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	<title>bioRxiv Channel: Somatic Cell Genome Editing Program</title>
	<link>https://biorxiv.org</link>
	<description>
	This feed contains articles for bioRxiv Channel "Somatic Cell Genome Editing Program"
	</description>

		<items>
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		</rdf:Seq>
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	<prism:publicationName>bioRxiv</prism:publicationName>
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	<title>bioRxiv</title>
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	<link>https://biorxiv.org</link>
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	<item rdf:about="https://biorxiv.org/cgi/content/short/864199v1?rss=1">
<title>
<![CDATA[
Efficient Homology-directed Repair with Circular ssDNA Donors 
]]>
</title>
<link>
https://biorxiv.org/cgi/content/short/864199v1?rss=1"
</link>
<description><![CDATA[
While genome editing has been revolutionized by the advent of CRISPR-based nucleases, difficulties in achieving efficient, nuclease-mediated, homology-directed repair (HDR) still limit many applications. Commonly used DNA donors such as plasmids suffer from low HDR efficiencies in many cell types, as well as integration at unintended sites. In contrast, single-stranded DNA (ssDNA) donors can produce efficient HDR with minimal off-target integration. Here, we describe the use of ssDNA phage to efficiently and inexpensively produce long circular ssDNA (cssDNA) donors. These cssDNA donors serve as efficient HDR templates when used with Cas9 or Cas12a, with integration frequencies superior to linear ssDNA (lssDNA) donors. To evaluate the relative efficiencies of imprecise and precise repair for a suite of different Cas9 or Cas12a nucleases, we have developed a modified Traffic Light Reporter (TLR) system [TLR-Multi-Cas Variant 1 (MCV1)] that permits side-by-side comparisons of different nuclease systems. We used this system to assess editing and HDR efficiencies of different nuclease platforms with distinct DNA donor types. We then extended the analysis of DNA donor types to evaluate efficiencies of fluorescent tag knock-ins at endogenous sites in HEK293T and K562 cells. Our results show that cssDNA templates produce efficient and robust insertion of reporter tags. Targeting efficiency is high, allowing production of biallelic integrants using cssDNA donors. cssDNA donors also outcompete lssDNA donors in template-driven repair at the target site. These data demonstrate that circular donors provide an efficient, cost-effective method to achieve knock-ins in mammalian cell lines.
]]></description>
<dc:creator>Iyer, S.</dc:creator>
<dc:creator>Mir, A.</dc:creator>
<dc:creator>Ibraheim, R.</dc:creator>
<dc:creator>Lee, J.</dc:creator>
<dc:creator>VegaBadillo, J.</dc:creator>
<dc:creator>Roscoe, B.</dc:creator>
<dc:creator>Zhu, L. J.</dc:creator>
<dc:creator>Liu, P.</dc:creator>
<dc:creator>Luk, K.</dc:creator>
<dc:creator>Mintzer, E.</dc:creator>
<dc:creator>de Brito, J. S.</dc:creator>
<dc:creator>Zamore, P.</dc:creator>
<dc:creator>Sontheimer, E. J.</dc:creator>
<dc:creator>Wolfe, S.</dc:creator>
<dc:date>2019-12-05</dc:date>
<dc:identifier>doi:10.1101/864199</dc:identifier>
<dc:title><![CDATA[Efficient Homology-directed Repair with Circular ssDNA Donors]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory Press</dc:publisher>
<prism:publicationDate>2019-12-05</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://biorxiv.org/cgi/content/short/538421v1?rss=1">
<title>
<![CDATA[
Delivering genes across the blood-brain barrier: LY6A, a novel cellular receptor for AAV-PHP.B capsids 
]]>
</title>
<link>
https://biorxiv.org/cgi/content/short/538421v1?rss=1"
</link>
<description><![CDATA[
The engineered AAV-PHP.B family of adeno-associated virus efficiently delivers genes throughout the mouse central nervous system. To guide their application across disease models, and to inspire the development of translational gene therapy vectors useful for targeting neurological diseases in humans, we sought to elucidate the host factors responsible for the CNS tropism of AAV-PHP.B vectors. Leveraging CNS tropism differences across mouse strains, we conducted a genome-wide association study, and rapidly identified and verified LY6A as an essential receptor for the AAV-PHP.B vectors in brain endothelial cells. Importantly, this newly discovered mode of AAV binding and transduction is independent of other known AAV receptors and can be imported into different cell types to confer enhanced transduction by the AAV-PHP.B vectors.
]]></description>
<dc:creator>Huang, Q.</dc:creator>
<dc:creator>Chan, K. Y.</dc:creator>
<dc:creator>Tobey, I. G.</dc:creator>
<dc:creator>Chan, Y. A.</dc:creator>
<dc:creator>Poterba, T.</dc:creator>
<dc:creator>Boutros, C. L.</dc:creator>
<dc:creator>Balazs, A. B.</dc:creator>
<dc:creator>Daneman, R.</dc:creator>
<dc:creator>Bloom, J. M.</dc:creator>
<dc:creator>Seed, C.</dc:creator>
<dc:creator>Deverman, B. E.</dc:creator>
<dc:date>2019-02-01</dc:date>
<dc:identifier>doi:10.1101/538421</dc:identifier>
<dc:title><![CDATA[Delivering genes across the blood-brain barrier: LY6A, a novel cellular receptor for AAV-PHP.B capsids]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory Press</dc:publisher>
<prism:publicationDate>2019-02-01</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://biorxiv.org/cgi/content/short/640169v1?rss=1">
<title>
<![CDATA[
Methods Matter -- Standard Production Platforms For Recombinant AAV Can Produce Chemically And Functionally Distinct Vectors 
]]>
</title>
<link>
https://biorxiv.org/cgi/content/short/640169v1?rss=1"
</link>
<description><![CDATA[
Different manufacturing approaches have been used in the production of recombinant adeno-associated virus (rAAV). The two leading approaches are transiently transfected human HEK293 cells and live baculovirus infection of Sf9 insect cells. Unexplained differences in vector performance have been seen clinically and preclinically. Thus, we performed for the first time a highly controlled comparative production analysis varying only the host cell species but keeping all other rAAV production parameters the same. We demonstrate that host cell species is critical for determining vector potency. Given these key findings, we then sought to deeply characterize differences in rAAVs when produced by these two manufacturing platforms with multiple analytical approaches including: proteomic profiling by mass spectrometry, isoelectric focusing, cryo-EM, denaturation assays, genomic and epigenomic sequencing of packaged genomes, human cytokine profiling, and comparative functional transduction assessments in vitro and in vivo, including in humanized liver mice. Using these tools weve made two major discoveries: 1) rAAV capsids have post-translational modifications (PTMs) including glycosylation, acetylation, phosphorylation, methylation and deamidation, and these PTMs differ between platforms; 2) rAAV genomes are methylated during production, and these methylation marks are also differentially deposited between platforms. In addition, our data also demonstrate that host cell protein impurities differ between platforms and can have their own PTMs including potentially immunogenic N-linked glycans. We show that human-produced rAAVs are more potent than baculovirus-Sf9 vectors in various cell types in vitro (P < 0.05-0.0001), in various mouse tissues in vivo (P < 0.03-0.0001), and in human liver in vivo (P < 0.005). Collectively, our findings were reproducible across vendors, including commercial manufacturers, academic core facilities, and individual laboratory preparations. These vector differences may have clinical implications for rAAV receptor binding, trafficking, expression kinetics, expression durability, vector immunogenicity as well as cost considerations.
]]></description>
<dc:creator>Rumachik, N. G.</dc:creator>
<dc:creator>Malaker, S. A.</dc:creator>
<dc:creator>Poweleit, N.</dc:creator>
<dc:creator>Maynard, L. H.</dc:creator>
<dc:creator>Adams, C. M.</dc:creator>
<dc:creator>Leib, R. D.</dc:creator>
<dc:creator>Cirolia, G.</dc:creator>
<dc:creator>Thomas, D.</dc:creator>
<dc:creator>Stamnes, S.</dc:creator>
<dc:creator>Holt, K.</dc:creator>
<dc:creator>Sinn, P.</dc:creator>
<dc:creator>May, A. P.</dc:creator>
<dc:creator>Paulk, N. K.</dc:creator>
<dc:date>2019-05-17</dc:date>
<dc:identifier>doi:10.1101/640169</dc:identifier>
<dc:title><![CDATA[Methods Matter -- Standard Production Platforms For Recombinant AAV Can Produce Chemically And Functionally Distinct Vectors]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory Press</dc:publisher>
<prism:publicationDate>2019-05-17</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://biorxiv.org/cgi/content/short/2020.10.09.333997v1?rss=1">
<title>
<![CDATA[
Precision Cas9 Genome Editing in vivo with All-in-one, Self-targeting AAV Vectors 
]]>
</title>
<link>
https://biorxiv.org/cgi/content/short/2020.10.09.333997v1?rss=1"
</link>
<description><![CDATA[
Adeno-associated virus (AAV) vectors are important delivery platforms for therapeutic genome editing but are severely constrained by cargo limits, especially for large effectors like Cas9s. Simultaneous delivery of multiple vectors can limit dose and efficacy and increase safety risks. The use of compact effectors has enabled single-AAV delivery of Cas9s with 1-3 guides for edits that use end-joining repair pathways, but many precise edits that correct disease-causing mutations in vivo require homology-directed repair (HDR) templates. Here, we describe single-vector, [~]4.8-kb AAV platforms that express Nme2Cas9 and either two sgRNAs to produce segmental deletions, or a single sgRNA with an HDR template. We also examine the utility of Nme2Cas9 target sites in the vector for self-inactivation. We demonstrate that these platforms can effectively treat two disease models [type I hereditary tyrosinemia (HT-I) and mucopolysaccharidosis type I (MPS-I)] in mice. These results will enable single-vector AAVs to achieve diverse therapeutic genome editing outcomes.
]]></description>
<dc:creator>Ibraheim, R.</dc:creator>
<dc:creator>Tai, P. W. L.</dc:creator>
<dc:creator>Mir, A.</dc:creator>
<dc:creator>Javeed, N.</dc:creator>
<dc:creator>Wang, J.</dc:creator>
<dc:creator>Rodriguez, T. C.</dc:creator>
<dc:creator>Nelson, S.</dc:creator>
<dc:creator>Khokhar, E.</dc:creator>
<dc:creator>Mintzer, E.</dc:creator>
<dc:creator>Maitland, S.</dc:creator>
<dc:creator>Cao, Y.</dc:creator>
<dc:creator>Tsagkaraki, E.</dc:creator>
<dc:creator>Wolfe, S. A.</dc:creator>
<dc:creator>Wang, D.</dc:creator>
<dc:creator>Pai, A. A.</dc:creator>
<dc:creator>Xue, W.</dc:creator>
<dc:creator>Gao, G.</dc:creator>
<dc:creator>Sontheimer, E. J.</dc:creator>
<dc:date>2020-10-09</dc:date>
<dc:identifier>doi:10.1101/2020.10.09.333997</dc:identifier>
<dc:title><![CDATA[Precision Cas9 Genome Editing in vivo with All-in-one, Self-targeting AAV Vectors]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory Press</dc:publisher>
<prism:publicationDate>2020-10-09</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://biorxiv.org/cgi/content/short/2021.03.08.434470v1?rss=1">
<title>
<![CDATA[
Genome-wide annotation of gene regulatory elements linked to cell fitness 
]]>
</title>
<link>
https://biorxiv.org/cgi/content/short/2021.03.08.434470v1?rss=1"
</link>
<description><![CDATA[
Noncoding regulatory elements control gene expression and thus govern nearly all biological processes. Epigenomic profiling assays have identified millions of putative regulatory elements, but systematically determining the function of those regulatory elements remains a substantial challenge. Here we adapt CRISPR screening by epigenetic repression to screen all 111,619 putative non-coding regulatory elements defined by open chromatin sites in human K562 leukemia cells for their role in regulating essential cellular processes and proliferation. In an initial screen containing 1,084,704 gRNAs, we implemented an analysis framework to quantify perturbation effects, and nominate 1,108 regulatory elements that strongly impact cell fitness. We tested 8,845 of the primary screen elements in a secondary screen, evaluated their cell-type specificity in a second cancer cell line, and then used a single-cell RNA-seq CRISPR screen to discover 63 connections between distal regulatory elements and target genes. This comprehensive and quantitative genome-wide map of essential gene regulatory elements presents a framework for extensive characterization of noncoding regulatory elements that drive complex cell phenotypes and for prioritizing non-coding genetic variants that may contribute to common traits and disease risk.
]]></description>
<dc:creator>Klann, T.</dc:creator>
<dc:creator>Barrera, A.</dc:creator>
<dc:creator>Ettyreddy, A.</dc:creator>
<dc:creator>Rickels, R.</dc:creator>
<dc:creator>Bryois, J.</dc:creator>
<dc:creator>Jiang, S.</dc:creator>
<dc:creator>Adkar, S.</dc:creator>
<dc:creator>Iglesias, N.</dc:creator>
<dc:creator>Sullivan, P.</dc:creator>
<dc:creator>Reddy, T. E.</dc:creator>
<dc:creator>Crawford, G. E.</dc:creator>
<dc:creator>Gersbach, C.</dc:creator>
<dc:date>2021-03-09</dc:date>
<dc:identifier>doi:10.1101/2021.03.08.434470</dc:identifier>
<dc:title><![CDATA[Genome-wide annotation of gene regulatory elements linked to cell fitness]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory Press</dc:publisher>
<prism:publicationDate>2021-03-09</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://biorxiv.org/cgi/content/short/2021.03.08.434430v1?rss=1">
<title>
<![CDATA[
Transgenic mice for in vivo epigenome editing with CRISPR-based systems 
]]>
</title>
<link>
https://biorxiv.org/cgi/content/short/2021.03.08.434430v1?rss=1"
</link>
<description><![CDATA[
The discovery, characterization, and adaptation of the RNA-guided clustered regularly interspersed short palindromic repeat (CRISPR)-Cas9 system has greatly increased the ease with which genome and epigenome editing can be performed. Fusion of chromatin-modifying domains to the nuclease-deactivated form of Cas9 (dCas9) has enabled targeted gene activation or repression in both cultured cells and in vivo in animal models. However, delivery of the large dCas9 fusion proteins to target cell types and tissues is an obstacle to widespread adoption of these tools for in vivo studies. Here we describe the generation and validation of two conditional transgenic mouse lines for targeted gene regulation, Rosa26:LSL-dCas9-p300 for gene activation and Rosa26:LSL-dCas9-KRAB for gene repression. Using the dCas9p300 and dCas9KRAB transgenic mice we demonstrate activation or repression of genes in both the brain and liver in vivo, and T cells and fibroblasts ex vivo. We show gene regulation and targeted epigenetic modification with gRNAs targeting either transcriptional start sites (TSS) or distal enhancer elements, as well as corresponding changes to downstream phenotypes. These mouse lines are convenient and valuable tools for facile, temporally controlled, and tissue-restricted epigenome editing and manipulation of gene expression in vivo.
]]></description>
<dc:creator>Gemberling, M.</dc:creator>
<dc:creator>Siklenka, K.</dc:creator>
<dc:creator>Rodriguez, E.</dc:creator>
<dc:creator>Eisinger, K.</dc:creator>
<dc:creator>Barrera, A.</dc:creator>
<dc:creator>Liu, F.</dc:creator>
<dc:creator>Kantor, A.</dc:creator>
<dc:creator>Li, L.</dc:creator>
<dc:creator>Cigliola, V.</dc:creator>
<dc:creator>Hazlett, M.</dc:creator>
<dc:creator>Williams, C.</dc:creator>
<dc:creator>Bartelt, L.</dc:creator>
<dc:creator>Bodle, J.</dc:creator>
<dc:creator>Daniels, H.</dc:creator>
<dc:creator>Rouse, C.</dc:creator>
<dc:creator>Hilton, I.</dc:creator>
<dc:creator>Madigan, V.</dc:creator>
<dc:creator>Asokan, A.</dc:creator>
<dc:creator>Ciofani, M.</dc:creator>
<dc:creator>Poss, K.</dc:creator>
<dc:creator>Reddy, T. E.</dc:creator>
<dc:creator>West, A.</dc:creator>
<dc:creator>Gersbach, C.</dc:creator>
<dc:date>2021-03-08</dc:date>
<dc:identifier>doi:10.1101/2021.03.08.434430</dc:identifier>
<dc:title><![CDATA[Transgenic mice for in vivo epigenome editing with CRISPR-based systems]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory Press</dc:publisher>
<prism:publicationDate>2021-03-08</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://biorxiv.org/cgi/content/short/354480v1?rss=1">
<title>
<![CDATA[
5′ Modifications Improve Potency and Efficacy of DNA Donors for Precision Genome Editing 
]]>
</title>
<link>
https://biorxiv.org/cgi/content/short/354480v1?rss=1"
</link>
<description><![CDATA[
Nuclease-directed genome editing is a powerful tool for investigating physiology and has great promise as a therapeutic approach to correct mutations that cause disease. In its most precise form, genome editing can use cellular homology-directed repair (HDR) pathways to insert information from an exogenously supplied DNA repair template (donor) directly into a targeted genomic location. Unfortunately, particularly for long insertions, toxicity and delivery considerations associated with repair template DNA can limit HDR efficacy. Here, we explore chemical modifications to both double-stranded and single-stranded DNA-repair templates. We describe 5'-terminal modifications, including in its simplest form the incorporation of triethylene glycol (TEG) moieties, that consistently increase the frequency of precision editing in the germlines of three animal models (Caenorhabditis elegans, zebrafish, mice) and in cultured human cells.
]]></description>
<dc:creator>Ghanta, K. S.</dc:creator>
<dc:creator>Dokshin, G. A.</dc:creator>
<dc:creator>Mir, A.</dc:creator>
<dc:creator>Krishnamurthy, P. M.</dc:creator>
<dc:creator>Gneid, H.</dc:creator>
<dc:creator>Edraki, A.</dc:creator>
<dc:creator>Watts, J. K.</dc:creator>
<dc:creator>Sontheimer, E. J.</dc:creator>
<dc:creator>Mello, C. C.</dc:creator>
<dc:date>2018-06-22</dc:date>
<dc:identifier>doi:10.1101/354480</dc:identifier>
<dc:title><![CDATA[5′ Modifications Improve Potency and Efficacy of DNA Donors for Precision Genome Editing]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory Press</dc:publisher>
<prism:publicationDate>2018-06-22</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://biorxiv.org/cgi/content/short/2020.12.15.422970v1?rss=1">
<title>
<![CDATA[
Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice 
]]>
</title>
<link>
https://biorxiv.org/cgi/content/short/2020.12.15.422970v1?rss=1"
</link>
<description><![CDATA[
Prime editors (PEs) mediate genome modification without utilizing double-stranded DNA breaks or exogenous donor DNA as a template. PEs facilitate nucleotide substitutions or local insertions or deletions within the genome based on the template sequence encoded within the prime editing guide RNA (pegRNA). However, the efficacy of prime editing in adult mice has not been established. Here we report an NLS-optimized SpCas9-based prime editor that improves genome editing efficiency in both fluorescent reporter cells and at endogenous loci in cultured cell lines. Using this genome modification system, we could also seed tumor formation through somatic cell editing in the adult mouse. Finally, we successfully utilize dual adeno-associated virus (AAVs) for the delivery of a split-intein prime editor and demonstrate that this system enables the correction of a pathogenic mutation in the mouse liver. Our findings further establish the broad potential of this genome editing technology for the directed installation of sequence modifications in vivo, with important implications for disease modeling and correction.
]]></description>
<dc:creator>Liu, P.</dc:creator>
<dc:creator>Liang, S.</dc:creator>
<dc:creator>Zheng, C.</dc:creator>
<dc:creator>Mintzer, E.</dc:creator>
<dc:creator>Zhao, Y. G.</dc:creator>
<dc:creator>Ponnienselvan, K.</dc:creator>
<dc:creator>Mir, A.</dc:creator>
<dc:creator>Sontheimer, E. J.</dc:creator>
<dc:creator>Gao, G.</dc:creator>
<dc:creator>Flotte, T. R.</dc:creator>
<dc:creator>Wolfe, S.</dc:creator>
<dc:creator>Xue, W.</dc:creator>
<dc:date>2020-12-16</dc:date>
<dc:identifier>doi:10.1101/2020.12.15.422970</dc:identifier>
<dc:title><![CDATA[Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory Press</dc:publisher>
<prism:publicationDate>2020-12-16</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://biorxiv.org/cgi/content/short/2021.04.01.438130v1?rss=1">
<title>
<![CDATA[
Diverse ATPase proteins in mobilomes constitute a large potential sink for prokaryotic host ATP 
]]>
</title>
<link>
https://biorxiv.org/cgi/content/short/2021.04.01.438130v1?rss=1"
</link>
<description><![CDATA[
Prokaryote mobilome genomes rely on host machineries for survival and replication. Given that mobile genetic elements (MGEs) derive their energy from host cells, we investigated the diversity of ATP-utilizing proteins in MGE genomes to determine whether they might be associated with proteins that could suppress related host proteins that consume host energy. A comprehensive search of 353 huge phage genomes revealed that up to 9% of the proteins have ATPase domains. For example, ATPase proteins constitute ~3% of the genomes of Lak phages with ~550 kbp genomes that occur in the microbiomes of humans and other animals. Statistical analysis shows the number of ATPase proteins increases linearly with genome length, consistent with a large sink for host ATP during replication of megaphages. Using metagenomic data from diverse environments, we found 505 mobilome proteins with ATPase domains fused to diverse functional domains. Among these composite ATPase proteins, 61.6% have known functional domains that could contribute to host energy diversion during the mobilome life cycle. As many have domains that are known to interact with nucleic acids and proteins, we infer that numerous ATPase proteins are used during replication and for protection from host immune systems. We found a set of uncharacterized ATPase proteins with nuclease and protease activities, displaying unique domain architectures that are energy intensive based on the presence of multiple ATPase domains. In many cases, these composite ATPase proteins genomically co-localize with small proteins in genomic contexts that are reminiscent of toxin-antitoxin systems and phage helicase-antibacterial helicase systems. Small proteins that function as inhibitors may be a common strategy for control of cellular processes, thus could inspire the development of new nucleic acid and protein manipulation tools, with diverse biotechnological applications.
]]></description>
<dc:creator>Shim, H.</dc:creator>
<dc:creator>Shivram, H.</dc:creator>
<dc:creator>Lei, S.</dc:creator>
<dc:creator>Doudna, J. A.</dc:creator>
<dc:creator>Banfield, J.</dc:creator>
<dc:date>2021-04-01</dc:date>
<dc:identifier>doi:10.1101/2021.04.01.438130</dc:identifier>
<dc:title><![CDATA[Diverse ATPase proteins in mobilomes constitute a large potential sink for prokaryotic host ATP]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory Press</dc:publisher>
<prism:publicationDate>2021-04-01</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://biorxiv.org/cgi/content/short/2021.05.18.444740v1?rss=1">
<title>
<![CDATA[
The FusX TALE Base Editor (FusXTBE) for rapid mitochondrial DNA programming of human cells in vitro and zebrafish disease models in vivo 
]]>
</title>
<link>
https://biorxiv.org/cgi/content/short/2021.05.18.444740v1?rss=1"
</link>
<description><![CDATA[
Functional analyses of mitochondria have been hampered by few effective approaches to manipulate mtDNA and a lack of existing animal models. Recently a TALE-derived base editor was shown to induce C-to-T (or G-to-A) sequence changes in mtDNA. We report here the FusX TALE Base Editor (FusXTBE) to facilitate broad-based access to TALE mitochondrial base editing technology. TALE Writer is a de novo in silico design tool to map potential mtDNA base editing sites. FusXTBE was demonstrated to function with comparable activity to the initial base editor in human cells in vitro. Zebrafish embryos were used as a pioneering in vivo test system, with FusXTBE inducing 90+% editing efficiency in mtDNA loci, the first example of majority mtDNA heteroplasmy induction in any system. Gene editing specificity as precise as a single nucleotide was observed in vivo for a protein-coding gene. Non-destructive genotyping enables single animal mtDNA analyses for downstream biological functional genomics applications. FusXTBE is a new gene editing toolkit for exploring important questions in mitochondrial biology and genetics.
]]></description>
<dc:creator>Sabharwal, A.</dc:creator>
<dc:creator>Kar, B.</dc:creator>
<dc:creator>Castillo, S. R.</dc:creator>
<dc:creator>Holmberg, S. R.</dc:creator>
<dc:creator>Kendall, B. L.</dc:creator>
<dc:creator>Cotter, R. P.</dc:creator>
<dc:creator>WareJoncas, Z.</dc:creator>
<dc:creator>Clark, K. J.</dc:creator>
<dc:creator>Ekker, S. C.</dc:creator>
<dc:date>2021-05-20</dc:date>
<dc:identifier>doi:10.1101/2021.05.18.444740</dc:identifier>
<dc:title><![CDATA[The FusX TALE Base Editor (FusXTBE) for rapid mitochondrial DNA programming of human cells in vitro and zebrafish disease models in vivo]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory Press</dc:publisher>
<prism:publicationDate>2021-05-20</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://biorxiv.org/cgi/content/short/2023.03.20.533459v1?rss=1">
<title>
<![CDATA[
Self-delivering CRISPR RNAs for AAV Co-delivery and Genome Editing in vivo 
]]>
</title>
<link>
https://biorxiv.org/cgi/content/short/2023.03.20.533459v1?rss=1"
</link>
<description><![CDATA[
Guide RNAs offer programmability for CRISPR-Cas9 genome editing but also add challenges for delivery. Chemical modification, which has been key to the success of oligonucleotide therapeutics, can enhance the stability, distribution, cellular uptake, and safety of nucleic acids. Previously, we engineered heavily and fully modified SpyCas9 crRNA and tracrRNA, which showed enhanced stability and retained activity when delivered to cultured cells in the form of the ribonucleoprotein complex. In this study, we report that a short, fully stabilized oligonucleotide (a "protecting oligo"), which can be displaced by tracrRNA annealing, can significantly enhance the potency and stability of a heavily modified crRNA. Furthermore, protecting oligos allow various bioconjugates to be appended, thereby improving cellular uptake and biodistribution of crRNA in vivo. Finally, we achieved in vivo genome editing in adult mouse liver and central nervous system via co-delivery of unformulated, chemically modified crRNAs with protecting oligos and AAV vectors that express tracrRNA and either SpyCas9 or a base editor derivative. Our proof-of-concept establishment of AAV/crRNA co-delivery offers a route towards transient editing activity, target multiplexing, guide redosing, and vector inactivation.
]]></description>
<dc:creator>Zhang, H.</dc:creator>
<dc:creator>Kelly, K.</dc:creator>
<dc:creator>Lee, J.</dc:creator>
<dc:creator>Echeverria, D.</dc:creator>
<dc:creator>Cooper, D.</dc:creator>
<dc:creator>Panwala, R.</dc:creator>
<dc:creator>Chen, Z.</dc:creator>
<dc:creator>Gaston, N.</dc:creator>
<dc:creator>Newby, G. A.</dc:creator>
<dc:creator>Xie, J.</dc:creator>
<dc:creator>Liu, D. R.</dc:creator>
<dc:creator>Gao, G.</dc:creator>
<dc:creator>Wolfe, S. A.</dc:creator>
<dc:creator>Khvorova, A.</dc:creator>
<dc:creator>Watts, J. K.</dc:creator>
<dc:creator>Sontheimer, E. J.</dc:creator>
<dc:date>2023-03-20</dc:date>
<dc:identifier>doi:10.1101/2023.03.20.533459</dc:identifier>
<dc:title><![CDATA[Self-delivering CRISPR RNAs for AAV Co-delivery and Genome Editing in vivo]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory Press</dc:publisher>
<prism:publicationDate>2023-03-20</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://biorxiv.org/cgi/content/short/2023.05.01.538906v1?rss=1">
<title>
<![CDATA[
Orthogonal CRISPR screens to identify transcriptional and epigenetic regulators of human CD8 T cell function 
]]>
</title>
<link>
https://biorxiv.org/cgi/content/short/2023.05.01.538906v1?rss=1"
</link>
<description><![CDATA[
The clinical response to adoptive T cell therapies is strongly associated with transcriptional and epigenetic state. Thus, technologies to discover regulators of T cell gene networks and their corresponding phenotypes have great potential to improve the efficacy of T cell therapies. We developed pooled CRISPR screening approaches with compact epigenome editors to systematically profile the effects of activation and repression of 120 transcription factors and epigenetic modifiers on human CD8+ T cell state. These screens nominated known and novel regulators of T cell phenotypes with BATF3 emerging as a high confidence gene in both screens. We found that BATF3 overexpression promoted specific features of memory T cells such as increased IL7R expression and glycolytic capacity, while attenuating gene programs associated with cytotoxicity, regulatory T cell function, and T cell exhaustion. In the context of chronic antigen stimulation, BATF3 overexpression countered phenotypic and epigenetic signatures of T cell exhaustion. CAR T cells overexpressing BATF3 significantly outperformed control CAR T cells in both in vitro and in vivo tumor models. Moreover, we found that BATF3 programmed a transcriptional profile that correlated with positive clinical response to adoptive T cell therapy. Finally, we performed CRISPR knockout screens with and without BATF3 overexpression to define co-factors and downstream factors of BATF3, as well as other therapeutic targets. These screens pointed to a model where BATF3 interacts with JUNB and IRF4 to regulate gene expression and illuminated several other novel targets for further investigation.
]]></description>
<dc:creator>McCutcheon, S.</dc:creator>
<dc:creator>Swartz, A.</dc:creator>
<dc:creator>Brown, M.</dc:creator>
<dc:creator>Barrera, A.</dc:creator>
<dc:creator>McRoberts Amador, C.</dc:creator>
<dc:creator>Siklenka, K.</dc:creator>
<dc:creator>Humayun, L.</dc:creator>
<dc:creator>Isaacs, J.</dc:creator>
<dc:creator>Reddy, T. E.</dc:creator>
<dc:creator>Nair, S.</dc:creator>
<dc:creator>Antonia, S.</dc:creator>
<dc:creator>Gersbach, C. A.</dc:creator>
<dc:date>2023-05-01</dc:date>
<dc:identifier>doi:10.1101/2023.05.01.538906</dc:identifier>
<dc:title><![CDATA[Orthogonal CRISPR screens to identify transcriptional and epigenetic regulators of human CD8 T cell function]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory Press</dc:publisher>
<prism:publicationDate>2023-05-01</prism:publicationDate>
<prism:section></prism:section>
</item>
</rdf:RDF>
