High-Throughput Library Transgenesis in Caenorhabditis elegans via Transgenic Arrays Resulting in Diversity of Integrated Sequences (TARDIS)

Reviewer #1 (Public Review):

This work describes a novel high-throughput approach to diverse transgenesis which the authors have named TARDIS for Transgenic Arrays Resulting in Diversity of Integrated Sequences. The authors describe the general approach: the generation of a synthetic 'landing pad' for transgene insertion (as previously reported by this group) that has a split selection hygromycin resistance gene, meaning that only perfect integration with the insert confers resistance to the otherwise lethal hygromycin drug. The authors then demonstrate two possible applications of the technology: individually barcoded lineages for lineage tracing and transcriptional reporter lines generated by inserting multiple promoters. In both cases, the authors did a limited 'proof of concept' study including many important controls, showcasing the potential of the method. The conclusions for applications of this method in C. elegans are supported by the data and the authors discuss important observations and considerations. In the discussion, the authors expand the application of the method beyond C. elegans, which is speculative at this point given that a nontrivial aspect of the success of the method in worms is the self-assembly of DNA into heritable extrachromosomal arrays (a feature that is absent in most other systems).

Reviewer #2 (Public Review):

This paper explores the possibility of integrating diverse and multiple DNA fragments in the genome taking advantage of plasmids in arrays, and CRISPR-Cas.

Since the efficiency of integration in the genome is low, they, as others in the field, use selection markers to identify successful events of integration. The use of these selection markers is common and diverse, but they use a couple of distinct strategies of selection to:

– Introduce bar codes in the genome of individuals at one specific genomic site (gene for Hygromycin resistance with bar code in an intron with homology arms to complete a functional gene);

– Introduce promoters at two specific genomic landing pads downstream of fluorescent reporters.

The strengths of the study rely on the clever design of the selection markers, which enrich the collection of this type of markers. The weaknesses are the lack of novelty in the field in theoretical or practical terms. In fact, they do not show any innovative application of these approaches. Moreover, they show a limited number of experiments in the manuscript, or at least insufficient in my opinion for an article that is based on a methodology.

This work adds to other recent studies, e.g. from Nonet, Mouridi et al., and Malaiwong et al, that use the integration of single and multiple/diverse DNA sequences in the C. elegans genome, and thus is not as groundbreaking as claimed. The real test of this method will be its use to address biological questions.