A high-efficiency and versatile CRISPR/Cas9-mediated HDR-based biallelic editing system

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.     

Target binding and residence: a new determinant of DNA double-strand break repair pathway choice in CRISPR/Cas9 genome editing

The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)is widely used for targeted genomic and epigenomic modifications and imaging in cells and organisms,and holds tremendous promise in clinical applications.The efficiency and accuracy of the technology are partly determined by the target binding affinity and residence time of Cas9-single-guide RNA(sgRNA)at a given site.However,little attention has been paid to the effect of target binding affinity and residence duration on the repair of Cas9-induced DNA double-strand breaks(DSBs).We propose that the choice of DSB repair pathway may be altered by variation in the binding affinity and residence duration of Cas9-sgRNA at the cleaved target,contributing to significantly heterogeneous mutations in CRISPR/Cas9 genome editing.Here,we discuss the effect of Cas9-sgRNA target binding and residence on the choice of DSB repair pathway in CRISPR/Cas9 genome editing,and the opportunity this presents to optimize Cas9-based technology.     

人类首例基因编辑婴儿试验:伦理失范和善治的迫切性（英文）

近年来,中国生物医学研究快速发展的同时,也在伦理治理方面带来了挑战。贺建奎公开声称其胚胎基因编辑婴儿诞生后,立即招致广泛的批评、谴责和激烈的伦理和法律大辩论。以生育为目的的胚胎基因编辑操作违反了公认的伦理规范,具体包括:较低的科学价值、不合理的风险-受益比、伦理审查不合规、并非真正的知情同意等。这说明主要依靠科研人员自律的机构伦理治理体系是成问题的,为此需要在不同层面上改进伦理治理水平,强化技术和伦理指南和法规。