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WO2025054224A2 - 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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WO2025054224A2
WO2025054224A2 PCT/US2024/045238 US2024045238W WO2025054224A2 WO 2025054224 A2 WO2025054224 A2 WO 2025054224A2 US 2024045238 W US2024045238 W US 2024045238W WO 2025054224 A2 WO2025054224 A2 WO 2025054224A2
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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.
  • 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.
  • 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.
  • 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.
  • 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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