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WO2025054224A9 - Dépistage in vivo à haute teneur et haute résolution pour analyser des fonctions géniques - Google Patents

Dépistage in vivo à haute teneur et haute résolution pour analyser des fonctions géniques Download PDF

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WO2025054224A9
WO2025054224A9 PCT/US2024/045238 US2024045238W WO2025054224A9 WO 2025054224 A9 WO2025054224 A9 WO 2025054224A9 US 2024045238 W US2024045238 W US 2024045238W WO 2025054224 A9 WO2025054224 A9 WO 2025054224A9
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aav
cells
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Xin Jin
Xinhe Zheng
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Scripps Research Institute
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Definitions

  • the invention provides methods for analyzing functions of a plurality of target genes in one or more specific cell types in vivo.
  • the methods entail (1) introducing a library of AAV vectors encoding genetic perturbations for the plurality of target genes into a CRISPR-Cas9 expressing transgenic system, (2) identifying one or more cells of a specific cell type from the transgenic system that express a genetic perturbation and display a specific phenotype, and (3) determining (a) the genetic perturbation encoded by the AAV vector introduced into each of the one or more cells and (b) the corresponding perturbed gene, thereby correlating the corresponding perturbed gene with the phenotype displayed by each of the one or more cells.
  • the employed transgenic system is a developing embryo of a Cas9-expressing transgenic animal
  • the library of AAV vectors are vectors of serotype AAV-SCH9 or AAV2.NN.
  • the employed library of AAV vectors are AAV-pRep2-SCH9 vectors or AAV-pRep2-SCH9 (repeat 136bp) vectors.
  • the library of AAV vectors are administered to the animal embryo around a development stage that is equivalent to mouse embryonic day 11.5 (E11.5), 12.5 (E12.5), 13.5 (E13.5), 14.5 (E14.5), or 15.5 (E15.5).
  • the library of AAV vectors is administered in utero into the brain (lateral ventricles) of the embryo.
  • the employed transgenic system is a postnatal or adult Cas9-expressing transgenic animal
  • the library of AAV vectors are vectors of serotype AAV-PHP.eB or AAV.PHP.S.
  • the library of AAV vectors is administered to the animal at an age that is equivalent to mouse age of from about postnatal day 10 (P10) to about 18 months old.
  • the library of AAV vectors are administered retroorbitally to the animal.
  • the specific cell type to be analyzed is newborn neuron or progenitor cell thereof obtained from a brain tissue.
  • the brain tissue is from neocortex, olfactory bulb, striatum, hippocampus, thalamus, or cerebellum.
  • the target genes to be analyzed are known or suspected to be associated with a neurodevelopmental disorder.
  • each of the genetic perturbations to be introduced into the specific cell types in vivo contains one or more guide RNAs (gRNAs) for introducing genomic changes via CRISPR-Cas9 gene editing in the coding region of a target gene.
  • gRNAs guide RNAs
  • the one or more gRNAs each target a sequence at the 5’ end of a target gene’s coding region.
  • the AAV vectors further encode a transposable element that is flanked with the gRNAs, and the library of AAV vectors are introduced into the transgenic system in the presence of a transposase that recognizes the transposable element and inserts it into the host genome.
  • the transposase is encoded by a second AAV vector co-introduced with the library of AAV vectors into the transgenic system.
  • the employed transposase is Transposase hypPB.
  • Some methods of the invention can additionally entail enriching the identified one or more cells that express one or more genetic perturbations and display a specific phenotype.
  • the one or more gRNAs’ coding sequence is operatively linked to a reporter gene in the AAV vectors.
  • the one or more cells expressing a genetic perturbation is identified by detecting expression of the reporter gene.
  • the genetic perturbation encoded by the AAV vector introduced into each of the one or more cells is determined via capturing the one or more gRNAs via single-cell RNA sequencing (scRNA-seq).
  • the one or more gRNAs are functional for gene editing and can be directly captured in scRNA-seq.
  • Some methods of the invention utilize AAV-SCH9 or AAV2.NN vectors.
  • the library of AAV vectors is administered to the animal embryo around a development stage that is equivalent to mouse embryonic day 11.5 (E11.5), 12.5 (E12.5), 13.5 (E13.5), 14.5 (E14.5), or 15.5 (E15.5).
  • Some methods of the invention utilize AAV- PHP.eB or AAV.PHP.S vectors.
  • the library of AAV vectors are administered to the animal retroorbitally in an age from postnatal day 10 (P10) to 18 months old.
  • the invention provides methods for analyzing functions of a plurality of target genes in one or more specific cell types in vivo.
  • These methods involve: (1) co-introducing into a developing embryo of a Cas9-expressing transgenic animal (i) an AAV vector encoding a transposase, and (ii) a library of AAV vectors each encoding (a) one or more guide RNAs (gRNAs) for one of the target genes and (b) a transposable element that is recognized by the transposase and that is flanked with the gRNAs; wherein the AAV vectors are of serotype AAV-SCH9 or AAV2.NN; (2) identifying one or more cells of a specific cell type from the embryo that express the gRNAs and display a specific phenotype, and (3) determining the gRNAs encoded by each of the AAV vectors introduced into the one or more cells and the corresponding perturbed gene; thereby correlating the specific phenotype with the function of each of the perturbed target genes.
  • gRNAs guide RNAs
  • B-C Immunofluorescence analysis of brain sections two days after AAV library administration: (B) co-stained with markers of newborn projection neurons (TBR1 and CTIP2) and intermediate progenitors (TBR2), in dorsal cortical laminar and ganglionic eminence (GE); (C) quantification of percentage of GFP + cells co-expressing neuronal and progenitor markers including TBR1, TBR2 and CTIP2. (D) Heatmap of proportion of 86 AAV serotype abundance in AAV library, 48 hours post transduction in HT22 cells and in embryonic mouse brain; each row represents the abundance of an AAV serotype.
  • A Schematics of a secondary AAV serotype screen: 14 AAV serotypes were barcoded and introduced in pool in utero at E13.5, followed by scRNA- seq 48 hours later.
  • B Uniform Manifold Approximation and Projection (UMAP) visualization of 11 major cell populations identified (left) from sorted (GFP + ) and unsorted cells (right); cell types include: upper and deep layer projection neurons (ULPN, DLPN), migrating neurons (Mig. neurons), apical progenitors (Api.
  • AAV serotype barcode expression in each cell type Each row represents an AAV barcode, and each AAV serotype is associated with a distinct set of 3 barcodes (BCa, BCb and BCc). Each column represents barcode expression in a cell, arranged by cell types.
  • FIG. 1 AAV-SCH9-GFP-KASH or lentiviral reporter (GFP) administered at E13.5 resulted in diverse brain region labeling at P7. Scale bars indicate 500 ⁇ m (left in D and top in F) or 50 ⁇ m (right in D, E, and bottom in F).
  • Figure 3. hypPB transposon enhanced and stabilized expression in embryonic and adult brains and peripheral nervous systems.
  • A Schematics of the molecular design to enhance transgene expression and prevent loss due to cell division and differentiation. Shaded box indicates transposon flanked by the inverted repeats (IR).
  • B Timelapse imaging showed co-transfection of hypPB increased the expression of the GFP transgene in vitro.
  • C-E Transposon stabilized expression in vivo across embryonic brain, adult brain, and adult dorsal root ganglion using three targeting vectors: AAV-SCH9, AAV9-PHP.eB and AAV9-PHP.S.
  • AAV9-PHP.eB labeled cortical neurons with increased expression intensity with hypPB: average fluorescence intensity (X axis) across cortical layers (Y axis), from ventricular zone (0) to pia (1) (n 3 animals/condition).
  • H Whole genome sequencing of AAV-SCH9 transduced cells in vivo showed the genomic regions of integration events. Left: each line indicates a unique hybrid read between mouse genome and transposon, illustrated as a wheel plot. Right: percentage of integration events occurred in intergenic and coding regions in the genome.
  • I Example reads that captured the junction of mouse genome and transposon, indicating the integration sites. Top: schematic illustration of the transposon sequence (SEQ ID NOs:27 and 37).
  • C UMAP plot of filtered cells, with each cell colored by gRNA identity estimated by DemuxEM with down-sampling (top) and batch/channel (bottom).
  • D Top: schematics of 5’ and 3’ scRNA-seq capture mechanism. Bottom: comparing gRNA capture rate by measuring percentage of cells in each cell type with gRNA UMI number greater than 5 in 5’ and 3’ scRNA-seq, with cell types on the X axis, percentage of cells assigned gRNA identity on the Y axis.
  • E Bar plot showing percentage of cells assigned to one or more gRNA across major cell types, color represents gRNA identity assignment.
  • F Percentage of insertion/deletion reads in Foxg1 gRNAs targeting loci by Foxg1 perturbation comparing to Non-Targeting control 2 (NT2) controls, extracted from scRNA-seq data.
  • G Heatmap showing cell type proportion changes by each perturbation. The rings highlight FDR adjusted P-value ⁇ 0.05.
  • H Dot plot of number of differentially expressed genes (DEG) and cell number (size of dots) across cell type- perturbation combinations. The boxes highlight the robust changes of ⁇ 10 DEGs supported by at least 2 gRNAs.
  • the conventional AAV expression tends to be transient, at a relatively low level, with subsequently dilutions through cell divisions, which together poses a challenge to accurately recover the perturbation identity of sparsely labeled cells in pooled assays like Perturb-seq. See, e.g., Lang et al., 2019; Nat. Commun.10, 3415.
  • the slow onset of AAV-mediated expression – often taking days or even weeks – is suboptimal for studying dynamic gene functions in fast-evolving cellular contexts such as neurodevelopment. For example, peak AAV-transgene expression commonly occurs after more than seven days, a timespan that encompasses the entire window for corticogenesis.
  • the present invention provides AAV-based high-content in vivo screen platforms that overcome these limitations.
  • the generalizable platforms for in vivo Perturb-seq are able to target diverse tissue types and cell types with high content phenotypic readout with single cell resolution.
  • the invention is derived in part from studies undertaken by the inventors to develop an AAV-based platform for massively parallel in vivo Perturb-seq to target a broad spectrum of tissues and cell types with gene expression-based characterization at single cell resolution.
  • the inventors identified specific AAV serotypes including AAV-SCH9, which enable swift and robust transgene delivery in newborn neurons and progenitors within 48 hours post transduction (versus 2-3 weeks by the currently available methods).
  • the identified AAV vectors were further synergized with a transposon system to ensure rapid and sustained expression of gRNAs in both target cells and their daughter cells, facilitating efficient gRNA captures in the single-cell gene expression-based analysis, with >82% cells recovered with perturbation identities.
  • the inventors additionally uncovered the cell-type specific impact of perturbations on neuronal subtype proportions.
  • the AAV-based in vivo Perturb-seq platforms as exemplified herein achieved 10-fold higher labeling in embryonic brains (two batches for 50,075 cells) and can label >6% of brain cells, surpassing the lentiviral efficacy of ⁇ 0.1% as reported in Jin et al. (Science 2020; 370, eaaz6063), permitting in vivo analysis of over 30,000 cells in a single trial.
  • conventional methods employing lentiviral vectors typically can only analyze a few thousand cells in vivo from one batch, e.g., 17 batches 46,770 cells as described in Jin et al., 2020.
  • the current invention represents significant improvement and technological advantages over other known in vivo Perturb-seq screens that rely on other viral vectors, e.g., the lentiviral vector based screen as described in Jin et al., 2020; and US Patent Application Publication NO.2021/0172017A1.
  • the Perturb- seq platforms described herein provides a flexible approach to interrogate gene function across diverse cell types in vivo, translating gene variants to their causal functions.
  • this invention is not limited to the particular methodology, protocols, and reagents described as these may vary.
  • AAV refers to adeno-associated virus, and may be used to encompass the naturally occurring wild-type virus itself or derivatives thereof.
  • An AAV vector is a small single-stranded DNA viral vector containing icosahedral protein capsids. The term covers all subtypes, serotypes and pseudotypes, and both naturally occurring and recombinant forms, except where required otherwise.
  • Pseudotyped AAV refers to an AAV that contains capsid proteins from one serotype and a viral genome including 5'-3' ITRs of a second serotype.
  • the abbreviation "rAAV” refers to recombinant adeno-associated viral particle or a recombinant AAV vector (or "rAAV vector").
  • AAV virus or “AAV viral particle” refers to a viral particle composed of at least one AAV capsid protein (preferably by all of the capsid proteins of a wild-type AAV) and an encapsidated polynucleotide. If the particle comprises a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell), it is typically referred to as "rAAV”.
  • rAAV heterologous polynucleotide
  • Cas9 CRISPR genome editing system requires the Cas9 DNase obtained from Streptococcus pyogenes and a guide RNA (gRNA) for targeting the Cas9 DNase activity to complementary genomic sequences.
  • the gRNA is made up of two parts: crispr RNA (crRNA), a 17-20 nucleotide sequence complementary to the target DNA, and a trans-activating crispr RNA (tracrRNA), which serves as a binding scaffold for the Cas nuclease.
  • crRNA crispr RNA
  • tracrRNA trans-activating crispr RNA
  • the cleavage site in the target DNA is also preceded by a short protospacer-adjacent motif (PAM).
  • PAM protospacer-adjacent motif
  • PAM Protospacer adjacent motif
  • Cas9 In bacteria, Cas9 relies on RNase III to excise crRNAs from a CRISPR array.
  • Protospacer adjacent motif is a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system.
  • PAM is a component of the invading virus or plasmid, but is not a component of the bacterial CRISPR locus. Cas9 will not successfully bind to or cleave the target DNA sequence if it is not followed by the PAM sequence.
  • PAM is an essential targeting component (not found in bacterial genome) which distinguishes bacterial self from non-self DNA, thereby preventing the CRISPR locus from being targeted and destroyed by nuclease.
  • the canonical PAM is the sequence 5'-NGG-3' where "N” is any nucleobase followed by two guanine (“G") nucleobases.
  • Guide RNAs gRNAs
  • the canonical PAM is associated with the Cas9 nuclease of Streptococcus pyogenes (designated SpCas9), whereas different PAMs are associated with the Cas9 proteins of the bacteria Neisseria meningitidis, Treponema denticola, and Streptococcus thermophilus.
  • 5'-NGA-3' can be a highly efficient non-canonical PAM for human cells, but efficiency varies with genome location. Attempts have been made to engineer Cas9s to recognize different PAMs to improve ability of CRISPR-Cas9 to do gene editing at any desired genome location. Cas9 of Francisella novicida recognizes the canonical PAM sequence 5'-NGG-3', but has been engineered to recognize the PAM 5'-YG-3' (where "Y" is a pyrimidine), thus adding to the range of possible Cas9 targets.
  • Protospacers are spacer sequences in CRISPR loci in a bacterium that were inserted into a CRISPR locus by invading viral or plasmid DNA.
  • Cas9 nuclease attaches to tracrRNA:crRNA which guides Cas9 to the invading protospacer sequence.
  • Cas9 will not cleave the protospacer sequence unless there is an adjacent PAM sequence.
  • the spacer in the bacterial CRISPR loci will not contain a PAM sequence, and will thus not be cut by the nuclease.
  • a “host cell” or “target cell” refers to a living cell into which a heterologous polynucleotide sequence is to be or has been introduced.
  • the living cell includes both a cultured cell and a cell within a living organism.
  • heterologous polynucleotide sequence means for introducing the heterologous polynucleotide sequence into the cell are well known, e.g., transfection, electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, and/or the like.
  • the heterologous polynucleotide sequence to be introduced into the cell is a replicable expression vector or cloning vector.
  • host cells can be engineered to incorporate a desired gene on its chromosome or in its genome. Many host cells that can be employed in the practice of the present invention (e.g., CHO cells) serve as hosts are well known in the art.
  • the host cell is a mammalian cell.
  • operably linked or “operably associated” refers to functional linkage between genetic elements that are joined in a manner that enables them to carry out their normal functions. For example, a gene is operably linked to a promoter when its transcription is under the control of the promoter and the transcript produced is correctly translated into the protein normally encoded by the gene.
  • a gRNA-encoding sequence is operably linked to a reporter gene if the gRNA is co-produced with a transcript encoded by the reporter gene.
  • a “substantially identical” nucleic acid or amino acid sequence refers to a polynucleotide or amino acid sequence which comprises a sequence that has at least 75%, 80% or 90% sequence identity to a reference sequence as measured by one of the well-known programs described herein (e.g., BLAST) using standard parameters.
  • the sequence identity is preferably at least 95%, more preferably at least 98%, and most preferably at least 99%.
  • the subject sequence is of about the same length as compared to the reference sequence, i.e., consisting of about the same number of contiguous amino acid residues (for polypeptide sequences) or nucleotide residues (for polynucleotide sequences).
  • Polynucleotide sequences are no less substantially identical if they are composed of RNA or DNA, despite the chemical differences between RNA and DNA, and the presence of uracil in RNA instead of thymidine in DNA. [0032] Sequence identity can be readily determined with various methods known in the art.
  • the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).
  • Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • complementary refers to a nucleotide or nucleotide sequence that hybridizes to a given nucleotide or nucleotide sequence.
  • nucleotide A is complementary to T and vice versa
  • nucleotide C is complementary to G and vice versa.
  • nucleotide A is complementary to the nucleotide U and vice versa
  • nucleotide C is complementary to the nucleotide G and vice versa.
  • “Paired” and “unpaired” refer to Watson-Crick base pairs, in either the context of DNA, RNA or a DNA-RNA hybrid.
  • a sequence is said to be paired with its reverse complement.
  • a cell has been “transformed” or “transfected” by exogenous or heterologous polynucleotide (or “a transgene” or “a target gene” as used interchangeably herein) when such polynucleotide has been introduced inside the cell.
  • the transforming polynucleotide may or may not be integrated (covalently linked) into the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming polynucleotide may be maintained on an episomal element such as a plasmid.
  • a stably transformed cell is one in which the transforming polynucleotide has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming polynucleotide.
  • a “clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • Transposons (transposable elements or jumping genes) refer to DNA sequences that can move and integrate to different locations within the genome.
  • TEs transposable elements or jumping genes
  • DNA transposons can move in the DNA of an organism via a single-or double-stranded DNA intermediate.
  • DNA transposons have been found in both prokaryotic and eukaryotic organisms. They can make up a significant portion of an organism's genome, particularly in eukaryotes.
  • DNA transposons can be autonomous or non-autonomous. Autonomous transposons encode their own transposase enzyme that facilitates the jumping of the gene while non-autonomous transposons require the transposase activity of another transposable element. DNA transposons are delineated by flanking terminal repeats that mark the location that the transposase excises the DNA. These DNA elements then re-integrate at a different location within the genome.
  • a "vector" or “construct” is a non-naturally occurring nucleic acid with or without a carrier that can be introduced into a cell, or has been introduced into a cell.
  • Vectors that have been introduced into a cell include transfected plasmids and integrated DNA molecules, including those resulting from retroviral integration, integration of an AAV vector, and integration by homologous recombination.
  • Vectors capable of directing the expression of heterologous polynucleotide or transgene sequences encoding for one or more polypeptides are referred to as "expression vectors" or "expression constructs".
  • the cloned transgene sequence or open reading frame (ORF) is usually placed under the control of (i.e., operably linked to) certain regulatory sequences such as promoters, enhancers and polynucleotide switch sequences.
  • AAV vector based Perturb-seq platforms for analyzing gene functions in vivo provides in vivo Perturb-seq platforms to analyze target genes in diverse tissue types and cell types with high content phenotypic readout and single cell resolution.
  • the in vivo systems of the invention utilize a library AAV vectors of specific serotypes for delivering genetic perturbations to modulate one or more target genes of interest in specific cell or tissue types, e.g., central and peripheral nervous system, in an animal embryo or an adult or aged animal.
  • Adeno-associated virus (AAV) is a small, nonenveloped virus that was adapted for use as a gene transfer vehicle.
  • AAV vectors refer to recombinant adeno-associated viruses that are derived from nonpathogenic parvoviruses. They evoke essentially no cellular immune response, and produce transgene expression lasting months in most systems. Like adenovirus, adeno-associated virus vectors also have the capability to infect replicating and nonreplicating cells and are believed to be nonpathogenic to humans. Delivery of heterologous polynucleotide sequences via recombinant AAV can provide for safe, unobtrusive and sustained expression (> 2 years) of high levels of protein therapeutics.
  • AAV vector serotypes suitable for the invention were identified through a barcoded screen of phylogenetically diverse AAVs in vivo.
  • Serotypes of AAV vectors refer to classifications of AAV variants based on their molecular composition and their targeting specificity in tissues and cell types. To date, there are at least 11 number of AAV serotypes, including AAV1, 2, 4, 5, 6, 7, 8, 9 (see, e.g., Issa et al., Cells 2023; 12(5):785).
  • AAV-SCH9 is an AAV serotype that is a hybrid of AAV2, 8, and 9 and originally made to transduce adult neural stem cells.
  • Detailed structural information of AAV-SCH9 is provided in, e.g., Ojala et al., Mol. Ther.2018; 26: 304-319.
  • Specific AAV-SCH9 and AAV2.NN vectors can be obtained from commercial vendors, e.g., Addgene (Watertown, MA).
  • AAV9-PHP.eB and AAV9-PHP.S exemplified herein can also be obtained commercially, e.g., from vendors such as Addgene (Watertown, MA).
  • the in vivo Perturb-Seq platforms described herein provide a flexible and generalizable strategy to efficiently target selective cell types in vivo, and perform scalable genetic perturbation screens with reliable phenotypic readout using high-throughput single-cell omic methods.
  • the employed AAV vectors are intended to deliver genetic perturbations to target genes in tissues in animal embryos.
  • the employed AAV vector is AAV-Rep2-SCH9.
  • the employed AAV vector is AAV2.NN.
  • the employed AAV vectors are intended to deliver genetic perturbations to target genes in tissues in adult or aged animals, e.g., adult brains and peripheral nerve systems.
  • the employed AAV vector is AAV-PHP.eB.
  • the employed AAV vector is AAV9-PHP.S.
  • Detailed structural information of these specific AAV vectors are known in the art. See, e.g., Ojala et al., Mol. Ther., 2018; 26:304-319; and Chan et al., Nat. Neurosci., 2017; 20:1172-1179.
  • the AAV vectors can be administered to a transgenic animal system (e.g., an embryo or an adult animal) that expresses a CRISSPR-Cas gene editing system.
  • the library of AAV vectors is administered to a developing embryo of a CRISSPR-Cas9 transgenic mouse.
  • these embodiments can employ the AAV-Rep2-SCH9 vector or the AAV2.NN.
  • these vectors can be administered in utero to the brain (e.g., lateral ventricle) at any time that is equivalent to a mouse embryo developing stage between E11.5 and E19.5.
  • the vectors are administered to the embryo during the E11.5-15.5 period or the E13.5-17.5 period.
  • the library of AAV vectors is administered to a developed adult animal of a CRISSPR-Cas9 transgenic mouse.
  • Some of these embodiments can employ the AAV-PHP.eB vector or the AAV9-PHP.S vector.
  • the AAV vectors are administered to the animal at an age that is equivalent to mouse age of from about postnatal day 10 (P10) to about 18 months old. In some of these embodiments, the vectors are administered to the adult mice via retroorbital injection, as exemplified herein.
  • the genetic perturbations encoded by each of the AAV vectors contain gRNAs that can guide the target genes for modulation (e.g., cleavage) by a CRISPR gene editing system. At least one gRNA targeting a gene of interest (“target gene”) is encoded by the AAV vectors. In some embodiments, two or more gRNAs designed for targeting each gene of interest are included in the library of AAV vectors.
  • the two or more gRNAs targeting one gene can be encoded by one AAV vector.
  • the multiple gRNAs targeting the same gene can be encoded by different AAV vectors in the library.
  • the animal or animal embryo employed in the screen is derived from a transgenic non-human animal that is engineered to express the CRISPR editing system.
  • the gene editing system expressed by the animal is CRISPR is CRISPR-Cas9.
  • each of the gRNAs can be put under the control of a different promoter, including, e.g., human U6 polymerase III promoter as exemplified herein.
  • a reporter gene such as GFP as exemplified herein can be operably linked to the gRNA-coding sequences in the AAV vectors.
  • the reporter gene allows enrichment of cells expressing the corresponding gRNAs after the AAV vectors are introduced into the animal embryos or developed animals.
  • delivery of the vectors into the CRSIPR-expressing transgenic system e.g., a mouse embryo
  • expression and detection of the genetic perturbations e.g., gRNAs for specific target genes
  • identification e.g., by reporter gene expression
  • enrichment of cells perturbed cells
  • analyses of phenotype- correlating genes in the perturbed cells e.g., single-cell transcriptomic analysis
  • phenotype- correlating genes in the perturbed cells e.g., single-cell transcriptomic analysis
  • the AAV vectors delivering genetic perturbations used in the in vivo screen platforms can be further coupled with a transposon system. As described in detail below, this is to ensure rapid, cell-type specific, and reliable expression of gRNAs in target cells as well as their daughter cells for highly efficient gRNA captures in the single-cell transcriptomic analysis. Due to incorporation of a transposon system, the AAV-based in vivo screen platforms of the invention can efficiently and economically target and perturb genes in both embryonic and adult contexts. This represents a significant improvement over genetic screens utilizing lentiviral or any other viral vectors, e.g., the lentiviral vector-based system as described in US Patent Application Publication No.2021/0172017.
  • the coding sequence for a genetic perturbation e.g., gRNA-coding sequence
  • any other operably linked sequences e.g., a reporter gene
  • Methods for producing AAV vectors are well-known in the art.
  • AAV vectors have also been used in many reported studies for gene therapy in research and clinical environment.
  • the AAV viral vectors and related reagents suitable for the invention can be obtained commercially.
  • the specific AAV vectors for practicing the invention can be based on the pAAV-MCS construct that is available from Agilent Technologies (Santa Clara, CA).
  • the genetic perturbations delivered by the AAV vectors in the in vivo Perturb-seq platforms of the invention include gRNAs to guide target genes for genetic modulation.
  • a transgenic animal system expressing a CRISPR gene editing functionality is used to express the genetic perturbations and examine their effect on target genes.
  • the transgenic animal system can express the CRISSPR-Cas9 gene editing functionality.
  • the genetic perturbations encoded by the AAV vectors can be gRNAs that are designed to subject one or more target genes to the activities of the CRISPPR-Cas system in the transgenic animal.
  • the library of AAV vectors to be introduced into the transgenic system encode at least one gRNA that is designed for each of target genes.
  • two or more gRNAs are encoded by the library of AAV vectors for each of the target genes.
  • 3 or 4 gRNAs are encoded by the library of AAV vectors for each of the target genes.
  • the multiple gRNAs designed for a specific target gene are encoded by multiple AAV vectors.
  • the multiple gRNAs designed for a specific target gene are encoded by one AAV vector.
  • Any transgenic systems with CRISPPR-Cas functionality can be employed in the practice of the invention. In general, these include any transgenic animals or embryos at various development stages that are engineered to express a Cas enzyme and optionally other components required for the CRISPR functionality.
  • the transgenic systems can constitutively or inducibly expresses the Cas enzyme and/or the other components in some or all of their cells.
  • the Cas enzyme expressed in the transgenic systems is a Cas Type I, II, III, IV, or V protein.
  • the Cas enzyme (e.g., Cas9) is expressed by the transgenic systems while the other components can be separately provided, e.g., by another vector to be introduced into the transgenic systems (embryos or developed animals).
  • another vector to be introduced into the transgenic systems embryonic or developed animals.
  • the genetic perturbations encoded by the vectors are gRNAs that are designed to subject the target genes to CRISPPR- Cas9 gene editing in the transgenic system.
  • each AAV vector encodes one or more gRNAs that can direct a target gene to the enzymatic activity of Cas9.
  • Methods well-known in the art for genome-scale screening of perturbations in single cells using the CRISPR-Cas9 gene editing system can be readily employed and modified as necessary in the practice of the invention.
  • transgenic systems described in any of these reports may be employed and adapted for use in the practice of the invention.
  • transgenic mice expressing Cas9 can be used in the practice of the invention.
  • Cas9-expressing transgenic mice can be readily obtained from commercial vendors, e.g., the Jackson Laboratory (Bar Harbor, ME).
  • V. Transposon system co-delivered with AAV vectors [0048]
  • the AAV-based in vivo Perturb-seq platforms of the invention can additionally incorporate a transposon system.
  • co- introduction of AAV vectors encoding gRNAs for perturbing targeting genes and a transposon system can substantially boost gRNA expression level and enable gRNA capture with sparse scRNA-seq readout.
  • transposon element effectively enhances and stabilizes expression of the gRNAs in embryonic and adult brains and peripheral nerve systems. It ensures rapid, cell-type specific, and reliable expression of gRNAs in target cells as well as their daughter cells for highly efficient gRNA captures in the single-cell transcriptomic analysis.
  • Various transposon systems can be incorporated into the AAV-based in vivo screen platforms of the invention.
  • the employed transposon system is a DNA transposon. Integration of a DNA transposon system with the genetic perturbation delivering AAV vectors can be performed in accordance with the specific protocols exemplified or methods well known in the art.
  • DNA transposon systems well known in the art can also be employed and modified for use in the practice of the present invention. These include a wide range of individual DNA transposons from different families and donor organisms that have been characterized in detail in the art, e.g., Tol2 originating from medeka fish, Sleeping Beauty - synthetic sequences derived from transposons found in the white cloud minnow, Atlantic salmon and rainbow trout, and piggyBac isolated from the cabbage looper moth.
  • the piggyBac DNA transposon encodes a transposase (iPB) that is highly active in mammalian cells and integrates at the conserved TTAA tetranucleotide sequence.
  • PB transposase There is also a murine codon-optimized PB transposase (mPB) that was 20 times more efficient than iPB in mouse embryonic stem cells. Further, a hyperactive form of PB transposase (hypPB) with several amino acid substitutions on mPB is known in the art. This DNA transposon system is able to increase relative integration frequency by nine times over mPB in mouse embryonic stem cells. Detailed technical information of this transposon system is provided in, e.g., Yusa et al., Proc Natl Acad Sci USA.2011; 108:1531–1536. In addition to the PB transposase and derivatives, other transposon systems well-known in the art can also be employed in the practice of the invention.
  • the transposase Upon binding, the transposase cuts out the transposon sequence from the surrounding genomic DNA of the host cell.
  • the formed complex consisting of the mobilized transposon DNA fragment and the still bound transposases is now able to change its position to a new location in the cell genome.
  • the transposases open the genomic DNA backbone at the new locus and insert the transposon fragment.
  • the ligation of the open DNA ends is mediated by cellular key factors of the non- homologous end joining pathway (NHEJ) within the double strand break (DSB) repair system. Employment of any of these transposon systems in the practice of the invention can be readily carried out in accordance with the teachings provided in the art.
  • the transposon system can be introduced into the transgenic animal system in the same AAV vectors that encode the genetic perturbations.
  • a dual vector system can be used.
  • the coding sequence of the genetic perturbation e.g., gRNA
  • a separate vector e.g., a second AAV vector
  • the employed DNA transposon system includes transposase hypPB (e.g., hyperactive piggyBac) and a transposon sequence recognized by the transposase (e.g., inverted terminal repeat sequences).
  • the PiggyBac (PB) transposon is a highly useful transposon for genetic engineering of a wide variety of species. It can efficiently transpose between vectors and chromosomes via a "cut and paste" mechanism.
  • the PB transposase recognizes transposon- specific inverted terminal repeat sequences (ITRs) located on both ends of the transposon vector and efficiently moves the contents from the original sites and integrates them into TTAA chromosomal sites.
  • ITRs transposon-specific inverted terminal repeat sequences
  • the PiggyBac transposon system enables genes of interest between the two ITRs (e.g., gRNA-encoding sequence) in the PB vector (e.g., AAV vectors exemplified herein) to be easily mobilized into target genomes.
  • PB vector e.g., AAV vectors exemplified herein
  • incorporation of this transposon system into the AAV-based Perturb-seq platform is able to overcome the challenge of gRNA dilution and significantly improve genetic perturbation and labeling efficiency in vivo.
  • a library of AAV vectors encoding genetic perturbations (e.g., gRNAs) specific for one or more target genes are introduced into the transgenic animal or animal embryo, cells expressing the genetic perturbations can be identified and isolated from the live animal. The identified cells can be further enriched, before being subject to examination for their biological and cellular properties including phenotypic characteristics.
  • examination of the perturbed cells for modified or altered biological and cellular properties can include measuring genomic, genetic, proteomic, epigenetic and/or phenotypic differences in single cells.
  • the specific genetic perturbations expressed in individual cells can then be correlated with the biological properties (e.g., phenotypic changes) revealed from the examination for the different cells.
  • phenotypic changes resulting from the genetic perturbations in the identified cells relate to changes in gene or protein expression.
  • a plurality of target genes can be perturbed in single cells, and gene expressions in the perturbed cells are then analyzed (e.g., via scRNA-seq).
  • perturb-seq is used to discover novel gene and drug targets to allow treatment of various diseases in which the target genes are involved.
  • Various tools are available to examine cells expressing the genetic perturbations and exhibiting changes in certain gene or protein expressions.
  • a sequencing analysis can be used to identify cell types and modulated genes in the perturbed cells.
  • analysis of phenotypic changes due to the introduced genetic perturbations (e.g., editing of a specific target gene) in the identified cells and correlation of a perturbation with an observed phenotypic change in single cells can be performed via single-cell RNA sequencing (scRNA-seq).
  • the gRNAs encoded by the AAV vectors are functional for gene editing and also can be directly captured in the scRNA-seq technology. This is to facilitate correlation of an observed phenotype with a specific perturbed gene in the identified cells.
  • the scRNA-seq analysis aims to detect gRNAs in the identified cells by sequencing transcripts expressed from the AAV vectors.
  • a given gRNA and a corresponding specific barcode are co-expressed from the same AAV vector, and detection of the gRNA is realized by detection of the specific barcode via RNA sequencing.
  • phenotypic analyses of the identified cells entail determining cell types and corresponding perturbations (e.g., gRNA sequences encoded by the AAV vectors) via single cell RNA (scRNA) sequencing of the identified and optionally further enriched perturbed cell population.
  • scRNA sequencing of a population of cells can be readily performed in accordance with experimental protocols that are routinely practiced in the art. See, e.g., Picelli et al., Nat.
  • phenotypic analyses of the identified and enriched perturbed cells involve high-throughput scRNA sequencing. This can be performed with the specific techniques exemplified herein and/or experimental procedures well-known in the art. See, e.g., Rosenberg et al., Science 2018; 360: 176-182; Vitak et al., Nat. Methods 2017; 14: 302-308; Cao et al., Science 2017; 357: 661-667; Gierahn et al., Nat.
  • phenotypic analyses of the identified and enriched perturbed cells involve can be accomplished by single nucleus RNA sequencing. This can be performed using standard protocols that have been reported in the literature.
  • Some embodiments of the invention utilize a CRISPPR-Cas9 gene editing system to perturb target genes in a CRISPPR-expressing transgenic system described herein.
  • transgenic system e.g., Cas9 transgenic mice
  • specific experimental protocols exemplified herein. Additional guidance for performing genome-scale screening of perturbations in single cells using CRISPR-Cas9 have been provided in the art.
  • Example 1 Barcoded AAV screens identify serotypes efficiently expressed in the developing brain [0057]
  • Existing Perturb-seq systems relies on lentiviral vectors, which have relatively low packaging yield and limited tissue penetration in vivo.
  • AAV vectors are advantageous for in vivo studies due to their high production yields and capsid engineering potential. However, most AAVs take weeks to reach prime expression; and there is no documented AAV to efficiently transduce newborn neurons and progenitors in the developing brain, which requires a faster expression onset.
  • AAV serotype targeting developing brain in vivo we constructed a barcoded library of 86 phylogenetically diverse AAV serotypes; each expresses a green fluorescent protein (GFP) with a unique DNA barcode upstream of the polyadenylation initiation sites (Fig.1, Panel A).
  • GFP green fluorescent protein
  • This library consists of engineered serotypes that were reported or published previously, including AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6.2, AAV7, AAV8, AAV9, AAV10, AAV 12 and AAV13; several of these serotypes are commonly used for in vivo studies with minimal toxicity as previously reported (Table 1). Table 1.
  • AAV serotypes lists and barcodes in the 86-AAV and 14-AAV libraries.
  • IP intermediate progenitor
  • AAV-SCH9 rapidly transduces developing brains within 48 hours [0059]
  • To identify the AAV serotypes that are enriched in GFP + cells we purified the cells from the neocortex (in vivo) and HT22 cells (in vitro) at 24- and 48-hours post- transduction and quantified the barcode abundance using next-generation sequencing (Fig.1, Panel D). Compared to the initial distribution in the AAV library, 48 hours post-transduction, both in vivo and in vitro, we detected significant shifts in the barcode distribution.
  • AAV serotypes were enriched in vitro (AAV-P1529, AAV2-NN, AAV2-P1583 and AAV-DJ), several serotypes were enriched in vivo (AAV1, AAV2-P1558, AAV-Hu48.2 and AAV2-7M8), and several were enriched in both (AAV- SCH9, AAV-SCH9repeat136bp, AAV2-P1576, AAV2-P1579 and AAV2-P1596) (Fig.1, Panel D; Table 1). This likely reflects a shared pattern of AAV tropism for newborn neurons and progenitors in vivo and in vitro, as well as a distinct pattern of enrichment in vivo.
  • PC1 explained 63% variance of the data and separated in vitro data (24 and 48h), in vivo data (48hr), from in vivo data (24h) and the initial AAV library.
  • PC2 (10% variance) separated in vivo (48h) data from all other samples. Consistent with the in vivo tissue analysis, it takes 48h to observe the change of barcode distribution in vivo, while the impact is quicker and more consistent at 24h and 48h in vitro.
  • AAV-SCH9 it constituted 2.3% of the initial AAV library and 48 hours after in vivo transduction, it constituted 27.4% of the enriched population.
  • Differential expression analysis revealed that AAV-SCH9 was the most significant hit from the in vivo experiment, followed by a variant of the same serotype (AAV-SCH9-repeat136bp). Both AAV-SCH9 and its variant showed significant enrichment in vitro 24 hours post transduction that persisted at 48 hours (Fig.1, Panel G).
  • AAV-SCH9 we also quantified the relative expression dynamics of several top hits, including AAV-SCH9, by comparing the abundance of the barcodes in 24 and 48-hour post-transduction (Fig.1, Panel F).
  • Fig.1, Panel F We also quantified the relative expression dynamics of several top hits, including AAV-SCH9, by comparing the abundance of the barcodes in 24 and 48-hour post-transduction (Fig.1, Panel F).
  • AAV9-PHP.eB and AAV-DJ showed only minimal expression in vivo, with 0.2-fold and 1.1-fold changes relative to their compositions in the initial AAV library, respectively (Table 1; and Fig.1, Panel D).
  • AAV-SCH9 demonstrated a 11.5-fold increase in expression abundance, making it an optimal choice for perturbing and studying gene function in a dynamic developmental context when cells are actively differentiating and maturing.
  • Example 3 Diverse cell type tropisms of AAV serotypes in vivo with single-cell resolution [0063]
  • AAV-SCH9 We identified several serotypes, including AAV-SCH9, that exhibit efficient transduction of the developing brain and neuronal cell lines by bulk measurement, but their precise cell type-specificity were still unclear.
  • each AAV was designed to express a GFP reporter with a set of 3 unique barcodes upstream of the polyadenylation initiation sites and then pooled them with equal titer (Fig.2, Panel A).
  • scRNA-seq droplet-based single-cell RNA-seq
  • Fig.2, Panels A-B we assigned the AAV serotype identities by using the barcodes, captured in a dial-out PCR library (Fig.2, Panel C).
  • neocortical cells for further analysis (7,630 cells from the FACS-enriched GFP + population, and 7,205 cells without enrichment) (Fig.2, Panel B).
  • Fig.2, Panel B We partitioned the cells into major cell types and annotated them based on known marker gene expressions (Table 3) (Di Bella et al., 2021; La Manno et al., 2021; Tasic et al., 2018). These cells were clustered into 11 cell types including upper and deep layer projection neurons (ULPN, DLPN), migrating neurons (Mig. neurons), apical progenitors (Api.
  • prog intermediate progenitors (IP), interneurons derived from the medial ganglionic eminence (IN-MGE), interneurons derived from the non-medial ganglionic eminence (IN-non-MGE), Cajal-Retzius cells (CR), fibroblast (Fibro), mural cells (Mural), and microglia (Mg).
  • IP intermediate progenitors
  • I-MGE medial ganglionic eminence
  • I-non-MGE interneurons derived from the non-medial ganglionic eminence
  • Cajal-Retzius cells CR
  • fibroblast Fibro
  • mural cells Mural
  • Mg microglia
  • AAV-SCH9 (BC1), AAV2-NN (BC2), AAV-SCH9-G160D (BC4) and AAV2-7M8 (BC10) (Fig.2, Panel C).
  • AAV-SCH9 transduced populations we detected 12.0% upper layer projection neurons, 56.5% deep layer projection neurons, 10.1% MGE-derived interneurons, 7.9% migrating neurons, 2.8% apical progenitors, and 1.3% intermediate progenitors (Tables 2-3).
  • Table 2 Summaries of scRNA-seq experiments (detailed information about each batch of Perturb-seq) Table 3.
  • AAV-SCH9 Cell type classification and differential expressed genes in 14-AAV library E16.5 scRNA-seq data Example 4. In situ characterizations of AAV-SCH9 reveal its fast-acting dynamics and broad neuronal labeling in the developing brain [0066] Using bulk sequencing and scRNA-seq, we identified and validated AAV-SCH9 as an effective vector to rapidly ( ⁇ 48 hours) transduce embryonic cortical tissues in vivo. Previously, AAV-SCH9 has been reported to target subventricular adult neural stem cells (Ojala et al., 2018).
  • AAV-SCH9 may directly transduce neurons, or first transduce progenitors which then differentiate into neurons.
  • the E13.5 transduced cells were enriched in bin 2 (46%) and bin 3 (23%) with CTIP2 expression, indicating their deep layer cortical sub-cerebral projection neuron identities (Arlotta et al., 2005; Chen et al., 2008).
  • the E17.5-transduced cells were broadly distributed in the later-born ULPNs in bin 1 (22%) and Layer 5 DLPNs in bin 2 (31%), supporting that AAV-SCH9 could label newborn neurons. (Fig.2, Panel E).
  • AAV-SCH9 can transduce additional regions, including olfactory bulb, striatum, hippocampus, thalamus, and cerebellum, with labeling density suitable for future cross-region Perturb-seq studies (Fig.2, Panel F).
  • lentiviral transduction using optimal, >9 ⁇ 10 9 U/mL high- titer vector: there were much fewer GFP + cells in most regions, especially cerebellum, thalamus, and striatum (Fig.2, Panel F), possibly due to limited tissue penetration in vivo.
  • gRNA and fluorophore expression from AAV will be diluted, and eventually lost, upon cell division and growth.
  • a dual-vector system including a hypPB (hyperactive piggyBac) transposase and a transposon with inverted repeat flanking the gRNA and fluorophore (Moudgil et al., 2020) (Fig.3, Panel A).
  • hypPB Upon transduction, hypPB can integrate the transgene into the nuclear genome for inheritance in the daughter cells, allowing consistent expression rather than transient, episomal expression (Fig.3, Panel A).
  • Fig.3, Panel A We first tested the design in vitro and transfected HT22 cells followed by time- lapse imaging. In the presence of hypPB, we detected more GFP + cells (Fig.3, Panel B) and enhanced GFP expression levels with faster onset; the expression was resistant to cell passages over the course of six days. hypPB allowed faster expression onset within a few hours post-transfection which would be highly advantageous for labeling and perturbing cells during the dynamic developmental process in vivo. Example 6.
  • hypPB improves transgene expression in vivo through embryonic transduction
  • AAV-SCH9 containing a transposon with or without an AAV-SCH9-hypPB in utero at E14.5 and performed immunofluorescence analysis at P7.
  • hypPB expression did not introduce overt toxicity in development (Fig.3, Panel C) as we measured the brain sizes and gliosis markers GFAP and IBA1 expression changes in the presence of hypPB.
  • hypPB could amplify AAV transgene expression in postmitotic neurons in adult central and peripheral nervous systems, where AAV transgene levels have been observed to be relatively low compared to genomic expression (Lang et al., 2019).
  • AAV9-PHP.eB for neurons and glia in the brain
  • AAV9-PHP.S for peripheral neurons, both through non-invasive retro-orbital administrations (Chan et al., 2017).
  • hypPB In adult brain, hypPB generally increased GFP expression levels (Fig.3, Panel D).
  • somatosensory cortex In the somatosensory cortex, average fluorescence intensity was 1.7-fold higher across laminar layers with hypPB (Fig.3, Panel G). Furthermore, by extracting nuclei from cortex and performing flow cytometry analysis, we found that the AAV9-PHP.eB transposon system can label 6.5-7.0% of total nuclei in the cortex, 4.0-fold higher than without hypPB. This indicates hypPB can enhance transgene expression in adult, post-mitotic cells. Notably, different from the AAV-SCH9 delivery, elevated levels of gliosis markers IBA1 and GFAP were detected in the retro-orbital AAV9-PHP.eB-hypPB transduction conditions.
  • Example 8 hypPB transposon integrates into the host genome with random insertions
  • Genome integration of the transgenes, via lentiviral vectors or transposons, could trigger unwanted changes in the genome and cellular activities. For instance, integrations in coding regions could profoundly influence function of nearby genes.
  • the 5’ method benefits from a higher concentration of gRNA-specific primers in solution (rather than on the beads) during reverse transcription, which is expected to give rise to a higher gRNA capture rate.
  • the 5’ and 3’ scRNA-seq gene expression analysis resulted in similar cell clustering and data quality (Table 5).
  • 3’ scRNA-seq we detected only 0.1% of reads assigned to gRNA from the library, whereas the 5’ scRNA-seq gave rise to 52.9% of reads assigned to gRNA. This is consistent with reliable assignment of the gRNA to the cell barcodes and high detection of gRNA levels from 5’ scRNA-seq.
  • Tcf4 and Nr2f1 are broadly expressed in most cell types, whereas Foxg1 and Tbr1 expressions are restricted to subclasses of neurons including deep layer projection neurons and immature neurons (Di Bella et al., 2021; La Manno et al., 2021).
  • the isocortex and hippocampal formation was micro-dissected and dissociated; the BFP-expressing cells were enriched and processed for droplet-based 5’ scRNA-seq with direct capture of gRNA (Replogle et al., 2020).
  • our system already achieves the collection of >10-fold more BFP + perturbed cells than the conventional lentiviral labeling, which significantly streamlines the experiment. Table 6.
  • gRNA1 of Foxg1 induced several distinct 1-base pair insertions in 39% of the reads, frameshift mutations leading to premature transcriptional termination downstream. This is likely an underestimation of the perturbation effects, as nonsense mediated decay should degrade much of the mutated mRNA.
  • different gRNAs, targeting the same gene may yield variable phenotypic outcomes due to their differential efficacies in gene editing, introducing potential phenotypic heterogeneity.
  • wild-type transcripts were detectable in our data, suggesting that in vivo Perturb-seq could allow the analysis of both full knockout and heterozygous loss-of-function effects across different cells collected.
  • Tbr1 perturbation is associated with a reduction in the proportion of deep layer excitatory neurons including L6 CT (1.9-fold) and L6 IT (3.6- fold). This is consistent with the known role of Tbr1 in maintaining L6 neuronal identity as previously reported (Bedogni et al., 2010; Fazel Darbandi et al., 2018), and our analysis provides a refined annotation of its cell type-specific effects.
  • Foxg1 in enforcing L6 CT neuronal identity (Liu et al., 2022b); Tbr1 loss-of-function causing defects in L6 CT neurons that acquire both L5- and L6-like identity and electrophysiological properties (Bedogni et al., 2010; Fazel Darbandi et al., 2018).
  • Foxg1 is critical for cell fate specification across neuronal cell types through regulations of transcription factor networks to maintain L6 neuronal identity (Liu et al., 2022b).
  • the Foxg1-DE genes in the four deep layer neuron cell types the only shared target is Stxbp6 (Syntaxin binding protein 6) whose expression is upregulated upon the perturbation in L5 PT, L5 IT, L5 PT, and L6 CT neurons (Fig.4, Panel I).
  • Stxbp6 Taxin binding protein 6
  • Fig.4, Panel I the only shared target is Stxbp6 (Syntaxin binding protein 6) whose expression is upregulated upon the perturbation in L5 PT, L5 IT, L5 PT, and L6 CT neurons.
  • the Foxg1-knockout induced changes of transcription factors are highly specific to the cell types.
  • the DE genes observed in L6 CT are largely absent in L5 IT, L5 PT or L5 NP cell types (Fig.4, Panel I).
  • mice [0098] C57BL/6J, Cas9, and CD-1 mice: [0099] All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committees (IACUC) of The Scripps Research Institute. E15 to P9 mice of varying sex and weight were used in the scRNA-seq experiments and mice ranging from E15 to adult were used in the immunohistochemistry experiments. All mice were kept in standard conditions (a 12-h light/dark cycle with ad libitum access to food and water).
  • IACUC Institutional Animal Care and Use Committees
  • HT22 and HEK293FT cell lines [00101] Mammalian cell culture experiments were performed in the HT-22 mouse hippocampal neuronal cell line (Millipore Sigma, #SCC129) or HEK293FT cell line (Thermo Fisher Scientific, #R70007) grown in DMEM (Thermo Fisher Scientific, #11965092) with 25mM high glucose, 1mM sodium pyruvate and 4mM L-Glutamine (Thermo Fisher Scientific, #11995073), additionally supplemented with 1 ⁇ penicillin–streptomycin (Thermo Fisher Scientific, #15140122), and 5-10% fetal bovine serum (Thermo Fisher Scientific, #16000069).
  • HT-22 cells were maintained at confluency below 80% and HEK293FT cells were maintained at confluency below 90%.
  • Method details [00103] Mammalian cell culture and time lapse imaging: All transfections were performed with PEI (Polysciences, #24765-1) in 96-well plates unless otherwise noted. Cells were plated at approximately 10,000 cells per well 16–20 hrs before transfection to ensure 50-60% confluency at the time of transfection. For each well on the plate, transfection plasmids were combined with OptiMEM I Reduced Serum Medium (Thermo Fisher, #31985070) with PEI to a total of 20 ⁇ l. This solution was added directly to the media dropwise.
  • HEK293FT cells were washed once with PBS, followed by trypsinization and resuspension in PBS supplemented with 0.04% BSA (NEB, #B9000S). Cells were FACS purified at 4°C and collected in Trizol (Thermo Fisher Scientific, #10296010) with 5,000-50,000 cells per sample.
  • the 2- ⁇ CT method was used for the analysis of qPCR data and normalized to GAPDH expression.
  • AAV vector construction and production Viral vectors and plasmids were constructed as previously reported (Jin et al., 2020).
  • the backbone plasmid contains the human U6 promoter to express one gRNA, and the EF1 ⁇ promoter to express a fluorescent protein conjugated to the nuclear membrane localized domain KASH. Cloning of the vectors was done individually and confirmed by Sanger sequencing.
  • the gRNA designs were defined using the online tool at benchling.com and the full sequences of the gRNAs used in this work are listed in Table 6.
  • AAV production and titration was performed by the viral vector core facility at Sanford Burnham Prebys and UCI Center for Neural Circuit Mapping viral core.
  • AAV administration AAV (0.5-1.5 ⁇ L per embryo) was administered in utero to the lateral ventricle at E13.5-17.5 in CD1, C57BL/6J or Cas9 transgenic mice (Jax#026179) (Platt et al., 2014) for immunohistochemistry analysis and scRNA-seq.
  • Adult mice were injected retro-orbitally with AAV (50-100 ⁇ L and ⁇ 1-4e 11 viral genome per animal) and perfused for immunohistochemistry experiments and nuclei flow cytometry.
  • AAV library barcode extraction At 24- or 48-hours post transduction, HT-22 cells and mouse primary cortical cells were purified with FACS.
  • Genomic DNA was extracted from approximately 3,000 purified cells by QuickExtract DNA Extraction Solution (Lucigen, #NC0302740) following the manufacture’s protocol.
  • the AAV serotype library was lysed by DNase I digestion and Proteinase K digestion.
  • PCRs with genomic DNA were performed with NEBNext High-Fidelity 2X PCR Master Mix (New England BioLabs, #M0541L) with the following primers: 5’-ctttccctacacgacgctcttcccgatct-gacgagtcggatctcccttt-3’ (SEQ ID NO:2), and 5’-gactggagttcagacgtgtgctcttccgatct-gcgatgcaatttcctcattt-3’ (SEQ ID NO:3).
  • Amplicons were amplified to include adaptors and sequenced on iSeq 1000 or MiSeq platforms (> 2 million reads per sample). BCL files were converted to FASTQ files using bcl2fastq (Illumina).
  • Immunofluorescent staining of brain sections and whole-mount DRGs Embryonic brains were directly harvested after decapitation and frozen immediately on dry ice in OCT. Continuous sets of 15-20 ⁇ m tissue sections were prepared on a cryostat, followed by the fixation for 15 min with 4% paraformaldehyde in PBS on ice.
  • Postnatal pups and adult mice were anesthetized and transcardially perfused with ice-cold PBS followed by ice-cold 4% paraformaldehyde in PBS. Dissected brains were postfixed overnight in 4% paraformaldehyde at 4 °C. Postnatal and adult brains were embedded in 2% agar and 60- 100 ⁇ m tissue sections were collected on a vibratome.
  • the primary antibodies and dilutions were: Chicken anti-GFP antibody (ab16901, 1:500; Millipore), Rabbit anti-GFP antibody (A-11122, 1:500; Invitrogen), Rabbit anti-RFP (600-401-379, 1:500; Rockland), Rabbit anti-Tbr1 (ab31940, 1:500: Abcam), Rabbit anti- Tbr2 (ab183991, 1:500: Abcam), Rat anti-Ctip2 (ab18465, 1:1000, Abcam), Rabbit anti-Pax6 (Cat#901302, 1:500, BioLegend), Rabbit anti-HA tag (5017, 1:500; Cell Signaling), Rat anti- HA tag (11867423001, 1:500, Roche), Chicken anti-GFAP (ab4674, 1:500, Abcam) and Goat anti-Iba1 (ab5076, 1:500, Abcam).
  • Tissue Dissociation and FACS-enrichment for genomic analysis Tissue dissociation was performed with the Papain Dissociation kit (Worthington, #LK003150) in a modification of a previously described protocol (Jin et al., 2020). Briefly, young mice were anesthetized then disinfected with 70% ethanol and decapitated. The brains were quickly extracted and gently dabbed with a PBS-soaked Kimwipe (Kimberly-Clark) to remove the meninge and fibroblasts.
  • Cortices were micro-dissected in ice-cold dissection medium (Hibernate A medium (Thermo Fisher Scientific, #A1247501) with B27 supplement (Thermo Fisher Scientific, #17504044) and Trehalose (Sigma Aldrich, Cat# T9531) under a dissecting microscope. Microdissected cortices were transferred into papain solution with DNase in a cell culture dish and cut into small pieces with a razor blade. The dish was then placed onto a digital rocker in a cell culture incubator for 30 mins with rocking speed at 30 rpm at 37°C.
  • Hibernate A medium Thermo Fisher Scientific, #A1247501
  • B27 supplement Thermo Fisher Scientific, #17504044
  • Trehalose Sigma Aldrich, Cat# T9531
  • the digested tissues were collected into a 15 mL tube and triturated with a 10 mL low bind plastic pipette 20 times and the cell suspension was carefully transferred to a new 15 mL tube.
  • 2.7 mL of EBSS, 3 mL of reconstituted Worthington inhibitor solution, and DNase solution were added to the 15 mL tube and mixed gently.
  • Cells were pelleted by centrifugation at 300 g for 5 mins at 4°C, followed by washing with 8ml cold dissection medium at 200g for 5 min at 4°C.
  • the scRNA-seq libraries were constructed using the Chromium Next GEM Single Cell 3' Solution v3.1 kit with Feature Barcode Technology or the Chromium Next GEM Single Cell 5’ Solution v2 kit with Feature Barcode Technology (10x Genomics) following the manufacturer’s protocol.
  • the gene expression library was sequenced with NextSeq500 high-output 75-cycle kits (Illumina) with sequencing saturation to ensure greater than 20,000 reads coverage per cell (R1: 26 base pair, R2: 46 base pair).
  • the CRISPR gRNA screening library was sequenced with Illumina iSeq100300-cycle (R1: 151 base pair, R2: 151 base pair) and Nextseq500 mid-output 150-cycle kits (Illumina) (R1: 73 base pair, R2: 74 base pair).
  • Illumina Nextseq500 mid-output 150-cycle kits
  • AAV barcode enrichment from scRNA-seq library Following whole transcriptome amplification (WTA) in the 10x Chromium library construction, a fraction of the WTA product was used to amplify AAV serotype barcodes as well as cell barcodes using a dial-out PCR strategy.
  • the final dial-out library was sequenced along with transcriptome library with NextSeq500 high-output 75-cycle kits (Illumina) flow cell (R1: 26 base pair, R2: 46 base pair).
  • Quantification and Statistical Analysis All images were analyzed with ImageJ (NIH), Photoshop (Adobe) and Illustrator (Adobe). Cells were counted manually from blinded files using ImageJ CellCount function.
  • AAV barcode analysis in the primary serotype screen FASTQ files of Illumina libraries were mapped to AAV barcodes using a custom script. Briefly, FASTQ that began with the correct initial primer sequence were kept.
  • the barcode sequence following the initial primer sequence was compared to our list of AAV barcodes and assigned to matching AAV barcodes with Levenshtein distance less than 2.
  • the barcode counts matrix was analyzed using DESeq2 v1.40.2 (Love et al., 2014).
  • Volcano plots were produced using the R package EnhancedVolcano v1.18.0 and heatmaps with pheatmap v1.0.12.
  • Transposon integration site analysis A custom reference genome was created by appending the transposon reporter plasmid to the mm39 mouse genome as an additional chromosome. FASTQ files were aligned to the custom genome using bwa mem (v0.7.17) (Li and Durbin, 2010) using the SP5M flags. The resulting files were filtered for reads that aligned to the piggybac plasmid and their pairs using SAMtoolsTM v1.15.1. After checking the distribution along the plasmid of these reads, they were further filtered down to reads aligning to the ends of the insert (position 1653-2053 and 6096-6496) within the plasmid.
  • the filtered files were then parsed using PairtoolsTM v0.3.0 and default settings. They were then filtered for junctions (pair_type of UU, UR, or RU with one end aligning to the mouse genome) that were then validated manually.
  • junctions junctions (pair_type of UU, UR, or RU with one end aligning to the mouse genome) that were then validated manually.
  • the annotatePeak function from the R package ChIPseekerTM (v1.36.0) (Yu et al., 2015) was used with the TSS set as +/- 3,000 bp.
  • the R package Circlize v0.4.15 was used to create a Circos Plot to visualize integration sites.
  • the AAV barcodes or gRNA reads were quantified at the single cell level with the feature-ref flag in Cell Ranger.
  • Cell type classification and cell identity annotation For the AAV serotype secondary screen and comparing 5’ vs 3’ scRNA-seq, filtered count matrices from Cell Ranger were loaded into R v4.3.0 with the Read10X command from Seurat v4.3.0.9003 (Hao et al., 2021) and loaded into Seurat object with CreateSeuratObject, filtering out cells with ⁇ 500 genes or mitochondrial count percent > 25%. The data were log normalized and the 2,000 most variable features were selected by FindVariableFeatures.
  • Perturb-seq data processing cell type classification, cell identity annotation, and perturbation identity annotation:
  • the filtered count matrices from Cell Ranger were loaded into R v4.0.3 with the Read10X command from Seurat v4.0.0 (Hao et al., 2021) and loaded into a Seurat object with CreateSeuratObject, filtering out cells with ⁇ 500 genes.
  • a UMAP was calculated on this reduction using RunUMAP, and clustering was performed on this reduction with FindNeighbors and FindClusters with otherwise default settings.
  • the UMI count matrix for the gRNAs produced by Cell Ranger was also added to this Seurat object as an additional assay.
  • Quality control (QC) metrics for each channel were calculated with CellLevel_QC tool (Github) and loaded into the metadata for the Seurat object. The % mitochondrial reads was also calculated for each cell.
  • Azimuth v0.3.2 was used to produce an initial annotation of the data using a single cell reference from the Allen brain atlas (Yao et al., 2021). Clusters with high percent intronic reads or high doublet scores were removed, as were cells with >20% intronic reads or >10% mitochondrial reads. Clusters were then labeled with cell type using the cell type labels from Azimuth and by comparing DE genes from our dataset to DE genes from the Allen brain atlas dataset (DE genes between clusters were calculated with presto v1.0.0).
  • the pipeline takes in the BAM file produced by Cell Ranger, extracts unmapped reads with SAMtools v1.8 (using the command “samtools view -f 4 -b”) (Li et al., 2009), extracts UMIs and CBCs with UMI-tools v1.0.1 (Smith et al., 2017), maps these reads to the sequences for GFP and BFP (including 5’ and 3’ UTR regions) with Minimap2 v2.11 (using the arguments -ax sr) (Li, 2018), and transforms them into a BAM file with SAM-tools view -b.
  • Unmapped reads were discarded and the name of the contig mapped to (GFP or BFP) for the remaining reads mapped to was added to the BAM file as an additional tag (XT tag) with awk.
  • Non-neuronal cells (cells not labeled as Excitatory, Inhibitory, or Cajal-Retzius neurons) were removed from the Seurat object, as were cells with ⁇ 3% intronic reads.
  • Excitatory and Inhibitory cells with ⁇ 3000 genes were filtered out, as were Cajal-Retzius cells with ⁇ 2,000 genes.
  • All cells that were not assigned to exactly one guide by DemuxEM were removed as well. This Seurat object was used for downstream analysis.
  • Detecting insertions and deletions in targeted regions For each targeted gene, the reads overlapping it in each 10x channel were extracted from the Cell Ranger BAM files with SAM-tools view and combined into one BAM file with SAM-tools concat, which was sorted and indexed with SAM-tools.
  • the sinto filterbarcode command (Stuart et al., 2021) was used to split the BAM file into one BAM file for each perturbation, consisting of cells in the final analysis assigned to each gRNA (excluding cell barcodes that occur in multiple 10x channels). The resulting BAM files were indexed.
  • Non-Targeting control 2 compared each of the perturbation/control groups containing other gRNAs to this group.
  • Statistics for these pairwise composition comparisons were computed using the propeller.ttest function from speckle (R package v0.99.7) (Phipson et al., 2022).
  • the batch (10x channel) was additionally considered as another fixed effect to the linear model.
  • Cell types and clusters with less than 200 cells overall were excluded from this analysis. Results are collected and visualized together using ComplexHeatmap (R package v2.14.0) (Gu et al., 2016).
  • the Fezf2-Ctip2 genetic pathway regulates the fate choice of subcortical projection neurons in the developing cerebral cortex. Proc Natl Acad Sci U S A 105, 11382-11387. 10.1073/pnas.0804918105. Chen, H.Y., Bohlen, J.F., and Maher, B.J. (2021). Molecular and Cellular Function of Transcription Factor 4 in Pitt-Hopkins Syndrome. Dev Neurosci 43, 159-167. 10.1159/000516666.
  • Spatial CRISPR genomics identifies regulators of the tumor microenvironment. Cell 185, 1223-1239 e1220. 10.1016/j.cell.2022.02.015.
  • Neonatal Tbr1 Dosage Controls Cortical Layer 6 Connectivity. Neuron 100, 831-845 e837. 10.1016/j.neuron.2018.09.027.
  • Virology 280 124-131.10.1006/viro.2000.0743. Hou, P.S., hAilin, D.O., Vogel, T., and Hanashima, C. (2020). Transcription and Beyond: Delineating FOXG1 Function in Cortical Development and Disorders. Front Cell Neurosci 14, 35.10.3389/fncel.2020.00035. Jang, M.J., Coughlin, G.M., Jackson, C.R., Chen, X., Chuapoco, M.R., Vendemiatti, J.L., Wang, A.Z., and Gradinaru, V. (2023). Spatial transcriptomics for profiling the tropism of viral vectors in tissues.
  • a transcription factor atlas of directed differentiation Cell 186, 209-229 e226.10.1016/j.cell.2022.11.026. Kalamakis, G., and Platt, R.J. (2023). CRISPR for neuroscientists. Neuron. 10.1016/j.neuron.2023.04.021. Kaplanis, J., Samocha, K.E., Wiel, L., Zhang, Z., Arvai, K.J., Eberhardt, R.Y., Gallone, G., Lelieveld, S.H., Martin, H.C., McRae, J.F., et al. (2020). Evidence for 28 genetic disorders discovered by combining healthcare and research data.
  • FOXG1 sequentially orchestrates subtype specification of postmitotic cortical projection neurons. Sci Adv 8, eabh3568.10.1126/sciadv.abh3568. Love, M.I., Huber, W., and Anders, S. (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15, 550.10.1186/s13059-014-0550- 8.
  • Zenodo (doi.org/10.5281/zenodo.8239932).
  • Perez, B.A. Shutterly, A., Chan, Y.K., Byrne, B.J., and Corti, M. (2020).
  • AAV- Sleeping Beauty composite system bioRxiv.10.1101/2023.03.14.532651. Ye, L., Park, J.J., Dong, M.B., Yang, Q., Chow, R.D., Peng, L., Du, Y., Guo, J., Dai, X., Wang, G., et al. (2019).
  • In vivo CRISPR screening in CD8 T cells with AAV-Sleeping Beauty hybrid vectors identifies membrane targets for improving immunotherapy for glioblastoma. Nat Biotechnol 37, 1302-1313.10.1038/s41587-019-0246-4.

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Abstract

La présente invention concerne des dépistages in vivo à haute teneur et haute résolution pour analyser des fonctions d'une pluralité de gènes. Dans les dépistages fonctionnels de l'invention, des perturbations génétiques sont appliquées à un système transgénique exprimant CRISPR par des vecteurs AAV spécifiques qui sont complètement compatibles avec des plateformes Perturbb-seq. Les dépistages permettent des analyses de génomique fonctionnelle à travers divers tissus, types de cellules et organismes modèles in vivo, avec une lecture unicellulaire à haut débit.
PCT/US2024/045238 2023-09-08 2024-09-05 Dépistage in vivo à haute teneur et haute résolution pour analyser des fonctions géniques Pending WO2025054224A2 (fr)

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