FusX: A Rapid One-Step Transcription Activator-Like Effector Assembly System for Genome Science

Alvin, C. H.; McNulty, Melissa; Poshusta, Tanya L.; Campbell, Jarryd M.; Martínez‐Gálvez, Gabriel; Argue, David P.; Lee, Han B.; Urban, Mark D.; Bullard, Cassandra E.; Blackburn, Patrick R.; Man, Toni K.; Clark, Karl J.; Ekker, Stephen C.

doi:10.1089/hum.2015.172

Cited by 48 publications

(51 citation statements)

References 52 publications

(58 reference statements)

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“…The Ekker lab takes scalable assembly of TALENs a step further, reducing the hands-on time for construction of new reagents to a single-day, single-tube reaction via the FusX system. 2 Furthermore, the FusX method of TALEN construction can be automated using liquid handling robots and simple inventory management programming that won't break the laboratory budget.…”

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confidence: 99%

Gene Editing: Practical Application in the Lab

Geurts

Ekker

2016

Human Gene Therapy

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confidence: 99%

Gene Editing: Practical Application in the Lab

Geurts

Ekker

2016

Human Gene Therapy

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“…TALEN constructs were generated using the FusX kit (Addgene # 1000000063) as previously described(30). In short, RCIscript-GoldyTALEN was linearized with BsmBI (NEB R0580.…”

Section: Methodsmentioning

confidence: 99%

Toward Precision Molecular Surgery: Robust, Selective Induction of Microhomology-mediated End Joining in vivo

Ata

Ekstrom

Martínez‐Gálvez

et al. 2018

Preprint

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One key problem in precision genome editing is the resultant unpredictable plurality of sequence outcomes at the site of targeted DNA double-strand breaks (DSBs). This is due to the typical activation of the versatile Non-homologous End Joining (NHEJ) pathway. Such unpredictability limits the utility of somatic gene editing for applications including gene therapy and functional genomics. For germline editing work, the accurate reproduction of identical alleles using NHEJ is a labor intensive process. In this study, we propose inducing Microhomology-mediated End Joining (MMEJ) as a viable solution for improving somatic sequence homogeneity in vivo, capable of generating a single predictable allele at high rates (56% ~ 86% of the entire mutant allele pool). Using a combined dataset from zebrafish (Danio rerio) in vivo and human HeLa cell in vitro as a training dataset, we identified specific contextual sequence determinants surrounding genomic DSBs for robust MMEJ pathway activation. We then applied our observation and prospectively designed MMEJ-inducing sgRNAs against a variety of proof-of-principle genes and demonstrated a high level of mutant allele homogeneity at these loci. F0 mutant zebrafish embryos and larvae generated with these gRNAs faithfully recapitulated previously reported, recessive loss-of-function phenotypes. We also provide a novel algorithm MENTHU (http://genesculpt.org/menthu/) for improved prediction of candidate MMEJ loci, suitable for both targeted and genome-wide applications. We believe that this MMEJ-centric approach will have a broad impact on genome engineering and its applications. For example, whereas somatic mosaicism hinders efficient recreation of a knockout mutant allele at base pair resolution via the standard NHEJ-based approach, we demonstrate that F0 founders transmitted the identical MMEJ allele of interest at high rates. Most importantly, the ability to directly dictate the reading frame of an endogenous target will have important implications for gene therapy applications in human genetic diseases.Author SummaryNew gene editing tools precisely break DNA at pre-defined genomic locations, but cells repair these lesions using diverse pathways that often lead to unpredictable outcomes in the resulting DNA sequences. This sequence diversity in gene editing outcomes represents an important obstacle to the application of this technology for human therapies. Using a vertebrate animal as a model system, we provide strong evidence that we can overcome this obstacle by selectively directing DNA repair of double-stranded breaks through a lesser-described pathway termed Microhomology-mediated End Joining (MMEJ). Unlike other, better-understood pathways, MMEJ uses recurring short sequence patterns surrounding the site of DNA breakage. This enables the prediction of repair outcomes with improved accuracy. Importantly, we also show that preferential activation of MMEJ is compatible with effective gene editing. Finally, we provide a simple algorithm and software for designing DNA-breaking reagents that have high chance of activating the MMEJ pathway. We believe that the MMEJ-centric approach to be broadly applicable for a variety of gene editing applications both within the laboratory and for human therapies.Author ContributionHA contributed in Conceptualization, Data Curation, Formal Analysis, Investigation, Funding Acquisition, Methodology, Validation, Visualization, Writing – Original draft preparation, and Writing – Review and Editing. TLE contributed in Data Curation, Investigation, Writing – Original draft preparation, and Writing – Review and Editing. GMG contributed in Software, Validation, and Writing. CMM contributed in Software Validation, and Writing. AVD contributed in Investigation, Methodology, Validation, and Writing – Review and Editing. KJS contributed in Investigation and Writing – Review and Editing. ACM contributed in Conceptualization, Data Curation, Investigation, and Writing – Review and Editing. DD contributed in Funding Acquisition, Resources, and Writing – Review and Editing. KJC contributed in Conceptualization, Funding Acquisition, Resources, Supervision, and Writing – Review and Editing. SCE contributed in Conceptualization, Funding Acquisition, Project Administration, Resources, Supervision, Writing – Review and Editing.

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“…One option for mtDNA targeting includes CRISPR/Cas9; however, targeting to mitochondria would require effective gRNA importation, a hurdle yet unresolved 1,17 . TALE proteins are traditionally more difficult to assemble than CRISPR/Cas9, but single-reaction synthesis technology makes TALEs readily accessible for genome engineering applications 18 . Importantly, TALE use in mtDNA engineering only require a single mitochondrial targeting sequence (S Figure 1) that was developed from an in vivo protein trapping method 19 .…”

Section: Textmentioning

confidence: 99%

“…The final mitoTALE backbone plasmids used in this paper will be made available at Addgene. To target a specific sequences in the genome, RVDs were cloned into the GoldyTALE backbone using the FusX assembly system 18 . The RVDs used were HD=C, NN=G, NI=A, NG=T.…”

Section: Mitotale-nickase and -Nuclease Assemblymentioning

confidence: 99%

Engineering targeted deletions in the mitochondrial genome

Campbell

Perales‐Clemente

Ata

et al. 2018

Preprint

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ParagraphMitochondria are a network of critical intracellular organelles with diverse functions ranging from energy production to cell signaling. The mitochondrial genome (mtDNA) consists of 37 genes that support oxidative phosphorylation and are prone to dysfunction that can lead to currently untreatable diseases. Further characterization of mtDNA gene function and creation of more accurate models of human disease will require the ability to engineer precise genomic sequence modifications. To date, mtDNA has been inaccessible to direct modification using traditional genome engineering tools due to unique DNA repair contexts in mitochondria 1 . Here, we report a new DNA modification process using sequence-specific transcription activator-like effector (TALE) proteins to manipulate mtDNA in vivo and in vitro for reverse genetics applications. First, we show mtDNA deletions can be induced in Danio rerio (zebrafish) using site-directed mitoTALEnickases (mito-nickases). Using this approach, the protein-encoding mtDNA gene nd4 was deleted in injected zebrafish embryos. Furthermore, this DNA engineering system recreated a large deletion spanning from nd5 to atp8, which is commonly found in human diseases like Kearns-Sayre syndrome (KSS) and Pearson syndrome. Enrichment of mtDNA-deleted genomes was achieved using targeted mitoTALE-nucleases (mitoTALENs) by co-delivering both mito-nickases and mitoTALENs into zebrafish embryos. This combined approach yielded deletions in over 90% of injected animals, which were maintained through adulthood in various tissues. Subsequently, we confirmed that large, targeted deletions could be induced with this approach in human cells. In addition, we show that, when provided with a single nick on the mtDNA light strand, the binding of a terminal TALE protein alone at the intended recombination site is sufficient for deletion induction. This "block and nick" approach yielded engineered mitochondrial molecules with single nucleotide precision using two different targeted deletion sites. This precise seeding method to engineer mtDNA variants is a critical step for the exploration of mtDNA function and for creating new cellular and animal models of mitochondrial disease.

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FusX: A Rapid One-Step Transcription Activator-Like Effector Assembly System for Genome Science

Cited by 48 publications

References 52 publications

Gene Editing: Practical Application in the Lab

Gene Editing: Practical Application in the Lab

Toward Precision Molecular Surgery: Robust, Selective Induction of Microhomology-mediated End Joining in vivo

Engineering targeted deletions in the mitochondrial genome

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