Person: Joung, Keith
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Joung
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Keith
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Joung, Keith
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Publication Transcriptome-Wide Off-Target RNA Editing Induced by CRISPR-Guided DNA Base Editors(Springer Science and Business Media LLC, 2019-04-17) Grünewald, Julian; Zhou, Ronghao; Garcia, Sara; Iyer, Sowmya; Lareau, Caleb; Aryee, Martin; Joung, KeithCRISPR-Cas base editor technology enables targeted nucleotide alterations and is being rapidly deployed for research and potential therapeutic applications. The most widely used base editors induce DNA cytosine (C) deamination with rat APOBEC1 (rAPOBEC1) enzyme, which is targeted by a linked Cas protein-guide RNA (gRNA) complex. Previous studies of cytosine base editor (CBE) specificity have identified off-target DNA edits in human cells. Here we show that a CBE with rAPOBEC1 can cause extensive transcriptome-wide RNA cytosine deamination in human cells, inducing tens of thousands of C-to-uracil (U) edits with frequencies ranging from 0.07% to 100% in 38% - 58% of expressed genes. CBE-induced RNA edits occur in both protein-coding and non-protein-coding sequences and generate missense, nonsense, splice site, 5’ UTR, and 3’ UTR mutations. We engineered two CBE variants bearing rAPOBEC1 mutations that substantially decrease the numbers of RNA edits (reductions of >390-fold and >3,800-fold) in human cells. These variants also showed more precise on-target DNA editing and, with the majority of gRNAs tested, editing efficiencies comparable to those observed with wild-type CBE. Finally, we show that recently described adenine base editors (ABEs) can also induce transcriptome-wide RNA edits. These results have important implications for the research and therapeutic uses of base editors, illustrate the feasibility of engineering improved variants with reduced RNA editing activities, and suggest the need to more fully define and characterize the RNA off-target effects of deaminase enzymes in base editor platforms.Publication Pathways Disrupted in Human ALS Motor Neurons Identified through Genetic Correction of Mutant SOD1(Elsevier BV, 2014) Kiskinis, Evangelos; Sandoe, Jackson L; Williams, Lauren; Boulting, Gabriella; Moccia, Robert; Wainger, Brian; Han, Steve Sang-woo; Peng, Theodore; Thams, Sebastian; Mikkilineni, Shravani; Mellin, Cassidy; Merkle, Florian; Davis-Dusenbery, Brandi N; Ziller, Michael; Oakley, Derek; Ichida, Justin; Di Costanzo, Stefania; Atwater, Nick; Maeder, M; Goodwin, Marcus; Nemesh, James; Handsaker, Robert; Paull, Daniel; Noggle, Scott; McCarroll, Steven; Joung, Keith; Woolf, Carl; Brown, Robert H; Eggan, KevinDirect electrical recording and stimulation of neural activity using micro-fabricated silicon and metal micro-wire probes have contributed extensively to basic neuroscience and therapeutic applications; however, the dimensional and mechanical mismatch of these probes with the brain tissue limits their stability in chronic implants and decreases the neuron–device contact. Here, we demonstrate the realization of a three-dimensional macroporous nanoelectronic brain probe that combines ultra-flexibility and subcellular feature sizes to overcome these limitations. Built-in strains controlling the local geometry of the macroporous devices are designed to optimize the neuron/probe interface and to promote integration with the brain tissue while introducing minimal mechanical perturbation. The ultra-flexible probes were implanted frozen into rodent brains and used to record multiplexed local field potentials and single-unit action potentials from the somatosensory cortex. Significantly, histology analysis revealed filling-in of neural tissue through the macroporous network and attractive neuron–probe interactions, consistent with long-term biocompatibility of the device.Publication Translating the Genomics Revolution: The Need for an International Gene Therapy Consortium for Monogenic Diseases(Nature Publishing Group, 2013) Tremblay, Jacques P; Xiao, Xiao; Aartsma-Rus, Annemieke; Barbas, Carlos; Blau, Helen M; Bogdanove, Adam J; Boycott, Kym; Braun, Serge; Breakefield, Xandra; Bueren, Juan A; Buschmann, Michael; Byrne, Barry J; Calos, Michele; Cathomen, Toni; Chamberlain, Jeffrey; Chuah, Marinee; Cornetta, Kenneth; Davies, Kay E; Dickson, J George; Duchateau, Philippe; Flotte, Terence R; Gaudet, Daniel; Gersbach, Charles A; Gilbert, Renald; Glorioso, Joseph; Herzog, Roland W; High, Katherine A; Huang, Wenlin; Huard, Johnny; Joung, Keith; Liu, Depei; Liu, Dexi; Lochmüller, Hanns; Lustig, Lawrence; Martens, Jeffrey; Massie, Bernard; Mavilio, Fulvio; Mendell, Jerry R; Nathwani, Amit; Ponder, Katherine; Porteus, Matthew; Puymirat, Jack; Samulski, Jude; Takeda, Shin'ichi; Thrasher, Adrian; VandenDriessche, Thierry; Wei, Yuquan; Wilson, James M; Wilton, Steve D; Wolfe, John H; Gao, GuangpingPublication Engineered zinc finger nickases induce homology-directed repair with reduced mutagenic effects(Oxford University Press, 2012) Ramirez, Cherie Lynn; Certo, Michael T.; Mussolino, Claudio; Goodwin, Matthew J.; Cradick, Thomas J.; McCaffrey, Anton P.; Cathomen, Toni; Scharenberg, Andrew M.; Joung, KeithEngineered zinc finger nucleases (ZFNs) induce DNA double-strand breaks at specific recognition sequences and can promote efficient introduction of desired insertions, deletions or substitutions at or near the cut site via homology-directed repair (HDR) with a double- and/or single-stranded donor DNA template. However, mutagenic events caused by error-prone non-homologous end-joining (NHEJ)-mediated repair are introduced with equal or higher frequency at the nuclease cleavage site. Furthermore, unintended mutations can also result from NHEJ-mediated repair of off-target nuclease cleavage sites. Here, we describe a simple and general method for converting engineered ZFNs into zinc finger nickases (ZFNickases) by inactivating the catalytic activity of one monomer in a ZFN dimer. ZFNickases show robust strand-specific nicking activity in vitro. In addition, we demonstrate that ZFNickases can stimulate HDR at their nicking site in human cells, albeit at a lower frequency than by the ZFNs from which they were derived. Finally, we find that ZFNickases appear to induce greatly reduced levels of mutagenic NHEJ at their target nicking site. ZFNickases thus provide a promising means for inducing HDR-mediated gene modifications while reducing unwanted mutagenesis caused by error-prone NHEJ.Publication Improved Somatic Mutagenesis in Zebrafish Using Transcription Activator-Like Effector Nucleases (TALENs)(Public Library of Science, 2012) Moore, Finola E.; Reyon, Deepak; Sander, Jeffry D.; Martinez, Sarah A.; Blackburn, Jessica S.; Khayter, Cyd; Ramirez, Cherie Lynn; Joung, Keith; Langenau, DavidZinc Finger Nucleases (ZFNs) made by Context-Dependent Assembly (CoDA) and Transcription Activator-Like Effector Nucleases (TALENs) provide robust and user-friendly technologies for efficiently inactivating genes in zebrafish. These designer nucleases bind to and cleave DNA at particular target sites, inducing error-prone repair that can result in insertion or deletion mutations. Here, we assess the relative efficiencies of these technologies for inducing somatic DNA mutations in mosaic zebrafish. We find that TALENs exhibited a higher success rate for obtaining active nucleases capable of inducing mutations than compared with CoDA ZFNs. For example, all six TALENs tested induced DNA mutations at genomic target sites while only a subset of CoDA ZFNs exhibited detectable rates of mutagenesis. TALENs also exhibited higher mutation rates than CoDA ZFNs that had not been pre-screened using a bacterial two-hybrid assay, with DNA mutation rates ranging from 20%–76.8% compared to 1.1%–3.3%. Furthermore, the broader targeting range of TALENs enabled us to induce mutations at the methionine translation start site, sequences that were not targetable using the CoDA ZFN platform. TALENs exhibited similar toxicity to CoDA ZFNs, with >50% of injected animals surviving to 3 days of life. Taken together, our results suggest that TALEN technology provides a robust alternative to CoDA ZFNs for inducing targeted gene-inactivation in zebrafish, making it a preferred technology for creating targeted knockout mutants in zebrafish.Publication Evaluation of OPEN Zinc Finger Nucleases for Direct Gene Targeting of the ROSA26 Locus in Mouse Embryos(Public Library of Science, 2012) Hermann, Mario; Rector, Kyle; Ruiz, Joseph; Becher, Burkhard; Bürki, Kurt; Khayter, Cyd; Aguzzi, Adriano; Buch, Thorsten; Pelczar, Pawel; Maeder, Morgan L.; Joung, KeithZinc finger nucleases (ZFNs) enable precise genome modification in a variety of organisms and cell types. Commercial ZFNs were reported to enhance gene targeting directly in mouse zygotes, whereas similar approaches using publicly available resources have not yet been described. Here we report precise targeted mutagenesis of the mouse genome using Oligomerized Pool Engineering (OPEN) ZFNs. OPEN ZFN can be constructed using publicly available resources and therefore provide an attractive alternative for academic researchers. Two ZFN pairs specific to the mouse genomic locus gt(ROSA26)Sor were generated by OPEN selections and used for gene disruption and homology-mediated gene replacement in single cell mouse embryos. One specific ZFN pair facilitated non-homologous end joining (NHEJ)-mediated gene disruption when expressed in mouse zygotes. We also observed a single homologous recombination (HR)-driven gene replacement event when this ZFN pair was co-injected with a targeting vector. Our experiments demonstrate the feasibility of achieving both gene ablation through NHEJ and gene replacement by HR by using the OPEN ZFN technology directly in mouse zygotes.Publication Highly Efficient Generation of Heritable Zebrafish Gene Mutations Using Homo- and Heterodimeric TALENs(Oxford University Press, 2012) Cade, Lindsay; Reyon, Deepak; Hwang, Woong Y.; Tsai, Shengdar Q.; Patel, Samir; Khayter, Cyd; Joung, Keith; Sander, Jeffry D.; Peterson, Randall; Yeh, Jing-RueyTranscription activator-like effector nucleases (TALENs) are powerful new research tools that enable targeted gene disruption in a wide variety of model organisms. Recent work has shown that TALENs can induce mutations in endogenous zebrafish genes, but to date only four genes have been altered, and larger-scale tests of the success rate, mutation efficiencies and germline transmission rates have not been described. Here, we constructed homodimeric TALENs to 10 different targets in various endogenous zebrafish genes and found that 7 nuclease pairs induced targeted indel mutations with high efficiencies ranging from 2 to 76%. We also tested obligate heterodimeric TALENs and found that these nucleases induce mutations with comparable or higher frequencies and have better toxicity profiles than their homodimeric counterparts. Importantly, mutations induced by both homodimeric and heterodimeric TALENs are passed efficiently through the germline, in some cases reaching 100% transmission. For one target gene sequence, we observed substantially reduced mutagenesis efficiency for a variant site bearing two mismatched nucleotides, raising the possibility that TALENs might be used to perform allele-specific gene disruption. Our results suggest that construction of one to two heterodimeric TALEN pairs for any given gene will, in most cases, enable researchers to rapidly generate knockout zebrafish.Publication Identification of promoter targets of enhancers by epigenetic knockdown using TAL DNA binding proteins(BioMed Central, 2013) Mendenhall, Eric M; Williamson, Kaylyn; Reyon, Deepak; Joung, Keith; Bernstein, BradleyPublication Zinc Finger Database (ZiFDB): A Repository for Information on C2H2 Zinc Fingers and Engineered Zinc-Finger Arrays(Oxford University Press, 2009) Fu, Fengli; Sander, Jeffry D.; Maeder, Morgan; Thibodeau-Beganny, Stacey; Joung, Keith; Dobbs, Drena; Miller, Leslie; Voytas, Daniel F.Zinc fingers are the most abundant DNA-binding motifs encoded by eukaryotic genomes and one of the best understood DNA-recognition domains. Each zinc finger typically binds a 3-nt target sequence, and it is possible to engineer zinc-finger arrays (ZFAs) that recognize extended DNA sequences by linking together individual zinc fingers. Engineered zinc-finger proteins have proven to be valuable tools for gene regulation and genome modification because they target specific sites in a genome. Here we describe ZiFDB (Zinc Finger Database; http://bindr.gdcb.iastate.edu/ZiFDB), a web-accessible resource that compiles information on individual zinc fingers and engineered ZFAs. To enhance its utility, ZiFDB is linked to the output from ZiFiT—a software package that assists biologists in finding sites within target genes for engineering zinc-finger proteins. For many molecular biologists, ZiFDB will be particularly valuable for determining if a given ZFA (or portion thereof) has previously been constructed and whether or not it has the requisite DNA-binding activity for their experiments. ZiFDB will also be a valuable resource for those scientists interested in better understanding how zinc-finger proteins recognize target DNA.Publication Rapid Mutation of Endogenous Zebrafish Genes Using Zinc Finger Nucleases Made by Oligomerized Pool ENgineering (OPEN)(Public Library of Science, 2009) Foley, Jonathan E.; Yeh, Jing-Ruey; Maeder, Morgan L.; Reyon, Deepak; Sander, Jeffry D.; Peterson, Randall; Joung, KeithBackground: Customized zinc finger nucleases (ZFNs) form the basis of a broadly applicable tool for highly efficient genome modification. ZFNs are artificial restriction endonucleases consisting of a non-specific nuclease domain fused to a zinc finger array which can be engineered to recognize specific DNA sequences of interest. Recent proof-of-principle experiments have shown that targeted knockout mutations can be efficiently generated in endogenous zebrafish genes via non-homologous end-joining-mediated repair of ZFN-induced DNA double-stranded breaks. The Zinc Finger Consortium, a group of academic laboratories committed to the development of engineered zinc finger technology, recently described the first rapid, highly effective, and publicly available method for engineering zinc finger arrays. The Consortium has previously used this new method (known as OPEN for Oligomerized Pool ENgineering) to generate high quality ZFN pairs that function in human and plant cells. Methodology/Principal Findings: Here we show that OPEN can also be used to generate ZFNs that function efficiently in zebrafish. Using OPEN, we successfully engineered ZFN pairs for five endogenous zebrafish genes: tfr2, dopamine transporter, telomerase, hif1aa, and gridlock. Each of these ZFN pairs induces targeted insertions and deletions with high efficiency at its endogenous gene target in somatic zebrafish cells. In addition, these mutations are transmitted through the germline with sufficiently high frequency such that only a small number of fish need to be screened to identify founders. Finally, in silico analysis demonstrates that one or more potential OPEN ZFN sites can be found within the first three coding exons of more than 25,000 different endogenous zebrafish gene transcripts. Conclusions and Significance: In summary, our study nearly triples the total number of endogenous zebrafish genes successfully modified using ZFNs (from three to eight) and suggests that OPEN provides a reliable method for introducing targeted mutations in nearly any zebrafish gene of interest.