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

Tardigrades are small aquatic invertebrates known for their remarkable tolerance to diverse extreme stresses. To elucidate the in vivo mechanisms underlying this extraordinary resilience, methods for genetically manipulating 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 that led to the generation of gene-edited tardigrade individuals. Drawing inspiration from the direct parental CRISPR (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. By 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 is the first example of heritable gene editing in the entire phylum of Tardigrada. This establishment of a straightforward method for generating homozygous knockout/knock-in individuals not only facilitates in vivo analyses of the molecular mechanisms underpinning extreme tolerance, but also opens up avenues for exploring various topics, including Evo-Devo, in tardigrades. Author summary: Tardigrades, tiny aquatic invertebrates also known as water bears, are celebrated for their extraordinary resilience to various extreme stresses like dehydration, radiation, and unusual ranges of temperature and pressure. Understanding the molecular mechanisms of this resilience not only satisfies scientific curiosity but also holds promise for the development of innovative technologies for the dry preservation of biomaterials like biomedicines and vaccines. However, the lack of a heritable genome manipulation technology has hindered in vivo analyses of these mechanisms. This study addresses this longstanding challenge in the field. Employing an extremotolerant parthenogenetic tardigrade species, we established conditions that enable the efficient production of gene-manipulated individuals. Using these conditions, the simple injection of Cas9 genome-editing components into parental females leads to the generation of knockout/knock-in progeny. Unlike similar approaches in other animals, we obtained mutant progeny predominantly carrying a single type of mutation, namely, homozygous mutants, which significantly facilitates downstream analyses. This is the first report of a heritable gene-editing method in the entire group of tardigrades. The establishment of this straightforward method for generating gene-manipulated tardigrades not only facilitates in vivo analyses of the molecular mechanisms underpinning extreme tolerance, but also opens up avenues for exploring various topics, including Evo-Devo. [ABSTRACT FROM AUTHOR]