CRISPR/Cas12a (formerly Cpf1), an RNA-guided endonuclease of the Class II Type V-A CRISPR system, provides a promising tool for genome engineering. Over 10 Cas12a orthologues have been identified and employed for gene editing in human cells. However, the functional diversity among emerging Cas12a orthologues remains poorly explored. Here, we report a high-throughput comparative profiling of editing activities across 16 Cas12a orthologues in human cells by constructing genome-integrated, self-cleaving, paired crRNA–target libraries containing >40,000 guide RNAs. Three Cas12a candidates exhibited promising potential owing to their compact structures and editing efficiency comparable with those of AsCas12a and LbCas12a, which are well characterized. We generated three arginine substitution variants (3Rv) via structure-guided protein engineering: BsCas12a-3Rv (K155R/N512R/K518R), PrCas12a-3Rv (E162R/N519R/K525R), and Mb3Cas12a-3Rv (D180R/N581R/K587R). All three Cas12a variants showed enhanced editing activities and expanded targeting ranges (NTTV, NTCV, and TRTV) compared with the wild-type Cas12a effectors. The base preference analysis among the three Cas12a variants revealed that PrCas12a-3Rv shows the highest activity at target sites with canonical PAM TTTV and non-canonical PAM TTCV, while Mb3Cas12a-3Rv exhibits recognition features distinct from the others by accommodating for more nucleotide A at position $-3$ for PAM TATV and at position $-4$ for PAM ATCV. Thus, the expanded Cas12a toolbox and an improved understanding of Cas12a activities should facilitate their use in genome engineering.

Glucotoxicity-induced pancreatic β-cell failure contributes to the development of type 2 diabetes mellitus (T2DM). Accumulating evidence reveals that miRNAs play a critical role in regulating pancreatic β-cell function and survival. In this study, we employed a self-assembled cell microarray (SAMcell)-based functional screening assay to identify miRNAs that are capable of regulating the dysfunction of β-cells induced by glucotoxicity. Among 62 conserved miRNAs we tested, miR-190 was identified as a candidate regulator that could effectively restore insulin expression in NIT-1 cells under high-glucose (HG) stimulation. Further analyses demonstrated that miR-190 was significantly down-regulated in HG-treated NIT-1 cells, as well as in the pancreas of diabetic mice. Mechanistic studies showed that Cybb is the direct target gene of miR-190, which encodes the gp91phox protein, a subunit of the NOX2 complex. Furthermore, both miR-190 overexpression and Cybb knockdown inhibited apoptosis and improved glucose-stimulated insulin secretion (GSIS) in HG-stimulated NIT-1 cells by attenuating the excessive production of reactive oxygen species (ROS). More importantly, a targeted delivery of mPEG-PCL-g-PDMAEMA nanoparticles/miR-190 complexes (PECgD NPs/miR-190) to the pancreas significantly ameliorated hyperglycemia, decreased fasting serum insulin levels, and improved glucose tolerance in diabetic mice. Taken together, our findings suggest that the miR-190/Cybb axis plays an important role in glucotoxicity-induced pancreatic β-cell failure. Restoring miR-190 expression levels may be a possible therapeutic strategy to protect β-cells in T2DM.

Background: Patient-derived tumor cell cluster (PTC)-based drug sensitivity assays show promise for enabling precision drug selection; however, their predictive value in advanced non-small cell lung cancer (NSCLC) requires further elucidation. Methods: To assess the concordance between PTC-based drug sensitivity and clinical outcomes, we conducted a single-center prospective cohort study at Peking University Third Hospital from August 2021 to August 2024. We enrolled 38 consecutive patients, from whom 44 fresh tumor specimens were obtained for PTC-based drug sensitivity assays and compared with paired clinical outcomes of chemotherapy and targeted therapy regimens. Concordance was assessed using the Kappa statistic and receiver operating characteristic curve analysis. A Cox proportional-hazard model was used to identify prognostic factors for progression-free survival (PFS). Results: We observed an 81.8% (36/44) concordance between the PTC killing rate (>0% vs 0%) and the best overall clinical response (disease controlled vs disease progression per RECIST v1.1) (Kappa = 0.484, area under the curve (AUC) = 0.740). The concordance with the local response of the biopsied lesions was even higher, at 87.5% (35/40) (Kappa = 0.593, AUC = 0.841). A PTC killing rate >0% was correlated with significantly longer PFS (8.6 vs 2.0 months, P < .001) and emerged as an independent predictor of PFS in multivariate analysis (hazard ratio (HR) 3.507, 95% confidence interval (CI) 1.289-9.536). Exploratory subgroup analysis revealed concordance rates of 73.7% (14/19) for malignant effusion-derived PTCs and 88.0% (22/25) for tumor tissue-derived PTCs; 95.2% (20/21) for first-line therapy and 69.6% (16/23) for later-line therapies; and 80.0% (12/15) for targeted therapy and 85.2% (23/27) for chemotherapy. Notably, the concordance rate reached 100% (14/14) in patients receiving chemotherapy plus immune checkpoint inhibitors. Conclusions: These findings validate the predictive value of the PTC-based drug sensitivity assay in guiding personalized treatment for patients with advanced NSCLC and support its clinical translation. Registration: Chinese Clinical Trial Registry, Registration No.: ChiCTR2100048791, https://www.chictr.org.cn/showproj.html?proj=129885 .