A new method for generating homozygous knockout/knock-in individuals in the extremotolerant parthenogenetic tardigrade Ramazzottius varieornatus using DIPA-CRISPR has been developed. This method involves injecting a concentrated Cas9 ribonucleoprotein (RNP) solution into the body cavity of parental females shortly before their first oviposition. This approach successfully generated gene-edited G0 progeny, with most carrying the edited allele in a homozygous form. By co-injecting single-stranded oligodeoxynucleotides (ssODNs) with Cas9 RNPs, homozygous knock-in G0 progeny were also generated, and these edited alleles were inherited by G1/G2 progeny. This is the first example of heritable gene editing in the entire phylum of Tardigrada. The establishment of this straightforward method not only facilitates in vivo analyses of the molecular mechanisms underlying extreme tolerance but also opens up avenues for exploring various topics, including Evo-Devo, in tardigrades. The study demonstrated that DIPA-CRISPR worked in R. varieornatus, and that the simple injection of Cas9 RNPs into parental tardigrades with appropriate conditions is sufficient to obtain homozygous knockout/knock-in individuals. This method enables efficient gene editing in tardigrades, which are known for their extreme resilience to various environmental stresses. The study also showed that gene-editing efficiency was comparable between knockout and knock-in trials, and that HDR was likely the dominant repair mode in the germ cells of this tardigrade species. The findings suggest that the method is effective for generating gene-edited individuals in various invertebrate species, including other tardigrades. The study provides a valuable tool for understanding the molecular mechanisms underlying the extreme tolerance of tardigrades and for exploring Evo-Devo-related topics.A new method for generating homozygous knockout/knock-in individuals in the extremotolerant parthenogenetic tardigrade Ramazzottius varieornatus using DIPA-CRISPR has been developed. This method involves injecting a concentrated Cas9 ribonucleoprotein (RNP) solution into the body cavity of parental females shortly before their first oviposition. This approach successfully generated gene-edited G0 progeny, with most carrying the edited allele in a homozygous form. By co-injecting single-stranded oligodeoxynucleotides (ssODNs) with Cas9 RNPs, homozygous knock-in G0 progeny were also generated, and these edited alleles were inherited by G1/G2 progeny. This is the first example of heritable gene editing in the entire phylum of Tardigrada. The establishment of this straightforward method not only facilitates in vivo analyses of the molecular mechanisms underlying extreme tolerance but also opens up avenues for exploring various topics, including Evo-Devo, in tardigrades. The study demonstrated that DIPA-CRISPR worked in R. varieornatus, and that the simple injection of Cas9 RNPs into parental tardigrades with appropriate conditions is sufficient to obtain homozygous knockout/knock-in individuals. This method enables efficient gene editing in tardigrades, which are known for their extreme resilience to various environmental stresses. The study also showed that gene-editing efficiency was comparable between knockout and knock-in trials, and that HDR was likely the dominant repair mode in the germ cells of this tardigrade species. The findings suggest that the method is effective for generating gene-edited individuals in various invertebrate species, including other tardigrades. The study provides a valuable tool for understanding the molecular mechanisms underlying the extreme tolerance of tardigrades and for exploring Evo-Devo-related topics.