A high-efficiency and versatile CRISPR/Cas9-mediated HDR-based biallelic editing system / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9), the third-generation genome editing tool, has been favored because of its high efficiency and clear system composition. In this technology, the introduced double-strand breaks (DSBs) are mainly repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways. The high-fidelity HDR pathway is used for genome modification, which can introduce artificially controllable insertions, deletions, or substitutions carried by the donor templates. Although high-level knock-out can be easily achieved by NHEJ, accurate HDR-mediated knock-in remains a technical challenge. In most circumstances, although both alleles are broken by endonucleases, only one can be repaired by HDR, and the other one is usually recombined by NHEJ. For gene function studies or disease model establishment, biallelic editing to generate homozygous cell lines and homozygotes is needed to ensure consistent phenotypes. Thus, there is an urgent need for an efficient biallelic editing system. Here, we developed three pairs of integrated selection systems, where each of the two selection cassettes contained one drug-screening gene and one fluorescent marker. Flanked by homologous arms containing the mutated sequences, the selection cassettes were integrated into the target site, mediated by CRISPR/Cas9-induced HDR. Positively targeted cell clones were massively enriched by fluorescent microscopy after screening for drug resistance. We tested this novel method on the amyloid precursor protein (APP) and presenilin 1 (PSEN1) loci and demonstrated up to 82.0% biallelic editing efficiency after optimization. Our results indicate that this strategy can provide a new efficient approach for biallelic editing and lay a foundation for establishment of an easier and more efficient disease model.

Research progress of interbody fusion cage and its materials / 中国组织工程研究

BACKGROUND:With the rapid development of technology, most interbody fusion cages have been applied in clinic, but some are stil in experimental stage. Al fusion cages hold their own distinct characteristics as wel as advantages and shortcomings. OBJECTIVE:To clarify the development course of interbody fusion cages and analyze their types in detail, thereby paving ways for its improvement. METHODS:A computer-based search for literatures in CNKI and PubMed databases published from January 1989 to March 2016 was performed using the keywords of“interbody fusion cage, PEEK, TMC”in English and Chinese, respectively. A total of 106 articles were retrieved and 38 articles were in accordance with the inclusion criteria. RESULTS AND CONCLUSION:Intervertebral fusion is a major method to treat spinal degenerative diseases. There are a variety of fusion cages in clinic, such as pyramesh, polyetheretherketone and absobable fusion cages. Implants used for fusion cages include autogenous, al ogenic and artificial bones as wel as bone morphogenetic protein. We introduce the development process of fusion cages in detail, emphasize the commonly used fusion cages and analyze its development in future. In the end, we give our opinions about the development of intervertebral fusion cages.