Single-step generation of homozygous knock-out/knock-in individuals in an extremotolerant parthenogenetic tardigrade using DIPA-CRISPR

Abstract

Tardigrades are small aquatic invertebrates known for their remarkable tolerance to diverse extreme stresses. To elucidate the in vivo mechanisms underlying this extraordinary resilience, the genetic manipulation methods in tardigrades have long been desired. Despite our prior success in somatic cell gene-editing by microinjecting Cas9 ribonucleoproteins (RNPs) into the body cavity of tardigrades, the generation of gene-edited individuals remained elusive. In this study, employing an extremotolerant parthenogenetic tardigrade species, Ramazzottius varieornatus, we established conditions conductive to generating gene-edited tardigrade individuals. Drawing inspiration from the direct parental CRIPSR (DIPA-CRISPR) technique employed in several insects, we simply injected a concentrated Cas9 RNP solution into the body cavity of parental females shortly before their initial oviposition. This approach yielded gene-edited G0 progeny. Notably, only a single allele was predominantly detected at the target locus for each G0 individual, indicative of homozygous mutations. Through co-injecting single-stranded oligodeoxynucleotides (ssODNs) with Cas9 RNPs, we achieved the generation of homozygously knocked-in G0 progeny and these edited-alleles were inherited by G1/G2 progeny. This establishment of a simple method for generating homozygous knock-out/knock-in individuals not only facilitates in vivo analyses of molecular mechanisms underpinning extreme tolerance but also opens avenues for exploring various topics, including Evo-Devo, in tardigrades.

Expression patterns of FGF and BMP pathway genes in the tardigradeHypsibius exemplaris

“…There has been recent progress towards development of genetic tools for H. exemplaris by CRISPR/Cas9-based genome editing of adults with 10-20% efficient genomic deletion and via a vector-based expression system that worked mosaically in adult cells at 70-89% adult transfection efficiency and rare germline transmission (Kumagai et al, 2022; Tanaka et al, 2022). Recently, there has been progress in another tardigrade species, Ramazzottius varieornatus , in which CRISPR injected directly into the maternal body cavity exhibited germline transmission with low efficiency (3-4%) (Kondo et al, 2024). We anticipate improvements to these techniques are soon to follow for efficient germline transmission, which would facilitate asking questions about development.…”

Sexual dimorphism in the tardigradeParamacrobiotus metropolitanustranscriptome

“…We also provide evidence for a new DMRT subfamily that may contribute to sex differentiation in this family. The 3090/3093 complex DMRT paralogs may be initial candidates for disruption or gene editing for evaluation their relationships with sex determination (79,(96)(97)(98). Future studies utilizing high-quality genomes and careful physiological experiments are required to reveal sex determination cues not only in this species but also in other tardigrades.…”

Single-animal, single-tube RNA extraction for quantitative analysis of transcripts in the tardigradeHypsibius exemplaris

“…Then vortex the sample at max speed (approximately 3200 RPM) until the sample is completely thawed for at least 15 seconds. 16. Repeat steps 14-15 five more times.…”

“…Stable genetic manipulation of these remarkable animals, including transgenesis and germline gene modification, has remained elusive until very recently 15, 16 . As such, most experimentation to reveal molecular mechanisms of extremotolerance is performed through transcriptional profiling 4, 5, 17, 18 .…”