Methods to regulate gene expression programs in bacterial cells are limited by the absence of effective gene activators. To address this challenge, we have developed synthetic bacterial transcriptional activators in E. coli by linking activation domains to programmable CRISPR-Cas DNA binding domains. Effective gene activation requires target sites situated in a narrow region just upstream of the transcription start site, in sharp contrast to the relatively flexible target site requirements for gene activation in eukaryotic cells. Together with existing tools for CRISPRi gene repression, these bacterial activators enable programmable control over multiple genes with simultaneous activation and repression. Further, the entire gene expression program can be switched on by inducing expression of the CRISPR-Cas system. This work will provide a foundation for engineering synthetic bacterial cellular devices with applications including diagnostics, therapeutics, and industrial biosynthesis.

Low targeting efficiency is one of the biggest limitations for nanoparticulate drug delivery system-based cancer therapy. In this study, an efficient approach for tumor-targeted drug delivery was developed with mesenchymal stem cells as the targeting vehicle and a silica nanorattle as the drug carrier. A silica nanorattle-doxorubicin drug delivery system was efficiently anchored to mesenchymal stem cells (MSCs) by specific antibody-antigen recognitions at the cytomembrane interface without any cell preconditioning. Up to 1500 nanoparticles were uploaded to each MSC cell with high cell viability and tumor-tropic ability. The intracellular retention time of the silica nanorattle was no less than 48 h, which is sufficient for cell-directed tumor-tropic delivery. In vivo experiments proved that the burdened MSCs can track down the U251 glioma tumor cells more efficiently and deliver doxorubicin with wider distribution and longer retention lifetime in tumor tissues compared with free DOX and silica nanorattle-encapsulated DOX. The increased and prolonged DOX intratumoral distribution further contributed to significantly enhanced tumor-cell apoptosis. This strategy has potential to be developed as a robust and generalizable method for targeted tumor therapy with high efficiency and low systematic toxicity.

Supplementary Figure 1: CRISPRa at the endogenous gene target ldhA does not follow predicted trends.Eight scRNA target sites (L1-L8) upstream of the ldhA promoter were selected. Three of the target sites (L5-L7) were within the 40 bp window where CRISPRa is effective (-100 to -60). While L1, L4, and L5 resulted in weak increases in gene expression, there was no apparent relationship between the position of the sites and ldhA expression levels. Gene expression was measured using RT-qPCR. Fold activation represents expression levels relative to an off-target control (hAAVS1). Bars indicate the average values between three technical replicates and black dots indicate the values of individual replicates. Error bars indicate the standard error of the mean between three technical replicates. S3 Supplementary Figure 2: CRISPRa activity depends on the target sequence on the scRNA. Reporter cassettes that differ only by the sequence of the 20 base scRNA target site give a broad range of gene expression levels, demonstrating that the sequence of the scRNA target site can have a substantial effect on CRISPRa. Three new reporter plasmids were constructed where the J306 target site, located at -81 from the TSS, on the J3-J23117-mRFP1 reporter was replaced by the J104, J106, and J108 sequence. Activation at each promoter was tested when CRISPRa was targeted to their cognate scRNA site. The off-target negative control (OT) represents a strain expressing the original reporter with the J306 site and the CRISPRa components to target an offtarget site (J206). Fluorescence/OD 600 values were measured using a plate reader. Bars indicate the average values between three biological replicates. Black dots indicate the values of individual biological replicates. Error bars indicate the standard deviation between biological replicates. S4 S5 Supplementary Figure 3: The sharp positioning dependence of CRISPRa is observed across multiple promoters. A) The sharp positioning requirements of CRISPRa are not significantly affected by the location or composition of the inserted sequence. Reporters were based on the J1-J23117-mRFP1 (Supplementary Figure 3A) with base shifts introduced in different ways. In the J1 reporter A, bases were inserted upstream of the -35 region. In the J1 reporter B, a different sequence was

One of the current challenges in biomedicine is to develop safe and effective nanomedicines for selective tumor therapy. [1] Recently, near-infrared (NIR) light absorbing plasmonic nanomaterials have attracted intensive attention for their hyperthemia therapy to kill tumorigenic cells without damaging normal cells, such as gold nanorods, [2] gold nanocages, [3] Au x Ag 1-x dendrites, [4] gold nanoshells on polystyrene spheres, [5] assembled gold nanoparticles [6] and many multifunctional nanocomposites. [7] Our previous study reported a novel material of gold nanoshells on drug-loaded silica nanorattles which can combine the hyperthermia with chemotherapy to optimize cancer therapy whose synergistic effects are greater than the two individual treatments alone. [8] Despite the successful application of many gold-based NIR absorbing materials in cancer therapy, most studies of them rely on the passive targeting effect (the so-called enhanced permeability and retention, EPR effect) to direct nanocarriers at tumor sites through the enhanced permeability of tumor vasculature and the decreased draining efficacy of tumor lymphatics. [9,10] The lack of cell specific interactions may decrease the therapeutic efficacy and thus need a relatively long NIR light irradiation time (30 min in vitro, e.g.), [11] a high NIR laser light irradiation intensity (35 W cm -2 in vitro, e.g.), [12] or repeated injections and NIR laser light irradiations [8] as the previous reports. Furthermore, not all tumors exhibit EPR effect which can enhance the preferential accumulation of nanoparticles in the tumor. [13,14] For clinical applications, a more effective drug delivery strategy should be developed to promote the binding and internalization of nanocarrier through their specific interactions with the receptors expressed on the cell surface of interest. [15] Transferrin (abbreviated hereinafter as Tf) is one of the widely used targeting ligand. Because of rapid cell division and stringent demand for iron (for heme synthesis), many cancer cell types have abundant expression of Tf receptors (TfRs).[16] Tf has been used to enhance the cellular uptake of gold nanoparticles [17] and quantum dots/rods. [18] To the best of our knowledge, Tf-conjugated gold-based nanoparticles for both hyperthermia and chemotherapy of cancer cells have been rarely reported. Most importantly, there are few comparative studies on in vivo excretion/clearance, safety, and efficacy of targeted conjugated and non-conjugated nanotherapeutics, while elucidating these in vivo studies is considered very important regarding the safety and validity of novel nanocarrier systems. [19] In view of this pressing need for more efficient treatment modalities, we engineered a kind of Tf and PEG functionalized gold nanoshells on silica nanorattles (pGSNs-Tf) for ablation of breast carcinoma in the present study. We hypothesized that the targeting pGSNs-Tf can preferentially bind to breast cancer cells rather than normal cells and then increase the efficiency of thermo-chemotherapy ab...

In bacterial systems, CRISPR-Cas transcriptional activation (CRISPRa) has the potential to dramatically expand our ability to regulate gene expression, but we currently lack a complete understanding of the rules for designing effective guide RNA target sites. We have identified multiple features of bacterial promoters that impose stringent requirements on bacterial CRISPRa target sites. Most importantly, we found that shifting a gRNA target site by 2-4 bases along the DNA target can cause a nearly complete loss in activity. The loss in activity can be rescued by shifting the target site 10-11 bases, corresponding to one full helical turn. Practically, our results suggest that it will be challenging to find a gRNA target site with an appropriate PAM sequence at precisely the right position at arbitrary genes of interest. To overcome this limitation, we demonstrate that a dCas9 variant with expanded PAM specificity allows activation of promoters that cannot be activated by S. pyogenes dCas9. These results provide a roadmap for future engineering efforts to further expand and generalize the scope of bacterial CRISPRa.

Stromal cell-derived factor 1 (SDF-1) is a chemokine that can be expressed in injured cardiomyocytes after myocardial infarction (MI). By combining with its receptor CXCR4, SDF-1 induced stem and progenitor cells migration. CXCR7, a novel receptor for SDF-1, has been identified recently. We aimed to explore the roles of SDF-1/CXCR4 and SDF-1/CXCR7 pathway and their crosstalk in CSCs migration. In the present study, CXCR4 and CXCR7 expression were identified in CSCs. Transwell assay showed that SDF-1 caused CSCs migration in a dose- and time-dependent manner, which could be significantly suppressed by CXCR4 or CXCR7 siRNA. Phospho-ERK, phospho-Akt and Raf-1 significantly elevated in CSCs with SDF-1 stimulation. Knockdown of CXCR4 or CXCR7 significantly decreased phospho-ERK or phospho-Akt, respectively, and eventually resulted in the inhibition of CSCs migration. Moreover, western blot showed that MK2206 (Akt inhibitor) increased the expression of phospho-ERK and Raf-1, whereas PD98059 (ERK inhibitor) had no effect on phospho-Akt and Raf-1. GW5074 (Raf-1 inhibitor) upregulated the expression of phospho-ERK, but had no effect on phospho-Akt. The present study indicated that SDF-1/CXCR7/Akt and SDF-1/CXCR4/ERK pathway played important roles in CSCs migration. Akt phosphorylation inhibited Raf-1 activity, which in turn dephosphorylated ERK and negatively regulated CSCs migration.

No abstract

Methods for implementing dynamically-controlled multi-gene programs could expand capabilities to engineer metabolism for efficiently producing high-value compounds. This work explores whether CRISPRi repression can be tuned in E. coli through the regulated expression of the CRISPRi machinery. When dCas9 is not limiting, variations in sgRNA expression alone can lead to CRISPRi repression levels ranging from 5- to 300-fold. Titrating sgRNA expression over a 2.5-fold range results in 16-fold changes in reporter gene expression. Many different classes of genetic controllers can generate 2.5-fold differences in transcription, suggesting they may be integrated into dynamically-regulated CRISPRi circuits. Finally, CRISPRi cannot be reversed for up to 12 hours by expressing a competing sgRNA later in the growth phase, indicating that CRISPR-Cas:DNA interactions can be persistent in vivo. Collectively, these results identify genetic architectures for tuning CRISPRi repression through regulated sgRNA expression and suggest that dynamically-regulated CRISPRi systems targeting multiple genes may be within reach.