Tissue-specific genome editing in <i>Ciona</i> embryos by CRISPR/Cas9

Stolfi, Alberto; Gandhi, Shashank; Salek, Farhana; Christiaen, Lionel

doi:10.1242/dev.114488

Cited by 136 publications

(193 citation statements)

References 49 publications

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“…We therefore also performed analysis of the same crispants using Sanger sequencing of a limited number of individual clones from PCR products, as routinely reported to assess mutagenesis efficiency (Auer et al, 2014a;Jao et al, 2013;Stolfi et al, 2014;Varshney et al, 2015). For analyzed targets with good sequence coverage (n=12 or more), our analysis revealed that limited Sanger sequencing data strongly correlate with our MiSeq data: for all analyzed loci, Sanger sequencing of subcloned PCR fragments (1) reliably established a mutagenesis efficiency estimate ( Fig.…”

Section: Crispants Replicate Loss-of-function Phenotypesmentioning

confidence: 57%

“…S5). This observation reveals that randomly generated in-frame alleles, which potentially maintain open reading frame integrity (yet nonetheless might impact amino acid residues important for protein function), are a major uncontrolled variable in the use of crispants induced by any method to directly assess loss-of-function phenotypes, both on a whole-embryo (Shah et al, 2015; this study) and on a tissue-specific (Ablain et al, 2015;Stolfi et al, 2014) scale.…”

Section: Crispants Replicate Loss-of-function Phenotypesmentioning

confidence: 97%

“…Somatic and tissue-specific mutagenesis has recently been reported for Ciona embryos (Stolfi et al, 2014), is possible for assessing tumorigenesis in mice (Platt et al, 2014), and is achievable using mosaic transgene injection in zebrafish (Ablain et al, 2015). Reproducible and significant phenotype penetrance and expressivity in a given cell type or on a whole-embryo scale requires a mutagenesis efficiency close to or reaching saturation, ideally by providing a limited number of alleles.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.

et al. 2016

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CRISPR-Cas9 enables efficient sequence-specific mutagenesis for creating somatic or germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-sgRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we apply in vitro assembled, fluorescent Cas9-sgRNA RNPs in solubilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. MiSeq-based sequence analysis of targeted loci in individual embryos using CrispRVariants, a customized software tool for mutagenesis quantification and visualization, reveals efficient biallelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis exposes loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show that targeting of non-coding elements in gene regulatory regions using saturating mutagenesis uncovers functional control elements in transgenic reporters and endogenous genes in injected embryos. Our results establish that optimally solubilized, in vitro assembled fluorescent Cas9-sgRNA RNPs provide a reproducible reagent for direct and scalable loss-of-function studies and applications beyond zebrafish experiments that require maximal DNA cutting efficiency in vivo.

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Section: Crispants Replicate Loss-of-function Phenotypesmentioning

confidence: 57%

Section: Crispants Replicate Loss-of-function Phenotypesmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

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Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.

et al. 2016

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“…In recent years, electroporation was successfully adapted to achieve tissue-targeted gene manipulation with plasmids that express gene editing tools like the CRISPR/Cas9 components under the control of tissue specific drivers 21,22 . Such approaches will quickly advance our understanding of the dynamics of GRNs and the potentially conserved signaling mechanisms, including transcription factor codes involved in tissue formation and the stepwise exit from pluripotency.…”

Section: Versatility and Potential Of Electroporation For Genetic Engmentioning

confidence: 99%

Embryo Microinjection and Electroporation in the Chordate <em>Ciona intestinalis</em>

Kari

Zeng

Zitzelsberger

et al. 2016

JoVE

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Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance to man of conventional invertebrate model systems and the complexity of genomes in vertebrates are critical challenges to modeling human normal and pathological conditions. The chordate Ciona intestinalis is an invertebrate chordate that emerged from a common ancestor with the vertebrates and may represent features at the interface between invertebrates and vertebrates. A common body plan with much simpler cellular and genomic composition should unveil gene regulatory network (GRN) links and functional genomics readouts explaining phenomena in the vertebrate condition. The compact genome of Ciona, a fixed embryonic lineage with few divisions and large cells, combined with versatile community tools foster efficient gene functional analyses in this organism. Here, we present several crucial methods for this promising model organism, which belongs to the closest sister group to vertebrates. We present protocols for transient transgenesis by electroporation, along with microinjection-mediated gene knockdown, which together provide the means to study gene function and genomic regulatory elements. We extend our protocols to provide information on how community databases are utilized for in silico design of gene regulatory or gene functional experiments. An example study demonstrates how novel information can be gained on the interplay, and its quantification, of selected neural factors conserved between Ciona and man. Furthermore, we show examples of differential subcellular localization in embryonic cells, following DNA electroporation in Ciona zygotes. Finally, we discuss the potential of these protocols to be adapted for tissue specific gene interference with emerging gene editing methods. The in vivo approaches in Ciona overcome major shortcomings of classical model organisms in the quest of unraveling conserved mechanisms in the chordate developmental program, relevant to stem cell research, drug discovery, and subsequent clinical application.

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“…The Cripsr-Cas9 system was used in a study using the ascidian Ciona intestinalis (Stolfi et al 2014). This study, from Lionel Christiaen's group, reported the success of tissue-specific genome editing in this species.…”

Section: Introductionmentioning

confidence: 99%

Aquatic Model Organisms in Neurosciences: The Genome-Editing Revolution

Joly¹

2017

Research and Perspectives in Neurosciences

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The use of aquatic model organisms has been greatly diversified in laboratories. Zebrafish is the most advanced aquatic species for the use of Crispr-Cas9 in laboratories. Because of the simplicity and broad applicability of this later system, knock-out is now efficiently performed at medium scale. Forward genetics in zebrafish can now be performed by CRISPR-based F0 screening using high speed and high content phenotyping for example by confocal imaging. As zebrafish, marine model organisms have the prominent advantage to be transparent, all the more at young stages (embryos and larvae) or when fixed samples are cleared by novel methods. The Cripsr-Cas9 system is routinely used in the ascidian Ciona intestinalis. It also starts to be used in many other marine models, such as the medusa Clythia hemispherica. We provide at the end of this review a list of aquatic model species and some examples of questions on the origin of our nervous system that can be coped with these models, where the possibility to perform genome editing would constitute a major advance.

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Tissue-specific genome editing in Ciona embryos by CRISPR/Cas9

Cited by 136 publications

References 49 publications

Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.

Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.

Embryo Microinjection and Electroporation in the Chordate <em>Ciona intestinalis</em>

Aquatic Model Organisms in Neurosciences: The Genome-Editing Revolution

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