Single-stranded HDR templates with truncated Cas12a-binding sequences improve knock-in efficiencies in primary human T cells

CRISPR-Cas12a gene editing offers an alternative to Cas9-based methods, providing better targeting of AT-rich regions, simplified guide RNA manufacturing, and high specificity. However, the efficacy of donor-based editing is subject to various factors, with template format playing a crucial role. Currently, the predominant non-viral template format for homology-directed repair (HDR) after nuclease-induced DNA breaks is double-stranded DNA, which is toxic when transfected at high doses. Others have demonstrated that using single-stranded DNA (ssDNA) with flanking double-stranded Cas-target-sequences (CTS) as a template for Cas9-mediated gene editing can mitigate this toxicity and increase knock-in efficiency. Here, we investigate CTS design for AsCas12a Ultra by exploring PAM orientation and binding requirements. Additionally, we rule out ssDNase activity of AsCas12a under cell-physiological Mg2(+) conditions. Finally, we showcase the advantage of ssDNA donors with CTS (ssCTS) at high doses for delivering clinically relevant transgenes of varying sizes into three TCR-CD3 complex genes (TRAC, CD3ζ, CD3ε), achieving up to 90% knock-in rates for a 0.8kb-insert at the CD3ε locus. Long-read sequencing confirmed higher HDR rates and revealed that CTS reduced partial integration events compared to unmodified ssDNA. Overall, AsCas12a and ssCTS represent a platform for highly efficient knock-in in primary human T cells with minimal toxicity

Internal oxidation of Nb-Zr alloys over the range 1555-1768°C at low oxygen pressures

Three Nb alloys, containing 1 w/oZr, 2.5 w/oZr, and 10W-2.5Zr, were internally oxidized in the range of 1555 to 1768°C at oxygen pressures ranging from 5 x 10-5 to 1 x 10-3 torr. Linear kinetics were measured suggesting that oxygen arrival at the surface and not oxygen diffusion in the substrate was rate controlling. Both tetragonal and monoclinic ZrO2 formed, the tetragonal form being favored by high nucleation rates (lower temperatures), lower alloy content, and location in the reaction zone (small particles near the surface). Monoclinic ZrO2 formed at higher temperatures and deeper within the reaction zone where larger precipitates formed. The high solubility product of ZrO2 in Nb-Zr alloys gives rise to non-Wagnerian behavior so that the solute is not precipitated out at the reaction front, additional precipitation occurring after the reaction front has passed. This causes a variation in the precipitate volume fraction with distance in the zone. Experimental observations are discussed in terms of various models for non-classical internal oxidation

Decline of Some West-Central Florida Anuran Populations in Response to Habitat Degradation

Recent reports have suggested that a global decline in amphibian populations has taken place during the past few decades. Urban development is thought to affect the richness and abundances of species and, therefore, could be an important cause of decline. We estimated the richness and abundances of anurans in wetlands at a residential development and in similar wetlands at a nearby undeveloped park. The residential development originally was pine flatwoods habitat, as is the undeveloped park curiently. We also compared the anuran species\u27 composition of the park in 1992 with the composition in 1974. Both richness and abundances of anurans in the residential development were different than those in the undeveloped park. Employing the same amount of sampling effort at both sites, we trapped or observed 11 species at the developement and 15 species at the park, and trapped 569 individuals at the development and 1224 individuals at the park. The anuran species richness at the undeveloped park in 1992 was nearly the same as in 1974; a single rare species apparently was not present in 1992. Of the 15 species present in both surveys, 14 showed higher abundances in 1992 than in 1974. We suggest that the current differences between the residential development and the park have resulted from degradation of both the uplands used by many species during the dry season and the temporary wetlands used by many species for reproduction. Four species especially sensitive to such degradation, Bufo quercicus, Scaphiopus h. holbrookii, Hyla femoralis, and H. gratiosa, were the species missing from the residential development. Not all species of anurans typical of pine flatwoods appeared to be affected adversely by development. Three species of ranids, Rana utricularia, R. grylio, and R. catesbeiana, were found in higher abundances at the residential development than at the park. These ranid species breed in a wide variety of aquatic systems, including the permanent bodies of water that are now abundant in the development, and probably use the uplands less than other anurans. If amphibian decline is international in scope, then the decline could be attributable either to global changes caused by humans, or to local, but widespread, environmental degradation, or to a combination of factors. While much recent popular focus has been on potential global causes of decline, we believe that this emphasis may have caused attention to be taken away from local causes that, as our study demonstrated, may be at least as important. We suggest that in many places, local environmental degradation is insidiously chipping away at amphibian diversity, and that more emphasis should be placed on these local causes than is now the case