A central goal in the development of genome engineering technology is to reduce the time and labor required to produce custom genome modifications. Here we describe a new selection strategy for producing fluorescent protein (FP) knock-ins using CRISPR/Cas9-triggered homologous recombination. We have tested our approach in Caenorhabditis elegans. This approach has been designed to minimize hands-on labor at each step of the procedure. Central to our strategy is a newly developed self-excising cassette (SEC) for drug selection. SEC consists of three parts: a drug-resistance gene, a visible phenotypic marker, and an inducible Cre recombinase. SEC is flanked by LoxP sites and placed within a synthetic intron of a fluorescent protein tag, resulting in an FP-SEC module that can be inserted into any C. elegans gene. Upon heat shock, SEC excises itself from the genome, leaving no exogenous sequences outside the fluorescent protein tag. With our approach, one can generate knock-in alleles in any genetic background, with no PCR screening required and without the need for a second injection step to remove the selectable marker. Moreover, this strategy makes it possible to produce a fluorescent protein fusion, a transcriptional reporter and a strong loss-of-function allele for any gene of interest in a single injection step.KEYWORDS CRISPR/Cas9; homologous recombination; gene tagging; Caenorhabditis elegans; self-excising cassette A common goal in biological and biomedical research is to visualize the localization of a protein of interest within a cell or organism. This is often accomplished by fusing GFP or another fluorescent protein (FP) to the protein of interest. In the nematode Caenorhabditis elegans, GFP fusions were historically generated by injecting plasmids into the gonad of the adult hermaphrodite worm, resulting in the formation of extrachromosomal arrays (Mello et al. 1991). However, the resulting fusion proteins were typically strongly overexpressed in somatic tissues and silenced in the germline. Microparticle bombardment allowed the generation of low-copy transgenes that in some cases more closely recapitulated endogenous expression levels (Praitis et al. 2001;Sarov et al. 2012), but this technique is inefficient, time consuming, and difficult and requires expensive equipment and materials. More recently, we and others have reported CRISPR/Cas9-based approaches that together can be used to make essentially any desired change to the C. elegans genome, including insertion of GFP into endogenous loci Dickinson et al. 2013;Lo et al. 2013;Chiu et al. 2013;Cho et al. 2013;Katic and Großhans 2013;Tzur et al. 2013;Waaijers et al. 2013;Chen et al. 2013;Zhao et al. 2014;Kim et al. 2014;Arribere et al. 2014;Paix et al. 2014;Ward 2015;Farboud and Meyer 2015). The resulting GFP knock-in strains express 100% labeled protein under the control of all native regulatory elements, resulting in endogenous levels and patterns of expression in all cases reported to date (Dickinson et al. 2013;Kim et al. 2014).Our publis...

Neuroectodermal signalling centres induce and pattern many novel vertebrate brain structures but are absent, or divergent, in invertebrate chordates. This has led to the hypothesis that signalling centre genetic programs were first assembled in stem vertebrates, which potentially drove morphological innovations. However, this scenario presumes that extant cephalochordates accurately represent ancestral chordate characters, which has not been tested using close chordate outgroups. Here, we report that genetic programs homologous to three vertebrate signalling centres; the anterior neural ridge, zona limitans intrathalamica, and isthmic organizer are present in the hemichordate Saccoglossus kowalevskii. Fgf8/17/18, sfrp1/5, hh, and wnt1 are expressed in vertebrate-like arrangements in hemichordate ectoderm, and homologous genetic mechanisms regulate ectodermal patterning in both animals. We propose these genetic programs were components of an unexpectedly complex, ancient genetic regulatory scaffold for deuterostome body patterning that degenerated in amphioxus and ascidians, but was retained to pattern divergent structures in hemichordates and vertebrates.

Acorn worms, also known as enteropneust (literally, ‘gut-breathing’) hemichordates, are marine invertebrates that share features with echinoderms and chordates. Together, these three phyla comprise the deuterostomes. Here we report the draft genome sequences of two acorn worms, Saccoglossus kowalevskii and Ptychodera flava. By comparing them with diverse bilaterian genomes, we identify shared traits that were probably inherited from the last common deuterostome ancestor, and then explore evolutionary trajectories leading from this ancestor to hemichordates, echinoderms and chordates. The hemichordate genomes exhibit extensive conserved synteny with amphioxus and other bilaterians, and deeply conserved non-coding sequences that are candidates for conserved gene-regulatory elements. Notably, hemichordates possess a deuterostome-specific genomic cluster of four ordered transcription factor genes, the expression of which is associated with the development of pharyngeal ‘gill’ slits, the foremost morphological innovation of early deuterostomes, and is probably central to their filter-feeding lifestyle. Comparative analysis reveals numerous deuterostome-specific gene novelties, including genes found in deuterostomes and marine microbes, but not other animals. The putative functions of these genes can be linked to physiological, metabolic and developmental specializations of the filter-feeding ancestor.