the enzyme is a uracil-specific excision reagent (USER) enzyme.Attorney Docket No.43487-1046601 46. The method of any of claims 37-45, wherein the TSO hybridized to the TSO- tagged probe is displaced by hybridization of the capture sequence of the barcoded oligonucleotide to the TSO-tagged probe.

47. The method of any of claims 33-46, wherein the gRNA-targeting probe comprises a 5’ overhang.

48. The method of claim 47, wherein the 5’ overhang of the gRNA-targeting probe comprises a barcode sequence, optionally wherein the barcode sequence is a sample-specific barcode sequence.

49. The method of claim 47 or 48, wherein the 5’ overhang of the gRNA-targeting probe comprises one or more functional sequences, optionally wherein the one or more functional sequences of the 5’ overhang of the gRNA-targeting probe comprise a primer hybridization sequence, a sequencing primer binding site, or complement thereof.

50. The method of any of claims 33-49, wherein the gRNA-targeting probe hybridizes to a sequence in the gRNA that is at least 10bp, at least 20bp, at least 30bp, or at least 40bp away from the spacer sequence.

51. The method of any of claims 33-50, wherein the gRNA-targeting probe hybridizes to a sequence in the constant region of the gRNA that is non-structured and / or that does not form a secondary structure of the scaffold sequence via base-pairing.

52. A method comprising: providing a gRNA-expressing cell comprising a gRNA having a spacer sequence and a constant region comprising a scaffold sequence, wherein the gRNA comprises a 5’ monophosphate; contacting the gRNA-expressing cell with a gRNA ligation adapter comprising a functional region and a 3’ ligation end; ligating the 3’ ligation end of the gRNA ligation adapter to the gRNA, thereby generating a tagged gRNA comprising the functional region; generating a partition comprising 1) the gRNA-expressing cell, and 2) a plurality of barcoded oligonucleotides comprising a partition-specific barcode and a capture sequence; hybridizing the constant region of the tagged gRNA to the capture sequence of a barcoded oligonucleotide of the plurality of barcoded oligonucleotides; and extending the barcoded oligonucleotide using the tagged gRNA as template, thereby generating a barcoded spacer oligonucleotide comprising the partition-specific barcode, aAttorney Docket No.43487-1046601 sequence complementary to the spacer sequence, and a sequence complementary to the functional region.

53. The method of claim 52, wherein the constant region of the gRNA comprises a capturing sequence, and wherein the constant region of the tagged gRNA is hybridized via the capturing sequence to the capture sequence of the barcoded oligonucleotide.

54. The method of claim 53, wherein the capturing sequence is at the 3’ end of the constant region of the gRNA.

55. The method of claim 53, wherein the capturing sequence is within and / or flanked by the scaffold sequence of the gRNA.

56. The method of any of claims 53-55, wherein the capturing sequence is complementary to the capture sequence.

57. A method comprising: providing a gRNA-expressing cell comprising a gRNA having a spacer sequence and a constant region comprising a scaffold sequence, wherein the gRNA comprises a 5’ monophosphate; contacting the gRNA-expressing cell with a gRNA ligation adapter comprising a 3’ ligation end, and a functional region comprising a capturing sequence; ligating the 3’ end of the gRNA ligation adapter to the gRNA, thereby generating a tagged gRNA; contacting the tagged gRNA with a primer that hybridizes to the constant region of the gRNA, and extending the primer using the tagged gRNA as template, thereby generating a tagged gRNA complement that comprises a sequence complementary to the spacer sequence and a complement of the capturing sequence; generating a partition comprising 1) the gRNA-expressing cell, and 2) a plurality of barcoded oligonucleotides comprising a partition-specific barcode and a capture sequence; hybridizing the complement of the capturing sequence in the tagged gRNA complement to the capture sequence of a barcoded oligonucleotide of the plurality of barcoded oligonucleotides; and extending the barcoded oligonucleotide using the tagged gRNA complement as template and / or extending the tagged gRNA complement using the barcoded oligonucleotide as template, thereby generating a barcoded spacer oligonucleotide comprising the partition-specific barcode or a complement thereof, and the sequence of the spacer sequence or a complement thereof.Attorney Docket No.43487-1046601 58. The method of claim 57, wherein the primer that hybridizes to the constant region of the gRNA comprises a 5’ overhang.

59. The method of claim 58, wherein the 5’ overhang of the primer that hybridizes to the constant region of the gRNA comprises a barcode sequence.

60. The method of claim 58 or 59, wherein the 5’ overhang of the primer that hybridizes to the constant region of the gRNA comprises a sample-specific barcode sequence.

61. The method of any of claims 58-60, wherein the 5’ overhang of the primer that hybridizes to the constant region of the gRNA comprises one or more functional sequences.

62. The method of claim 61, wherein the one or more functional sequences of the 5’ overhang of the primer that hybridizes to the constant region of the gRNA comprise a primer hybridization sequence, a sequencing primer binding site, or complement thereof.

63. The method of any of claims 52-62, wherein the gRNA ligation adapter comprises the functional region; a 5’ hybridizing region that hybridizes to the gRNA; and a self-hybridizing region, wherein the self-hybridizing region comprises a first sequence and second sequence that hybridize to one another, wherein the second sequence of the self-hybridizing region comprises the 3’ ligation end, and wherein the 3’ ligation end is configured to be ligated to the 5’ end of the gRNA upon hybridization of the 5’ hybridizing region to the gRNA.

64. The method of claim 63, wherein the gRNA ligation adapter comprises a first gRNA ligation adapter nucleic acid molecule and a second gRNA ligation adapter nucleic acid molecule.

65. The method of claim 64, wherein: the first gRNA ligation adapter nucleic acid molecule comprises the 5’ hybridizing region that hybridizes to the gRNA, and the first sequence of the self-hybridizing region; and the second gRNA ligation adapter nucleic acid molecule comprises the functional region and the second sequence of the self-hybridizing region comprising the 3’ ligation end.

66. The method of any of claims 52-63, wherein the gRNA ligation adapter is a single molecule gRNA ligation adapter.

67. The method of claim 66, wherein the single molecule gRNA ligation adapter comprises in the 5’ to 3’ direction: the 5’ hybridizing region, the first sequence of the self-Attorney Docket No.43487-1046601 hybridizing region, the functional region, and the second sequence of the self-hybridizing region comprising the 3’ ligation end that is configured to be ligated to the 5’ end of the gRNA upon hybridization of the 5’ hybridizing region to the gRNA.

68. The method of claim 66 or 67, wherein the single molecule gRNA ligation adapter has a stem-loop structure.

69. The method of claim 68, wherein the functional region is in the loop of the stem- loop structure.

70. The method of any of claims 52-69, wherein the functional region comprises a barcode sequence.

71. The method of any of claims 52-70, wherein the functional region comprises a sample-specific barcode sequence.

72. The method of any of claims 52-71, wherein the functional region comprises one or more functional sequences, optionally wherein the one or more functional sequences of the functional region comprise a primer hybridization sequence, a sequencing primer binding site, or complement thereof.

73. The method of any of claims 52-72, wherein the method further comprises sequencing the barcoded spacer oligonucleotide to determine the sequence of the spacer sequence and the partition-specific barcode, and associating the spacer sequence with the partition-specific barcode.

74. The method of any of claims 52-73, wherein the gRNA ligation adapter comprises a polymerase block site that is configured to terminate 3’ extension of a polynucleotide by a polymerase using the gRNA ligation adapter as template.

75. The method of claim 74, wherein the polymerase block site is 5’ of the functional region and / or 3’ of the first sequence of the self-hybridizing region.

76. The method of claim 74 or 75, wherein the polymerase block site comprises an abasic site.

77. The method of claim 76, wherein the polymerase block site comprises uracil, and the uracil is removed to generate the abasic site.Attorney Docket No.43487-1046601 78. The method of claim 77, wherein the uracil is removed by contacting the uracil with a Uracil-DNA Glycosylase (UDG) enzyme or a Uracil-Specific Excision Reagent (USER) enzyme.

79. The method of any of claims 74-78, wherein the polymerase block site terminates extension of the barcoded oligonucleotide using the tagged gRNA as template.

80. The method of any of claims 74-79, wherein the polymerase block site is 5’ of the capturing sequence in the gRNA ligation adapter.

81. The method of any of claims 74-78 and 80, wherein the polymerase block site terminates extension of the primer that hybridizes to the constant region of the gRNA during the generation of the tagged gRNA complement.

82. The method of any of claims 52-81, wherein the method comprises modifying a pre-modified gRNA to generate the gRNA comprising the 5’ monophosphate.

83. The method of claim 82, wherein the pre-modified gRNA comprises a 5’ triphosphate, and the method comprises modifying the 5’ triphosphate to generate the 5’ monophosphate.

84. The method of claim 82 or 83, wherein the method comprises contacting the pre- modified gRNA with an enzyme to generate gRNA comprising the 5’ monophosphate.

85. The method of claim 84, wherein the enzyme is RNA 5’ Pyrophosphohydrolase (RppH).

86. The method of any of claims 63-85, wherein the 5’ hybridizing region hybridizes to the spacer sequence of the gRNA.

87. The method of any of claims 63-85, wherein the 5’ hybridizing region hybridizes to the constant region of the gRNA.

88. The method of any of claims 63-85, wherein the 5’ hybridizing region hybridizes to the spacer sequence of the gRNA and the constant region of the gRNA.

89. The method of any of claims 63-88, wherein the 5’ hybridizing region comprises a non-specific hybridization region.Attorney Docket No.43487-1046601 90. The method of claim 89, wherein the non-specific hybridization region comprises a sequence of residues capable of hybridizing to different spacer sequences.

91. The method of claim 89 or 90, wherein the non-specific hybridization region comprises inosine residues.

92. The method of any of claims 89-91, wherein the non-specific hybridization region comprises a sequence of inosine residues capable of hybridizing to different spacer sequences.

93. The method of any of claims 63-92, wherein the 5’ hybridizing region comprises a sequence that is complementary to a portion of the constant region of the gRNA.

94. The method of claim 93, wherein the sequence that is complementary to a portion of the constant region of the gRNA is at the 5’ end of the 5’ hybridizing region.

95. The method of any of claims 63-94, wherein the 5’ hybridizing region comprises a non-hybridizing portion and a hybridizing portion.

96. The method of claim 95, wherein the non-hybridizing portion comprises a carbon spacer.

97. The method of claim 95 or 96, wherein the hybridizing portion hybridizes to at least a portion of the gRNA spacer and / or at least a portion of the constant region of the gRNA.

98. A method comprising: providing a gRNA-expressing cell comprising a gRNA having a spacer sequence and a constant region comprising a scaffold sequence; contacting the gRNA-expressing cell with a gRNA ligation adapter comprising a capturing sequence and a 5’ ligation end; ligating the 5’ ligation end of the gRNA ligation adapter to the gRNA, thereby generating a tagged gRNA comprising the capturing sequence; generating a partition comprising 1) the gRNA-expressing cell, and 2) a plurality of barcoded oligonucleotides comprising a partition-specific barcode and a capture sequence; hybridizing the capturing sequence to the capture sequence of a barcoded oligonucleotide of the plurality of barcoded oligonucleotides; and using the barcoded oligonucleotide and the tagged gRNA to generate a barcoded spacer oligonucleotide comprising 1) the partition-specific barcode or a complement thereof, and 2) a sequence of the spacer or a complement thereof.Attorney Docket No.43487-1046601 99. The method of claim 98, wherein the method comprises extending the barcoded oligonucleotide using the tagged gRNA as template, thereby generating a barcoded spacer oligonucleotide comprising the partition-specific barcode and a sequence complementary to the spacer sequence.

100. The method of claim 98 or 99, wherein the 5’ ligation end of the gRNA ligation adapter is ligated to the gRNA prior to generating the partition.

101. The method of claim 98 or 99, wherein the 5’ ligation end of the gRNA ligation adapter is ligated to the gRNA after generating the partition.

102. The method of any of claims 98-101, wherein the gRNA ligation adapter comprises: the capturing sequence; a 3’ hybridizing region that hybridizes to the gRNA; and a self-hybridizing region, wherein the self-hybridizing region comprises a first sequence and second sequence that hybridize to one another, wherein the second sequence of the self- hybridizing region comprises the 5’ ligation end, and wherein the 5’ ligation end is configured to be ligated to the 3’ end of the gRNA upon hybridization of the 3’ hybridizing region to the gRNA.

103. The method of any of claims 98-102, wherein the gRNA ligation adapter comprises a first gRNA ligation adapter nucleic acid molecule and a second gRNA ligation adapter nucleic acid molecule.

104. The method of claim 103, wherein: the first gRNA ligation adapter nucleic acid molecule comprises the 3’ hybridizing region that hybridizes to the gRNA and the first sequence of the self-hybridizing region; and the second gRNA ligation adapter nucleic acid molecule comprises the capturing sequence and the second sequence of the self-hybridizing region comprising the 5’ ligation end.

105. The method of any of claims 98-102, wherein the gRNA ligation adapter is a single molecule gRNA ligation adapter.

106. The method of claim 105, wherein the single molecule gRNA ligation adapter comprises in the 3’ to 5’ direction: the 3’ hybridizing region, the first sequence of the self- hybridizing region, the capturing sequence, and the second sequence of the self-hybridizingAttorney Docket No.43487-1046601 region comprising the 5’ ligation end that is configured to be ligated to the 3’ end of the gRNA upon hybridization of the 3’ hybridizing region to the gRNA.

107. The method of claim 105 or 106, wherein the single molecule gRNA ligation adapter has a stem-loop structure.

108. The method of claim 107, wherein the capturing sequence is in the loop of the stem-loop structure.

109. The method of any of claims 98-108, wherein the 5’ ligation end of the gRNA ligation adapter comprises a 5’ monophosphate.

110. The method of any of claims 98-109, wherein the gRNA ligation adapter further comprises a sample-specific barcode sequence, and wherein the barcoded spacer oligonucleotide further comprises the sample-specific barcode sequence or a complement thereof.

111. The method of any of claims 98-109, wherein the constant region of the gRNA further comprises a functional sequence.

112. The method of claim 111, wherein the functional sequence is at the 5’ end of the constant region of the gRNA.

113. The method of claim 111, wherein the functional sequence is within and / or flanked by the scaffold sequence of the gRNA.

114. The method of any of claims 111-113, wherein the functional sequence comprises a primer hybridization sequence, a sequencing primer binding site, or a complement thereof.

115. The method of any of claims 102-114, wherein the 3’ hybridizing region hybridizes to the spacer sequence of the gRNA.

116. The method of any of claims 102-115, wherein the 3’ hybridizing region hybridizes to the constant region of the gRNA.

117. The method of any of claims 102-116, wherein the 3’ hybridizing region hybridizes to the spacer sequence of the gRNA and the constant region of the gRNA.

118. The method of any of claims 102-117, wherein the 3’ hybridizing region comprises a non-specific hybridization region.Attorney Docket No.43487-1046601 119. The method of claim 118, wherein the non-specific hybridization region comprises a sequence of residues capable of hybridizing to different spacer sequences.

120. The method of claim 118 or 119, wherein the non-specific hybridization region comprises inosine residues.

121. The method of any of claims 118-120, wherein the non-specific hybridization region comprises a sequence of inosine residues capable of hybridizing to different spacer sequences.

122. The method of any of claims 102-121, wherein the 3’ hybridizing region comprises a sequence that is complementary to a portion of the constant region of the gRNA.

123. The method of claim 122, wherein the sequence that is complementary to a portion of the constant region of the gRNA is at the 3’ end of the 3’ hybridizing region.

124. The method of any of claims 102-123, wherein the 3’ hybridizing region comprises a non-hybridizing portion and a hybridizing portion.

125. The method of claim 124, wherein the non-hybridizing portion comprises a carbon spacer.

126. The method of claim 124 or 125, wherein the hybridizing portion hybridizes to at least a portion of the gRNA spacer and / or at least a portion of the constant region of the gRNA.

127. The method of any of claims 1-126, wherein the method further comprises sequencing the barcoded spacer oligonucleotide or a derivative thereof.

128. The method of claim 127, wherein the method comprises analyzing the results of the sequencing to determine the sequence of the spacer sequence.

129. The method of claim 127 or 128, wherein the method comprises analyzing the results of the sequencing to determine the presence and / or abundance of the gRNA in the gRNA- expressing cell.

130. The method of any of claims 1-129, wherein the partition comprises the gRNA- expressing cell and no other cells.

131. The method of any of claims 1-130, wherein the method comprises removing unhybridized probes from the gRNA-expressing cell.Attorney Docket No.43487-1046601 132. The method of any of claims 1-131, wherein the method comprises performing one or more wash steps to remove unhybridized probes.

133. The method of any of claims 1-132, wherein the method comprises performing one or more wash steps prior to generating the partition.

134. The method of any of claims 1-133, wherein the method further comprises: contacting the gRNA-expressing cell with a ligatable probe pair comprising a first ligatable probe and a second ligatable probe that hybridize to a target nucleic acid in the gRNA- expressing cell; ligating the first ligatable probe to the second ligatable probe using the target nucleic acid as template to generate a ligated probe pair; and using the ligated probe pair and a second barcoded oligonucleotide of the plurality of barcoded oligonucleotides to generate a barcoded analyte oligonucleotide comprising a sequence of the ligated probe pair or complement thereof, and the partition-specific barcode or a complement thereof.

135. The method of claim 134, wherein the method comprises sequencing the barcoded spacer oligonucleotide or a derivative thereof and the barcoded analyte oligonucleotide or a derivative thereof.

136. The method of claim 135, wherein the method comprises analyzing the results of the sequencing to determine the sequence of the spacer sequence.

137. The method of claim 135 or 136, wherein the method comprises analyzing the results of the sequencing to determine the presence and / or abundance of the gRNA and / or the target nucleic acid in the gRNA-expressing cell.

138. The method of any of claims 134-137, wherein the first ligatable probe comprises a 3’ overhang and a 5’ hybridizing region that hybridizes to the target nucleic acid, and the second ligatable probe comprises a 5’ overhang and a 3’ hybridizing region that hybridizes to the target nucleic acid.

139. The method of any of claims 134-138, wherein the ligated probe pair comprises a sequence that is complementary to and / or indicative of the target nucleic acid.Attorney Docket No.43487-1046601 140. The method of any of claims 134-139, wherein the barcoded analyte oligonucleotide comprises a sequence that is complementary to and / or indicative of the target nucleic acid.

141. The method of any of claims 138-140, wherein the method comprises hybridizing a sequence of the 3’ overhang of the ligated probe pair to the second barcoded oligonucleotide, and extending the 3’ end of the ligated probe pair and / or extending the 3’ end of the barcoded oligonucleotide to generate the barcoded analyte oligonucleotide.

142. The method of any of claims 134-141, wherein the target nucleic acid is not a gRNA.

143. The method of any of claims 134-142, wherein the target nucleic acid is an mRNA.

144. The method of any of claims, wherein the method further comprises: contacting the gRNA-expressing cell with a plurality of ligatable probe pairs that hybridize to a plurality of different target nucleic acids in the cell; ligating the plurality of ligatable probe pairs using the plurality of different target nucleic acids as templates to generate a plurality of ligated probe pairs; and using the plurality of ligated probe pairs and the plurality of barcoded oligonucleotides to generate a plurality of barcoded analyte oligonucleotides; wherein a barcoded analyte oligonucleotide of the plurality of barcoded analyte oligonucleotides comprises a sequence of a ligated probe pair of the plurality of ligated probe pairs or a complement thereof and a sequence of the partition-specific barcode or complement thereof.

145. The method of claim 144, wherein a barcoded analyte oligonucleotide of the plurality of barcoded analyte oligonucleotides comprises a sequence of a target nucleic acid of the plurality of different target nucleic acids or a complement thereof and a sequence of the partition-specific barcode or complement thereof.

146. The method of claim 144 or 145, wherein the method further comprises sequencing the plurality of barcoded analyte oligonucleotides or derivatives thereof.

147. The method of claim 146, wherein the method further comprises analyzing the results of the sequencing to determine the presence and / or abundance of the different target nucleic acids in the gRNA-expressing cell.Attorney Docket No.43487-1046601 148. The method of any of claims 1-147, wherein the method is performed in parallel for a plurality of gRNA-expressing cells, wherein different partitions are generated for different gRNA-expressing cells of the plurality of gRNA-expressing cells, and wherein barcoded spacer oligonucleotides comprising partition-specific barcodes are generated from the different gRNA- expressing cells.

149. The method of claim 148, wherein barcoded analyte oligonucleotides are generated from the different gRNA-expressing cells.

150. The method of claim 148 or 149, wherein the method comprises sequencing the barcoded spacer oligonucleotides or derivatives thereof and / or the barcoded analyte oligonucleotides or derivatives thereof.

151. The method of claim 150, wherein the method comprises analyzing the results of the sequencing to determine the presence and / or abundance of one or more gRNAs and one or more target nucleic acids in the different gRNA-expressing cells of the plurality of gRNA- expressing cells.

152. The method of any of claims 1-151, wherein the method comprises: contacting the gRNA-expressing cell with a ligatable probe pair comprising 1) a first ligatable probe having a 3’ overhang, and a 5’ hybridizing region that hybridizes to a target nucleic acid in the cell, and 2) a second ligatable probe having a 3’ hybridizing region that hybridizes to the target nucleic acid in the cell, and a 5’ overhang; ligating the 5’ hybridizing region of the first ligatable probe to the 3’ hybridizing region of the second ligatable probe using the target nucleic acid as template, thereby generating a ligated probe pair comprising a sequence complementary to and / or indicative of the target nucleic acid; hybridizing a sequence of the 3’ overhang to the capture sequence of a barcoded oligonucleotide of the plurality of barcoded oligonucleotides in the partition; extending the 3’ end of the ligated probe pair to incorporate a sequence complementary to the barcoded oligonucleotide and / or extending the 3’ end of the barcoded oligonucleotide to incorporate a sequence complementary to the ligated probe pair, thereby generating a barcoded analyte oligonucleotide comprising: the sequence of the ligated probe pair or complement thereof, and the sequence of the barcoded capture oligonucleotide or complement thereof.

153. The method of claim 152, wherein the method further comprises sequencing the barcoded analyte oligonucleotide to determine the sequence complementary to and / or indicativeAttorney Docket No.43487-1046601 of the target nucleic acid and the sequence of the partition-specific barcode, and associating the target nucleic acid with the partition-specific barcode.

154. The method of claim 152 or 153, wherein the 3’ overhang of the first ligatable probe and / or the 5’ overhang of the second ligatable probe comprise a barcode sequence.

155. The method of any of claims 152-154, wherein the 3’ overhang of the first ligatable probe and / or the 5’ overhang of the second ligatable probe comprise a sample-specific barcode sequence.

156. The method of any of claims 152-155, wherein the 3’ overhang of the first ligatable probe and / or the 5’ overhang of the second ligatable probe comprise one or more functional sequences.

157. The method of claim 156, wherein the one or more functional sequences of the 3’ overhang of the first ligatable probe and / or the 5’ overhang of the second ligatable probe comprise a primer hybridization sequence, a sequencing primer binding site, or complement thereof.

158. The method of any of claims 152-157, wherein the first ligatable probe is ligated to the second ligatable probe in the partition.

159. The method of any of claims 152-157, wherein the first ligatable probe is ligated to the second ligatable probe prior to generating the partition.

160. The method of any of claims 1-159, wherein the plurality of barcoded oligonucleotides comprise one or more functional sequences.

161. The method of claim 160, wherein the one or more functional sequences of the plurality of barcoded oligonucleotides comprise a primer hybridization sequence, a sequencing primer binding site, or complement thereof.

162. The method of any of claims 1-161, wherein barcoded oligonucleotides of the plurality of barcoded oligonucleotides comprise unique molecular identifier (UMI) sequences.

163. The method of any of claims 152-162, wherein the method comprises sequencing the barcoded analyte oligonucleotide and the barcoded spacer oligonucleotide, thereby determining the presence of the target analyte and the presence of the gRNA having the spacer sequence in the same cell.Attorney Docket No.43487-1046601 164. The method of claim 163, wherein the barcoded spacer oligonucleotide and barcoded analyte oligonucleotide are amplified and / or sequenced outside of the partition.

165. The method of any of claims 1-164, wherein the method is performed in parallel for a plurality of gRNA-expressing cells, wherein different partitions are generated for different gRNA-expressing cells of the plurality of gRNA-expressing cells, and wherein barcoded spacer oligonucleotides comprising partition-specific barcodes are generated from the different gRNA- expressing cells.

166. The method of claim 165, wherein barcoded analyte oligonucleotides comprising partition-specific barcodes are generated from the different gRNA-expressing cells.

167. The method of claim 165 or 166, wherein the method comprises sequencing the one or more barcoded spacer oligonucleotides and / or the one or more barcoded analyte oligonucleotides from the different gRNA-expressing cells.

168. The method of any of claims 165-167, wherein for the gRNA expressing cells, the presence and / or abundance of one or more gRNA spacer sequences is determined.

169. The method of any of claims 165-168, wherein for the gRNA expressing cells, the presence and / or abundance of one or more target nucleic acids is determined.

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