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.

A CRISPR/Cas9-Gal4BD donor adapting system for enhancing homology-directed repair.

The fast-rising CRISPR-derived gene editing technologies has been widely used in the fields of life science and biomedicine, as well as plant and animal breeding. However, the efficiency of homology-directed repair (HDR), an important strategy for gene knock-in and base editing, remains to be improved. In this study, we came up with the term Donor Adapting System (DAS) to summarize those CRISPR/Cas9 systems modified with adaptor for driving aptamer-fused donor DNA. A set of CRISPR/Cas9-Gal4BD DAS was designed in our study. In this system, Gal4 DNA binding domain (Gal4BD) is used as adaptor to fuse with Cas9 protein, and Gal4 binding sequence (Gal4BS) is used as aptamer to bind to the double-stranded DNA (dsDNA) donor, in order to improve the HDR efficiency. Preliminary results from the HEK293T-HDR.GFP reporter cell line show that the HDR editing efficiency could be improved up to 2-4 times when donor homologous arms under certain length (100-60 bp). Further optimization results showed that the choice of fusion port and fusion linker would affect the expression and activity of Cas9, while the Cas9-Gal4BD fusion with a GGS5 linker was the prior choice. In addition, the HDR efficiency was likely dependent on the aptamer-dsDNA donor design, and single Gal4BD binding sequence (BS) addition to the 5'-end of intent dsDNA template was suggested. Finally, we achieved enhanced HDR editing on the endogenous AAVS1 and EMX1 sites by using the CRISPR/Gal4BD-Cas9 DAS, which we believe can be applied to facilitate animal molecular design breeding in the future.

Synthesis of In Situ ZrB2-SiC-ZrC Coating on ZrC-SiC Substrate by Reactive Plasma Spraying.

In situ synthesis feasibility of ZrB2-SiC-ZrC composite coatings on ZrC ceramics by reactive plasma spraying (RPS) was investigated. To help to understand the phase evolution during plasma spraying process, reaction behavior in the ZrH2-Si-B4C system was explored carefully by differential scanning calorimetry. The results indicated that the phase transformation sequence in the ZrH2-Si-B4C system could be described as ZrH1.66, Zr3O, ZrC, ZrB2, Zr2Si, ZrSi, and SiC. The prior formation of ZrC was due to high diffusion rate of C atoms from B4C. ZrB2 was produced above 1100 °C. As the temperature increased, SiC were finally formed by the reaction of ZrC with ZrSi and B4C. The RPS composite coatings mainly consisted of ZrB2, SiC, and ZrC phases, except for a small fraction of ZrO2 phase. The microstructural characterization exhibited more dense melted splats, which appears to increase gradually with the increase in spraying currents and distances. The coatings had typical lamellar structure and adhered to the substrate well. The microhardness values were higher than 1000 HV1, but there were few variations with varying spraying currents and distances.