Strategies for Optimization of the Clustered Regularly Interspaced Short Palindromic Repeat-Based Genome Editing System for Enhanced Editing Specificity.

The clustered regularly interspaced short palindromic repeats (CRISPR) system is inarguably the most valuable gene editing tool ever discovered. Currently, three classes of CRISPR-based genome editing systems have been developed for gene editing, including CRISPR/CRISPR associate system (Cas) nucleases, base editors, and prime editors. Ever-evolving CRISPR technology plays an important role in medicine; however, the biggest obstacle to its use in clinical practice is the induction of off-target effects (OTEs) during targeted editing. Therefore, continuous improvement and optimization of the CRISPR system for reduction of OTEs is a major focus in the field of CRISPR research. This review aims to provide a comprehensive guide for optimization of the CRISPR-based genome editing system.

Development of a Self-Restricting CRISPR-Cas9 System to Reduce Off-Target Effects.

Development of the CRISPR-Cas9 gene-editing system has given rise to a new era of gene editing with wide applications in biology, medicine, agriculture, and other fields. However, the overexpression of Cas9 nuclease causes off-target effects and may trigger an immune response in vivo. Therefore, we constructed a self-restricting CRISPR-Cas9 system, where the target gene sequence corresponding to the guide RNA (gRNA) is inserted on either end of the Cas9 promoter. When double-strand breaks (DSBs) are induced in the target gene sequence, the Cas9 promoter is cut off and transcription ceases. With this system, expression of Cas9 protein at 60 h after transfection is only 10% that of the wild-type system, with about 70% promoter deletion efficiency. The target site editing efficiency and homologous recombination efficiency of the self-restricting system remain at about 50% and 30%, respectively, while the frequency of off-target indel formation decreased by 76.7%. Further, the number of indel types was also reduced from 13 to 2. Because this system does not include additional gRNA sequences, the possibility of introducing new off-target mutations is decreased. Importantly, this system is composed of a single plasmid, which could potentially be easily introduced in vivo using a viral vector or nanoparticles.

[Distribution characteristics of organochlorine pesticides in river surface sediments in Song-Liao watershed].

With gas chromatography-electron capture detector (GC-ECD), this paper analyzed 13 kinds of organochlorinae pesticides (OCP) in surface sediments collected from 22 sites of Song-Liao water system. The results showed that the total concentration of OCP varied from 3.06 to 23.24 ng x g(-1) in Daliao River system and from 4.26 to 18.45 ng x g(-1) in Songhuajiang River system. The most dominant OCP pollutant in the sediments in Song-Liao Watershed was HCH, but there existed different pollution tendency in the two subsystems. In Songhuajiang River, there were also relatively high concentrations of DDT. High concentration gamma-HCH was observed among the isomers of HCH in the whole watershed, which exceeded the initial affecting concentration and could potentially do toxic effect on the water creatures.

[Contamination characters of polycyclic aromatic hydrocarbons in Daliao River system of China].

With GC/MS, a quantitative study was made on the polycyclic aromatic hydrocarbons (PAHs) in the surface water and suspended particulates in Daliao River system, with their spatial distribution and sources investigated. The results showed that the total PAHs concentration ranged from 946.1 to 13 448.5 ng x L(-1) in surface water, and from 317.5 to 238 518.7 ng x g in suspended particulates. The PAHs concentration was decreased in the order of Taizi River > Daliao River > Hunhe River. The PAHs in surface water were dominated by 3-5 rings, while those in suspended particulates were dominated by 2 rings. The calculated ratios of selected PAHs suggested that the discharges from municipal, petrochemical and steel industrial wastewater, and the atmospheric fallout might be the important PAHs sources, and the contamination sources of PAHs were the mixture of petrogenic and pyrolytic inputs. In comparing with other river and marine systems in the world, Daliao River system had higher concentrations of PAHs in its surface water and suspended particulates, and thus, presented certain ecological risk.

[Activities of circulating endothelial progenitor cells in patients with in-stent restenosis].

OBJECTIVE: To observe the number and activities of circulating endothelial progenitor cells (EPCs) in patients with in-stent restenosis. METHODS: Peripheral blood samples were collected from 15 patients with angiographically restenosis, and 17 baseline characteristics-matched patients without angiographically restenosis (control group). Mononuclear cells were isolated by Ficoll density-gradient centrifugation and plated on dishes coated with human fibronectin. After 7 days in culture, the nature of EPCs was characterized with anti-CD34 and anti-KDR, specific surface antibodies of EPC, and confirmed further with the use of fluorescein isothiocyanate-labeled ulex europaeus agglutinin-I (FITC-UEA-I) and DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine percolate)-labeled acetylated low-density lipoprotein (DiI-acLDL) by laser scanning confocal microscopy. The number of EPCs was counted in a blinded manner. EPCs were inoculated onto the culture plate and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide assay was used to measure the A value by enzyme labeling instrument to evaluate the proliferation. The migration of EPCs was assayed by scratch assay. EPC adhesion was performed by replating cells on fibronectin-coated dishes and then counting the adherent cells. Results The number of EPCs of the patients with in-stent restenosis was 4.97 +/- 1.42/well, significantly lower than that of the control group (17.2 +/- 3.90/well, P = 0.001). MTT assay showed that the proliferative activities of the in-stent restenosis group was 1.37 +/- 0.32 times the baseline value, significantly lower than that of the control group (2.01 +/- 0.62, P < 0.05). The number of migrating EPCs of the in-stent restenosis group was remarkably lower than that of the control group. There was no significant difference in the adherent activity between the two groups. Conclusion The number, proliferation activity, and migration activity of the EPCs patients with in-stent restenosis are all significantly lower, which may contribute to the mechanism of in-stent restenosis.