The CRISPR/Cas9 system has ushered in a new era of targeted genetic manipulations. Here, we report the use of CRISPR/Cas9 to induce double-stranded breaks in the genome of the sea squirt Ciona intestinalis. We use electroporation to deliver CRISPR/Cas9 components for tissue-specific disruption of the Ebf (Collier/Olf/ EBF) gene in hundreds of synchronized Ciona embryos. Phenotyping of transfected embryos in the 'F0' generation revealed that endogenous Ebf function is required for specification of Isletexpressing motor ganglion neurons and atrial siphon muscles. We demonstrate that CRISPR/Cas9 is sufficiently effective and specific to generate large numbers of embryos carrying mutations in a targeted gene of interest, which should allow for rapid screening of gene function in Ciona.

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The advent of CRISPR/Cas9 has made genome editing possible in virtually any organism, including those not previously amenable to genetic manipulations. Here, we present an optimization of CRISPR/Cas9 for application to early avian embryos with improved efficiency via a three-fold strategy. First, we employed Cas9 protein flanked with two nuclear localization signal sequences for improved nuclear localization. Second, we used a modified guide RNA (gRNA) scaffold that obviates premature termination of transcription and unstable Cas9-gRNA interactions. Third, we used a chick-specific U6 promoter that yields 4-fold higher gRNA expression than the previously utilized human U6. For rapid screening of gRNAs for in vivo applications, we also generated a chicken fibroblast cell line that constitutively expresses Cas9. As proof of principle, we performed electroporation-based loss-of-function studies in the early chick embryo to knock out Pax7 and Sox10, key transcription factors with known functions in neural crest development. The results show that CRISPR/Cas9-mediated deletion causes loss of their respective proteins and transcripts, as well as predicted downstream targets. Taken together, the results reveal the utility of this optimized CRISPR/Cas9 method for targeted gene knockout in chicken embryos in a manner that is reproducible, robust and specific.

The CRISPR/Cas9 system has emerged as an important tool for various genome engineering applications. A current obstacle to high throughput applications of CRISPR/Cas9 is the imprecise prediction of highly active single guide RNAs (sgRNAs). We previously implemented the CRISPR/Cas9 system to induce tissue-specific mutations in the tunicate Ciona. In the present study, we designed and tested 83 single guide RNA (sgRNA) vectors targeting 23 genes expressed in the cardiopharyngeal progenitors and surrounding tissues of Ciona embryo. Using high-throughput sequencing of mutagenized alleles, we identified guide sequences that correlate with sgRNA mutagenesis activity and used this information for the rational design of all possible sgRNAs targeting the Ciona transcriptome. We also describe a one-step cloning-free protocol for the assembly of sgRNA expression cassettes. These cassettes can be directly electroporated as unpurified PCR products into Ciona embryos for sgRNA expression in vivo, resulting in high frequency of CRISPR/Cas9-mediated mutagenesis in somatic cells of electroporated embryos. We found a strong correlation between the frequency of an Ebf loss-of-function phenotype and the mutagenesis efficacies of individual Ebf-targeting sgRNAs tested using this method. We anticipate that our approach can be scaled up to systematically design and deliver highly efficient sgRNAs for the tissue-specific investigation of gene functions in Ciona.

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The CRISPR/Cas9 system has emerged as an important tool for various genome engineering applications. A current obstacle to high throughput applications of CRISPR/Cas9 is the imprecise prediction of highly active single guide RNAs (sgRNAs). We previously implemented the CRISPR/Cas9 system to induce tissue-specific mutations in the tunicate Ciona. In the present study, we designed and tested 83 single guide RNA (sgRNA) vectors targeting 23 genes expressed in the cardiopharyngeal progenitors and surrounding tissues of Ciona embryo. Using high-throughput sequencing of mutagenized alleles, we identified guide sequences that correlate with sgRNA mutagenesis activity and used this information for the rational design of all possible sgRNAs targeting the Ciona transcriptome. We also describe a one-step cloning-free protocol for the assembly of sgRNA expression cassettes. These cassettes can be directly electroporated as unpurified PCR products into Ciona embryos for sgRNA expression in vivo, resulting in high frequency of CRISPR/Cas9-mediated mutagenesis in somatic cells of electroporated embryos.We found a strong correlation between the frequency of an Ebf loss-of-function phenotype and the mutagenesis efficacies of individual Ebf-targeting sgRNAs tested using this method. We anticipate that our approach can be scaled up to systematically design and deliver highly efficient sgRNAs for the tissue-specific investigation of gene functions in Ciona.

Introduction Hookahs (water pipes) are rapidly increasing in popularity worldwide. Evidence suggests that although perceived as safer than cigarette smoke, hookah smoke may be as, or even more, dangerous as cigarette smoke. Methods Air samples from 33 homes—11 where only hookah-smoking occurred, 12 with only cigarettes and 10 with no smoking—were collected to analyse concentrations of particulate matter (PM2.5), black carbon, elemental and organic carbon and carbon monoxide (CO). Air quality was assessed in rooms where smoking occurred and in an adjacent room. Results Hookah and cigarette smoking impaired home air quality. The rooms in which hookahs were smoked showed the highest concentrations for all pollutants. CO was significantly greater in the rooms where hookahs were smoked than in the cigarette-smoking rooms and the non-smoking households (p<0.05). In addition, CO levels in the rooms adjacent to where hookah was smoked were 2.5-fold to 4-fold greater than those in the smoking and non-smoking rooms of the cigarette homes (p<0.05). PM2.5 levels were also elevated in hookah homes compared to cigarette and non-smoking homes, although not significantly different. Conclusions This study, the first of its kind, demonstrates potentially hazardous levels of home air pollution in rooms where hookahs are being smoked as well as in adjacent rooms. These levels were greater than those in cigarette smoking homes, raising concerns about potential negative health effects on all individuals living in homes where hookahs are smoked.

Please cite this article as: Tang, W., Li, Y., Gandhi, S., Bronner, M., Multiplex clonal analysis in the chick embryo using retrovirally-mediated combinatorial labeling, Developmental Biology (2019), doi: https:// Abstract 10 Lineage analysis plays a central role in exploring the developmental potential of stem and progenitor cell populations. In higher vertebrates, a variety of techniques have been used to label individual cells or cell populations, including interspecies grafting, intracellular microinjection, and Cre-mediated recombination. However, these approaches often suffer from difficulties in progenitor cell targeting, low cellular resolution and/or ectopic labeling. To 15 circumvent these issues, here we utilize replication incompetent avian (RIA) retroviruses to deliver combinations of fluorescent proteins into distinct cellular compartments in chick embryos.In particular, RIA-mediated lineage tracing is optimal for long term mapping of dispersing cell populations like the neural crest. Using this tool, we confirm that trunk neural crest cells are multipotent. Furthermore, our RIA vector is engineered to be fully adaptable for other purposes 20 such as cell fate analysis, gene perturbation studies and time-lapse imaging. Taken together, we present a novel approach of multiplex lineage analysis that can be applied to normal and perturbed development of diverse cell populations in avian embryos.