nCas9 Engineering for Improved Target Interaction Presents an Effective Strategy to Enhance Base Editing.

Base editors (BEs) are a recent generation of genome editing tools that couple a cytidine or adenosine deaminase activity to a catalytically impaired Cas9 moiety (nCas9) to enable specific base conversions at the targeted genomic loci. Given their strong application potential, BEs are under active developments toward greater levels of efficiency and safety. Here, a previously overlooked nCas9-centric strategy is explored for enhancement of BE. Based on a cytosine BE (CBE), 20 point mutations associated with nCas9-target interaction are tested. Subsequently, from the initial positive X-to-arginine hits, combinatorial modifications are applied to establish further enhanced CBE variants (1.1-1.3). Parallel nCas9 modifications in other versions of CBEs including A3A-Y130F-BE4max, YEE-BE4max, CGBE, and split-AncBE4max, as well as in the context of two adenine BEs (ABE), likewise enhance their respective activities. The same strategy also substantially improves the efficiencies of high-fidelity nCas9/BEs. Further evidence confirms that the stabilization of nCas9-substrate interactions underlies the enhanced BE activities. In support of their translational potential, the engineered CBE and ABE variants respectively enable 82% and 25% higher rates of editing than the controls in primary human T-cells. This study thus demonstrates a highly adaptable strategy for enhancing BE, and for optimizing other forms of Cas9-derived tools.

Click-Reaction-Mediated Chemotherapy and Photothermal Therapy Synergistically Inhibit Breast Cancer in Mice.

The development of functional materials for tumor immunogenicity enhancement is desirable for overcoming the low therapeutic efficiency and easy metastasis during tumor treatments. Herein, the thermoresponsive nanoparticles composed of photothermal agent (PTA) and click reactive reagent are developed for enhanced immunotherapy application. A Ni-bis(dithiolene)-containing PTA with intense near-infrared absorption and efficient photothermal conversion is developed for thermoresponsive nanoparticles construction. The generated heat by encapsulated PTA further induces the phase transition of thermoresponsive nanoparticles with the release of chemotherapy reagent to react with the amino groups on functional proteins, realizing PTT and chemotherapy simultaneously. Moreover, the immunogenic cell death (ICD) of cancer cells evoked by PTT could be further enhanced by the released reactive reagent. As a result, the synergistic effect of photothermal treatment and reaction-mediated chemotherapy can suppress the growth of a primary tumor, and the evoked ICD could further activate the immune response with the suppression of a distant tumor. This synergistic treatment strategy provides a reliable and promising approach for cancer immunotherapy in clinic.

NIR luminogen for low-temperature photothermal therapy by triggering HSP90α down-regulation.

A near-infrared (NIR) luminogen TST was designed and used to efficiently trigger HSP90α protein knockdown through photo-thermal conversion based on a gene interference strategy, by which in vitro and in vivo tumor ablation were significantly acquired at low-temperature.

Precise tumor immune rewiring via synthetic CRISPRa circuits gated by concurrent gain/loss of transcription factors.

Reinvigoration of antitumor immunity has recently become the central theme for the development of cancer therapies. Nevertheless, the precise delivery of immunotherapeutic activities to the tumors remains challenging. Here, we explore a synthetic gene circuit-based strategy for specific tumor identification, and for subsequently engaging immune activation. By design, these circuits are assembled from two interactive modules, i.e., an oncogenic TF-driven CRISPRa effector, and a corresponding p53-inducible off-switch (NOT gate), which jointly execute an AND-NOT logic for accurate tumor targeting. In particular, two forms of the NOT gate are developed, via the use of an inhibitory sgRNA or an anti-CRISPR protein, with the second form showing a superior performance in gating CRISPRa by p53 loss. Functionally, the optimized AND-NOT logic circuit can empower a highly specific and effective tumor recognition/immune rewiring axis, leading to therapeutic effects in vivo. Taken together, our work presents an adaptable strategy for the development of precisely delivered immunotherapy.

Map-based cloning of a new total loss-of-function allele of OsHMA3 causes high cadmium accumulation in rice grain.

Rice (Oryza sativa) is a major dietary source of the toxic metal cadmium (Cd). Reducing Cd transfer from soil to the rice grain is important for food safety. Rice cultivars vary widely in their Cd accumulation, but the genetic basis for this variation is not fully understood. Based on field and pot experiments comparing 26 rice cultivars, we identified a cultivar with high Cd accumulation in grain (BG367, coded as W4) and a cultivar with low grain Cd accumulation (Huajingxian 74, coded as W0). W4 showed a higher Cd translocation from roots to shoots than W0. Using chromosome single segment substitution lines derived from the two cultivars, we mapped a quantitative trait locus for Cd accumulation in grain to a 400 kb region in chromosome 7. Using yeast expression assays and transgenic complementation, we identified OsHMA3 as the causal gene at this locus. Compared with OsHMA3W0, OsHMA3W4 has a deletion of 14 amino acids predicted to be in the ATP binding domain. OsHMA3W4 showed a complete loss of transport activity for Cd in yeast assays. Taking our findings together, we have identified a new allele of OsHMA3 with a total loss-of-function, resulting in greatly elevated Cd translocation to rice shoots and grain.

Polymorphism of follicle stimulating hormone beta (FSHß) subunit gene and its association with litter traits in giant panda.

The different SSCP patterns of the follicle stimulating hormone beta (FSHß) gene amplified by three pairs of primers were sequenced. Comparisons among the three nucleotide sequences of three genotypes indicated that three base substitutions (A213T, A91G, and A89C) were detected in FSHß gene, which A213T substitution led to one amino acids mutation (Lys > Met), and the other two substitutions were synonymous mutations. The AA, AB and BB genotypes patterns obtained by FSHß primer1 had evident relation with the litter traits, but the SSCP genotypes patterns obtained by FSHß primer2 and primer3 had no evident relation with the litter traits in giant panda. The giant panda with AA and AB genotype had the largest litter size and multiparity rate compared with the BB genotypes (P < 0.05). We speculated that the giant pandas with the A allele have better litter traits than those with the B allele.

Relationship of the estrogen surge and multiple mates to cub paternity in the giant panda (Ailuropoda melanoleuca): implications for optimal timing of copulation or artificial insemination.

The effectiveness of ex situ breeding programs for endangered species can be limited by challenges in mimicking mating competitions that naturally occur among multiple mates in the wild. The objective of this study was to evaluate the impact of timed natural matings and/or artificial inseminations in the context of the urinary estrogen surge on cub production in the giant panda (Ailuropoda melanoleuca). We used a large cohort of giant pandas, including 12 females and 17 males. DNA paternity exclusion was used to pinpoint accurately the interval during the estrogen surge that coincided with the ideal sperm deposition time to produce offspring. Of the 31 cubs (in 19 pregnancies), 22 (71.0%; 15 pregnancies) were produced from matings occurring on the day of or the day after the maximal urinary estrogen peak. Sixteen of the 19 pregnancies (84.2%) produced at least one offspring sired by the first male mating with the dam. There was a preponderance of twins (12 of 19; 63.2%), and dual paternities were discovered in 3 of 12 twin sets (25%). These findings indicate a strong relationship between the excreted estrogen surge and sperm deposition to achieve pregnancy in the giant panda. To ensure the production of the most genetically diverse young, it is imperative that the most appropriate male mate first and on the day of or the day after the highest detected estrogen value. There is no advantage to increasing the number of copulations or mating partners within 1 day of the estrogen peak on the incidence of twinning, although this practice may increase the prevalence of dual paternity in cases of multiple births.