Multiplex gene function mapping via in vivo brain phenotyping

Abstract

The present invention provides high-content and high-resolution in vivo screens for mapping genes that are linked to changes in cytoarchitecture or spatial transcriptomics of brain cells (e.g., neurons). The multiplex gene mapping platform utilizes genetic perturbations that are delivered to a CRISPR-expressing transgenic system via viral vectors (e.g., lentiviral vectors or AAV vectors). Phenotypic changes in the cytoarchitecture or spatial transcriptomics resulting from genetic perturbations are examined in individual brain cells, which can then be correlated with specific target genes that are modulated by the genetic perturbations.

Description

PATENTAttorney Docket No.: 2265.1PCMultiplex Gene Function Mapping Via In Vivo Brain PhenotypingCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The subject patent application claims the benefit of priority to U.S. Provisional Patent Application Number 63 / 699,134 (filed September 25, 2024; now pending). The full disclosure of the priority application is incorporated herein by reference in its entirety and for all purposes.STATEMENT CONCERNING GOVERNMENT SUPPORT

[0002] This invention was made with government support under HG012819 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND OF THE INVENTION

[0003] The mammalian brain comprises an enormous number of neurons with significant diversity of morphology and connectivity. The cytoarchitectures (including anatomical positions, cellular morphology, synaptic structure, and projection / connectivity) of neurons are highly responsible for their cell-type-specific function, as abnormal morphological changes are strongly associated with brain dysfunction, including neurodevelopmental and degenerative diseases. These cytoarchitectures are broadly regulated by networks of transcription factors and other genes during development. However, to systemically screen and characterize the genetics-cytoarchitecture relationship remains challenging, due to the required tissue disruption in most phenotypic in vivo screening.SUMMARY OF THE INVENTION

[0004] The present disclosure relates to multiplex gene function mapping with brainwide, high-content cytoarchitectural or spatial transcriptomic phenotyping at single-cell resolution. In one aspect, the invention provides methods for identifying or mapping genes that are linked to or associated with a phenotypic change in the cytoarchitecture or spatial transcriptomics of brain cells. The methods entail (1) introducing a library of vectors encoding genetic perturbations for a plurality of target genes into a CRISPR-Cas9 expressing transgenic animal system, (2) examining cytoarchitecture or spatial transcriptomics of brain cells at one or more developmental stages of the transgenic animal system, (3) identifyingone or more brain cells of the transgenic system that express a genetic perturbation and display a phenotypic change of cytoarchitecture or spatial transcriptomics, and (4) determining the corresponding gene perturbed by the genetic perturbation that is encoded by the vector introduced into each of the one or more cells. This allows identification of one or more genes that are linked to or associated with the change in the cytoarchitecture or spatial transcriptomics of brain cells.

[0005] In some embodiments of the invention, the examined brain cells are neurons or glia. In some methods, the monitored phenotypic change in the cytoarchitecture is a change in the morphology of the brain cells. In various embodiments, the monitored change in the morphology of the brain cells is a change in cellular position, morphology, synaptic structure, spatial organization or projection. In some of these embodiments, the change in the morphology of the brain cells is examined via imaging of the whole brain or tissue sections of the transgenic animal system. In some other methods, the monitored phenotypic change is examined via determining spatial transcriptome of the brain cells. In some embodiments, the employed vectors are lentiviral vectors. In some embodiments, the employed vectors are adeno-associated virus (AAV) vectors.

[0006] In some methods of the invention, each of the genetic perturbations contains one or more guide RNAs (gRNAs) for introducing genomic changes via CRISPR-Cas9 gene editing in the coding region of a target gene. In some of these embodiments, the one or more gRNAs each target a sequence at the 5’ end of a target gene’s coding region. In various embodiments, each of the vectors can further express at least one reporter molecule or be labeled with a detectable agent. In some of these embodiments, the employed reporter molecule is GFP, HA, tdTomato, or Myc.

[0007] Some methods of the invention employ a transgenic animal system that is a developing embryo of a Cas9-expressing transgenic animal. Some other methods employ a transgenic animal system that is a postnatal or adult Cas9-expressing transgenic animal. In some embodiments, the library of vectors is administered to the transgenic animal system in utero to lateral ventricles of the embryo. In some other embodiments, the library of vectors is administered to the transgenic animal system via postnatal intravenous injection. In some methods, the library of vectors are administered to the animal embryo around a development stage that is equivalent to any mouse embryonic day between Day 11.5 (El 1.5) and Day 17.5 (El 7.5). In some of these embodiments, the development stage for administration of the vectors is equivalent to mouse embryonic day 13.5 (E13.5) or 15.5 (E15.5). In some methods,the phenotypic change is examined at one or more developmental stages that are equivalent to any mouse postnatal day between day 2 (P2) to day 60 (P60). In some of these embodiments, the one or more developmental stages are equivalent to mouse postnatal day 10 (PIO), 14 (P14), or 28 (P28).

[0008] In a related aspect, the present disclosure provides methods for correlating a phenotypic change in the cytoarchitecture or spatial transcriptomics brain cells with genetic perturbations of one or more target genes. These methods involve (1) introducing a library of viral vectors encoding genetic perturbations for a plurality of target genes into a CRISPR- Cas9 expressing transgenic system, (2) examining cytoarchitecture or spatial transcriptomics of brain cells at one or more developmental stages of the transgenic system, (3) identifying one or more brain cells of the transgenic system that express a genetic perturbation and display a phenotypic change of cytoarchitecture or spatial transcriptomics, and (4) determining the corresponding gene perturbed by the genetic perturbation that is encoded by the viral vector introduced into each of the one or more cells. This enables a correlation of the corresponding perturbed gene with the phenotypic change of cytoarchitecture or spatial transcriptomics displayed by each of the one or more cells.

[0009] Some of these methods are directed to neurons or glia in the brain. In some methods, the target genes to be perturbed are known or suspected to be associated with a specific disease or disorder. In some methods, the monitored phenotypic change is a change in the morphology of the brain cells. In various embodiments, the change in the morphology to be monitored is a change in cellular position, synaptic structure, spatial organization or projection. In some other methods, the phenotypic change to be monitored is a change in the spatial distribution of genomic content in the brain cells. This can be performed by, e.g., determining spatial transcriptome of the brain cells. In some embodiments, cytoarchitecture or morphology of brain cells can be examined via whole mount imaging or thick tissue section imaging of the brain across one or more brain regions. In some of these embodiments, the one or more brain regions to be imaged include, e.g., cortex, striatum, hippocampus, cerebellum, and olfactory bulb. In some methods, the employed vectors encoding the genetic perturbations are lentiviral vectors. In some methods, the employed vectors encoding the genetic perturbations are AAV vectors. In some methods of the invention, the employed vectors can further encode one or more reporter molecules. Examples of suitable reporter molecules include, e.g., GFP, HA, tdTomato, Myc, any other fluorophores, and enzymes producing detectable signals. In some other methods, the employed vectors can be labeledwith other types of detectable labels, e.g., fluorescence-enabling agents or fluorescence contrast agents.

[0010] A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and claims.DESCRIPTION OF THE DRAWINGS

[0011] Figure 1. Whole-brain viral-based cellular labeling platform for spatially resolved cytoarchitecture and transcriptome analysis. A. Schematics of the platform with cytoarchitectural analysis. The mouse embryos were injected in utero with lentiviral vectors expressing a labeling reporter (Tagl). The brains were harvested postnatally at P14 followed by tissue clearing, immunofluorescence staining, and light-sheet microscopy imaging to analyze the labeling pattern. B. Left, a maximum intensity projection image from a 1.6 mm thick plane of the whole mount dataset (sagittal view) showing a broad coverage across brain regions and cell types. Middle and righ zoomed-in images showing cellular morphology of labeled neurons in cortex (CTX), hippocampus (HPC), and striatum (STR). Zoomed-in areas are indicated by dashed boxes in the left MIP image. C. Zoomed-in images from this dataset showing cellular morphology of labeled astrocytes and oligodendrocytes. D. A zoomed-in image from this dataset showing the axonal projection from cortical neurons. E. An image of labeled neurons in a 100 nm brain section with clear dendritic spines, imaged by confocal 40x. F. Example images of cortical regions from 100 nm brain sections from P10, Pl 4, and P28 mice. G. Schematics of the platform with spatial transcriptome analysis. The mice were retro-orbitally injected with AAV vectors expressing Tagl. The brains were collected postnatally and subjected to Tagl immunofluorescence staining to visualize the brain-wide targeted cells. Spatial transcriptomics approaches (STARmap) was applied to profile cellular transcriptomes by mapping gene expression patterns. Four genes (Malatl, Caimi, Snap25, ActV) were presented as examples, each assigned a unique color combination across two imaging rounds.

[0012] Figure 2. Multiplex genetic screening using combinatorial optical labeling reveals perturbation-associated morphological changes. A. Schematics showing the strategy of the multiplex optical labeling and genetic perturbation in vivo. The mouse embryos were injected in utero with lentiviral vectors expressing a set of six labeling reporter (Tagl, Tag2, Tag3, Tagl-Tag2, Tagl-Tag3, Tag2-Tag3). The brains were harvested at P14 followed by tissue clearing, immunofluorescence staining, and imaging to analyze the perturbation-associatedcytoarchitectural changes. B. Top-left, a maximum intensity projection image from a 0.5 mm thick plane of a whole mount image showing cortical area with neurons labeled by different reporters. Others: zoomed-in images showing cortical neuron morphology revealed by each labeling reporter. C. Zoomed-in images from this dataset showing altered cellular morphology of a neuron with PertX perturbation compared to control.

[0013] Figure 3. A. Tracing and 3D dendritic morphology reconstruction of labeled neurons. B. A summary list of all morphometric measurements included in the analysis and example quantification for the two neurons in Fig. 2C and 3 A, as well as three other neurons. C. Left: UMAP plot showing clustering results based on morphometries of 156 neurons. Dots represent individual neurons. Dashed circles indicate the five example neurons in Fig. 3B. Right: Heatmap showing the distinct morphological signatures of the five clusters.

[0014] Figure 4. A. Engineered vector enables robust gRNA detection. Top-left: schematic representation of the polyadenylated gRNA (polyA-gRNA) expression construct (GFP reporter) in comparison to a conventional gRNA vector (RFP reporter). Bottom-left: HT22 cells were co-transfected with both constructs and analyzed via STARmap to quantify gRNA expression (white puncta). Dashed boxes indicate zoomed-in regions in the right panels. Right: quantitative comparison of gRNA detection efficiency between the two designs. B. Direct detection of gRNAs in vivo using STARmap. Left: schematics of AAV delivery of the polyA-gRNA construct (GFP reporter, co-expressing gRNAl and gRNA2) into embryonic mouse brains, followed by postnatal harvesting to achieve sparse, brain-wide labeling. Top-right: Schematics of the gRNA detection strategy, wherein each gRNA is recognized by a pair of probes targeting separate regions of the transcript. Bottom-right: example images showing GFP labeled cells in brain tissue and spatially resolved gRNA transcripts within GFP-positive neurons, demonstrating co-expression of gRNAl and gRNA2.DETAILED DESCRIPTION OF THE INVENTIONI. Overview

[0015] The present invention provides a multiplex gene function mapping platform that utilizes brain-wide, high-content cytoarchitectural or spatial transcriptomic phenotyping at single-cell resolution. The platform allows one to systemically screen and characterize the genetics to cytoarchitecture or spatial transcriptomics relationship. Specifically, the inventors developed an in vivo labeling and genetic screening approach termed Perturb-CAST(CytoArchitecture See Through), which utilizes whole mount imaging to analyze perturbation-linked cytoarchitectural changes or changes in spatial transcriptome of sparsely labeled cells in intact brains. Through a screening of human disease risk genes, the inventors revealed substantial cytoarchitectural or spatial transcriptomic changes associated with certain genetic perturbations associated with human disease and disorder. Collectively, this platform enables brain-wide, single-cell level multiplex gene function mapping with cytoarchitectural or spatial transcriptomic phenotyping across brain regions, cell types, and developmental timepoints.

[0016] This multiplex gene function mapping platform described herein enables dissection of gene function in vivo through CRISPR gene editing, embryonic gene delivery, tissue clearing, light sheet imaging, morphometric analysis, or spatial transcriptomic study. There are a number of technological advantages associated with the platform and related applications as described herein. The described platform and gene mapping approach can be used to dissect many genetic factors (multiplexed) in intact whole brains, which are comprised of distinct brain regions, without tissue sectioning or distortion. Existing methodologies in spatial omics often apply < 300 pm tissue sections whereas our platform can flexibly analyze both tissue sections and whole brains (>10 mm). The AAV or lentiviral vector-based labeling and perturbation platform allows a broad coverage across cell types (neurons, glia, etc.), brain regions (cortex, striatum, hippocampus, cerebellum, olfactory bulb, etc.), and developmental timepoints (from El 1.5 to adult). The gene mapping platform of the invention is capable of analyzing gene function in a multiplexing way (>60 perturbations as a theoretical limit), in contrast to the traditional approach (one at a time).

[0017] It is noted that, unless otherwise specified, this invention is not limited to the particular methodology, protocols, and reagents described as these may vary. Unless otherwise indicated, the practice of the present invention employs conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. For example, exemplary methods are described in the following references, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (3rded., 2001); Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003); Freshney, Culture of Animal Cells: A Manual of Basic Technique, Wiley-Liss, Inc. (4thed., 2000); and Weissbach & Weissbach, Methods forPlant Molecular Biology, Academic Press, NY, Section VIII, pp. 42 1-463, 1988. In addition, the following sections provide more detailed guidance for practicing the invention.II. Definitions

[0018] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. The following references provide one of skill with a general definition of many of the terms used in this invention: Academic Press Dictionary of Science and Technology, Morris (Ed.), Academic Press (1sted., 1992); Oxford Dictionary of Biochemistry and Molecular Biology, Smith et al. (Eds.), Oxford University Press (revised ed., 2000); Encyclopaedic Dictionary of Chemistry, Kumar (Ed.), Anmol Publications Pvt. Ltd. (2002); Dictionary of Microbiology and Molecular Biology, Singleton et al. (Eds.), John Wiley & Sons (3rded., 2002); Dictionary of Chemistry, Hunt (Ed.), Routledge (1sted., 1999); Dictionary of Pharmaceutical Medicine , Nahler (Ed.), Springer-Verlag Telos (1994); Dictionary of Organic Chemistry, Kumar and Anandand (Eds.), Anmol Publications Pvt. Ltd. (2002); and A Dictionary of Biology (Oxford Paperback Reference) , Martin and Hine (Eds.), Oxford University Press (4thed., 2000). Further clarifications of some of these terms as they apply specifically to this invention are provided herein.

[0019] As used herein, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, reference to "a protein" includes one or more proteins and equivalents thereof known to those skilled in the art, and so forth.

[0020] As used herein, 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 a recombinant adeno-associated viral particle or a recombinant AAV vector (or "rAAV vector"). An "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-typeAAV genome such as a transgene to be delivered to a mammalian cell), it is typically referred to as "rAAV".

[0021] 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. For Cas9 CRISPR systems, 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. In addition to the sequence complementarity, the cleavage site in the target DNA is also preceded by a short protospacer-adjacent motif (PAM). In bacteria, Cas9 relies on RNase III to excise crRNAs from a CRISPR array.

[0022] Protospacer adjacent motif (PAM) 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) can transport Cas9 to anywhere in the genome for gene editing, but no editing can occur at any site other than one at which Cas9 recognizes PAM.

[0023] 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 the 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.

[0024] Protospacers are spacer sequences in CRISPR loci in a bacterium that were inserted into a CRISPR locus by invading viral or plasmid DNA. On subsequent invasion, Cas9 nuclease attaches to tracrRNA: crRNA which guides Cas9 to the invading protospacersequence. But 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. But the protospacer in the invading virus or plasmid will contain the PAM sequence and will thus be cleaved by the Cas9 nuclease. For editing genes, guide RNAs (gRNAs) are synthesized to perform the function of the tracrRNA:crRNA complex in recognizing gene sequences having a PAM sequence at the 3 '-end.

[0025] Cytoarchitecture broadly refers to the arrangement of cells in, as well as cellular makeup of, a bodily tissue or structure. In particular, it refers to the arrangement of cells in the brain that are associated with particular functions. Unless otherwise noted, the term cytoarchitecture as used herein encompasses the various features related to structural arrangement of neurons within the central nervous system. These include cellular position, neuronal size, shape, cellular morphology, synaptic structure, packing density, projection, spatial organization and staining intensity, etc., which can be used to characterize a specific cytoarchitectural area, region, or trend.

[0026] Spatial transcriptome or spatial transcriptomics refers to gene expression patterns within the context of tissue architecture. By visualizing and quantifying gene expression across spatial locations of a tissue sample, spatial transcriptome provides information on genomic content in intact tissue sections. It allows one to better understand cell-cell interactions, identify unique cell presence, and gain deep insights into complex biological systems and diseases. Spatial transcriptome of a specific tissue can be examined via a number of assays or techniques routinely practiced in the art. These include, e.g., in situ hybridization, single-molecule fluorescence in situ hybridization (smFISH), laser capture microdissection (LCM) coupled with RNA sequencing, spatial transcriptomics (ST) platforms, and integrated systems that combine these different techniques. For example, one integrated method that has been developed for analyzing tissue spatial transcriptomes is spatially-resolved transcript amplicon readout mapping (STARmap), which is a 3D intacttissue RNA sequencing system that integrates hydrogel-tissue chemistry, targeted signal amplification, and in situ sequencing. Detailed guidance for utilizing the various methods for examining spatial transcriptome is provided in the literature. See, e.g., Williams et al., Genome Med. 14, 68, 2022; Pineiro et al., J. Invest. Dermatol. 142, 993-1001, 2022; and Wang et al., Science 361 (6400) :eaat5691, 2018.

[0027] A neurite refers to any part of a neuronal cell body that protrudes, such as axons or dendrites. It plays a fundamental role in brain development and function. A neurite is anytype of process or protrusion extending out from the cell body of a neuron. Neurites may differentiate into either dendrites or axons, which help give neurons their distinctive shape, and are required in order to send / receive signals to / from other parts of the nervous system. Axon refers to a portion of a nerve cell (neuron) that carries nerve impulses away from the cell body. A neuron typically has one axon that connects it with other neurons or with muscle or gland cells. Dendrites are projections from the cell body, or soma, of neurons, the cells of the nervous system. The main function of dendrites is to receive information from other neurons, called pre-synaptic neurons, or from the environment. Dendrites receive input from many other neurons and carry those signals to the cell body. The difference between axons and dendrites lies in their structure and function within neurons. Axons are long, single extensions that transmit electrical impulses away from the neuron's cell body, while dendrites are shorter, branched extensions that receive incoming signals from other neurons.

[0028] 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. 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. Often, the heterologous polynucleotide sequence introduced into the cell is a replicable expression vector or cloning vector. In some embodiments, 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. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (3rded., 2001); and Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003). In some preferred embodiments, the host cell is a mammalian cell.

[0029] The term “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. Similarly, 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 (e.g., a Tag).

[0030] 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%. In some embodiments, 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.

[0031] Sequence identity can be readily determined with various methods known in the art. For example, the BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, 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.

[0032] As used herein, “complementary” or “complement” refers to a nucleotide or nucleotide sequence that hybridizes to a given nucleotide or nucleotide sequence. For instance, for DNA, the nucleotide A is complementary to T and vice versa, and the nucleotide C is complementary to G and vice versa. For instance, in RNA, the nucleotide A is complementary to the nucleotide U and vice versa, and the nucleotide C is complementary to the nucleotide G and vice versa.

[0033] “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.

[0034] A cell has been “transduced” by viral vectors containing exogenous or heterologous polynucleotide (or “a transgene” or “a target gene” as used interchangeably herein) when such polynucleotide has been introduced inside the cell by viral vectors. After transduction, the polynucleotide in viral vectors will 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. With respect to eukaryotic cells, 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 (e.g. during division of neural progenitors in the brain). This stability is demonstrated by the ability of the eukaryotic cell to establish clones comprised of a population of daughter cells containing the viral polynucleotide. A “clone” is a population of cells derived from a single cell or common ancestor (e.g. neural progenitors) by mitosis.

[0035] 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 (e.g., a lentiviral vector), 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.III. Multiplex gene mapping platform for analyzing cytoarchitectural changes

[0036] The present invention provides in vivo platforms to analyze genetic perturbation mediated phenotypic changes in the cytoarchitecture or spatial transcriptomics of brain cells. In general, phenotypic changes in the brain cells that can be monitored and / or quantified with the platforms broadly encompass any alterations in the overall cytoarchitecture or morphology of an examined brain tissue, as well as alterations in the spatial distribution of genomic content (e.g., spatial transcriptome) across the tissue. In some embodiments, thephenotypic change to be monitored can be a change in any morphological or structural features of the examined brain tissue, e.g., cellular position, morphology, synaptic structure, spatial organization and projection, etc. In some embodiments, the phenotypic change to be monitored can be a change in the spatial transcriptome of the brain tissue. As described herein, the platforms enable multiplex gene mapping with high content phenotypic readout and single-cell resolution. Typically, the in vivo systems of the invention utilize a library of vectors (e.g., viral vectors) for delivering genetic perturbations to modulate one or more target genes of interest in the brain (including many brain regions, e.g., cortex, striatum, hippocampus, cerebellum, olfactory bulb, etc.) of a transgenic system (e.g., an animal embryo or an adult or aged animal). In some embodiments, the target genes to be modulated or perturbed are genes known or suspected to be associated with a specific disease or disorder, e.g., autism as exemplified herein. The genetic perturbations to be introduced into the brain cells (including neurons and glia) in vivo each contain one or more guide RNAs (gRNAs) for introducing genomic changes via CRISPR-Cas9 gene editing in the coding region of a target gene. Following introduction and expression of the genetic perturbations in the brain of the transgenic system, the brain tissues can be harvested and cleared at various timepoints before subjecting to examination for phenotypic changes in the cytoarchitecture or spatial transcriptomics. This includes a quantitative analysis of the phenotypic changes of individual cells (e.g., neurons) in the brain as exemplified herein. Cells with observed phenotypic changes can then be further deconvoluted to determine the specific genetic perturbation harbored by each of the cells and the corresponding genes perturbed thereby.

[0037] The in vivo phenotyping platforms of the invention can be readily employed in a number of applications. In some embodiments, they can be used in identifying or mapping genes that are linked to or associated with cytoarchitecture of brain cells, e.g., genes that function or play a role in maintaining the normal cytoarchitecture of brain cells (e.g., neurons or glia). In some embodiments, they can be used to correlate functions of one or more target genes of interest with specific cytoarchitectural changes of brain cells. The target genes to be examined in these methods can be those that are known or suspected to be linked to a specific disease or disorder. The diseases or disorders include, e.g., autism spectrum disorder, major depressive disorder, bipolar disorder, schizophrenia, obsessive-compulsive disorder, and attention-deficit / hyperactivity disorder. In some other embodiments, the in vivo phenotyping methods of the invention are suitable for capturing one or more changes of cytoarchitecture that are associated with or caused by a specific gene. The specific gene can be any gene thatis known or suspected to play a role in maintaining a cytoarchitectural or morphological feature of neuronal cells (e.g., cellular position, morphology, synaptic structure, spatial organization and projection, etc.). In mapping the genes in these applications, various phenotypic changes of cytoarchitecture or perturbation-associated cytoarchitectural changes in the brain cells (e.g., neurons) can be examined and quantified. As exemplified herein, the examined phenotypic changes include any changes in the morphology or overall cytoarchitecture of brain cells. See, e.g., Figures 2 and 3. In still some other embodiments, the in vivo phenotyping methods of the invention are suitable for identifying or mapping genes that are linked to or associated with spatial transcriptomics of brain cells. In these embodiments, the phenotypic change to be monitored can be a change in the spatially distributed genomic content of the examined brain tissue, e.g., an alteration in spatial transcriptome as exemplified herein (See, e.g., Figures 1 and 4).

[0038] In some embodiments, the employed vectors are lentivirus or other retrovirusbased vectors. As exemplified herein in the in vivo gene mapping platforms, lentiviral vectors are retroviral vectors that are able to transduce or infect both dividing and non-dividing cells and typically produce high viral titers. Therefore, they have certain advantages over other retroviral vectors. See, Trono D. (2002) Lentiviral vectors, New York: Spring-Verlag Berlin Heidelberg. Examples of well-known lentiviral based vectors include, e.g., lentiviral vector pLV2. Other lentiviral vectors that may be employed and modified for practicing the invention include, e.g., pLVX-Puro, pLVX-IRES-Neo, pLVX-IRES-Hyg, and pLVX-IRES- Puro. Lentivirus-based systems for Cas9 / gRNA expression, as required for the practice of the invention, are well known in the art. See, e.g., Carpenter et al., Methods. 2019; 156: 79-84; PCT publication WO2023 / 212396; and US patent application US20210172017. The various lentiviral vectors encoding genetic perturbations can be introduced into an appropriate host cell for producing viral particles. For example, the HEK293T cell line and other derivative packaging cell lines well known in the art (e.g., Lenti-X 293T cell line) may be employed in the invention. In addition to lentiviral based vectors and host cells, other retroviral based vectors and expression systems may also be employed in the practice of the methods of the invention. These include MMLV based vectors pQCXIN, pQCXIQ and pQCXIH, and compatible producer cell lines such as HEK 293 based packaging cell lines GP2-293, EcoPack 2-293 and AmphoPack 293, as well as NIH / 3T3 -based packaging cell line RetroPack PT67.

[0039] In some embodiments, AAV vectors may be used to introduce the genetic perturbations into the brain cells, as exemplified herein. 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. 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). General characteristics including structural information of well-known AAV serotypes are provided in, e.g., Srivastava, Curr Opin Virol. 2016; 21 : 75-80; Issa et al., Cells 2023; 12:785; Chen et al., Human Gene Therapy. 2020 ; 31 : 440-447; Pillay et al., J. Virol. 2017; 91 : e00391-17; Naso et al., BioDrugs. 2017; 31 : 317-334; Wang et al., Nat. Rev. Drug Discov. 2019; 18: 358-378; and Wu et al., Mol. Ther. 2006; 14: 316-327.

[0040] As demonstrated herein, the perturbation-expressing vectors (e.g., lentiviral vectors) can be administered to a transgenic animal system (e.g., an embryo or an adult animal) that expresses a CRISPR-Cas gene editing system. In some embodiments, the library of vectors is administered to a developing embryo of a CRISPR-Cas9 transgenic mouse. In some exemplified embodiments, 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 El 1.5 and E17.5. In some other embodiments, the library of vectors is administered (e.g., intravenous or retro-orbital injection) to a developed adult animal of a CRISPR-Cas9 transgenic mouse, as exemplified herein. In various embodiments, the 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 can be administered to the adult mice via retroorbital injection. Once the genetic perturbations are expressed in the transgenic system, cytoarchitecture and / or spatial transcriptomics of brain cells of interest (e.g., neurons) can be examined at one or more developing or growth stages after administration of the perturbation-expressing vectors.

[0041] In some preferred embodiments, animal embryos administered with the library of perturbation-expressing vectors can be analyzed for cytoarchitectural or spatial transcriptomic changes in individual neurons at one or more timepoints when the embryos develop into young postnatal or adolescent animals. Using mouse embryos as reference, the phenotypic examination can be performed at any developing stage that is equivalent to a mouse postnatal day that, e.g., falls between Day 2 (P2) to Day 60 (P60). As specific exemplifications, mouse embryos can be administered the genetic perturbations at embryonic day 13.5 (E13.5) or 15.5 (E15.5), and brain tissues can be harvested, cleared, then examined for phenotypic changes in the neurons at postnatal Day 10 (PIO), 14 (P14) and 28 (P28).

[0042] In some preferred embodiments, the genetic perturbations encoded by each of the 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 vectors. In some embodiments, two or more gRNAs designed for targeting each gene of interest are included in the library of vectors. In some embodiments, the two or more gRNAs targeting one gene can be encoded by one vector (e.g., a lentiviral vector) to increase the CRISPR editing efficiency in vivo. For example, the vectors can each contain two tandem gRNA-expressing cassettes that are conjugated by a tRNA linker, as exemplified herein. For each of these vectors, the two-encoded gRNAs target the same gene, or two different genes to be perturbed simultaneously. After transcription, the tRNA linker undergoes a self-cleavage process, resulting in two gRNA cassettes that can function independently. In some other embodiments, the multiple gRNAs targeting the same gene can be encoded by different vectors in the library. In some embodiments, 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. In some preferred embodiments, the gene editing system expressed by the animal is CRISPR is CRISPR-Cas9. In some embodiments, each of the gRNAs can be put under the control of a different promoter, including, e.g., human U6 polymerase III promoter. In some embodiments, a reporter gene such as HA, Myc, tdTomato, and GFP as exemplified herein can be operably linked to the gRNA-coding sequences in the vectors. In various other embodiments, the reporter gene can encode any labeling molecules known in the art that are suitable for expression in cells, e.g., other fluorophores such as mCherry, EYFP and ECFP, other enzymes such as luciferase, P- galactosidase (lacZ), and chloramphenicol acetyltransferase (CAT). In some other embodiments, the vectors can be labeled with a detectable agent such as a fluorescence-enabling agent or fluorescence contrast agent, e.g., indocyanine green (ICG), 5- aminolevulinic acid (5-ALA), and sodium fluorescein (SF).

[0043] The reporter gene or detectable label in the vectors allows identification and of cells expressing the corresponding gRNAs after the vectors are introduced into the animal embryos or developed animals. Unless otherwise described herein, delivery of the vectors into the CRISPR-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) and enrichment of cells (perturbed cells) expressing the genetic perturbations, and analyses of perturbation-associated phenotypes in the perturbed cells (e.g., single-cell cytoarchitectural analysis) can all be performed with techniques specifically exemplified herein and / or well known in the art. See, e.g., PCT publication W02024 / 081820; Carpenter et al., Methods. 2019; 156: 79-84; and US Patent Application Publication No. 2021 / 0172017.

[0044] In order to construct viral vectors (e.g., lentiviral vectors or AAV vectors) encoding genetic perturbations in the practice of the invention, the coding sequence for a genetic perturbation (e.g., gRNA-coding sequence) and any other operably linked sequences (e.g., a reporter gene) are often inserted into the viral genome in the place of certain viral sequences to produce a viral construct that is replication-defective. Methods for producing such vectors are well-known in the art. See, e.g., Carpenter et al., Methods. 2019; 156: 79- 84; US Patent Publications 20120295960, US20110117189, US20110293571; Ojala et al., Mol. Then, 2018; 26:304-319; Chan et al., Nat. Neurosci., 2017; 20: 1172-1179; Strobel et al., Am. J. Resp. Cell Mol. Biol. 53: 291-302, 2015; and Kotterman et al., Nat. Rev. Genet. 15:445-451, 2014.IV. Transgenic animal systems expressing CRISPR functionality

[0045] Typically, a transgenic animal system expressing a CRISPR gene editing functionality is used to express the genetic perturbations and examine their effect on target genes. As exemplified herein, the transgenic animal system can express the CRISPR-Cas9 gene editing functionality. In these embodiments, the genetic perturbations encoded by the vectors can be gRNAs that are designed to subject one or more target genes to the activities of the CRISPR-Cas system in the transgenic animal. Any transgenic systems with CRISPR- 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 toexpress a Cas enzyme and optionally other components required for the CRISPR functionality. The transgenic systems can constitutively or inducibly express the Cas enzyme and / or the other components in some or all of their cells. In various embodiments, the Cas enzyme expressed in the transgenic systems is a Cas Type I, II, III, IV, or V protein. In some embodiments, 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). A more detailed description of the various CRISPR-Cas gene editing systems that may be incorporated in the transgenic systems of the invention is provided in, e.g., US Patent Application Publication No. 2021 / 0172017.

[0046] In some preferred embodiments as exemplified herein, the genetic perturbations encoded by the vectors are gRNAs that are designed to subject the target genes to CRISPR / Cas9 gene editing in the transgenic system. In these embodiments, each viral vector (e.g., a lentiviral vector) encodes one or more gRNAs that can direct the enzymatic activity of Cas9 to a target gene. 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. See, e.g., Dixit et al., “Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens” 2016, Cell 167, 1853-1866; Adamson et al., “A Multiplexed SingleCell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response” 2016, Cell 167, 1867-1882; Feldman et al., Lentiviral co-packaging mitigates the effects of intermolecular recombination and multiple integrations in pooled genetic screens, bioRxiv 262121, doi: doi.org / 10.1101 / 262121; Datlinger, et al., 2017, Pooled CRISPR screening with single-cell transcriptome readout. Nature Methods. Vol. 14 No. 3 DOI: 10.1038 / nmeth.4177; Hill et al., On the design of CRISPR-based single cell molecular screens, Nat Methods. 2018; 15(4): 271-274; Replogle, et al., “Combinatorial single-cell CRISPR screens by direct guide RNA capture and targeted sequencing” Nat. Biotechnol. 2020; 38: 954-961; and PCT publication WO / 2017 / 075294. The transgenic systems described in any of these reports may be employed and adapted for use in the practice of the invention. In some preferred embodiments, transgenic mice expressing Cas9 can be used in the practice of the invention. As an example, Cas9-expressing transgenic mice can be readily obtained from commercial vendors, e.g., the Jackson Laboratory (Bar Harbor, ME).V. Correlating phenotypic changes with perturbed genes

[0047] Once a library of vectors (e.g., lentiviral vectors) encoding genetic perturbations (e.g., gRNAs) specific for one or more target genes are introduced into the transgenic animal or animal embryo, the brain or brain tissues of the animal can be harvested at desired timepoints, cleared, and then examined for phenotypic changes in the cytoarchitecture. In general, analysis of phenotypic changes (e.g., changes in cytoarchitecture or spatial transcriptomics) in the brain involves examination of individual neurons for both phenotypic changes and expression of the genetic perturbations.

[0048] Phenotypic changes in the brain cells (e.g., neurons and glia) that are examined in the methods of the invention can be any of the structural or morphological features that define cytoarchitecture and organization of the brain, as well as changes in spatial transcriptomics of the cells. These include changes in cellular properties or parameters related to cellular position, morphology, synaptic structure, spatial organization and projection. For example, the examined phenotypic changes can be different layer positions, altered apical dendrite length, or different dendritic complexity. Some of the phenotypic changes can be examined quantitatively. For example, morphological changes of the neurons can be quantitatively analyzed via tracing and 3D reconstruction of individual neurons, followed by examination of their morphological information. As exemplified herein, the morphometric properties to be quantified can include, e.g., y-extent, total dendritic length, total branching points, trunk length, tuft dendrite length, basal dendrite length, and oblique dendrite length. In some embodiments, cytoarchitectural changes can be examined via computation methods to characterize the phenotypic variation among a neuron population and changes associated with genetic perturbations. Such methods include, e.g., normalization, principal component analysis, KNN clustering, and UMAP visualization.

[0049] Preferably, cytoarchitectural changes in the harvested brain of the transgenic system are examined via a 3-dimentional whole mount means, e.g., whole mount imaging. Whole mount imaging involves the visualization of intact, three-dimensional (3D) samples, such as tissues or organs, without the need for sectioning. It offers a holistic view of the sample, capturing the spatial arrangement of cells, cellular components, and extracellular matrix in their native context. In some embodiments, phenotypic changes of the neurons can be examined by immunofluorescent staining of the harvested brain tissues and whole-mount light-sheet imaging, as exemplified herein. See also Schueth et al., Commun. Biol. 2023; 6: 170; Delage et al., J Cell Biol. 2023; 222: e202307143; and Timin et al., iScience 2023; 26:106452. In some other embodiments, examination of cytoarchitecture of the neurons in the harvested brain that express the genetic perturbations may be performed with high resolution diffusion MRI. See, e.g., Wang et al., Neuroimage. 2020; 216: 116876; and Wei et al., Neuroimage. 2016; 137, 107-115. In some embodiments, examination of phenotypic changes of the neurons can include volumetric and quantitative imaging of cytoarchitecture of the brain. See, e.g., Leahy et al., Biomed. Opt. Express2013; 4: 1978-90. In still some embodiments, cytoarchitecture of the neuronal cells in the harvested brain may be examined via x-ray phase-contrast tomography. See, e.g., Tbpperwien et al., Sci. Rep. 2017; 7: 42847 (2017).

[0050] The specific genetic perturbation expressed in an identified brain cell and the corresponding perturbed gene can be readily ascertained via routinely practiced methods. In some embodiments, a given gRNA and a corresponding specific barcode are co-expressed from the same viral vector, and detection of the gRNA is realized by detection of the specific Tags in the imaging. In some embodiments, the specific genetic perturbation and the corresponding perturbed gene in identified brain cells can be identified by single nucleus RNA sequencing. This can be performed using standard protocols that have been reported in the literature. See, e.g., Drokhlyansky et al., Cell 2020; 182: 1606-1622; Swiech et al., Nat. Biotechnol. 2014; 33: 102-106; Habib et al., Science 2016; 353: 925-928; Habib et al., Nat. Methods. 2017; 14: 955-958; and PCT publications WO2017 / 164936, WO2019 / 094984 and W02020 / 077236. In some embodiments, corresponding perturbations (e.g., gRNA sequences encoded by the viral vectors) expressed by the identified neurons can be identified via singlecell RNA (scRNA) sequencing. scRNA sequencing of a population of cells can be readily performed in accordance with experimental protocols that are routinely practiced in the art. In some embodiments, high-throughput scRNA sequencing can be used. 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; Picelli et al., Nat. Protoc. 2014; 9: 171-181; Ramskold et al., Nat. Biotechnol. 2012; 30: 777-782; Hashimshony et al., Cell Rep. 2012; 2: 666-673; Kalisky et al., Ann. Rev. Genetics 2011; 45: 431-445; Kalisky et al., Nat. Methods 2011; 8: 311-314; Islam et al., Genome Res. 2011; 21 : 1160-7; Tang et al., Nat. Protoc. 2010; 5: 516-535; and Tang et al., Nat. Methods 2009; 6: 377-382.EXAMPLES

[0051] The following examples are provided to further illustrate the invention but not to limit its scope. Other variants of the invention will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims.Example 1. Whole-brain, viral-based cellular labeling platform for tissue-clearing assisted cytoarchitecture analysis

[0052] We developed an viral -based whole-brain labeling platform to profile cytoarchitecture (including the cellular position, morphology, synaptic structure, projection, spatial organization and whole-brain organization) as well as spatial transcriptomes from the brain. First, we demonstrated its utility in analyzing cytoarchitecture. Lentiviral vectors expressing a cellular labeling reporter (tdTomato; Tagl) were delivered through in utero injection in lateral ventricles of mouse embryos at embryonic day 13.5 (E13.5) (Fig. 1, Panel A). The lentiviral vectors diffused in the cerebrospinal fluid upon injection and transduced progenitors surrounding all the ventricles. The capability of genome integration of lentiviral vectors ensures a high expression level of labeling reporters, which can be further inherited by the progenies. Consequently, by targeting the progenitors at an early development stage, we expect that all the neurons and glia derived from targeted progenitors to be labeled, providing a broad coverage of diverse cell types across different brain regions. The brains were harvested at postnatal day 14 (Pl 4) and cleared, followed by immunofluorescent staining and whole-mount light-sheet imaging (Fig. 1, Panel A).

[0053] The labeling pattern indicates that the viral targeting approach has a broad coverage of brain regions, including but not limited to cortex, hippocampus, striatum, and cerebellum (Fig. 1, Panel B). Zoomed-in images showed the refined local dendritic cytoarchitecture of the labeled neurons in different brain regions (Fig. 1, Panel B). Notably, our strategy labeled different layers of cortical neurons, to which the progenitors gave birth along the entire cortical development, showcasing the temporal coverage provided by the inheritable labeling of lentiviral strategy. Additionally, this strategy can also target other cell types, such as astrocytes and oligodendrocytes, underlying the potential of using this approach to study non-neuronal cells (Fig. 1, Panel C).

[0054] Besides local dendritic morphology, we detected that long-range axon projections (axon tracts connecting brain regions) were captured, underlying the use of this platform to trace long-range axons and map the projection of individual neurons (Fig. 1, Panel D).Furthermore, it will allow us to distinguish and quantify projection and connectivity changes associated with genetic perturbation. In images with higher magnification, we also observed clear labeling of dendritic spines (Fig. 1, Panel E). This indicates that our platform has the resolution to visualize subcellular structures, such as spines. By targeting mouse embryonic brains, we also make this platform compatible with in vivo models at early developmental stages, such as young postnatal and adolescent animals (Fig. 1, Panel F).

[0055] Second, we demonstrated the utility of this platform for spatial transcriptomics. Adeno-associated virus (AAV) expressing a cellular labeling reporter (tdTomato; Tagl) were administered in mice via intravenous injection postnatally, enabling brain-wide cell labeling (Fig. 1, Panel G, left). The brain tissue was then subjected to spatial transcriptomic profiling (for example, a methodology called STARmap) to obtain spatially resolved gene expression information (Fig. 1, Panel G, right).

[0056] Collectively, these data provide crucial evidence that our platform can capture whole-brain cytoarchitecture and spatial transcriptome at single-cell resolution with broad spatial-temporal coverage across brain regions, cell types, and developmental time points. This serves as a strong foundation for future analysis of cytoarchitectural change linked with genetic perturbations.Example 2. Enabling in vivo genetic screen with cytoarchitectural and spatial transcriptomic readouts.

[0057] Long lists of disease risk genes have been revealed by human genetics studies, which outstrip our ability to study their function individually at high resolution. Seeking convergent and divergent effects of these genes and investigating fundamental disease mechanisms have long been a primary goal in the field, which raised the need to study multiple genes in one animal or experiment.

[0058] To address the need, after establishing the whole-brain labeling platform, we further engineer it for compatibility with in vivo genetic screens, which require reliable readout of perturbations. Specifically, we implemented two key advancements: 1) we employed a combinatorial reporter strategy in which distinct perturbations are encoded by unique combinations of labeling reporters. Each reporter combination generates a distinguishable barcode that can be resolved by imaging. 2) we developed molecular tools to enhance gRNA expression level by using a n unpublished construct, enabling direct detectionof gRNAs using spatial transcriptomic approaches. These strategies are introduced separately below.

[0059] For strategy 1, we developed a toolbox of vectors co-expressing unique labeling reporters and gRNAs (Fig. 2, Panel A). We first adopted a set of three labeling reporters (Tagl, Tag2, and Tag3) that can be amplified by different antibodies in separate channels using immunohistochemistry. Further, we used a strategy of combinatorial labeling to increase the number of unique labeling reporters by combining the three reporters, and this could reach a theoretical limit of >60 perturbations with advanced lasers and microscopy (Fig. 2, Panel A). Eventually, this strategy ensures the perturbation identity can be readout by imaging and allows simultaneous targeting multiple genes in one animal.

[0060] To test whether the six labeling constructs can visualize cellular morphology with distinct perturbation identity in vivo, we performed a genetic screen targeting a set of ten autism spectrum disorder risk genes. We divided the ten genes into two groups to ensure that each group of genes can be targeted simultaneously together with controls (safe-targeting and non-targeting gRNAs, all controls shared one labeling reporter) in single animals. We constructed gRNAs targeting each gene into backbones with unique labeling reporters and then produced a pooled lentiviral vector library. We then delivered the library into Cas9 transgenic mouse embryos through in utero injection at El 5.5. The brain samples were harvested postnatally at P14, followed by tissue clearing, immunofluorescence, and imaging.

[0061] After optimization of the antibody choice and tissue processing, the six reporters showed similar capacity to capturing neuronal cytoarchitectural features, which surpassed the requirement to resolve cellular morphology (Fig. 2, Panel B). As a result of El 5.5 lentiviral delivery, we successfully labeled a group of cortical projection neurons with different labeling identities, corresponding to each genetic perturbation (Fig. 2, Panel B). Notably, our platform has clearly revealed that several neuropsychiatric risk genes led to altered cellular morphological changes in these neurons. As an example, we found that Layer 3 pyramidal neurons with GeneX perturbation showed longer apical dendrites (Fig. 2, Panel C).

[0062] To quantitatively analyze the morphological changes associated with perturbations, we performed tracing and 3D reconstruction of individual neurons (Fig. 3, Panel A) and extracted their morphological information into 19 cardinal morphometries including y-extent (distance between the soma and the top of tuft dendrites), total dendritic length, number of total dendritic segments, total branching points, total terminal points, maximum dendritic order, maximum apical dendritic order, maximum basal dendritic order,trunk length, number of total stems, tuft dendrite length, number of total tuft dendritic segments, tuft terminal points, basal dendrite length, number of total basal dendritic segments, basal terminal points, oblique dendrite length, number of total oblique dendritic segments, and oblique terminal points. (Fig. 3, Panel B). After tracing and quantifying the two example neurons showed in Fig. 2, Panel C and Fig. 3, Panel A, we validated the findings that neurons with GeneX perturbation showed longer apical dendrites by quantification results (reflecting by increased value of “y-extent” and “trunk length”). From there, we can perform normalization, principal component analysis, KNN clustering, and UMAP visualization (Fig. 3, Panel C); these computational approaches allow clustering of individual neurons based on their morphometries without a priori assumptions. These approaches will help us to understand how these neurons are distinct from each other morphologically. Moreover, these clusters can be annotated based on their perturbation identities to gain a comprehensive view of the altered spatial cytoarchitectural features across cell type and across genetic perturbations (e.g., Fig. 3, Panel B, first two columns).

[0063] We observed a variety of upper layer projection neurons with diverse morphology, including different layer positions, apical dendrite length, and dendritic complexity. These results showed the innate cytoarchitectural diversity of the upper layer neurons, which will be carefully analyzed quantitatively by this data analysis platform, another key innovation of this invention.

[0064] For strategy 2, we engineered the conventional gRNA expression vectors by incorporating a polyadenylation modification (polyA-gRNA) to stabilize gRNA transcripts. We transfected HT22 cells with either the polyA-gRNA construct (co-expressing green fluorescent protein reporter, GFP) and the conventional construct (co-expressing red fluorescent protein reporter, RFP) (Fig. 4, Panel A, left). We then subjected the cell culture sample to gRNA detection using STARmap. The quantification results showed that the polyA-gRNA strategy enhanced gRNA expression and detection by >12 folds (Fig. 4, Panel A, right). Further, we validated this strategy in vivo by administering AAV expressing polyA- gRNA constructs (co-express GFP, gRNAl and gRNA2) into embryonic mouse brains. We harvested the brains postnatally and successfully detected both gRNAl and gRNA2 transcripts at high abundancy via STARmap. Specifically, we observed gRNA signal are mostly restricted in GFP-positive cells, showcasing its specificity.Example 3. Some detailed experimental protocols and materials

[0065] Experimental animals: Mouse stains used in this invention are: Rosa26-Cas9 knockin on B6J (Platt et al., Neuron 2014; 159: 440-455) (Jackson Laboratories, Strain #:026179, RRID:IMSR JAX:026179); Cd-1 IGS (Charles River Laboratories, Strain #022). All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committees (IACUC) of The Scripps Research Institute. Mouse embryos from E13.5-E15.5 of varying sex and weight were used in the in utero injection experiments and mice ranging from P10 to P28 were used in the tissue clearing and immunohistochemistry experiments. All mice were kept in standard conditions (a 12-h light / dark cycle with ad libitum access to food and water).

[0066] Mammalian cell culture experiments were performed in the HEK293FT cell line (Thermo Fisher Scientific, #R70007) grown in DMEM (Thermo Fisher Scientific, #11965092) with 25mM high glucose, ImM sodium pyruvate and 4mM L-Glutamine (Thermo Fisher Scientific, #11995073), additionally supplemented with lx penicillinstreptomycin (Thermo Fisher Scientific, #15140122), and 5-10% fetal bovine serum (Thermo Fisher Scientific, #16000069). HEK293FT cells were maintained at confluency below 90%. HEK293FT cells were used for lentiviral vector production.

[0067] Lentiviral vector construction and production: viral vectors and plasmids were constructed as previously reported. See Jin et al., Science 2020; 370: eaaz6063. The backbone plasmid contains the human U6 promoter to express one gRNA cassette, or two gRNA cassettes conjugated by a tRNA linker. The backbone also contains an EFla promoter to express a labeling reporter (Tagl, Tag2, Tag3, or the combinations) conjugated to a membrane-bound reporter. See Veldman et al., 2020 Neuron; 108: 111-127. Cloning of the vectors was done individually and confirmed by Sanger sequencing or whole plasmid sequencing. The gRNA designs were defined using the online tool at benchling.com. Lentiviral vector production and titration was performed according to a published protocol. See Brown et al., 2020 STAR Protoc.; 1 : 100152.

[0068] In utero administration: lentiviral vectors or AAV (0.5-1.5 pL per embryo, with stock concentration at l-9xl08Unit / mL for lentivirus or l-9xl012Unit / mL for AAVs) were administered in utero to the lateral ventricles at El 1.5-17.5 in CD1 or Cas9 transgenic mice (Jax#026179). In vivo postnatal administration: AAV (10-100 pL per animal, with stock concentration l-9xl012Unit / mL) were administered through intravenous injections in CD1 or Cas9 transgenic mice (Jax#026179).

[0069] Tissue clearing and immunofluorescent staining: Postnatal mice from PIO to P28 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. Whole brain samples were cleared using SHIELD protocol. See Park et al., 2018 Nat Biotechnol.; 37: 73-83. After delipidation, the whole brain samples were washed 3 times (in total 24 hrs) in PBS with 0.3% Triton X-100 at 37 °C with shaking. The whole brain samples then incubated with blocking media (10% donkey serum, 5% bovine serum albumin, 0.3% Triton X-100 in PBS) for 48 hrs at 37 °C with shaking, then incubated with 1 pg / mL primary antibodies in the blocking media for 5 days at 37 °C with shaking. Samples were washed with PBS with 0.3% Triton X-100 5 times (in total 48 hrs) in PBS with 0.3% Triton X-100, at 37 °C with shaking, then incubated with 2 pg / mL secondary antibodies in blocking media for 5 days at 37 °C with shaking. Samples were washed with PBS with 0.3% Triton X-100 5 times (in total 48 hrs) in PBS with 0.3% Triton X-100, at 37 °C with shaking. After staining, samples were incubated in Easylndex (RI 1.52) for 24 hrs or until the samples were optically clear. Images were taken at LifeCanvas Technology LLC using a SmartSPIM lightsheet microscope. If the experiment focuses on certain brain regions (e.g. cortex, striatum, hippocampus, cerebellum, olfactory bulb, etc.), the whole brain samples can be dissected into smaller pieces for convenience (e.g., we dissected the brains into 1mm thick sections when local dendritic morphology is analyzed in the experiment). For smaller pieces of samples, the tissue clearing and staining procedures can be shortened (tissue clearing: according to the original protocol, washing: can be reduced to 12hrs, antibody incubation: can be reduced to 2 days at room temperature (RT), Easylndex incubation: can be reduced to 12 hrs). A Nikon AX Confocal Microscope with a 20x air objective was used for 1 mm think brain section imaging.

[0070] Neuron morphology 3D reconstruction and morphometries measurement: the images were imported into Imaris 10.2.0 for morphology analysis. The dendritic morphology traces of labeled neurons were then reconstructed using the Filament module semi- automatically. The morphological features were measured directly using the measurement functions of the Filament module in Imaris. The reconstruction and measurement were finished in a double-blinded approach. The perturbation identity for each labeled neuron was identified and annotated based on the expression of labeling reporters.

[0071] Spatial transcriptome analysis assay: the biological samples (cells or brian sections) were subjected to spatial transcriptome analysis via STARmap. See Wang et al.,Science 361 (6400) :eaat5691, 2018. The samples were fixed using 4% PFA for 1 h at RT, followed by permeabilization using 70% ethanol for 1-3 h at -20 °C. The samples were then washed three times using PBS with 0.1 % Tween-20 before incubation with hybridization probes at 40 °C with rocking for 24-36 h. The probes were designed to target either mouse genes or gRNAs. After hybridization, the samples were washed three times using PBS with 0.1% Tween-20. The samples were then incubated in ligation mix (1 :25 T4 ligase, ligation buffer, BSA) at RT for Ih, followed by incubation in rolling circle amplification (RCA) mix (1 :50 Phi29 polymerase, amplification mix, 5-(3-aminoallyl)-dUTP, dNTP, BSA) at 30 °C for 2h. Next, the samples were embedded in polyacrylamide gel and proceeded to imaging and sequencing. For each sequencing round, the samples were treated using stripping buffer (60% formamide, 0.1% TritonX-100) twice at RT for 10 min, followed by incubation in ligation mix with sequencing probes (target the barcode regions on RCA amplicons) at RT for 3 h to overnight.

[0072] Some cited references1. Platt RJ, Chen S, Zhou Y, Yim MJ, Swiech L, Kempton HR, Dahlman JE, Parnas O, Eisenhaure TM, Jovanovic M, Graham DB, Jhunjhunwala S, Heidenreich M, Xavier RJ, Langer R, Anderson DG, Hacohen N, Regev A, Feng G, Sharp PA, Zhang F. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell. 2014 Oct 9;159(2):440-55. doi: 10.1016 / j. cell.2014.09.014. Epub 2014 Sep 25. PMID: 25263330; PMCID: PMC4265475.2. Jin X, Simmons SK, Guo A, Shetty AS, Ko M, Nguyen L, Jokhi V, Robinson E, Oyler P, Curry N, Deangeli G, Lodato S, Levin JZ, Regev A, Zhang F, Arlotta P. In vivo Perturb-Seq reveals neuronal and glial abnormalities associated with autism risk genes. Science. 2020 Nov 27;370(6520):eaaz6063. doi: 10.1126 / science.aaz6063. PMID: 33243861; PMCID: PMC7985844.3. Veldman MB, Park CS, Eyermann CM, Zhang JY, Zuniga- Sanchez E, Hirano AA, Daigle TL, Foster NN, Zhu M, Langf elder P, Lopez IA, Brecha NC, Zipursky SL, Zeng H, Dong HW, Yang XW. Brainwide Genetic Sparse Cell Labeling to Illuminate the Morphology of Neurons and Glia with Cre-Dependent MORF Mice. Neuron. 2020 Oct 14; 108(1): 111- 127. e6. doi: 10.1016 / j.neuron.2020.07.019. Epub 2020 Aug 13. PMID: 32795398; PMCID: PMC7572760.4. Brown LY, Dong W, Kantor B. An Improved Protocol for the Production of Lentiviral Vectors. STAR Protoc. 2020 Oct 27;l(3):100152. doi: 10.1016 / j.xpro.2020.100152. PMID: 33377046; PMCID: PMC7757317.5. Park YG, Sohn CH, Chen R, McCue M, Yun DH, Drummond GT, Ku T, Evans NB, Oak HC, Trieu W, Choi H, Jin X, Lilascharoen V, Wang J, Truttmann MC, Qi HW, Ploegh HL, Golub TR, Chen SC, Frosch MP, Kulik HJ, Lim BK, Chung K. Protection of tissue physicochemical properties using polyfunctional crosslinkers. Nat Biotechnol. 2018 Dec 17:10.1038 / nbt.4281. doi: 10.1038 / nbt.4281. Epub ahead of print. PMID: 30556815; PMCID: PMC6579717.***

[0073] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0074] All publications, databases, GenBank sequences, patents, and patent applications cited in this specification are herein incorporated by reference as if each was specifically and individually indicated to be incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. A method for identifying or mapping genes that are linked to or associated with a phenotypic change in the cytoarchitecture or spatial transcriptomics of brain cells, comprising (1) introducing a library of vectors encoding genetic perturbations for a plurality of target genes into a CRISPR-Cas9 expressing transgenic animal system, (2) examining cytoarchitecture or spatial transcriptomics of brain cells at one or more developmental stages of the transgenic animal system, (3) identifying one or more brain cells of the transgenic system that express a genetic perturbation and display a phenotypic change of cytoarchitecture or spatial transcriptomics, and (4) determining the corresponding gene perturbed by the genetic perturbation that is encoded by the vector introduced into each of the one or more cells; thereby identifying one or more genes that are linked to or associated with the change in the cytoarchitecture or spatial transcriptomics of brain cells.

2. The method of claim 1, wherein the brain cells are neurons or glia.

3. The method of claim 1, wherein the phenotypic change in the cytoarchitecture is a change in the morphology of the brain cells.

4. The method of claim 3, wherein the change in the morphology of the brain cells is a change in cellular position, morphology, synaptic structure, spatial organization or projection.

5. The method of claim 3, wherein the change in the morphology of the brain cells is examined via imaging of the whole brain or tissue sections of the transgenic animal system.

6. The method of claim 1, wherein the phenotypic change is examined via determining spatial transcriptome of the brain cells.

7. The method of claim 1, wherein the vectors are lentiviral vectors or AAV vectors.

8. The method of claim 1, wherein each of the genetic perturbations comprises one or more guide RNAs (gRNAs) for introducing genomic changes via CRISPR-Cas9 gene editing in the coding region of a target gene.

9. The method of claim 8, wherein the one or more gRNAs each target a sequence at the 5’ end of a target gene’s coding region.

10. The method of claim 1, wherein each of the vectors further expresses one or more reporter molecules.

11. The method of claim 10, wherein the one or more reporter molecules are GFP, HA, tdTomato, and Myc.

12. The method of claim 1, wherein the transgenic animal system is (1) a developing embryo of a Cas9-expressing transgenic animal, or (2) a postnatal or adult Cas9- expressing transgenic animal.

13. The method of claim 12, wherein the library of vectors is administered in utero to lateral ventricles of the embryo or via postnatal intravenous injection.

14. The method of claim 12, wherein the library of vectors are administered to the animal embryo around a development stage that is equivalent to any mouse embryonic day between Day 11.5 (El 1.5) and Day 17.5 (E17.5).

15. The method of claim 14, wherein the development stage that is equivalent to mouse embryonic day 13.5 (E13.5) or 15.5 (E15.5)16. The method of claim 12, wherein the phenotypic change is examined at one or more developmental stages that are equivalent to any mouse postnatal day between day 2 (P2) to day 60 (P60).

17. The method of claim 16, wherein the one or more developmental stages are equivalent to mouse postnatal day 10 (P10), 14 (P14), or 28 (P28).

18. A method for correlating a phenotypic change in the cytoarchitecture or spatial transcriptomics brain cells with genetic perturbations of one or more target genes, comprising (1) introducing a library of viral vectors encoding genetic perturbations for a plurality of target genes into a CRISPR-Cas9 expressing transgenic system, (2) examining cytoarchitecture or spatial transcriptomics of brain cells at one or more developmental stages of the transgenic system, (3) identifying one or more brain cells of the transgenic system that express a genetic perturbation and display a phenotypic change of cytoarchitecture or spatial transcriptomics, and (4) determining the corresponding gene perturbed by the genetic perturbation that is encoded by the viral vector introduced into each of the one or more cells; thereby correlating the corresponding perturbed gene with the phenotypic change of cytoarchitecture or spatial transcriptomics displayed by each of the one or more cells.

19. The method of claim 18, wherein the brain cells are neurons or glia.

20. The method of claim 18, wherein the phenotypic change of cytoarchitecture is a change in the morphology of the brain cells.

21. The method of claim 20, wherein the change in the morphology is a change in cellular position, synaptic structure, spatial organization or projection.

22. The method of claim 18, wherein the phenotypic change is examined via determining spatial transcriptome of the brain cells.

23. The method of claim 18, wherein cytoarchitecture of brain cells is examined via whole mount imaging or thick tissue section imaging of the brain across one or more brain regions.

24. The method of claim 23, wherein the one or more brain regions comprise cortex, striatum, hippocampus, cerebellum, and olfactory bulb.

25. The method of claim 18, wherein the viral vectors are lentiviral vectors or AAV vectors.

26. The method of claim 18, wherein the viral vectors further express one or more reporter molecules.

27. The method of claim 26, wherein the one or more reporter molecules comprise GFP, HA, tdTomato, and Myc.

28. The method of claim 18, wherein the target genes are known or suspected to be associated with a specific disease or disorder.

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