The genome of the crustacean Parhyale hawaiensis: a model for animal development, regeneration, immunity and lignocellulose digestion

Damian Kao; Alvina G. Lai; Evangelia Stamataki; Silvana Rosic; Nikolaos Konstantinides; Erin Jarvis; Alessia Di Donfrancesco; Natalia Pouchkina-Stantcheva; Marie Sémon; Marco Grillo; Heather Bruce; Suyash Kumar; Igor Siwanowicz; Andy Le; Andrew Lemire; Michael B. Eisen; Cassandra Extavour; William E. Browne; Carsten Wolff; Michalis Averof; Nipam H. Patel; Peter Sarkies; Anastasios Pavlopoulos; A. Aziz Aboobaker

Preliminary evidence regarding the presence of PIWI proteins and other piRNA pathway proteins also 542 suggests that the piRNA pathway is likely active in Parhyale, although piRNAs themselves await to be 543 surveyed. The opportunity to study these piRNA, miRNA and siRNA pathways in a genetically tractable Figure 15A ). 558 We used genome wide bisulfite sequencing to confirm the presence and also assess the distribution of 559 CpG dinucleotide methylation. Our results indicated that 20-30% of Parhyale DNA is methylated at CpG 560 dinucleotides ( Figure 15B ). The Parhyale methylation pattern is similar to that observed in vertebrates, The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/065789 doi: bioRxiv preprint Parhyale has already emerged as a powerful model for developmental genetic research where the 572 expression and function of genes can be studied in the context of stereotyped cellular processes and with 573 a single-cell resolution. Several experimental approaches and standardized resources have been 574 established to study coding and non-coding sequences (Table 1 ). These functional studies will be 575 enhanced by the availability of the assembled and annotated genome presented here. As a first application 576 of these resources, we tested the efficiency of the CRISPR/Cas system for targeted genome editing in 577 Parhyale [17] [18] [19] [20] [21] [22] . In these studies, we targeted the Distal-less patterning gene (called PhDll-e) [24] that 578 has a widely-conserved and highly-specific role in animal limb development [154] . 579 We first genotyped our wild-type laboratory culture and found two PhDll-e alleles with 23 SNPs and 1 PhDll-e alleles modified by Cas9 in their germlines. We tested this by genotyping individual G1s from 596 two of these crosses and found that embryos bearing truncated limbs were homozygous for 597 loss-of-function alleles with out-of-frame deletions, while their wild-type siblings carried one The non-homologous end joining (NHEJ) repair mechanism operating in the injected cells can be 601 exploited not only for gene knock-out experiments described above, but also for CRISPR knock-in 602 approaches where an exogenous DNA molecule is inserted into the targeted locus in a 603 homology-independent manner. This homology-independent approach could be particularly useful for 604 Parhyale that exhibits high levels of heterozygosity and polymorphisms in the targeted laboratory The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/065789 doi: bioRxiv preprint designed in such a way that upon its linearization by the same sgRNA and Cas9 and its integration into 608 the PhDll-e locus in the appropriate orientation and open reading frame, it would restore the endogenous 609 PhDll-e coding sequence in a bicistronic mRNA also expressing a nuclear fluorescent reporter. Among The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/065789 doi: bioRxiv preprint these topics will benefit enormously from the standardized genome-wide resources reported here.