Comparison and optimization of different CRISPR/Cas9 donor-adapting systems for gene editing.

Gene knock-in in mammalian cells usually uses homology-directed repair (HDR) mechanism to integrate exogenous DNA template into the target genome site. However, HDR efficiency is often low, and the co-localization of exogenous DNA template and target genome site is one of the key limiting factors. To improve the efficiency of HDR mediated by CRISPR/Cas9 system, our team and previous studies fused different adaptor proteins with SpCas9 protein and expressed them. By using their characteristics of binding to specific DNA sequences, many different CRISPR/SpCas9 donor adapter gene editing systems were constructed. In this study, we used them to knock-in eGFP gene at the 3'-end of the terminal exon of GAPDH and ACTB genes in HEK293T cells to facilitate a comparison and optimization of these systems. We utilized an optimized donor DNA template design method, validated the knock-in accuracy via PCR and Sanger sequencing, and assessed the efficiency using flow cytometry. The results showed that the fusion of yGal4BD, hGal4BD, hLacI, hTHAP11 as well as N57 and other adaptor proteins with the C-terminus of SpCas9 protein had no significant effect on its activity. At the GAPDH site, the donor adapter systems of SpCas9 fused with yGal4BD, hGal4BD, hLacI and hTHAP11 significantly improved the knock-in efficiency. At the ACTB site, SpCas9 fused with yGal4BD and hGal4BD significantly improved the knock-in efficiency. Furthermore, increasing the number of BS in the donor DNA template was beneficial to enhance the knock-in efficiency mediated by SpCas9-hTHAP11 system. In conclusion, this study compares and optimizes multiple CRISPR/Cas9 donor adapter gene editing systems, providing valuable insights for future gene editing applications.

Proximal femoral nail antirotation and third-generation Gamma nail: which is a better device for the treatment of intertrochanteric fractures?

<p><b>INTRODUCTION</b>Proximal femoral nail antirotation (PFNA) and third-generation Gamma nail (Gamma 3) are widely used in the treatment of intertrochanteric fractures. However, it remains unclear which device achieves better clinical and radiographic outcomes when treating intertrochanteric fractures.</p><p><b>METHODS</b>This study comprised 239 patients with intertrochanteric fractures treated with either PFNA or Gamma 3 for a minimum of 12 months. During surgery, the operative time, image intensifier time and amount of blood loss were recorded. Following surgery, we assessed reduction quality and implant position. At the final follow-up, postoperative complications, including femoral shaft fracture, cutout, reoperation, pneumonia, urinary tract infection, cerebral infarction, cardiac infarction and decubital ulcer, were recorded. In addition, walking ability was assessed using the Parker-Palmer mobility score.</p><p><b>RESULTS</b>No difference was found in the operative time, image intensifier time and amount of blood loss between patients treated with PFNA and those treated with Gamma 3. The reduction quality of fractures treated with Gamma 3 was better than those treated with PFNA. However, there were no significant differences in implant position, walking ability and postoperative complications between the two groups. Although Gamma 3 resulted in better reduction quality, it did not provide any advantages in walking ability and postoperative complications when compared with PFNA.</p><p><b>CONCLUSION</b>Therefore, we conclude that both PFNA and Gamma 3 are safe and reliable devices for the treatment of intertrochanteric fractures.</p>