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Background CRISPR/Cas9 systems have been repurposed as canonical genome editing tools in a variety of species, but no application for the model strain Rhodobacter sphaeroides 2.4.1 was unveiled. Results Here we showed two kinds of programmable base editing systems, cytosine base editors (CBEs) and adenine base editors (ABEs), generated by fusing endonuclease Cas9 variant to cytosine deaminase PmCDA1 or heterodimer adenine deaminase TadA–TadA*, respectively. Using CBEs, we were able to obtain C-to-T mutation of single and double targets following the first induction step, with the efficiency of up to 97% and 43%; while the second induction step was needed in the case of triple target, with the screening rate of 47%. Using ABEs, we were only able to gain A-to-G mutation of single target after the second induction step, with the screening rate of 30%. Additionally, we performed a knockout analysis to identify the genes responsible for coenzyme Q10 biosynthesis and found that ubiF, ubiA, ubiG, and ubiX to be the most crucial ones. Conclusions Together, CBEs and ABEs serve as alternative methods for genetic manipulation in Rhodobacter sphaeroides and will shed light on the fundamental research of other bacteria that are hard to be directly edited by Cas9-sgRNA.

Transcription activator-like effectors (TALEs) are becoming powerful DNA-targeting tools in a variety of mammalian cells and model organisms. However, generating a stable cell line with specific gene mutations in a simple and rapid manner remains a challenging task. Here, we report a new method to efficiently produce monoclonal cells using integrated TALE nuclease technology and a series of high-throughput cell cloning approaches. Following this method, we obtained three mTOR mutant 293T cell lines within 2 months, which included one homozygous mutant line.

Cell metabolism is critical for cancer cell transformation and progression. In this study, we have developed a novel method, named Met-express, that integrates a cancer gene co-expression network with the metabolic network to predict key enzyme-coding genes and metabolites in cancer cell metabolism. Met-express successfully identified a group of key enzyme-coding genes and metabolites in lung, leukemia, and breast cancers. Literature reviews suggest that approximately 33-53% of the predicted genes are either known or suggested anti-cancer drug targets, while 22% of the predicted metabolites are known or high-potential drug compounds in therapeutic use. Furthermore, experimental validations prove that 90% of the selected genes and 70% of metabolites demonstrate the significant anti-cancer phenotypes in cancer cells, implying that they may play important roles in cancer metabolism. Therefore, Met-express is a powerful tool for uncovering novel therapeutic biomarkers.

Kinases become one of important groups of drug targets. To identify more kinases being potential for cancer therapy, we developed an integrative approach for the large-scale screen of functional genes capable of regulating the main traits of cancer metastasis. We first employed self-assembled cell microarray to screen functional genes that regulate cancer cell migration using a human genome kinase siRNA library. We identified 81 genes capable of significantly regulating cancer cell migration. Following with invasion assays and bio-informatics analysis, we discovered that 16 genes with differentially expression in cancer samples can regulate both cell migration and invasion, among which 10 genes have been well known to play critical roles in the cancer development. The remaining 6 genes were experimentally validated to have the capacities of regulating cell proliferation, apoptosis and anoikis activities besides cell motility. Together, these findings provide a new insight into the therapeutic use of human kinases. From the Clinical Editor This team of authors have utilized a self-assembled cell microarray to screen genes that regulate cancer cell migration using a human genome siRNA library of kinases. They validated previously known genes and identified novel ones that may serve as therapeutic targets.

MicroRNAs (miRNAs) are a group of endogenous, small noncoding RNAs of approximately 22 nucleotides in lengths. As a new class of signaling modulators, miRNAs have attracted great attention for their unique features, including multitarget regulation, tissue specificity, and evolutionary conservation. These small endogenous RNAs are able to interact with many important genes and play critical roles in a wide range of biological processes, including cell proliferation and differentiation. Strikingly, miRNAs are frequently dysregulated in human cancers. A number of studies have shown that miRNAs are involved in cancer pathogenesis by regulating oncogenes or tumor suppressor genes. Here, we review recent studies of miRNAs in cancer development and discuss their potential applications in cancer therapeutics.

MicroRNAs, a group of endogenous non-coding single-strand RNAs typically of 21~23nt in length, have been convincingly shown to play a critical role in a wide variety of physiological processes such as cell proliferation, differentiation, apoptosis, tumor metastasis, etc. Here we incorporated a microfluidic device with a miRNA detection method based on ramification amplification (RAM) to increase the detection throughput and reduce the labor cost. Four different reactions could be performed in a microfluidic device: (i) reverse-transcription of miRNA, (ii) annealing of circular probe (C-Probe), (iii) C-Probe ligation, and (iv) Ramification amplification. Through monitoring the intensity of fluorescent signal, tens of miRNA can be quantified in a chip in parallel. Thus, these primary results demonstrate that this new device has advantages in throughput and cost, and it might also be used for the detection of miRNA in dangerous samples, such as virus because of its automation control system.

Recent effective use of TAL Effectors (TALEs) has provided an important approach to the design and synthesis of sequence-specific DNA-binding proteins. However, it is still a challenging task to design and manufacture effective TALE modulators because of the limited knowledge of TALE-DNA interactions. Here we synthesized more than 200 TALE modulators and identified two determining factors of transcription activity in vivo: chromatin accessibility and the distance from the transcription start site. The implementation of these modulators in a gain-of-function screen was successfully demonstrated for four cell lines in migration/invasion assays and thus has broad relevance in this field. Furthermore, a novel TALE-TALE modulator was developed to transcriptionally inhibit target genes. Together, these findings underscore the huge potential of these TALE modulators in the study of gene function, reprogramming of cellular behaviors, and even clinical investigation.

The chemically-synthesized siRNA duplex has become a powerful and widely used tool for RNAi loss-of-function studies, but suffers from a high off-target effect problem. Recently, endoribonulease-prepared siRNA (esiRNA) has been shown to be an attractive alternative due to its lower off-target effect and cost effectiveness. However, the current manufacturing method for esiRNA is complicated, mainly in regards to purification and normalization on a large-scale level. In this study, we present a magnetic bead-integrated chip that can immobilize amplification or transcription products on beads and accomplish transcription, digestion, normalization and purification in a robust and convenient manner. This chip is equipped to manufacture ready-to-use esiRNAs on a large-scale level. Silencing specificity and efficiency of these esiRNAs were validated at the transcriptional, translational and functional levels. Manufacture of several normalized esiRNAs in a single well, including those silencing PARP1 and BRCA1, was successfully achieved, and the esiRNAs were subsequently utilized to effectively investigate their synergistic effect on cell viability. A small esiRNA library targeting 68 tyrosine kinase genes was constructed for a loss-of-function study, and four genes were identified in regulating the migration capability of Hela cells. We believe that this approach provides a more robust and cost-effective choice for manufacturing esiRNAs than current approaches, and therefore these heterogeneous RNA strands may have utility in most intensive and extensive applications.

SummaryThe transcription activator-like (TAL) effector targets specific host promoter through its central DNA-binding domain, which comprises multiple tandem repeats (TALE repeats). Recent structural analyses revealed that the TALE repeats form a superhelical structure that tracks along the forward strand of the DNA duplex. Here, we demonstrate that TALE repeats specifically recognize a DNA-RNA hybrid where the DNA strand determines the binding specificity. The crystal structure of a designed TALE in complex with the DNA-RNA hybrid was determined at a resolution of 2.5 Å. Although TALE repeats are in direct contact with only the DNA strand, the phosphodiester backbone of the RNA strand is inaccessible by macromolecules such as RNases. Consistent with this observation, sequence-specific recognition of an HIV-derived DNA-RNA hybrid by an engineered TALE efficiently blocked RNase H-mediated degradation of the RNA strand. Our study broadens the utility of TALE repeats and suggests potential applications in processes involving DNA replication and retroviral infections.