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WO2025027136A1 - Criblage crispr unicellulaire de perturbations géniques multiples in vivo - Google Patents

Criblage crispr unicellulaire de perturbations géniques multiples in vivo Download PDF

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WO2025027136A1
WO2025027136A1 PCT/EP2024/071816 EP2024071816W WO2025027136A1 WO 2025027136 A1 WO2025027136 A1 WO 2025027136A1 EP 2024071816 W EP2024071816 W EP 2024071816W WO 2025027136 A1 WO2025027136 A1 WO 2025027136A1
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grna
cell
promoter
gene
vector
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António José SANTINHA
Randall Jeffrey Platt
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Eidgenoessische Technische Hochschule Zurich ETHZ
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Eidgenoessische Technische Hochschule Zurich ETHZ
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    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]

Definitions

  • the present invention relates to a method allowing for a single-cell-based analysis of multiple CRISPR- mediated gene perturbations in a single organism.
  • the method comprises the administration of a plurality of expression vectors each encoding a gRNA into an organism expressing a Cas enzyme, and allows for analysis of the resulting phenotype on a single-cell level.
  • a general framework for direct in vivo single-cell screening could broadly facilitate mechanistic studies of health and disease as well as enable the systematic interrogation of the vast and growing catalog of disease- associated risk alleles in disease-relevant cells and tissues.
  • Genomics studies have identified thousands of genetic variants associated with human disease and interrogating them in in vivo models is essential to understanding their causality, function, and pathology as well as developing new diagnostics and therapeutics.
  • US 2020 018 746 A1 discloses a method for perturbation of disease-implicated genes in 3D tissues composed of human induced neuronal cells and astrocytic cells.
  • US 2021 172 017 A1 discloses that Perturb-seq and single-cell sequencing allow the reconstruction of a cellular network or circuit.
  • WO 2019 113 499 A1 discloses the use of AAV in a Perturb-seq method.
  • WO 2015 089 462 A1 discloses SpCas9- mediated in vivo genome editing in the brain. Still, high-throughput, phenotype-rich, and broadly- applicable in vivo methods are urgently needed.
  • AAV-Perturb-seq an adeno-associated virus (AAV)-based single-cell or -nuclei CRISPR screening method that is simple to implement, tunable, and broadly applicable for in vivo functional genomics studies.
  • AAV adeno-associated virus
  • gRNA guide RNA
  • AAV-Perturb-seq using either gene editing in LSL-Cas9 mice or transcriptional inhibition in dCas9-KRAB mice, to systematically interrogate the genotype-phenotype landscape of individual genes linked to 22q1 1.2 deletion syndrome (22q1 1.2 DS, also known as DiGeorge syndrome), a complex genetic disorder affecting numerous organs including the brain, where dysfunction is typically clinically expressed as schizophrenia or autism spectrum disorder (ASD).
  • the objective of the present invention is to provide means and methods to analyse multiple gRNA-mediated gene perturbations on a single-cell level in vivo in a single organism.
  • a first aspect of the invention relates to a method for analyzing multiple gene perturbations in vivo in a tissue of interest; said method comprising the steps: a. providing an organoid or a non-human organism; b. administering a plurality of viral gRNA-delivering nucleic acid expression vectors to the organoid or organism, each vector comprising: i. inverted-terminal repeats (ITRs); ii. a gRNA promoter;
  • gRNA guide-RNA
  • each vector comprises a different gRNA or gRNA combination; and iv. a terminator of transcription; wherein the organism expresses a Cas enzyme, or said vector additionally encodes a Cas enzyme under control of a promoter operable in said cell; c. isolating a sample of the tissue of interest from the organism, or a sample of the organoid; d. in a collection step, collecting cells or nuclei from the sample; e.
  • gRNA guide-RNA
  • a single-cell or single-nucleus assay comprising an analysis of the gene perturbation and comprising gRNA sequencing of each cell or nucleus, thereby generating a plurality of assay patterns related to expression of a defined gRNA or a defined gRNA combination.
  • references to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”
  • gene refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after being transcribed and translated.
  • ORF open reading frame
  • a polynucleotide sequence can be used to identify larger fragments or full-length coding sequences of the gene with which they are associated. Methods of isolating larger fragment sequences are known to those of skill in the art.
  • transgene in the context of the present specification relates to a gene or genetic material that has been transferred from one organism to another.
  • the term may also refer to transfer of the natural or physiologically intact variant of a genetic sequence into tissue of a patient where it is missing. It may further refer to transfer of a natural encoded sequence the expression of which is driven by a promoter absent or silenced in the targeted tissue.
  • a recombinant in the context of the present specification relates to a nucleic acid, which is the product of one or several steps of cloning, restriction and/or ligation and which is different from the naturally occurring nucleic acid.
  • a recombinant virus particle comprises a recombinant nucleic acid.
  • gene expression or expression may refer to either of, or both of, the processes - and products thereof - of generation of nucleic acids (RNA) or the generation of a peptide or polypeptide, also referred to transcription and translation, respectively, or any of the intermediate processes that regulate the processing of genetic information to yield polypeptide products.
  • the term gene expression may also be applied to the transcription and processing of a RNA gene product, for example a regulatory RNA or a structural (e.g. ribosomal) RNA. If an expressed polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. Expression may be assayed both on the level of transcription and translation, in other words mRNA and/or protein product.
  • nucleotides in the context of the present specification relates to nucleic acid or nucleic acid analogue building blocks, oligomers of which are capable of forming selective hybrids with RNA or DNA oligomers on the basis of base pairing.
  • nucleotides in this context includes the classic ribonucleotide building blocks adenosine, guanosine, uridine (and ribosylthymine), cytidine, the classic deoxyribonucleotides deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine and deoxycytidine.
  • nucleic acids such as phosphothioates, 2’0-methylphosphothioates, peptide nucleic acids (PNA; N-(2-aminoethyl)-glycine units linked by peptide linkage, with the nucleobase attached to the alpha-carbon of the glycine) or locked nucleic acids (LNA; 2’0, 4’C methylene bridged RNA building blocks).
  • PNA peptide nucleic acids
  • LNA locked nucleic acids
  • hybridizing sequence may be composed of any of the above nucleotides, or mixtures thereof.
  • nucleic acid expression vector in the context of the present specification relates to a plasmid, a viral genome or an RNA, which is used to transfect (in case of a plasmid or an RNA) or transduce (in case of a viral genome) a target cell with a certain gene of interest, or -in the case of an RNA construct being transfected- to translate the corresponding protein of interest from a transfected mRNA.
  • the gene of interest is under control of a promoter sequence and the promoter sequence is operational inside the target cell, thus, the gene of interest is transcribed either constitutively or in response to a stimulus or dependent on the cell’s status.
  • the viral genome is packaged into a capsid to become a viral vector, which is able to transduce the target cell.
  • fluorescent protein in the context of the present specification may relate, but is not limited to, a fluorescent protein selected from the group comprising: green fluorescent protein (GFP) from Aequorea victoria and derivatives thereof, such as enhanced blue fluorescent protein (EBFP), enhanced blue fluorescent protein 2 (EBFP2), azurite, mKalamal , Sirius; enhanced green fluorescent protein (EGFP), emerald, superfolder avGFP, T-sapphire; yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), citrine, venus, YPet, topaz, SYFP, mAmetrine enhanced cyan fluorescent protein (ECFP), mTurquoise, mTurquoise2, cerulean, CyPet, SCFP; fluorescent protein from Discosoma striata and derivatives thereof: mTagBFP,
  • GFP green fluorescent protein
  • EBFP enhanced blue fluorescent protein
  • EBFP2 enhanced blue fluorescent protein 2
  • EGFP enhanced green fluorescent protein
  • TagCFP AmCyan, Midoriishi Cyan, mTFP1
  • gRNA in the context of the present specification relates to a guide RNA which comprises a crRNA part and a tracrRNA part.
  • gRNA combination in the context of the present specification relates to multiple (a plurality of) gRNAs.
  • a gRNA combination consists of 2 to 1000 gRNAs.
  • a gRNA combination consists of 2 to 100 gRNAs.
  • a gRNA combination consists of 2 to 10 gRNAs.
  • gene perturbation in the context of the present specification relates to an alteration in gene expression induced by CRISPR/Cas and a gRNA.
  • a gene perturbation relates to the loss of expression of the gene targeted by the respective gRNA.
  • a gene perturbation relates to the inhibition of transcription of the target gene by the respective gRNA and Cas-effector (eg KRAB) complex.
  • a gene perturbation relates to the activation of transcription of the target gene by the respective gRNA and Cas-effector (eg VP64, HSF1 , p65) complex.
  • a gene perturbation relates to the base editing of the target gene by the respective gRNA and base editing complex.
  • a gene perturbation relates to the prime editing of the target gene by the respective prime editing gRNA and base editing complex.
  • Cas enzyme in the context of the present specification relates to an enzyme of the Cas family.
  • the Cas enzyme is a Cas enzyme of type II.
  • the Cas enzyme is a Cas9 enzyme of UniProt-ID Q99ZW2.
  • polypeptide part in the context of the present specification relates to one or several domains of a polypeptide, wherein the domain(s) are covalently linked to the rest of the polypeptide.
  • gRNA-delivering nucleic acid expression vector in the context of the present specification relates to a nucleic acid expression vector which encodes a gRNA, and optionally further elements.
  • a first aspect of the invention relates to a method for analyzing multiple (a plurality of) gene perturbations in vivo in a tissue of interest.
  • the method comprises the steps:
  • Step a providing an organoid or a non-human multi-cellular organism, particularly a non-human organism.
  • An organoid is a multi-cellular, three-dimensional, simplified version of an organ, which is generated in vitro.
  • the organoid can be composed of human or non-human cells.
  • the non-human organism is an animal or a plant or a fungus.
  • the non-human organism is an animal.
  • the non-human organism is an adult organism.
  • the non-human organism is an adult animal.
  • the non-human organism is an animal which is born (not an embryo, post-embryonic stage).
  • Step b administering a plurality of gRNA-delivering nucleic acid expression vectors to the organoid or organism, wherein each vector comprises the following elements: i. a gRNA promoter allowing for transcription inside a tissue of interest (particularly a Pol III RNA polymerase promoter); ii. at least one guide-RNA (gRNA) transcribable under control of said gRNA promoter, wherein each vector of the plurality comprises a different gRNA or gRNA combination (in case more than one gRNA is encoded); and
  • the organism expresses a Cas enzyme, or the gRNA-delivering nucleic acid expression vector additionally encodes a Cas enzyme under control of a promoter operable in said cell.
  • the Cas enzyme is encoded in the genome of the organism or organoid.
  • the Cas enzyme is delivered via a separate nucleic acid expression vector.
  • the gRNA-delivering nucleic acid expression vector is a viral vector.
  • each vector comprises the following elements: i. inverted-terminal repeats (ITRs) flanking the elements described below at the 3’ and the 5’ end (positioned 5'upstream and 3’ downstream of all elements encoded by the vector, i.e. the elements described below: the gRNA and its promoter, optionally a gene encoding a Cas enzyme, and other optional elements); ii. a gRNA promoter allowing for transcription inside a tissue of interest; (particularly a Pol III RNA polymerase promoter);
  • each vector of the plurality comprises a different gRNA or gRNA combination (in case more than one gRNA is encoded); and iv. a terminator of transcription in 3’ direction of the gRNA;
  • the expression vector is delivered via a peptide-based delivery system using cell-penetrating peptides.
  • the expression vector is delivered via a lipid-based delivery system via lipid nano-particles.
  • the expression vector is delivered via an inorganic delivery system using black phosphorus, graphene oxide, mesoporous silica nanoparticles, or gold nanoparticles.
  • the expression vector is delivered via a polymeric delivery system, wherein the expression vector is incorporated into a polymer.
  • the expression vector is delivered via electroporation.
  • Lan et al. (Mol Cancer 21 , 71 (2022)) reviews the non-viral delivery ways, and is incorporated by reference herein.
  • Step b1 keeping the organism under conditions allowing the organism to live or the organoid under conditions allowing the organoid to stay intact.
  • these conditions are physiological conditions.
  • these conditions are pathological conditions which impose a particular burden on the organism or organoid.
  • Step c isolating a sample of the tissue of interest from the organism, or a sample of the organoid. This sample may comprise a complete organ of the organism or only a subtraction of an organ.
  • Step d in a collection step, collecting cells or nuclei from the sample of the tissue of interest or of the organoid.
  • Step e in an analysis step, performing a single-cell or single-nucleus assay comprising an analysis of the gene perturbation and comprising gRNA sequencing of each cell or nucleus of the collected cells or nuclei, thereby generating a plurality of assay patterns related to expression of a defined gRNA or a defined gRNA combination.
  • the analysis of the gene perturbation is an assay or a combination of assays which captures a certain feature of the analyzed cell.
  • this assay it is possible to relate the perturbation of expression via the gRNA to a phenotype of the cell in vivo. There are multiple assays which can be performed on a single-cell level.
  • An assay pattern reflects the phenotype of the cell for a certain parameter.
  • the assay of the analysis step comprises single-cell or single-nucleus RNA sequencing.
  • Each cell has a unique barcode that is present in both the mRNA and gRNA fractions.
  • One uses this barcode to make connections between mRNA information and gRNA expression gRNA expression is an indirect way to identify the mutated gene).
  • gRNA expression is an indirect way to identify the mutated gene.
  • the gRNA indicates which gene was perturbed (knocked out, inhibited, activated) in that cell.
  • the mRNA information from that cell is informative about the impact of perturbing that gene.
  • the assay of the analysis step comprises single-cell or single-nucleus DNA sequencing.
  • the assay of the analysis step comprises single-cell quantification of surface proteins, particularly cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), and the assay patterns are protein patterns, particularly surface protein patterns.
  • Surface protein quantification is performed as described in Stoeckius, M. et al. Simultaneous epitope and transcriptome measurement in single cells. Nat Methods 14, 865-868 (2017).
  • the assay of the analysis step comprises single-cell or single-nucleus quantification of cytosolic and nuclear proteins.
  • This assay uses antibodies labelled with short oligonucleotides to indirectly quantify proteins of interest.
  • cells are treated with oligo-tagged antibodies that bind to the protein(s) of interest.
  • Cells are then used to prepare single-cell RNA-seq libraries with modified protocols that capture both mRNA and oligo-tags. Quantification of the oligo tags indirectly tells the quantity of protein present in the cell. Cytoplasmatic protein quantification is performed as described in Katzenelenbogen, Y. et al. Coupled scRNA-Seq and Intracellular Protein Activity Reveal an Immunosuppressive Role of TREM2 in Cancer. Cell 182, 872-885. e19 (2020).
  • the assay of the analysis step comprises single-cell or single-nucleus quantification of histone marks.
  • This assay uses an antibody binding to a histone modifying protein. After binding to the target protein, the antibody recruits a DNA cutting enzyme that cuts DNA around the specific histone modification. Deep sequencing of the cut DNA allows one to understand the precise original genomic localization of the histone modification. Bartosovic, M., Kabbe, M. & Castelo- Branco, G. Single-cell CUT&Tag profiles histone modifications and transcription factors in complex tissues. Nat Biotechnol 39, 825-835 (2021).
  • the assay of the analysis step comprises single-cell or single-nucleus mRNA sequencing, and the assay patterns are mRNA expression patterns.
  • the assay of the analysis step comprises transposase-accessible chromatin with sequencing (ATAC-seq), and the assay patterns are chromatin accessibility patterns.
  • the assay patterns are clustered by their type of cell or origin, thereby generating an assay profile for each cell type in the tissue of interest.
  • the assay patterns are clustered by their type of expressed gRNA, thereby generating an assay profile for each gene perturbed by a gRNA or a gRNA combination. Via clustering, the read-out of multiple cells of the same cell type and having the same gRNA perturbation can be combined to increase the signal-to-noise ratio.
  • each vector of the plurality of gRNA-delivering nucleic acid expression vectors comprises a reporter gene under control of a reporter gene promoter, wherein said reporter gene encodes a reporter protein, wherein said reporter protein is detectable when expressed inside a cell and enables selective isolation of cells that express said reporter protein in the collection step.
  • This reporter protein facilitates the analysis, because the cells which express a gRNA can be distinguished from non-expressing cells, e.g., via their fluorescence.
  • cells or nuclei are collected selectively from the tissue of interest that exhibit expression of said reporter protein.
  • the reporter protein is a fluorescent protein.
  • the collection step is performed via fluorescence-activated cell sorting (FACS) or fluorescence-activated nucleus sorting (FANS).
  • FACS fluorescence-activated cell sorting
  • FANS fluorescence-activated nucleus sorting
  • nuclei are collected.
  • the reporter protein comprises a polypeptide part localizing to a nucleus of a cell, particularly wherein the reporter protein comprises a polypeptide part interacting with a membrane of a nucleus, particularly wherein the reporter protein comprises a KASH (Klarsicht, ANC-1 , Syne Homology) domain.
  • the reporter protein comprises a polypeptide part localizing to a nucleus of a cell, particularly wherein the reporter protein comprises a NLS (nuclear localization sequence) domain.
  • whole cells are collected.
  • patterns are clustered first by their cell type.
  • said organism expresses a gene (from its genome or delivered via a vector) encoding a recombinase enzyme (under control of a promoter), and wherein activation of expression of said Cas enzyme is mediated via said recombinase enzyme.
  • the recombinase enzyme is a Cre enzyme.
  • an organism expressing a Cas enzyme on its genome has an LSL (lox-stop-lox) sequence between the promoter and the Cas coding sequence. This LSL sequence blocks expression of the Cas enzyme. Once the Cre protein is expressed, the Cre protein removes the LSL sequence and unblocks the expression of Cas enzyme.
  • Cre can be delivered via AAV, but it’s also possible to cross-breed the LSL-Cas animal to another animal that expresses Cre from its genome.
  • the recombinase enzyme is a Flp enzyme.
  • the promoter of the reporter/Cre is a pol-ll promoter.
  • the gRNA promoter is a pol-ll promoter. In certain embodiments, the gRNA promoter is a pol-lll promoter.
  • said reporter gene promoter is an RNA-polymerase II promoter.
  • the gRNA-delivering nucleic acid expression vector is a viral vector. In certain embodiments, the gRNA-delivering nucleic acid expression vector is selected from the group of an AAV vector, an adenoviral vector, a rabies vector, a Sindbis vector, and a lentiviral vector. In certain embodiments, the gRNA-delivering nucleic acid expression vector is an AAV vector.
  • the organism is an animal. In certain embodiments, the organism is a vertebrate. In certain embodiments, the organism is a mammal, particularly a mammal selected from the group of a rodent, a primate, an ungulate (particularly an artiodactyla or a perissodactyla), a lagomorph, a carnivore, an insectivore, and a chiroptera.
  • the Cas enzyme is Cas9.
  • the gene encoding the Cas enzyme is introduced into the germline of the organism.
  • the gene encoding the Cas enzyme is delivered via a (particularly viral) vector.
  • the gRNA promoter and the reporter gene promoter are two distinct promoters.
  • the gRNA promoter is a tissue-specific promoter.
  • the gRNA promoter is an inducible or conditional promoter. In certain embodiments, the gRNA promoter is a promoter selected from the group of a Tet-ON promoter, a Tet- OFF promoter, and a Cre-dependent promoter.
  • 5’ capture sequencing is performed.
  • Infected nuclei are subjected to a modified 5’ single-cell library preparation protocol to capture both mRNA and gRNA information.
  • a. The reaction is modified to include a gRNA-specific reverse transcription primer to capture gRNA alongside mRNA.
  • mRNA and gRNA molecules are separated to create two independent libraries.
  • the mRNA library is bigger than 300 bp, while the gRNA library is approximately 180 bp. We separate the 2 fractions by using a specific protocol (beads) that sequesters and removes molecules bigger than 300 bp (mRNA) and keeps the gRNA molecules. Both fractions are prepared separately during the following steps.
  • c. The mRNA library is processed accordingly to the kit’s instructions.
  • the gRNA library is PCR amplified and indexed prior to Illumina deep sequencing.
  • RNA-seq preparation kit For 5’ capture sequencing, a single-cell RNA-seq preparation kit is used that barcodes RNA at the 5’ (ex: 10xGenomics Chromium 5’ kit).
  • the kit’s protocol was modified to additionally include a reverse transcription primer specific to the gRNA. This primer mediates capture and barcoding of gRNA molecules from each single cell, alongside the protocol’s standard capture of mRNA.
  • capture sequencing comprises the following steps:
  • scRNA-seq 5 a scRNA-seq 5’ capturing protocol (e.g. from 10x Genomics), comprising the steps:
  • RNA molecules from the 5’ position • a scRNA-seq platform (e.g. from 10x Genomics) that barcode the RNA molecules from the 5’ position;
  • RT reverse transcriptase
  • RT reverse transcription
  • the invention can be described as follows: For a given tissue, we infect a percentage of cells with a nucleic expression vector (not all cells from the tissue are infected, but later cell or nuclei sorting permits focus only on infected cells with fluorescent protein expression). Each infected cell carries a perturbation in one or more genes. The number of genes tested in parallel can be between 2 and +20 000. We assume that a pool of single cells carrying the same perturbation is representative of the effect of that perturbation in that tissue and cell type.
  • the therapy in this case could be seemingly any therapeutic modality that modifies the target in the same way as the genetic perturbation.
  • the target was an enzyme
  • a small molecule drug inhibiting that enzyme could have the same capacity to rescue the disease state.
  • a further aspect of the invention relates to the use of a plurality of viral gRNA-delivering nucleic acid expression vectors in a method according to the first aspect; each vector comprising: i.) inverted-terminal repeats (ITRs); ii.) a gRNA promoter; iii.) at least one guide-RNA (gRNA) under control of said gRNA promoter, wherein each vector comprises a different gRNA or gRNA combination; and iv.) a terminator of transcription.
  • ITRs inverted-terminal repeats
  • gRNA guide-RNA
  • Our invention specifically relates to a method that allows for single-cell-based analysis of multiple CRISPR-mediated gene perturbations within a single organism in vivo. This capability is significantly different from US 2020 018 746 A1 , which focuses on 3D tissues and does not cover single-cell resolution in live animals.
  • AAV-Perturb-seq AAV-based single-cell CRISPR screening method, AAV-Perturb-seq, which is broadly applicable for in vivo functional genomics studies. Unlike the methods in US 2020 018 746 A1 , our approach ensures efficient gRNA expression and detection within single-cell libraries, optimized for large numbers of single nuclei from complex tissues isolated from animals.
  • the systemic delivery via intravenous injections of AAV vectors in our method enables targeting a wide range of tissues and cell types in animals of any age. This systemic delivery is tunable and provides broader application compared to the approaches described in US 2020 018 746 A1 .
  • Our invention allows for single-cell-based analysis of multiple CRISPR-mediated gene perturbations within a single organism in vivo. This enables detailed and comprehensive mapping of gene functions at a single-cell resolution in a living animal, a capability not emphasized or developed in US 2021 172 017 A1.
  • Our invention features systemic delivery of AAV vectors via intravenous injections, enabling the targeting of a wide range of tissues and cell types across the entire body in animals of any age. This broad targeting capability allows for more comprehensive studies of gene functions and interactions in various tissues simultaneously, which is not covered by US 2021 172 017 A1's approach. US 2021 172 017 A1 only covers in vitro applications.
  • Our invention involves a method for analyzing multiple gene perturbations in vivo in a tissue of interest, which includes the administration of a plurality of viral gRNA-delivering nucleic acid expression vectors.
  • This method allows for single-cell or single-nucleus assays to analyze gene perturbation, including gRNA sequencing of each cell or nucleus.
  • the specificity lies in the detailed steps for isolating tissues, collecting cells or nuclei, and performing various assays (e.g., RNA sequencing, DNA sequencing, protein quantification).
  • the invention includes detailed protocols for systemic delivery, enabling targeting of a wide range of tissues and cell types in animals of any age, and incorporates numerous specific assays (e.g., singlecell RNA-seq, CITE-seq, ATAC-seq). This broader scope is not suggested or implied by WO 2019 113 499 A1 .
  • the method involves administering a plurality of viral gRNA-delivering nucleic acid expression vectors and performing single-cell or single-nucleus assays to analyze gene perturbation at a high resolution.
  • This broad applicability to different tissues and comprehensive analysis distinguishes our invention from WO 2015 089 462 A1 , which focuses specifically on the brain and electrophysiological recording.
  • Our invention facilitates the targeting of multiple genes simultaneously using a library of gRNA- delivering vectors.
  • This method can perturb multiple genes in parallel, enabling high-throughput studies of complex genetic interactions across various tissues.
  • This capability for parallel perturbation and analysis of multiple genes is a significant advancement over the single-gene focus described in WO 2015 089 462 A1.
  • a method for analyzing multiple gene perturbations in vivo in a tissue of interest comprising the steps: a. providing an organoid or a non-human organism, particularly a non-human organism; b. administering a plurality of gRNA-delivering nucleic acid expression vectors to the organoid or organism, each vector comprising: i. a gRNA promoter ii. at least one guide-RNA (gRNA) under control of said gRNA promoter, wherein each vector comprises a different gRNA or gRNA combination; and
  • the assay of the analysis step comprises transposase-accessible chromatin with sequencing (ATAC-seq), and the assay patterns are chromatin accessibility patterns.
  • the assay of the analysis step comprises cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), and the assay patterns are protein patterns, particularly surface protein patterns.
  • the assay patterns are clustered by their type of cell or origin, thereby generating an assay profile for each cell type in the tissue of interest.
  • the assay patterns are clustered by their type of expressed gRNA, thereby generating an assay profile for each gene perturbed by a gRNA or a gRNA combination.
  • each vector of the plurality of gRNA-delivering nucleic acid expression vectors comprises a reporter gene under control of a reporter gene promoter, wherein said reporter gene encodes a reporter protein, wherein said reporter protein enables selective isolation of cells that express said reporter protein in the collection step.
  • the reporter protein comprises a polypeptide part localizing to a nucleus of a cell, particularly wherein the reporter protein comprises a polypeptide part interacting with a membrane of a nucleus, particularly wherein the reporter protein comprises a KASH domain.
  • the reporter protein comprises a polypeptide part localizing to a nucleus of a cell, particularly wherein the reporter protein comprises a NLS (nuclear localization sequence) domain.
  • the gRNA-delivering nucleic acid expression vector is a viral vector, particularly wherein the gRNA-delivering nucleic acid expression vector is an AAV vector, an adenoviral vector, or a lentiviral vector, more particularly wherein the gRNA-delivering nucleic acid expression vector is an AAV vector.
  • the organism is an animal, particularly wherein the organism is a vertebrate, more particularly wherein the organism is a mammal, most particularly a mammal selected from the group of a rodent, a primate, an ungulate, a lagomorph, a carnivore, an insectivore, and a chiroptera.
  • gRNA promoter is a tissue-specific promoter.
  • the gRNA promoter is an inducible or conditional promoter, particularly a promoter selected from the group of a Tet-ON promoter, a Tet-OFF promoter, and a Cre-dependent promoter.
  • a method for analyzing multiple gene perturbations in vivo in a tissue of interest comprising the steps: a. providing an organoid or a non-human organism, particularly a non-human organism; b. administering a plurality of viral gRNA-delivering nucleic acid expression vectors to the organoid or organism, each vector comprising: i. inverted-terminal repeats (ITRs); ii. a gRNA promoter;
  • gRNA guide-RNA
  • each vector comprises a different gRNA or gRNA combination; and iv. a terminator of transcription; wherein the organism expresses a Cas enzyme, or said vector additionally encodes a Cas enzyme under control of a promoter operable in said cell; c. isolating a sample of the tissue of interest from the organism, or a sample of the organoid; d. in a collection step, collecting cells or nuclei from the sample; e.
  • gRNA guide-RNA
  • an analysis step performing a single-cell or single-nucleus assay comprising an analysis of the gene perturbation and comprising gRNA sequencing of each cell or nucleus, thereby generating a plurality of assay patterns related to expression of a defined gRNA or a defined gRNA combination.
  • the assay of the analysis step comprises a method selected from the group of
  • the assay patterns are mRNA expression patterns
  • the assay patterns are protein patterns, particularly surface protein patterns.
  • each vector of the plurality of gRNA-delivering nucleic acid expression vectors comprises a reporter gene under control of a reporter gene promoter, wherein said reporter gene encodes a reporter protein, wherein said reporter protein enables selective isolation of cells that express said reporter protein in the collection step, particularly wherein in the collection step, cells or nuclei are collected selectively from the tissue of interest that exhibit expression of said reporter protein, more particularly wherein the reporter protein is a fluorescent protein.
  • the reporter protein comprises a polypeptide part localizing to a nucleus of a cell, particularly wherein
  • the reporter protein comprises a polypeptide part localizing to a nucleus of a cell, particularly wherein the reporter protein comprises a NLS (nuclear localization sequence) domain.
  • NLS nuclear localization sequence
  • the gRNA- delivering nucleic acid expression vector is an AAV vector, an adenoviral vector, a rabies vector, a Sindbis vector, or a lentiviral vector, more particularly wherein the gRNA-delivering nucleic acid expression vector is an AAV vector.
  • the organism is an animal, particularly wherein the organism is a vertebrate, more particularly wherein the organism is a mammal, most particularly a mammal selected from the group of a rodent, a primate, an ungulate, a lagomorph, a carnivore, an insectivore, and a chiroptera.
  • the Cas enzyme is Cas9.
  • the gene encoding the Cas enzyme is Cas9.
  • gRNA promoter and the reporter gene promoter are two distinct promoters.
  • the gRNA promoter is a tissue-specific promoter.
  • the gRNA promoter is an inducible or conditional promoter, particularly a promoter selected from the group of a Tet-ON promoter, a Tet-OFF promoter, and a Cre-dependent promoter.
  • Fig. 2 Perturbation of 22q11.2-linked genes Dgcr8, Dgcr14, Gnbll, and Ufdll result in strong transcriptional changes in adult brain cell types
  • a Schematic of the analysis pipeline (SH control: nuclei with control gRNAs targeting safe-harbor locus; P: perturbation; n: total number of perturbations; LFC: log fold change)
  • b Number of differentially expressed genes (DEG) for all perturbations in individual cell types. Dashed line indicates 5 DEGs with an adjusted p-value (p.adj) lower than 0.05.
  • Fig. 3 Perturbation of 22q11.2 genes results in the disruption of distinct sets of biological processes, a. Schematic representation of arrayed validation experiments, b. Pearson correlation and hierarchical clustering of transcriptional signatures (LFC values) mediated by Dgcr8, Dgcr14, or Gnbll perturbation in pooled screen and arrayed confirmation experiments for each neuron type. c. Heatmap showing the six transcriptional programs (grouped rows) altered in Dgcr8, Dgcr14, and Gnbll perturbed cells (columns) across cell types and experiments (screen or arrayed). Left: LFC values for each altered gene across neuron types and experiments.
  • LFC values transcriptional signatures
  • FIG. 6 AAV injection and nuclei isolation conditions
  • a Schematic representation of AAV genomes used to deliver and express mTagBFP, Venus, or mCherry under the control of the CBh promoter
  • b Schematic representation of the triple color experiment.
  • An equal-ratio mix of the three AAVs was injected in LSL-Cas9 animals with different doses (Low: 2.5 x 10 9 ; Medium: 5.0 x 10 9 ; High: 2.5 x 10 10 , total AAV particles),
  • c Percentage of infected nuclei (/.e., nuclei expressing at least one fluorescent protein) after systemic injection of different viral doses, d.
  • Percentage of infected nuclei expressing one, two, or the three FPs Data shown for injections with 5.0 x 10 9 and 2.5 x 10 10 total AAV particles, e. Fluorescence imaging of brain cells expressing GFP four weeks after systemic injection of 5.0 x 10 9 AAV particles, f. Flow cytometry gating strategy to sort GFP-positive nuclei.
  • FIG. 7 Astrocytes-specific pooled screen, a. Schematic representation of the AAV genome engineered to express, b. UMAP embedding of ⁇ 35 000 AAV.PHP.B-infected nuclei isolated from the mouse prefrontal cortex, c. Abundance of cell types in single-nucleus datasets generated from brain cells infected with CBh and GfaABCI D AAVs. d. Percentage of gRNAs detected per nucleus in Astrocytes, e. Number of DEGs for all perturbations in Astrocytes.
  • Fig. 8 In vivo CRISPR screening in a high-fat diet MASH model, a. Animals were injected with genetic interventions and exposed to HFD for five months, b. Bulk gRNA count analysis revealed genes involved in hepatocyte damage, c. Bulk gRNA count prioritizes interventions with therapeutic potential. Interventions highlighted in red are examples of positive controls known to have an effect and support the ability of our platform to pinpoint potential therapeutical targets.
  • FIG. 9 Microglia-specific AAV capsids, a. Average number of nuclei per perturbation across cell types using AAV. PHP. b for gRNA library delivery, as reported in Santinha et al. 2023. b. Experimental design to test microglia-specific AAV capsids. Viruses were tail vein injected (10 A 12 particles per animal), and the number of infected microglia was assessed three weeks later, c. Flow cytometry results for CD11 b positive microglia for two controls (PBS and PHP.eB) and four microglia-specific AAV capsids (AAV M1 - M4).
  • Example 1 In vivo single-nucleus pooled CRISPR screening in the adult brain enabled through systemic administration ofAAV.PHP.B and 5’ gRNA capture
  • AAV AAV transfer plasmids to independently express either mTagBFP, Venus, or mCherry under the control of a ubiquitous CBh promotor (Fig. 6a).
  • FP fluorescent protein
  • Fig. 6a Each fluorescent protein (FP) was additionally fused to a KASH domain which physically attaches proteins to the nuclear membrane, thus enabling nuclei sorting.
  • Example 2 AAV-Perturb-seg of 22g 11.2 DS genes yields a rich single-nucleus dataset spanning genes and brain cell types from adult mice
  • Example 3 Perturbation of Dgcr8, Dgcr14, Gnbll, or Ufdll result in strong transcriptional changes in prefrontal cortex neurons
  • ⁇ Ne developed a data analysis pipeline to associate gRNAs, and thus genetic perturbations, with cell type-specific transcriptional phenotypes (Fig. 2a).
  • Fig. 2a We create pseudobulk profiles by aggregating nuclei with the same perturbation and employ edgeR (Robinson, M. D et al., Bioinforma. Oxf. Engl. 26, I SOO (2010)) to calculate pairwise differential expression (DE) between control and each perturbation in superficial and deep layer excitatory neurons, interneurons, astrocytes, and oligodendrocytes.
  • edgeR Robotson, M. D et al., Bioinforma. Oxf. Engl. 26, I SOO (2010)
  • DE pairwise differential expression
  • Our choice of using pseudobulk profiles is supported by recent benchmarking studies indicating that commonly used single-cell-specific DE methods tend to identify differentially expressed genes (DEG) in the absence of biological differences.
  • Example 4 Altered transcriptional phenotypes are due to gene function and not a consequence of gene editing efficiency
  • LDA linear discriminant analysis
  • Example 6 Perturbation of 22g11.2-associated genes results in heterozygous and homozygous cells with similar transcriptional phenotypes
  • the control of zygosity is a general challenge in CRISPR screens, as the expression of Cas9 and gRNA can lead to three potential scenarios: 1) the cells are infected but not edited and are thus wild-type (WT); 2) the cells are infected and acquire a heterozygous mutation; or 3) the cells are infected and acquire a homozygous mutation. While WT cells do not contribute to the observed transcriptional phenotypes and are removed by our filtering strategy, it is unclear whether heterozygous and heterozygous mutations lead to the same transcriptional phenotype. This is especially important for modelling haploinsufficiency, as is the case for 22q11.2 DS.
  • CRISPR inhibition (CRISPRi)-mediated knockdown may reduce target gene expression to levels observed in a heterozygous condition and thus be used to simulate the phenotypes generated by haploinsufficiency.
  • CRISPRi CRISPR inhibition
  • CRISPRi-mediated Dgcr8 mRNA reduction was comparable to the values observed for 22q1 1 .2 DS, indicating our ability to model heterozygosity.
  • CRISPRi- and CRISPR- mediated Dgcr8 perturbation led largely to analogous transcriptional phenotypes.
  • Example 7 Perturbation of 22q11.2 DS genes results in the disruption of distinct sets of biological processes
  • Dgcr8 encodes for a component of the microprocessor complex involved in processing primary microRNA (miRNA) transcripts (pri-miRNAs) into precursor miRNAs (pre-miRNAs), which are ultimately further processed by Dicer into mature miRNAs, and has been extensively studied in the context of 22q1 1.2 DS. While we found that no biological pathways were disrupted in the Dgcr8 down-regulated genetic program, in the up-regulated genetic program we identified a disruption in genes related with miRNA-mediated RNA silencing (Fig. 3c), which included several long noncoding RNAs (IncRNA) such as Mirg and Spaca6.
  • IncRNAs encode pri-miRNAs and their up-regulation was previously reported in mouse models of Dgcr8 haploinsufficiency and 22q11.2 DS.
  • the accumulation of these pri-miRNAs implies that there is less mature miRNA being produced.
  • mature miRNAs negatively regulate gene expression, we would expect a concordant increase in the expression of genes targeted by the disrupted miRNAs.
  • miRNA-target enrichment analysis Licursi, V.
  • Dgcr14 encodes for the nuclear protein DGCR14, a component of C complex spliceosomes.
  • Gene ontology analysis of the down-regulated genetic program revealed the presence of genes connected with RNA binding (Fig. 3c). We found a specific enrichment for genes associated with regulation of RNA splicing and the spliceosome, supporting the involvement of Dgcr14 in RNA maturation processes.
  • Gnbl l encodes for a protein of unknown function46 that contains six WD40 repeats which facilitate protein-protein interactions and the formation of multiprotein complexes.
  • the down-regulated genetic program was enriched for genes involved in neuronal development, synaptic organization and function, and chemical transmission (Fig. 3c), including genes that encode for glutamatergic receptor subunits (Grial , Gria4, Grik3, Grin2a, and Grin2b), regulation of a prepulse inhibition phenotype (Ctnna2 and Nrxnl), and regulation of action potential (Ank3, Cnr1 , Fgf13, Foxpl , and Trpc4).
  • Grial , Gria4, Grik3, Grin2a, and Grin2b regulation of a prepulse inhibition phenotype
  • Ank3, Cnr1 , Fgf13, Foxpl , and Trpc4 regulation of action potential
  • AAV-Perturb-seq both confirms prior published work and provides new insights into the phenotypic landscape underlying 22q11 .2 genes.
  • our data reveals new pri-miRNA targets of Dgcr8, a disrupted balance between RNA transcription and splicing resulting from perturbation of Dgcr14, and broad dysfunction in neuronal communication linked to the synapse and glutamate signaling in Gnbl l-perturbed cells.
  • these 22q11.2 genes play an active role in mature neurons in the mouse brain, which may also contribute to 22q11 .2 DS symptomatology.
  • Example 8 Single-nucleus prefrontal cortex atlas of a 22ct11.2 DS mouse model
  • Hierarchical clustering of the bulk transcriptional profiles revealed a primary clustering driven by cell type followed by a second level clustering by genotype.
  • GSEA gene set enrichment analysis
  • Example 9 Transcriptional changes found in LgDel model neurons are partially explained by perturbation of Dgcr8, Dgcr14, and Gnb 11
  • ⁇ Ne set out to quantify the extent to which individual perturbations explain the transcriptional signature observed in LgDel neurons.
  • the Dgcr8 contribution was focused on up-regulated genes mostly associated with the accumulation of miRNA primary genes (Mirg, Spaca6, Mir9-3hg, and Mir181 a-1 hg).
  • the smaller Dgcr14 contribution included down-regulated spliceosomal genes Srsfl , Srsf2, and Srsf6, while the Gnbl l contribution was primarily related to down-regulation of genes involved with synapse signaling (Fig. 5).
  • An in vivo cell type-specific screen could in principle be achieved using cell type-specific delivery or expression as well as through physical enrichment of the cell type of interest.
  • NASH is characterized by an excessive accumulation of fat in hepatocytes. At least 20% of patients progress to severe liver disease, in which the excessive fat causes cell damage and initiates a cascade of inflammatory events - mediated by Kupfer cells and hepatic stellate cells (HSC) - that lead to tissue fibrosis and scarring. If not addressed, this progression can culminate in cirrhosis and liver failure, which are major risk factors for the most common liver cancer, hepatocellular carcinoma.
  • HSC hepatic stellate cells
  • AAV-Perturb-seq is particularly well-positioned to identify therapeutic targets for the disease.
  • NASH mouse model There are several types of murine models of NASH, ranging from genetic to chemical to dietarian. We chose to utilize a dietarian model based on a high-fat diet (HFD). This model has been reported to better mimic the pathological features observed in human patients.
  • HFD high-fat diet
  • AAV-Perturb-seq can prioritize interventions with therapeutic potential and set the stage for the identification of novel targets for the treatment of human diseases.
  • AAV-Perturb-seq offers, for the first time, the possibility of identifying genetic targets able to modulate microglia states and, consequently, disease progression.
  • AAV.PHP.b AAV capsid specifically developed to target neuronal cells in the mouse brain.
  • AAV serotype proved to infect all major brain cell types but to different extents ( Figure 9a).
  • Figure 9a Figure 9a.
  • cell type-specific delivery of gRNA libraries by using cell type-specific AAV capsids will permit better control over the number of cells per perturbation.
  • microglia are the cell type least abundant in an AAV-Perturb-seq experiment ( Figure 9a).
  • Figure 9a we set out to identify microglia-specific AAV capsids.
  • To test these capsids we produced AAV particles with four evolved capsids (M1 - M4) and included one neuron-specific capsid (AAV. PHP. eB) as a control.
  • These viruses carried a GFP transgene to report successful infections.
  • AAV-Perturb-seq a direct in vivo single-cell CRISPR screening method that is tunable, scalable, and broadly applicable for systematically interrogating genetic elements in vivo in high- throughput.
  • AAV.PHP.B encoding a library of CRISPR gRNAs targeting genes linked to 22q11.2 DS.
  • AAV-Perturb-seq offers for the first time the opportunity to directly interrogate multiple genes in several cell types at a single-cell level in the same animal without restriction to tissue or developmental time points, opening immense further possibilities for studying processes of health and disease in vivo.
  • Gnb //-perturbed neurons displayed altered gene expression related to synaptic signaling, strongly suggesting that heterozygous loss of Gnbll may result in impaired neuronal communication throughout development and contribute to the emergence of alterations in neuronal functioning. This hypothesis is further supported by the observation of reduced expression levels of Gnbll in postmortem brain samples of schizophrenia patients with and without 22q1 1 .2 deletion and by the deficits in synaptic signaling and behavior related to schizophrenia and ASD and found in Gnbll* 1 - mouse models.
  • a promising area for further study is determining whether 22q11.2 DS-associated neuronal and cognitive phenotypes can be rescued exclusively through restoring Dgcr8, Dgcr14, Gnbll, and/or Ufdll expression during or after development.
  • AAV-Perturb-seq will broadly enable the interrogation of genotype-phenotype landscapes directly in vivo in different tissues, cell types, developmental stages, and under different health and disease contexts.
  • the ability to interrogate complex in vivo biology at scale could lead to breakthroughs in our causal understanding of biological and disease mechanisms as well as our capacity to identify genetic interventions and targets for treating disease.
  • AAV Compared to lentivirus for in vivo delivery Considering contemporary research, AAV is the vastly preferred modality for in vivo delivery for several very good reasons. These advantages, as well as disadvantages, are thoroughly covered by many recent reviews on in vivo delivery (Asokan, A., et la., Molecular Therapy 20, 699-708 (2012); Mingozzi, F. et al., Nat Rev Genet 12, 341-355 (2011)) Below, we highlight the main advantages and disadvantages relevant for single-cell CRISPR screening.
  • AAVs can be injected systemically and infect seemingly any organ and cell type in a tunable way. If LV is injected systemically, it is only capable of infecting a few cells in the liver. If LV is injected within a compartment (e.g., via intraperitoneal, intrathecal, or intracerebroventricular injection) it mostly infects cells along the barrier without penetrating deeply into tissue. Thus, in vivo delivery of LV almost always reguires direct injection into the organ of interest.
  • AAVs When injected systemically, AAVs can infect almost any tissue or cell type in a tunable way. Unlike LV, which is difficult to modify and target to specific cell types, AAV is very easy to modify and preferentially target to specific cell types thanks to natural serotypes and engineered/evolved capsid variants. If natural AAV serotypes are injected systemically, they show unigue (i.e., tunable) biodistributions. AAV capsid proteins can further be engineered or evolved to enable the preferential targeting of (new) cell types of interest or passing of physiological barriers such as the blood brain barrier. In our study, we leveraged the evolved AAV capsid PHP.B, which enabled us to achieve brain-wide infection with a simple systemic (tail vein) injection.
  • AAVs do not generally reguire direct injection into tissue, thus avoiding the need for surgical procedures and complicated ethics approvals.
  • Direct injection into tissue often reguires a surgical procedure, which in the case of brain delivery is a craniotomy.
  • the drawbacks of this are plentiful.
  • Surgery, compared to systemic delivery, is labor intensive, reguires expert knowledge, low throughput, leads to increased mortality in experimental mice, reguires increased post-surgical monitoring, and involves a more elaborate ethics approval process (due to increase severity and stress to the animals).
  • AAVs do not generally reguire direct injection into tissue, thus avoiding tissue damage and confounding alterations to cell states. Direct injection into tissue causes damage, resulting in altered cell states. For example, along the injection track created during cranial/brain injections, it is extremely common to find reactive astrocytes and activated microglia, which can confound phenotypes of interest especially when using single cell methods.
  • AAV sparsely infects a large number of cells across a tissue. With direct injection of a virus into tissue, it is extremely difficult if not impossible to infect a large number of cells while controlling the MOI. Systemically injecting AAV made it simple for us to titrate the amount of virus to optimize the number of infected cells with a single infection.
  • LV has a narrow range of utility. Beyond what has already been discussed above, the only case that we are aware of where delivering LV in vivo is commonly done and useful is in the context of development where LV is injected before or shortly after birth. When LV is injected in utero or postnatally in the ventricle of the brain, it is possible to achieve brain-wide infection. Intracerebral ventricular injection into neonates can lead to brain-wide infection due to the fact that the blood brain barrier is immature. In utero injection into the brain of embryos within a pregnant mother can also lead to brain-wide infection due to radial glial progenitor cells of the ventricle wall being readily infected and differentiating to give rise to transgene-expressing daughter cells throughout the brain.
  • AA V is vastly superior to LV for in vivo delivery and AA V-Perturb-seq will therefore open new avenues for single-cell CRISPR screening in vivo.
  • ⁇ Ne created AAV transfer plasmids to test both 5’ and 3’ gRNA capture methods.
  • the gRNA sequence is present within an mRNA transcript and can be captured by conventional single-cell RNA-seq (scRNA-seq) 3’ capture methods.
  • the second strategy (pAS088) was designed to enable direct capture of the gRNA, wherein we designed an AAV transfer plasmid with independent gRNA and mRNA expression cassettes.
  • the gRNA sequence can be directly captured via scRNA-seq 5’ capture methods, e.g., as shown by Replogle et al (Replogle, J. M.
  • UMI proportion filter to correct for chimeric molecules and ambient RNA.
  • the number of detected UMIs in a nucleus correlates directly with the number of reads.
  • a nucleus with a higher number of UMIs is more likely to also have a higher number of UMIs coming from contaminating gRNA and mRNA.
  • a gRNA associated with a small number of UMIs that represent a small proportion of the total gRNA-UMIs identified in a nucleus most likely represents a chimeric read or RNA cross contamination.
  • Proportionbased filters have been used previously to address this issue. We incorporate this concept in our workflow but increased the stringency of the threshold compared to published work.
  • gRNAs ar filtered out from nuclei where the most abundant gRNA UMI count is 1 .3x higher than the UMI counts for the second most abundant gRNA.
  • Such a threshold only considers information about the two most expressed gRNAs and selects a gRNA label based on the highest expressed gRNA, even if there is a second gRNA with high counts. For instance, one cell containing 10 and 14 UMIs for two different gRNAs would be labeled as only having one gRNA.
  • Pseudobulk performs superior to single-cell specific methods in a true positive control.
  • Th e Lg D e I model carries a heterozygous deletion in chromosome 16 (eguivalent to human 22g1 1.2 locus). We hypothesized that this can be used as a control to test DE methods, as we know that genes within the locus are being expressed from only one copy, and thus, in general, their expression should be reduced to approximately 50%.
  • Our pseudobulk detects LFC values of approximately -1 (50% reduction) across deleted genes and cell types, while the logistic regression test typically applied to single-cell data presents smaller LFC values and high variance.
  • An in vivo cell type-specific screen could in principle be achieved using cell type-specific delivery or expression as well as through physical enrichment of the cell type of interest.
  • AAV genome plasmids (Fig. 1a Fig. 6a) were based on the Addgene plasmid #60231 (Platt, R. J. et al., Cell 159, 440-455 (2014)). To achieve widespread transgene expression, the hSyn promoter was replaced by the ubiquitous CBh promoter (pAS088). For the triple color experiments (Fig. 6a), the U6 expression cassette and Cre were removed, while eGFP was replaced by mTagBF2 (Addgene plasmid #55302), Venus (Addgene plasmid #22663), or mCherry (Addgene plasmid #27970) (pAS132, pAS133, pAS134).
  • the original U6 expression cassette was first removed by restriction digestion with Mlul (ThermoFisher) and Xbal (ThermoFisher) from upstream of the pol-ll promotor and cloned between the WPRE and poly(A) signal sequences (pAS006).
  • Mlul ThermoFisher
  • Xbal ThermoFisher
  • the plasmid backbone (2.5 pg) was digested with Bsmbl (ThermoFisher) for 1 hr at 37 °C followed by an inactivation step for 5 min at 80 °C.
  • the Gibson Assembly reaction was set as follows: 50 ng of digested plasmid backbone, 2 pL (200 fmoles of ssDNA oligos (stock at 100 mM), 10 pL NEBuilder® HiFi DNA Assembly Master Mix (NEB, E2621 L), and H2O up to 20 pL total reaction. The reaction was incubated for 1 hr at 50 °C.
  • Isopropanol purification was used to concentrate the cloned gRNA library by mixing the total Gibson Assembly reaction with 20 pL isopropanol, 0.2 pL GlycoBlue Coprecipitant (ThermoFisher, AM9515) and 0.4 pL NaCI solution (stock at 5 M). The precipitation reaction was incubated at room temperature (RT) for 15 min, followed by centrifugation at > 15,000 xg for 15 min at RT. The supernatant was discarded and the DNA pellet was washed with 1 mL ice-cold 80 % ethanol and finally resuspended in 10 pL TE buffer.
  • RT room temperature
  • gRNA libraries were amplified as previously described (Joung, J. et al., Nat. Protoc. 2017 124 12, 828-863 (2017)). Briefly, the plasmid library was electroporated into Endura ElectroCompetent cells (Lucigen, 60242-2) according to the manufacturer’s instructions, followed by 1 hr recovering period at 37 °C. Bacteria were grown on a bioassay plate (Merck, D4803-1 CS) for 14 hr at 37 °C. Colonies were harvested by scrapping the plate surface before plasmid isolation with QIAGEN Plasmid Maxi kit (QIAGEN, #12165) according to the manufacturer’s protocol.
  • the gRNA expression cassette was PCR amplified using KAPA HiFi ReadyMix with 100 ng of the final library as template and 0.5 pM of both custom Illumina P5 primer (AATG ATACG GCG ACCACCG AG ATCTACAC-N N N N N N N N- ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCTTTATATATCTTGTGGAAAGGACGAAACACC , SEQ ID NO 13) and P7 primer (CAAGCAGAAGACGGCATACGAGAT-NNNNNNNN- GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCCCGACTCGGTGCCACTTTTTCAA, SEQ ID NO 14).
  • PCR of the reaction mixture was performed as follows: (1) 95 °C for 3 min; (2) 98 °C for 20 s, 63 °C for 15 s, 72 °C 20 sec (18 cycles); (3) 72 °C for 2 min.
  • the PCR reaction purified with double-size 0.6x - 1.0x AMPURE bead selection (A63882, Beckman Coulter). Deep sequencing libraries were sequenced using a NextSeq 550 75 cycle kit with the following cycle distribution: 75 to read 1 , 8 to index 1 , and 8 to index 2.
  • AAVs were produced in HEK293T cells and purified by iodixanol gradient centrifugation. Briefly, HEK293T were expanded in DMEM (Merck) + 10% FBS (Merck) + 1 % HEPES (ThermoFisher). Twenty- four hours before the beginning of AAV production, cells were seeded in 15 cm dishes (HuberLab) at a density of 0.6 M cells per mL and a total of 20 mL medium per dish.
  • Cells were transiently transfected with 21 ug of an equal molar-ratio mix of the AAV genome, AAV serotype plasmid (AAV.PHP.B), and the adeno helper plasmid pAdDeltaF6 (Puresyn) using polyethyleneimine max (PEI Max).
  • AAV.PHP.B AAV serotype plasmid
  • Puresyn adeno helper plasmid pAdDeltaF6
  • Puresyn polyethyleneimine max
  • Harvested medium was mixed with 5 x AAV precipitation buffer (400 g PEG 8000, 146.1 g NaCI in 1 L H2O) and kept at 4°C. One day later, cells were mechanically dislodged and centrifuged at 800 xg for 15 min.
  • the resulting AAV solutions were aliquoted and flash-frozen in liquid nitrogen.
  • the AAV particle concentration was determine by ddPCR (BioRad). Briefly, 5 uL of isolated AAVs were diluted 10x in water and treated with DNAse I (NEB, M0303S) before preparing tenfold serial dilutions with ddPCR dilution buffer [Ultrapure Water with 2 ng/pL sheared salmon sperm DNA (Thermo Fisher Scientific, AM9680) and 0.05% Pluronic F-68 (Thermo Fisher Scientific, 24040032)].
  • the amplification reaction was performed as following: (1) 95 °C for 10 min; (2) 95 °C for 30 s, 60 °C for 1 min (42 cycles); (3) 72 °C for 15 s; (4) 98 °C for 10 min. Data were collected and analyzed with BioRad ddPCR apparatus to calculate number of viral particles per pL.
  • mice were kept under specific pathogen-free conditions on a standard light cycle.
  • Six to eight weeks old male Rosa26-LSL-Cas9 mice 1 were used unless otherwise indicated below.
  • Six to eight weeks old male dCas9-KRAB mice (JAX stock #030000) were used.
  • Eight weeks old male LgDel +/+ and LgDel +A mice 5 were used for the 22q11 .2 DS model snRNA-seq cell atlas.
  • Triple-color experiment We developed the triple color experiment to fine-tune AAV injection conditions (Fig. 6a).
  • the three AAV genomes were individually packaged into the AAV. PHP. B capsid and purified as indicated in “AAV production and purification”.
  • Different viral particle doses (low: 2.5 x 10 9 ; medium: 5.0 x 10 9 ; and high: 2.5 x 10 10 , total number of particles) were generated by pooling equal-portions of the three viruses. Animals were spit into cages accordingly to their experimental groups. After tail vein injection of 100 pL of the AAV mixtures into LSL-Cas9 mice, animals were kept for three weeks under standard conditions before tissue extraction and processing.
  • AAV particles carrying gRNAs to target 22q11 .2 locus genes were generated as indicated in “AAV production and purification”.
  • a single dose of 5.0 x 10 9 viral particles in 100 pL total volume was injected per mouse. Animals were kept for four weeks under normal conditions before brain tissue extraction and processing.
  • Virus carrying gRNAs to target validation genes were individually prepared as in “AAV production and purification”. Animals were spit into cages accordingly to their experimental groups before tail vein injection (100 pL) of 5.0 x 10 9 viral particles carrying unique gRNAs. Animals were kept for four weeks under standard conditions before tissue extraction and processing. Animals injected with Ufd1 /-targeting gRNAs presented comorbidities three weeks after injection and had to sacrificed at that time point.
  • mice were intravenously injected with a lethal dose of pentobarbital (100 mg/kg body weight) before transcardial perfusion with 15 mL of ice cold 1x PBS followed by 15 mL of ice cold artificial cerebrospinal fluid (aCSF, in mM: 87 NaCI, 2.5 KCI, 1 .25 NaH2PO4, 26 NaHCO3, 75 sucrose, 20 glucose, 1 CaCI2, 7 MgSO4).
  • the brain was removed, placed into a mouse brain matrix slicer (Zivic Instruments, BSMAS001-1), 1 mm slices were immediately snap-frozen, and the region of interest manually dissected into a frozen Eppendorf tube. Tissue samples were kept at -80 °C.
  • Nuclei isolation was performed with mechanical and chemical tissue dissociation procedures.
  • a tissue grinder (Sigma-Aldrich, D8938) was filled with 2 mL of ice-cold nuclei isolation buffer (NIB) (Sigma- Aldrich, NUC101-1 KT) and frozen pieces of tissue were directly placed inside the grinder.
  • NAB ice-cold nuclei isolation buffer
  • nuclei from different animals were isolated in individual grinders, except for the 22q11.2 pooled screen, in which tissue of 15 animals was joined into 3 grinders to reduce the number of isolations and the waiting time before subsequent procedures.
  • the tissue was mechanically disrupted with 25 strokes with pestle A followed by 25 strokes with pestle B.
  • the homogenized solution was transferred to a protein low-binding tube (Eppendorf, 0030122216), mixed with an additional 2 mL of NIB, incubated for 5 min, and immediately centrifuged at 500 xg for 5 min at 4 °C. Supernatant was discarded and the pellet was resuspended in 4 mL NIB, incubated for 5 min and centrifuged at 500 xg for 5 min at 4 °C.
  • the pellet was resuspended in 4 mL of nuclei wash buffer [NWF: 1 % BSA in 1x PBS, 50 U/mL Superasein RNA inhibitor (ThermoFisher, AM2694), and 50 U/mL Enzymatics RNA inhibitor (Enzymatics, Y9240L)] and centrifuged at 500 xg for 5 min at 4 °C. Finally, the nuclei pellet was resuspended in 1 mL NWF and filtered through a 30 pm cell strainer (Sysmex) into a new protein low-binding tube.
  • NWF 1 % BSA in 1x PBS
  • 50 U/mL Superasein RNA inhibitor ThermoFisher, AM2694
  • Enzymatics RNA inhibitor Enzymatics, Y9240L
  • Fluorescence-Activated Nucleus Sorting was performed to: 1) quantify infected nuclei in the triple-color experiment; 2) purify nuclei from debris to ensure a clean nuclei solution before snRNA-seq library preparation; 3) isolate GFP+ nuclei to prepare snRNA-seq libraries with nuclei from infected cells. Briefly, isolated nuclei solutions were spiked with 2 pL/mL of Vybrant DyeCycle Ruby Stain (ThermoFisher, V10273) and sorted in a MA900 apparatus (Sony). Singlet nuclei were gated based on the DNA dye signal as illustrated in Fig.
  • Vybrant DyeCycle Ruby Stain ThermoFisher, V10273
  • mice were sacrificed by intravenous injection with a lethal dose of pentobarbitol (100 mg/kg body weight), followed by perfusion with 15 mL of ice cold 1x PBS and 15 mL of 4% PFA in 1x PBS. Brain tissue was incubated in 4% PFA in 1x PBS overnight and subsequently transferred in 1x PBS with 30% sucrose where they were left until they sunk. Brains were then embedded in OCT and sections of 20 pm were cut on a cryotome. Imaging was performed in a LSM 900 apparatus (Zeiss).
  • Nuclei infected with AAV carrying the 3’ capture design were sequenced with a Chromium Single Cell 3' Reagent Kit v3 (1 Ox Genomics). The nuclei suspension was diluted to 1 ,000 nuclei/pL and processed accordingly to the kit’s protocol with 13 cycles of cDNA amplification and 14 cycles of sample indexing PCR.
  • PCR 1 with primers targeting the U6 promotor sequence (TTTCCCATGATTCCTTCATATTTGC, SEQ ID NO 3) and read 1 sequence (ACACTCTTTCCCTACACGACG, SEQ ID NO 4) was performed with 20 ng of full-length single-cell cDNA library as DNA template.
  • PCR 2 was performed with a forward primer targeting the U6 sequence immediately before the gRNA and containing the P7 adapter (GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGcTTGTGGAAAGGACGAAACAC, SEQ ID NO 5), a reverse P5 primer (AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACG, SEQ ID NO 6), and 2 pL of PCR 1 reaction as template.
  • a third PCR to index samples for deep sequencing used 2 pL of PCR 2 rection as template and was performed with a forward P7 index primer (CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTCTCGTGGGCTCGG, SEQ ID NO 7) and the P5 primer as reverse (same primer used in PCR 2). All primers were used at a final concentration of 0.3 pM.
  • Amplification reactions were performed as following: (1) 95 °C for 3 min; (2) 98 °C for 20 s, 65 °C for 15 s (72 °C for PCR 3), 72 °C 20 sec (number of cycles up to qPCR saturation); (3) 72 °C for 2 min.
  • Nuclei infected with the 5’ capture design (pAS088, preliminary experiments, pooled screen, and confirmation experiments) were sequenced with a Chromium Single Cell 5' Reagent Kit v1 (10x Genomics).
  • RT reverse transcription
  • gRNA-constant-region-targeting RT primer (0.15 pM, AAGCAGTGGTATCAACGCAGAGTACCAAGTTGATAACGGACTAGCC, SEQ ID NO 8) (Mimitou, E. P. et al., Nat. Methods 2019 165 16, 409-412 (2019); Replogle, J. M. et al., Nat. Biotechnol. 2020 388 38, 954-961 (2020)).
  • the reaction was purified with 0.6x SPRI beads (Beckman Coulter). At this point, longer cDNAs (more than 300 bp) from mRNA molecules bind to the beads, while the shorter cDNAs (approximately 200 bp) from gRNA sequences are free in the supernatant.
  • the preparation of gene expression libraries was performed as indicated by the kit’s protocol, with 14 cycles of sample indexing PCR. To recover the gRNA-cDNA sequences, the supernatant from the above step was purified with 1 .4x SPRI beads and eluted in 30 pL of ultra-pure water (ThermoFisher).
  • a 1 :10 diluted aliquot was loaded into Agilent Bioanalyzer High Sensitivity (Agilent) to confirm the presence of a gRNA band of - 180 bp.
  • the gRNA-cDNA library (30 ng) was subjected to a sample indexing PCR using KAPA HiFi ReadyMix and 1 pM of P5 primer (AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTC, SEQ ID NO 18) and P7 indexing primer binding to the gRNA constant region directly downstream of the spacer sequence (CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTCTCGTGGGCTCGGAGATGTGTATAAGAGA CAGTATTTCTAGCTCTAAAAC, SEQ ID NO 10).
  • Amplification reactions were performed as follows: (1) 95 °C for 3 min; (2) 98 °C for 20 s, 54 °C for 30 s, 72 °C 20 sec (15 cycles); (3) 72 °C for 5 min.
  • the final PCR reaction was cleaned and purified with double-size 0.6x - 1 .2x SPRI bead selection.
  • Gene expression and gRNA libraries (5% of flow cell) were sequenced with a NextSeq 550 75 cycle kit or a NovaSeq 100 cycle kit with the following cycle distribution: 26 to read 1 , 8 to index 1 , and 56 (NextSeq) or 91 (NovaSeq) to read 2.
  • PCR 1 was performed to specifically amplify -150 bp around the Cas9 cut site (keeping the cut site central in the amplicon) from genomic DNA (5 pL) with gene specific primers containing adapters (0.5 pM, fwd: ACACTCTTTCCCTACACGACGCTCTTCCGATCT (SEQ ID NO 19) + forward gene specific sequence; rev: GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC (SEQ ID NO 20) + reverse gene specific sequence).
  • PCR 1 amplification was performed as follows: (1) 95 °C for 3 min; (2) 98 °C for 20 s, primer set specific annealing temperature for 15 s, 72 °C 20 sec (15 cycles); (3) 72 °C for 2 min.
  • CAAGCAGAAGACGGCATACGAGAT-NNNNNNNN- GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC, SEQ ID NO 21) was performed as follows: (1) 95 °C for 3 min; (2) 98 °C for 20 s, 70 °C for 15 s, 72 °C 20 sec (15 cycles); (3) 72 °C for 2 min. Indexed samples were pooled, purified with PCR purification & concentration kit (Zymo Research, D4013), and loaded on a 2% E-Gel (Thermo Fisher Scientific, G402022).
  • the PCR product (-250 bp) was extracted from the agarose gel with QIAquick Gel Extraction Kit (QIAGEN, 28706X4) and sequenced using a NextSeq 550 150 cycle kit with the following cycle distribution: 150 to read 1 , 8 to index 1 , and 8 to index 2.
  • pseudobulk profiles were generated by aggregating raw UMI counts of nuclei from the same sample (/.e., same animal) and cell type. Differential gene expression of pseudobulk profiles was performed with the R package edgeR v3.36.0 (Robinson, M. D. et al., Bioinforma. Oxf. Engl. 26, 139-140 (2010)). For each cell type, we use the likelihood ratio test (egdeR-LRT) to calculate LFC and FDR values for each perturbation against SH control. The same process was used to compare LgDel +/_ against LgDel +/+ samples.
  • Deep sequencing libraries for indel analysis were generated as described in “Deep sequencing quantification of Indels” and analyzed with CRISPresso2 (Gene Set Knowledge Discovery with Enrichr Xie - 2021 - Current Protocols - Wiley Online Library.
  • the top 1000 up-regulated genes from each perturbation and cell type were uploaded as input to the online tool MIENTURNET (Licursi, V. et al., BMC Bioinformatics 20, 1-10 (2019)) using the miRTarBase (http://userver.bio.uniroma1 .it/apps/mienturnet/) reference dataset (Huang, H.-Y. et al., Nucleic Acids Res. 50, D222-D230 (2022)).
  • Licursi, V., Conte, F., Fiscon, G. & Paci, P. MIENTURNET An interactive web tool for microRNA- target enrichment and network-based analysis.
  • Licursi, V., Conte, F., Fiscon, G. & Paci, P. MIENTURNET An interactive web tool for microRNA- target enrichment and network-based analysis.

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Abstract

La présente invention concerne un procédé permettant une analyse unicellulaire de perturbations génétiques multiples à médiation CRISPR dans un seul et même organisme. Le procédé comporte l'administration d'une pluralité de vecteurs d'expression viraux codant chacun un ARNg dans un organisme exprimant une enzyme Cas, et permet l'analyse du phénotype résultant au niveau unicellulaire.
PCT/EP2024/071816 2023-08-01 2024-08-01 Criblage crispr unicellulaire de perturbations géniques multiples in vivo Pending WO2025027136A1 (fr)

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