The precise editing of genes mediated by CRISPR-Cas9 necessitates the application of donor DNA with appropriate lengths of homologous arms and fragment sizes. Our previous development, SSB/CRISPR-Cas9, has demonstrated high efficiency in homologous recombination and non-homologous end joining gene editing within bacteria. In this study, we optimized the lengths and sizes of homologous arms of the donor DNA within this system. Two sets of donor DNA constructs were generated: one set comprised donors with only 10–100 bp homologous arms, while the other set included donors with homologous arms ranging from 10–100 bp, between which was a tetracycline resistance expression cassette (1439 bp). These donor constructs were transformed into Escherichia coli MG1655 cells alongside pCas-SSB/pTargetF-lacZ. Notably, when the homologous arms ranged from 10 to 70 bp, the transformation efficiency of non-selectable donors was significantly higher than that of selectable donors. However, within the range of 10–100 bp homologous arm lengths, the homologous recombination rate of selectable donors was significantly higher than that of non-selectable donors, with the gap narrowing as the homologous arm length increased. For selectable donor DNA with homologous arm lengths of 10–60 bp, the homologous recombination rate increased linearly, reaching a plateau when the homologous arm length was between 60–100 bp. Conversely, for non-selectable donor DNA, the homologous recombination rate increased linearly with homologous arm lengths of 10–90 bp, plateauing at 90–100 bp. Editing two loci simultaneously with 100 bp homologous arms, whether selectable or non-selectable, showed no difference in transformation or homologous recombination rates. Editing three loci simultaneously with 100 bp non-selectable homologous arms resulted in a 45% homologous recombination rate. These results suggest that efficient homologous recombination gene editing mediated by SSB/CRISPR-Cas9 can be achieved using donor DNA with 90–100 bp non-selectable homologous arms or 60–100 bp selectable homologous arms.

This study develops a “Bio-Social-Cognitive Dynamic Interaction Model” to explore seasonal mental health risks in Chinese adolescents. Using data from cross-sectional surveys (N = 6,121), longitudinal tracking (N = 1,000), and RCTs (N = 200), we assess mental health via PHQ-9, GAD-7, and SADQ scales. We analyze multidimensional indicators (melatonin, vitamin D, academic stress) via SEM and ARIMA, considering school stage and gender. Results show winter has the highest depression and anxiety scores (PHQ-9: M = 14.5 ± 4.8), linked to vitamin D deficiency (β=-0.25, p < 0.001) and social stress. Spring academic stress affects emotional stability via melatonin and sleep issues (β = 0.15, p = 0.002), while summer anxiety is tied to social overload and body dissatisfaction (β = 0.10, p = 0.04). Winter light therapy increases vitamin D (Δ = 8.2 ng/mL, p < 0.001) and reduces depression (ΔPHQ-9=-3.1, p = 0.005). We recommend seasonal interventions like winter light therapy, flexible spring exams, and summer social media literacy programs, and emphasize year-round monitoring for effective risk mitigation.