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• W2955699710 abstract "•HiUGE, an AAV-based CRISPR-mediated method for universal insertional genome editing•Arrayed gRNAs and premade donors enable high-throughput endogenous protein labeling•“Plug-and-play” selection of donors at each target locus enables diverse applications•HiUGE provides a powerful method to analyze proteomic candidates in vitro and in vivo Analysis of endogenous protein localization, function, and dynamics is fundamental to the study of all cells, including the diversity of cell types in the brain. However, current approaches are often low throughput and resource intensive. Here, we describe a CRISPR-Cas9-based homology-independent universal genome engineering (HiUGE) method for endogenous protein manipulation that is straightforward, scalable, and highly flexible in terms of genomic target and application. HiUGE employs adeno-associated virus (AAV) vectors of autonomous insertional sequences (payloads) encoding diverse functional modifications that can integrate into virtually any genomic target loci specified by easily assembled gene-specific guide-RNA (GS-gRNA) vectors. We demonstrate that universal HiUGE donors enable rapid alterations of proteins in vitro or in vivo for protein labeling and dynamic visualization, neural-circuit-specific protein modification, subcellular rerouting and sequestration, and truncation-based structure-function analysis. Thus, the “plug-and-play” nature of HiUGE enables high-throughput and modular analysis of mechanisms driving protein functions in cellular neurobiology. Analysis of endogenous protein localization, function, and dynamics is fundamental to the study of all cells, including the diversity of cell types in the brain. However, current approaches are often low throughput and resource intensive. Here, we describe a CRISPR-Cas9-based homology-independent universal genome engineering (HiUGE) method for endogenous protein manipulation that is straightforward, scalable, and highly flexible in terms of genomic target and application. HiUGE employs adeno-associated virus (AAV) vectors of autonomous insertional sequences (payloads) encoding diverse functional modifications that can integrate into virtually any genomic target loci specified by easily assembled gene-specific guide-RNA (GS-gRNA) vectors. We demonstrate that universal HiUGE donors enable rapid alterations of proteins in vitro or in vivo for protein labeling and dynamic visualization, neural-circuit-specific protein modification, subcellular rerouting and sequestration, and truncation-based structure-function analysis. Thus, the “plug-and-play” nature of HiUGE enables high-throughput and modular analysis of mechanisms driving protein functions in cellular neurobiology. Selective labeling and manipulation of endogenous proteins are essential to delineating the molecular mechanisms of cell and organismal biology. Recent advances in exploratory proteomics and gene expression analysis generate sizable datasets that urgently require high-throughput and reliable methods for protein visualization and functional manipulation purposes. However, current techniques to enable these strategies are often inefficient or resource intensive. For example, due to the limited availability and the cost of antibodies, it is often not a viable option for single labs to immunolabel tens to hundreds of proteomic candidates. Further, a large fraction of available antibodies may have limited utility due to unsuspected cross-reactivity to other proteins, lot-to-lot variability in quality, and insufficient application-specific validation (Berglund et al., 2008Berglund L. Björling E. Oksvold P. Fagerberg L. Asplund A. Szigyarto C.A. Persson A. Ottosson J. Wernérus H. Nilsson P. et al.A genecentric Human Protein Atlas for expression profiles based on antibodies.Mol. Cell. Proteomics. 2008; 7: 2019-2027Crossref PubMed Scopus (503) Google Scholar, Bradbury and Plückthun, 2015Bradbury A. Plückthun A. Reproducibility: Standardize antibodies used in research.Nature. 2015; 518: 27-29Crossref PubMed Scopus (440) Google Scholar, Egelhofer et al., 2011Egelhofer T.A. Minoda A. Klugman S. Lee K. Kolasinska-Zwierz P. Alekseyenko A.A. Cheung M.S. Day D.S. Gadel S. Gorchakov A.A. et al.An assessment of histone-modification antibody quality.Nat. Struct. Mol. Biol. 2011; 18: 91-93Crossref PubMed Scopus (312) Google Scholar, Michel et al., 2009Michel M.C. Wieland T. Tsujimoto G. How reliable are G-protein-coupled receptor antibodies?.Naunyn Schmiedebergs Arch. Pharmacol. 2009; 379: 385-388Crossref PubMed Scopus (238) Google Scholar). Additionally, overexpression of recombinant constructs to map protein localization or conduct functional analyses is also resource intensive and can be highly sensitive to available cellular docking sites or unforeseen artifactual cellular effects associated with protein overexpression. CRISPR-associated endonuclease Cas9-based strategies have great promise to enable highly precise genome editing of mammalian cells to address many of the above limitations (Cong et al., 2013Cong L. Ran F.A. Cox D. Lin S. Barretto R. Habib N. Hsu P.D. Wu X. Jiang W. Marraffini L.A. Zhang F. Multiplex genome engineering using CRISPR/Cas systems.Science. 2013; 339: 819-823Crossref PubMed Scopus (9981) Google Scholar, Doudna and Charpentier, 2014Doudna J.A. Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9.Science. 2014; 346: 1258096Crossref PubMed Scopus (3581) Google Scholar, Hsu et al., 2014Hsu P.D. Lander E.S. Zhang F. Development and applications of CRISPR-Cas9 for genome engineering.Cell. 2014; 157: 1262-1278Abstract Full Text Full Text PDF PubMed Scopus (3606) Google Scholar, Jinek et al., 2012Jinek M. Chylinski K. Fonfara I. Hauer M. Doudna J.A. Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (9327) Google Scholar, Mali et al., 2013Mali P. Yang L. Esvelt K.M. Aach J. Guell M. DiCarlo J.E. Norville J.E. Church G.M. RNA-guided human genome engineering via Cas9.Science. 2013; 339: 823-826Crossref PubMed Scopus (6424) Google Scholar). CRISPR-Cas9 introduces double-strand breaks (DSBs) at guide RNA (gRNA) specified genomic sites. These genomic DSBs are repaired via two main pathways in cells; non-homologous end joining (NHEJ) is the prevailing pathway, while homology-directed repair (HDR) is reportedly less frequent, especially in non-dividing cells outside of the S and G2 phase of cell cycle (Heyer et al., 2010Heyer W.D. Ehmsen K.T. Liu J. Regulation of homologous recombination in eukaryotes.Annu. Rev. Genet. 2010; 44: 113-139Crossref PubMed Scopus (720) Google Scholar, Hsu et al., 2014Hsu P.D. Lander E.S. Zhang F. Development and applications of CRISPR-Cas9 for genome engineering.Cell. 2014; 157: 1262-1278Abstract Full Text Full Text PDF PubMed Scopus (3606) Google Scholar, Mao et al., 2008Mao Z. Bozzella M. Seluanov A. Gorbunova V. DNA repair by nonhomologous end joining and homologous recombination during cell cycle in human cells.Cell Cycle. 2008; 7: 2902-2906Crossref PubMed Scopus (409) Google Scholar, Saleh-Gohari and Helleday, 2004Saleh-Gohari N. Helleday T. Conservative homologous recombination preferentially repairs DNA double-strand breaks in the S phase of the cell cycle in human cells.Nucleic Acids Res. 2004; 32: 3683-3688Crossref PubMed Scopus (196) Google Scholar). Both HDR and NHEJ pathways are currently utilized to insert foreign DNA sequences into genes of interest (GOIs) for applications such as labeling of translated endogenous protein products. Examples of these methods include single-cell labeling of endogenous proteins (SLENDR and viral SLENDR [(v)SLENDR]) and homology-independent targeted integration (HITI), both of which have been shown to function in post-mitotic cells, including neurons. SLENDR is based on HDR, using oligonucleotides or adeno-associated virus (AAV) donors containing gene-specific homology sequences to facilitate insertion into GOIs (Mikuni et al., 2016Mikuni T. Nishiyama J. Sun Y. Kamasawa N. Yasuda R. High-throughput, high-resolution mapping of protein localization in mammalian brain by in vivo genome editing.Cell. 2016; 165: 1803-1817Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, Nishiyama et al., 2017Nishiyama J. Mikuni T. Yasuda R. Virus-mediated genome editing via homology-directed repair in mitotic and postmitotic cells in mammalian brain.Neuron. 2017; 96: 755-768.e755Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Alternatively, HITI leverages NHEJ to insert foreign sequences into a GOI. It utilizes donor vectors containing gene-specific gRNA recognition sequences, which simultaneously direct DSB cuts to the gene and donor vector, facilitating directional insertion during NHEJ (Suzuki et al., 2016Suzuki K. Tsunekawa Y. Hernandez-Benitez R. Wu J. Zhu J. Kim E.J. Hatanaka F. Yamamoto M. Araoka T. Li Z. et al.In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration.Nature. 2016; 540: 144-149Crossref PubMed Scopus (662) Google Scholar). Although SLENDR and HITI are flexible in their ability to modify proteins both in vitro and in vivo for neuronal applications, they both necessitate the cloning of gRNA vectors for the GOIs, as well as customized gene-specific donor vectors for each insertion. By requiring the generation of tailored donor vectors for each DSB cut site in each GOI, the scalability and throughput of testing multiple insertional sequences using either SLENDR or HITI are limited. Alternatively, CRISPaint pairs NHEJ with modular donor vectors that are linearized and integrated into GOIs and demonstrates improved throughput for insertional editing in cell lines (Schmid-Burgk et al., 2016Schmid-Burgk J.L. Höning K. Ebert T.S. Hornung V. CRISPaint allows modular base-specific gene tagging using a ligase-4-dependent mechanism.Nat. Commun. 2016; 7: 12338Crossref PubMed Scopus (85) Google Scholar). However, it requires specially prepared mini-circular vectors that are not compatible with viral delivery methods important for many in vivo applications, or the bacterial vector backbones are necessarily co-inserted into genomes, which can interfere long-term transgene expression (Chen et al., 2001bChen Z.Y. Yant S.R. He C.Y. Meuse L. Shen S. Kay M.A. Linear DNAs concatemerize in vivo and result in sustained transgene expression in mouse liver.Mol. Ther. 2001; 3: 403-410Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, Chen et al., 2003Chen Z.Y. He C.Y. Ehrhardt A. Kay M.A. Minicircle DNA vectors devoid of bacterial DNA result in persistent and high-level transgene expression in vivo.Mol. Ther. 2003; 8: 495-500Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar). These issues may limit its widespread utility, especially for applications where viral transduction is preferred in vitro or in vivo. Here, we describe homology-independent universal genome engineering (HiUGE), a new generalized method that obviates many of the constraints to the current state of the art. HiUGE is an AAV-mediated method that uses a two-vector modular approach for higher-throughput genomic knockin (KI) applications. The donor vector contains an insertional DNA fragment (payload) that is flanked on both ends by an artificial DNA sequence non-homologous to the target genome. This sequence is recognized by a donor-specific gRNA (DS-gRNA) that directs the Cas9-mediated autonomous excision and release of the payload. The payload can then be integrated across diverse GOIs, both in vitro and in vivo, at genomic loci specified by a separate panel of high-throughput and low-cost gene-specific gRNA (GS-gRNA) vectors. This design frees the donor vectors of any gene-specific sequences, rendering them universally compatible for virtually any CRISPR-Cas9-accessible genomic loci. Importantly, these AAV donors can be premade for ubiquitous applications, greatly simplifying strategies for insertional genome editing. We have tested payloads for a variety of applications and demonstrate their interchangeability within and between GOIs in both cells and tissues. These applications include antibody epitope and fluorescent protein labeling for localization mapping and dynamic visualization of endogenous proteins, protein subcellular rerouting and sequestration, protein truncation for structure-function relationship analysis, and neural-circuit-specific genome engineering. Because this method is highly modular, scalable, and suitable for in vitro as well as in vivo applications, it opens new avenues to pair higher-throughput proteomic and genomic applications with experimental validation and phenotypic screening to address molecular mechanisms of cellular neurobiology. Recent advances in proteomics and gene expression studies generate sizable protein and gene network datasets, which urgently require novel methods to analyze them on larger scales with greater precision. Higher-throughput genome engineering techniques targeting candidate proteins/genes could satisfy these needs. We thus designed a method utilizing the delivery of universal DNA inserts (payloads) that can ubiquitously integrate across genes to achieve this. In the HiUGE system (Figure 1A), a two-vector approach was used to deliver GS-gRNAs and universal payloads, with AAV as the delivery vehicle for its flexible use in vitro and in vivo. HiUGE GS-gRNA vectors are prepared by ligations of 23- to 24-mer oligonucleotides to the backbone vector, which is scalable to generate panels of gRNA expressing vectors targeting diverse GOIs. HiUGE donor vectors are autonomous, expressing a synthetic donor-specific gRNA (DS-gRNA) that is non-homologous to the targeted genome but directs Cas9-mediated self-cleavage and release of the universal payloads for genomic insertion. This design allows the separation of the donor vector construction from any specific sequence of genomic targets, thus enabling universal applications of the donor “toolkits.” Such utilities include localization mapping and functional manipulation of the endogenous proteins, by introducing tags at their carboxy- or amino-terminus (C- or N-term) within diverse CRISPR-Cas9-accessible genomic loci unlocked by the GS-gRNA “keys.” To test this concept, a dual-orientation payload design was used to facilitate the expression of a hemagglutinin (HA)-epitope tag following either forward or reverse integration into the C-term of proteins of interest (Figure 1B). We adopted a protocol to produce small-scale transduction-ready AAVs for higher-throughput use in vitro, complimenting traditional AAV purifications for use in vivo (Figure 1C; STAR Methods). As a proof of concept, small-scale AAVs of a GS-gRNA targeting the mouse Tubb3 gene and a dual-orientation HA-epitope HiUGE donor were used to co-transduce primary neurons prepared from neonatal pups of conditional Cas9 mice. The GS-gRNA AAV also expresses Cre-recombinase to induce Cas9-2A-GFP expression. Approximately 1 week following infection, immunofluorescent detection of HA-epitope showed successful labeling of endogenous βIII-tubulin, with localization characteristics specific to microtubules (Figures 1D and S1A). Western blot detection of the HA-epitope showed a single band (∼51 kDa) consistent with the predicted molecular mass of βIII-tubulin (Figure 1E). Genomic insertion of the payload was verified by sequencing the Tubb3 locus, confirming the successful HA-epitope integration (Figure 1F). In control experiments, the relative selectivity of frame-dependent labeling was tested by comparing the correct open reading frame (ORF) donor (ORF+1, in reference to the Cas9 cleavage locus specified by the Tubb3 gRNA) to out-of-frame donors (ORF+0 or ORF+2) (Figure S1B i–iii). HA-labeling in the correct ORF was substantially more efficient (Figure S1C), with occasional HA-positive cells observed from out-of-frame ORFs, indicating comparatively less frequent frameshifting insertion or deletion (indel) events during payload integration. Negative controls demonstrated the absolute requirement for both GS-gRNA and HiUGE donor vectors for HA-epitope KI (Figure S1B iv–vi). To demonstrate that donor vectors for all three ORFs are equally capable of facilitating HiUGE, three GS-gRNAs, one for each ORF, were designed to target the mouse Map2 gene. Pairing these GS-gRNA AAVs with HA-epitope donor AAVs in their corresponding ORFs resulted in comparable and efficient labeling (Figures S1D and S1E). An important consideration is that the HiUGE donor vectors should be suitable to target diverse coding sequences without introducing premature stop codons that prevent proper expression of the insert. This is achieved by using donor recognition sequences (DRSs) specifically designed such that premature stop codons cannot be introduced during genomic integration. We compared HA-epitope donor vectors with different DRSs and found similar cellular labeling efficiencies among them (Figures S2A–S2C), demonstrating flexibility in DRS usage. Also, we found that the cellular labeling efficiency for HiUGE was comparable to HITI (Figures S2D–S2I). We next tested whether a single HiUGE payload can be integrated across diverse genomic loci and label multiple target proteins for subcellular localization mapping. We used oligonucleotide ligation to rapidly construct an arrayed panel of GS-gRNA AAV vectors targeting the C terminus of 12 proteins, which exhibit previously described and highly patterned subcellular localizations in neurons and glia. These included proteins of the microtubule network (βIII-tubulin, Tubb3; microtubule-associated protein 2, Map2), the nucleus (methyl-CpG-binding protein 2, Mecp2; Chen et al., 2001aChen R.Z. Akbarian S. Tudor M. Jaenisch R. Deficiency of methyl-CpG binding protein-2 in CNS neurons results in a Rett-like phenotype in mice.Nat. Genet. 2001; 27: 327-331Crossref PubMed Scopus (1032) Google Scholar), the synaptic actin cytoskeleton (actin-related protein 2, Actr2; Kim et al., 2013Kim I.H. Racz B. Wang H. Burianek L. Weinberg R. Yasuda R. Wetsel W.C. Soderling S.H. Disruption of Arp2/3 results in asymmetric structural plasticity of dendritic spines and progressive synaptic and behavioral abnormalities.J. Neurosci. 2013; 33: 6081-6092Crossref PubMed Scopus (117) Google Scholar, Spence and Soderling, 2015Spence E.F. Soderling S.H. Actin out: regulation of the synaptic cytoskeleton.J. Biol. Chem. 2015; 290: 28613-28622Crossref PubMed Scopus (112) Google Scholar), clathrin-coated vesicles (clathrin light chain A, Clta), the axonal initial segment (AIS) (neuronal adhesion molecule, Nrcam; Ankyrin-G, Ank3; βIV-spectrin, Sptbn4; sodium channel subunit Nav1.2, Scn2a; Jones and Svitkina, 2016Jones S.L. Svitkina T.M. Axon initial segment cytoskeleton: architecture, development, and role in neuron polarity.Neural Plast. 2016; 2016: 6808293Crossref PubMed Scopus (48) Google Scholar), intermediate filaments (glial fibrillary acidic protein, Gfap), mitochondria (pyruvate dehydrogenase E1 component subunit alpha, Pdha1), and the distal tips of neurites (Doublecortin, Dcx; Francis et al., 1999Francis F. Koulakoff A. Boucher D. Chafey P. Schaar B. Vinet M.-C. Friocourt G. McDonnell N. Reiner O. Kahn A. et al.Doublecortin is a developmentally regulated, microtubule-associated protein expressed in migrating and differentiating neurons.Neuron. 1999; 23: 247-256Abstract Full Text Full Text PDF PubMed Scopus (828) Google Scholar, Gdalyahu et al., 2004Gdalyahu A. Ghosh I. Levy T. Sapir T. Sapoznik S. Fishler Y. Azoulai D. Reiner O. DCX, a new mediator of the JNK pathway.EMBO J. 2004; 23: 823-832Crossref PubMed Scopus (185) Google Scholar). Colony screening demonstrated that the cloning reactions were highly efficient (Figures S1F–S1H). Primary neuron-glia cultures of Cas9 mice were co-transduced with one of these GS-gRNA AAVs in combination with the same HA-epitope donor AAV (Figure 2A). Approximately 7 days following infection, we observed the predicted localization of the 12 protein targets by HA immunostaining (Figures 2B–2M). Further, genomic PCRs were performed for five targets to amplify the region around the edited junctions. HA-epitope integrations were confirmed (Figures S3A and S3B), with deep sequencing of the amplicons revealing the positional indel occurrences, the proportions of forward and reverse payload insertion without indels, and the proportions of allelic mutations without donor integration (Figure S3C). We also tested for the integration of donor payloads in top-ranked predicted off-target sites and found no evidence for payload insertion (Figures S3D and S3E). In addition, experimental genome-wide analysis was performed to detect potential off-target payload integrations, including those at nonpredicted sites. We found that while genomic off-target integrations can occur, their estimated frequencies are low (Figures S3F–S3H). In each instance, the observed off-target integration was into non-coding regions that would not result in unintended labeling of non-target proteins. Further, the cellular labeling efficiencies across various conditions were quantified for several AIS-specific targets, as each neuron typically has only one AIS, and their distinct expression pattern is suitable for easy visual identification and quantification. The ratios of HiUGE-labeled AIS structures compared to all AIS (stained with an antibody against the AIS-marker Ankyrin-G) were quantified (Figures S4A–S4C). Estimated efficiencies were shown across multiple viral doses (Sptbn4: 34%; Scn2a: 29% at the high dose) and across various ratios between the GS-gRNA and the donor (ratios around 1:1 are optimal). Further, we tested HiUGE-mediated labeling to visualize several members of the recently uncovered inhibitory postsynaptic density (iPSD) proteome (Uezu et al., 2016Uezu A. Kanak D.J. Bradshaw T.W. Soderblom E.J. Catavero C.M. Burette A.C. Weinberg R.J. Soderling S.H. Identification of an elaborate complex mediating postsynaptic inhibition.Science. 2016; 353: 1123-1129Crossref PubMed Scopus (162) Google Scholar). Spaghetti-monster HA (smFP-HA), a larger insert that exhibits enhanced antigenicity suitable for detecting low-expression proteins (Viswanathan et al., 2015Viswanathan S. Williams M.E. Bloss E.B. Stasevich T.J. Speer C.M. Nern A. Pfeiffer B.D. Hooks B.M. Li W.P. English B.P. et al.High-performance probes for light and electron microscopy.Nat. Methods. 2015; 12: 568-576Crossref PubMed Scopus (132) Google Scholar), was used as the payload (Figure 2A). These iPSD proteomic candidates included inhibitory synaptic protein 1 (Insyn1), inhibitory synaptic protein 2 (Insyn2), and Rho GTPase activating protein 32 (Arhgap32). Immunocytochemistry demonstrated that they were localized juxtaposed to the vesicular GABA transporter (VGAT) (Figures 2N–2P), confirming their presence at inhibitory synapses. In addition, three different GS-gRNAs (Table S1) were tested for Insyn1, and all of them yield comparable inhibitory synaptic labeling (Figures S4D–S4G), demonstrating the flexibility of GS-gRNA selection and the robustness of the labeling method. Finally, we performed mixed labeling of diverse protein targets by applying a single C-term HA-epitope donor with a mixture of GS-gRNA AAVs (Figures S4H and S4I). Immunocytochemistry revealed simultaneous labeling of the targeted proteins (βIV-spectrin, GFAP, and MeCP2) in a single experiment and provided a proof of concept for other applications that require simultaneous modification of multiple proteins. Next, we tested N-term modifications using a single Myc-epitope payload with an upstream stop codon cassette to constrain its expression to the N-term (Figure 2A). GS-gRNAs targeting the N-term of three proteins were tested, including proteins of the actin cytoskeleton (β-actin, Actb), nuclear envelope (lamin B1, Lmnb1), and neurofilaments (neurofilament medium, Nefm). Myc-epitope immunolabeling, consistent with the expected localization pattern, was observed for each target (Figures 2Q–2S). Further, like previous studies (Mikuni et al., 2016Mikuni T. Nishiyama J. Sun Y. Kamasawa N. Yasuda R. High-throughput, high-resolution mapping of protein localization in mammalian brain by in vivo genome editing.Cell. 2016; 165: 1803-1817Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), we tested selective dual labeling of different proteins with different tags. This is achieved by combining the N-term selective Myc-epitope payload with the HA-epitope HiUGE payload described above, which contains a stop codon cassette following the epitope tag that enforces its expression at the C-term (Figure S4J). These donors were co-infected with GS-gRNA AAVs targeting the N-term of dendritic MAP2 (Map2) and the C-term of AIS-enriched βIV-spectrin (Sptbn4). Co-staining for HA and Myc-epitopes revealed selective labeling for each protein, with instances of co-staining in single neurons (Figure S4K). Thus, HiUGE N- and C-term selective payloads can be used to co-label proteins simultaneously to reveal the spatial relationships of two endogenous proteins, which may be more useful than the mixed labeling of multiple proteins with identical tags described above. Together, using rapidly constructed GS-gRNA libraries, HiUGE integrates universal payloads into diverse protein coding regions in a highly specific and higher-throughput manner for mapping endogenous protein localizations. HiUGE is thus suitable for rapid and robust labeling of proteins, including post hoc localization analysis following large-scale proteomic studies. We next tested in vivo labeling by intracranial injections of HiUGE AAVs (Figure 3A). Approximately 2 weeks following infection, immunohistochemistry was performed. Images of sections from each injection revealed the expected HA immunoreactivity localized to the AIS (Sptbn4 and Scn2a; Figures 3B and 3C), microtubules (Tubb3; Figure 3D), and nuclei (Mecp2; Figure 3E). Of note, the detection for each of four targets was consistent across independent cells with no visible mis-localization, suggesting off-target protein coding integration was undetectable or rare. In addition, local injections of HiUGE AAVs into the motor cortex of adult Cas9 mice were performed, and the cellular labeling efficiencies for the AIS-localized targets at the injection sites were quantified (Sptbn4: 30%; Scn2a: 23%. Figures 3F–3J). These data demonstrated that a single HiUGE donor can be paired with multiple GS-gRNAs to modify diverse endogenous proteins in vivo. While the above data demonstrated that a single HiUGE payload could integrate across diverse gene targets in vitro and in vivo, an additional feature of the HiUGE method is that the donor payloads should be interchangeable for multiplexing and flexible selection of protein modifications. The ability to mix and match premade HiUGE donors would simplify the experimental selection of optimal epitopes, diverse fusion proteins, or fusion proteins with variable linkers. To demonstrate the interchangeability of HiUGE payloads, we modified proteins either in vitro or in vivo with a combination of epitope tags (HA, Myc, and/or V5) to create a mosaic labeling of infected cells (Figure 4A). Immunostaining of cultured neurons infected with a mixture of these HiUGE epitope payload AAVs paired with GS-gRNA AAV targeting the Tubb3 locus revealed a visual montage of multicolored βIII-tubulin labeling, with occasional double-labeled cells indicating the two alleles of Tubb3 were modified by different epitopes (Figure 4B, magenta neurons). Thus, after genomic cleavage directed by the GS-gRNA, different HiUGE payloads can be inserted interchangeably. Further, multiplexed labeling was also tested in vivo, where a mixture of HiUGE epitope payload AAVs were co-injected with the Tubb3 GS-gRNA AAV into neonatal pups intracranially. Two weeks following infections, immunohistochemistry revealed extensive multiplexed labeling of neurons throughout multiple brain regions of the cerebrum (Figure 4C). Higher magnification images (Figure 4D) demonstrated heterogeneous epitope labeling of ßIII-tubulin in neighboring neurons of the cortex, hippocampus, and thalamus, as well as projections representing thalamocortical connectivity, globus pallidus bundles, and corpus callosum fibers. Together, these data demonstrated the interchangeability of HiUGE donor payloads within a single gene locus, thus enabling flexible selection of diverse protein modifications. We further reasoned that it should be possible to limit the genome editing activity to specific neural circuits by utilizing a recently reported retrograde-transported AAV2-retro serotype (Tervo et al., 2016Tervo D.G. Hwang B.Y. Viswanathan S. Gaj T. Lavzin M. Ritola K.D. Lindo S. Michael S. Kuleshova E. Ojala D. et al.A designer AAV variant permits efficient retrograde access to projection neurons.Neuron. 2016; 92: 372-382Abstract Full Text Full Text PDF PubMed Scopus (622) Google Scholar). Injection of AAV2-retro GS-gRNA into a brain area containing axon terminals of a circuit could be paired with injection of AAV2/9 HiUGE donor into a specific projection region, allowing retrograde access of projection neurons from a target brain region for neural circuit-selective protein manipulations. We tested this possibility in the well-defined cortico-striatal circuit and the thalamocortical circuit. For the corticostriatal circuit, Tubb3 GS-gRNA AAV2-retro was injected to the striatum, whereas individual AAV2/9 HiUGE donor AAVs were injected laterally into either the primary motor cortex (MOp, HA-epitope) or secondary motor cortex (MOs, Myc-epitope) of adult conditional Cas9 mice (Figure 5A). Retrograde access to the projection neurons in the motor cortex was confirmed by positive GFP labeling, indicating Cre-dependent activation of Cas9-2A-GFP (Figure 5B). HiUGE" @default.
• W2955699710 created "2019-07-12" @default.
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• W2955699710 date "2019-08-01" @default.
• W2955699710 modified "2023-10-10" @default.
• W2955699710 title "Plug-and-Play Protein Modification Using Homology-Independent Universal Genome Engineering" @default.
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• W2955699710 doi "https://doi.org/10.1016/j.neuron.2019.05.047" @default.
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