Programmable Live-Cell CRISPR Imaging with Toehold-Switch-Mediated Strand Displacement

The widespread application of CRISPR-Cas9 has transformed genome engineering. Nevertheless, the precision to control the targeting activity of Cas9 requires further improvement. We report a toehold-switch-based approach to engineer the conformation of single guide RNA (sgRNA) for programmable activation of Cas9. This activation circuit is responsive to multiple inputs and can regulate the conformation of the sgRNA through toehold-switch-mediated strand displacement. We demonstrate the orthogonal suppression and activation of Cas9 with orthogonal DNA inputs. Combination of toehold switches leads to a variety of intracellular Cas9 activation programs with simultaneous and orthogonal responses, through which multiple genome loci are displayed in different colors in a controllable manner. This approach provides a new route for programing CRISPR in living cells for genome imaging and engineering.     

Antibacterial Activity of Pharmaceutical-Grade Rose Bengal: An Application of a Synthetic Dye in Antibacterial Therapies

Rose bengal has been used in the diagnosis of ophthalmic disorders and liver function, and has been studied for the treatment of solid tumor cancers. To date, the antibacterial activity of rose bengal has been sporadically reported; however, these data have been generated with a commercial grade of rose bengal, which contains major uncontrolled impurities generated by the manufacturing process (80–95% dye content). A high-purity form of rose bengal formulation (HP-RBf, >99.5% dye content) kills a battery of Gram-positive bacteria, including drug-resistant strains at low concentrations (0.01–3.13 μg/mL) under fluorescent, LED, and natural light in a few minutes. Significantly, HP-RBf effectively eradicates Gram-positive bacterial biofilms. The frequency that Gram-positive bacteria spontaneously developed resistance to HP-RB is extremely low (less than 1 × 10⁻¹³). Toxicity data obtained through our research programs indicate that HP-RB is feasible as an anti-infective drug for the treatment of skin and soft tissue infections (SSTIs) involving multidrug-resistant (MDR) microbial invasion of the skin, and for eradicating biofilms. This article summarizes the antibacterial activity of pharmaceutical-grade rose bengal, HP-RB, against Gram-positive bacteria, its cytotoxicity against skin cells under illumination conditions, and mechanistic insights into rose bengal’s bactericidal activity under dark conditions.     

Developing CRISPR/Cas9-Mediated Fluorescent Reporter Human Pluripotent Stem-Cell Lines for High-Content Screening

Application of the CRISPR/Cas9 system to knock in fluorescent proteins to endogenous genes of interest in human pluripotent stem cells (hPSCs) has the potential to facilitate hPSC-based disease modeling, drug screening, and optimization of transplantation therapy. To evaluate the capability of fluorescent reporter hPSC lines for high-content screening approaches, we targeted EGFP to the endogenous OCT4 locus. Resulting hPSC–OCT4–EGFP lines generated expressed EGFP coincident with pluripotency markers and could be adapted to multi-well formats for high-content screening (HCS) campaigns. However, after long-term culture, hPSCs transiently lost their EGFP expression. Alternatively, through EGFP knock-in to the AAVS1 locus, we established a stable and consistent EGFP-expressing hPSC–AAVS1–EGFP line that maintained EGFP expression during in vitro hematopoietic and neural differentiation. Thus, hPSC–AAVS1–EGFP-derived sensory neurons could be adapted to a high-content screening platform that can be applied to high-throughput small-molecule screening and drug discovery campaigns. Our observations are consistent with recent findings indicating that high-frequency on-target complexities appear following CRISPR/Cas9 genome editing at the OCT4 locus. In contrast, we demonstrate that the AAVS1 locus is a safe genomic location in hPSCs with high gene expression that does not impact hPSC quality and differentiation. Our findings suggest that the CRISPR/Cas9-integrated AAVS1 system should be applied for generating stable reporter hPSC lines for long-term HCS approaches, and they underscore the importance of careful evaluation and selection of the applied reporter cell lines for HCS purposes.     

黑胸大蠊浓核病毒基因组末端序列测定与分析

利用构建的黑胸大蠊浓核病毒(PfDNV)全基因组重组质粒(PfDNV-pUC119)以及一系列完整缺失克隆,通过全自动测序方法完成了PfDNV两末端各728 bp 和730 bp 核苷酸序列的测定. 通过计算机软件分析发现,PfDNV 末端201nt为倒置重复序列,其中最末端122nt为回文序列,能形成典型的发夹结构. 并与同科其它病毒末端序列进行了核苷酸序列同源性比较.     

Analysis of longitudinal multispin orders in strongly coupled spin systems

Longitudinal multispin orders provide an effective way for measurement of scalar couplings and also to probe molecular interactions and dynamics. Analysis of longitudinal orders has been made in strongly coupled AB and ABX spin systems to determine the dependence of strong coupling parameter on these orders. Experimental and simulated spectra of various longitudinal orders are illustrated for these spin systems. This general procedure can be extended to broad range of spin systems to understand the influence of strong coupling on longitudinal orders.     

Exact Distribution of the Distances between Any Occurrences of a Set of Words

The distribution of the distance between two (or more) successive occurrences of a specific word in a random sequence of letters is known under different models. In this paper, a more general problem is studied: the distribution of the distance between two (or more) successive occurrences of any word of a given set under a Markov model for the sequence. The generating function and a recurrence for obtaining the probabilities are given. These results are applied to study the distribution of the "CHI" motif in the genome sequence of Haemophilus influenzae.     

对DEPT实验技术的一点改进

将DEPT -1 3 5实验的 1 3 5° -脉冲分成一个 90°脉冲和一个 4 5°脉冲 ,通过相位循环 ,设计了两个新的DEPT实验分别称为DEPT-90 -4 5和DEPT -90 +4 5。DEPT -90 -4 5谱中只有 -CH3的吸收峰。DEPT -90 +4 5谱中没有 >CH -的吸收峰 ,其 -CH3为正信号 ,-CH2 -为负信号。实验结果显示 ,对比DEPT -90谱 ,这两个新的DEPT谱中完全没有所选基团之外的残余干扰信号 ,而且DEPT -90 -4 5谱比DEPT-90 +4 5谱更好。     

反BIRD在NMR谱编辑中的应用

建议了一个新的双线性旋转去偶序列 ,称为反BIRD (inverse -BIRD) ,其去偶作用与碳连质子的数目有关。将反BIRD结合到INEPT实验中 ,得到了INEPT -iBIRD实验技术 ,它可用于核磁共振碳谱的编辑与简化     

An α/β-HSQC-α/β Experiment for Spin-State Selective Editing of IS Cross Peaks

A generalized version of the TROSY experiment allows the spin-state selective editing of the four multiplet components of¹⁵N–¹H cross peaks of amide groups in proteins into four different subspectra, with no penalty in sensitivity. An improvement by in sensitivity results, if only two of the four multiplet components are selected. Use of the experiment for the measurement of¹J_HNcoupling constants is discussed. A water flip-back version of the experiment is demonstrated with a 45 kDa fragment of¹⁵N/²H labeledStaphylococcus aureusgyrase B.