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.

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