Generation of Human Isogenic Induced Pluripotent Stem Cell Lines with CRISPR Prime Editing.

We developed an efficient CRISPR prime editing protocol and generated isogenic-induced pluripotent stem cell (iPSC) lines carrying heterozygous or homozygous alleles for putatively causal single nucleotide variants at six type 2 diabetes loci (ABCC8, MTNR1B, TCF7L2, HNF4A, CAMK1D, and GCK). Our two-step sequence-based approach to first identify transfected cell pools with the highest fraction of edited cells significantly reduced the downstream efforts to isolate single clones of edited cells. We found that prime editing can make targeted genetic changes in iPSC and optimization of system components and guide RNA designs that were critical to achieve acceptable efficiency. Systems utilizing PEmax, epegRNA modifications, and MLH1dn provided significant benefit, producing editing efficiencies of 36-73%. Editing success and pegRNA design optimization required for each variant differed depending on the sequence at the target site. With attention to design, prime editing is a promising approach to generate isogenic iPSC lines, enabling the study of specific genetic changes in a common genetic background.

Hawaiian larval stomatopods: molecular and morphological diversity.

Estimating stomatopod species diversity using morphology alone has long been difficult; though over 450 species have been described, new species are still being discovered regularly despite the cryptic behaviors of adults. However, the larvae of stomatopods are more easily obtained due to their pelagic habitat, and have been the focus of recent studies of diversity. Studies of morphological diversity describe both conserved and divergent traits in larval stomatopods, but generally cannot be linked to a particular species. Conversely, genetic studies of stomatopod larvae using DNA barcoding can be used to estimate species diversity, but are generally not linked to known species by analyses of morphological characters. Here we combine these two approaches, larval morphology and genetics, to estimate stomatopod species diversity in the Hawaiian Islands. Over 22 operational taxonomic units (OTUs) were identified genetically, corresponding to 20 characterized morphological types. Species from three major superfamilies of stomatopod were identified: Squilloidea (4 OTUs, 3 morphotypes), Gonodactyloidea (9, 8), and Lysiosquilloidea (6, 7). Among these, lysiosquilloids were more diverse based on larval morphotypes and OTUs as compared to previously documented Hawaiian species (3), while squilloids had a lower diversity of species represented by collected larvae as compared to the seven species previously documented. Two OTUs / morphotypes could not be identified to superfamily as their molecular and morphological features did not closely match any available information, suggesting they belong to poorly sampled superfamilies. The pseudosquillid, Pseudosquillana richeri, was discovered for the first time from Hawai'i. This study contributes an updated estimate for Hawaiian stomatopod diversity for a total of 24 documented species, provides references for identification of larval stomatopods across the three major superfamilies, and emphasizes the lack of knowledge of species diversity in more cryptic stomatopod superfamilies, such as Lysiosquilloidea.