High Cleavage Efficiency Research Articles

Developing polycistronic expression tool in Yarrowia lipolytica

Unconventional oleaginous yeast Yarrowia lipolytica has gained widespread applications as a microbial cell factory for synthesizing various chemicals and natural products. The construction of efficient cell factories requires intricate metabolic engineering. However, multi-gene expression in Y. lipolytica is labor-intensive. To facilitate multi-gene expression, we developed the polycistronic expression tool using 2A peptides. We first compared different 2A peptides in Y. lipolytica and identified two 2A peptides with high cleavage efficiency: P2A and ERBV-1. The effect of 2A peptides on the expression level of upstream and downstream genes was then determined. Ultimately, we applied the identified 2A peptides to express four genes in canthaxanthin biosynthetic pathway within one expression cassette for canthaxanthin production. This study enriches the multi-gene expression tools of Y. lipolytica, which will facilitate the cell factory construction of Y. lipolytica.

Evaluation of culture methods and chemical reagent combinations on CRISPR/Cas9 gene editing systems by lipofection in pig zygotes.

The delivery of CRISPR/Cas ribonucleoprotein (RNP) complexes is gaining attention owing to its high cleavage efficiency and reduced off-target effects. Although RNPs can be delivered into porcine zygotes via electroporation with relatively high efficiency, lipofection-mediated transfection appears to be versatile because of its ease of use, low cost, and adaptation to high-throughput systems. However, this system requires improvements in terms of embryo development and mutation rates. Therefore, this study elucidated the effects of culture methods and reagent combinations on the CRISPR/Cas9 gene editing systems by using three lipofection reagents: Lipofectamine™ CRISPRMAX™ Cas9 Transfection Reagent (CM), Lipofectamine™ 2000 Transfection Reagent (LP), and jetCRISPR™ RNP Transfection Reagent (Jet). Porcine zona pellucida-free zygotes were incubated for 5h with Cas9, a guide RNA targeting CD163, and the above lipofection reagents. When examining the effect of culture methods using 4-well (multiple embryo culture) and 25-well plates (single embryo culture) on the efficiency of CM-mediated zygote transfection, the culture of embryos in 25-well plates significantly increased the blastocyst formation rate; however, there was no difference in mutation rates between the 4-well and 25-well plates. When assessing the effects of individual or combined reagents on the efficiency of zygote transfection, the mutation rate was significantly lower for individual LP compared to individual CM- and Jet-mediated transfections. Moreover, combinations of lipofection transfection reagents did not significantly increase the mutation rate or mutation efficiency.

Cas-Rainbow: Cas12a-driven single-reaction multiplex detection system

Recently, CRISPR/Cas system has garnered considerable attention owing to its high cleavage efficiency. However, it lacks the ability to differentiate between signal sources. To address this, we have developed a multiplex detection system called Cas-Rainbow. This system combines CRISPR/Cas with rolling circle amplification (RCA) and hybridization chain reaction (HCR) to enable the detection of multiple miRNAs using reporter probes labeled with different fluorescent groups. The RCA product can trigger the formation of HCR and activate the Cas12a/crRNA complex, achieving the simultaneous detection of multiple miRNAs. Detection limits of three miRNAs (miR-155, miR-10b, and miR-21) were 33 fM, 41 fM, and 79 fM, respectively. Cas-Rainbow system could effectively identify these three miRNAs in serum, distinguishing breast cancer patients from healthy donors with accuracy of 100 %. Moreover, this system exhibited exceptional performance in logic gate circuits, showcasing its remarkable versatility and robustness. In summary, our proposed method offers a new approach for multiplex detection based on the CRISPR/Cas system.

A fusion framework of deep learning and machine learning for predicting sgRNA cleavage efficiency

CRISPR/Cas9 system is a powerful tool for genome editing. Numerous studies have shown that sgRNAs can strongly affect the efficiency of editing. However, it is still not clear what rules should be followed for designing sgRNA with high cleavage efficiency. At present, several machine learning or deep learning methods have been developed to predict the cleavage efficiency of sgRNAs, however, the prediction accuracy of these tools is still not satisfactory. Here we propose a fusion framework of deep learning and machine learning, which first deals with the primary sequence and secondary structure features of the sgRNAs using both convolutional neural network (CNN) and recurrent neural network (RNN), and then uses the features extracted by the deep neural network to train a conventional machine learning model with LGBM. As a result, the new approach overwhelmed previous methods. The Spearman's correlation coefficient between predicted and measured sgRNA cleavage efficiency of our model (0.917) is improved by over 5% compared with the most advanced method (0.865), and the mean square error reduces from 7.89 × 10−3 to 4.75 × 10−3. Finally, we developed an online tool, CRISep (http://www.cuilab.cn/CRISep), to evaluate the availability of sgRNAs based on our models.

Y-shaped walker with abundant recognition domians mediated ultrasensitive electrochemical biosensor for miRNA-21 detection

Herein, an ultrasensitive electrochemical biosensor was constructed by a high-efficiency Y-shaped walker with abundant recognition domains (YSWARD) for microRNA-21 (miRNA-21) detection. Once target miRNA-21 induced the assembly of Y-shaped DNA nanostructure (YSDN) through a catalytic hairpin assessbly (CHA), amounts of the YSWARD could be synthesized with the assistance of padlock via rolling circle amplification (RCA). Impressively, the YSWARD exhibited much higher cleavage efficiency (2.81 × 109) which was at least ten times more than that of conventional Y-shaped walker with three recognition domains (2.36 × 108). As a proof of concept, the YSWARD was adopted to construct electrochemical biosensing platform that achieved ultrasensitive detection with a detection limit of 10.6 aM for target miRNA-21, which carved out promising potential for exploring novel DNA walker platforms to apply in sensitive detection of biomarkers and early diagnosis of diseases.

CNN-XG: A Hybrid Framework for sgRNA On-Target Prediction.

As the third generation gene editing technology, Crispr/Cas9 has a wide range of applications. The success of Crispr depends on the editing of the target gene via a functional complex of sgRNA and Cas9 proteins. Therefore, highly specific and high on-target cleavage efficiency sgRNA can make this process more accurate and efficient. Although there are already many sophisticated machine learning or deep learning models to predict the on-target cleavage efficiency of sgRNA, prediction accuracy remains to be improved. XGBoost is good at classification as the ensemble model could overcome the deficiency of a single classifier to classify, and we would like to improve the prediction efficiency for sgRNA on-target activity by introducing XGBoost into the model. We present a novel machine learning framework which combines a convolutional neural network (CNN) and XGBoost to predict sgRNA on-target knockout efficacy. Our framework, called CNN-XG, is mainly composed of two parts: a feature extractor CNN is used to automatically extract features from sequences and predictor XGBoost is applied to predict features extracted after convolution. Experiments on commonly used datasets show that CNN-XG performed significantly better than other existing frameworks in the predicted classification mode.

A combination of the modified catalytic core and conjugation of 3′-inverted deoxythymidine for a more efficient and nuclease-resistant 10-23 DNAzyme

10-23 DNAzyme is a catalytic DNA molecule capable of cleaving complementary RNA. Its high cleavage efficiency is being pursued by chemical modifications, for realizing its genetic therapeutics potential. The most efficient and nuclease-resistant DNAzyme was obtained in this study combined two modifications − 7-aminopropyl-8-aza-7-deaza-2′-deoxyadenosine (residue 1) at A9 and 3′-inverted deoxythymidine residue (iT) at 3′-end. Moreover, this combinatorial modification could be a universal approach for designing efficient and enzyme-resistant 10-23 DNAzyme against other RNA targets, and the catalytic core-modification could be further combined with other recognition arm modifications for practical applications as genetic therapeutics and biosensor tools.

Soluble expression of bioactive recombinant porcine-human chimeric uricase mutant employing MBP-SUMO fusion system

Urate oxidase is a promising biological medicine for hyperuricemia treatment, but immunogenicity obstructs the development of its clinical application. The recombinant porcine-human chimeric uricase mutant named dHU-wPU is a humanized chimeric uricase based on wild porcine uricase (wPU), which can effectively reduce the limitation of potential immunogenicity with a high homology (92.76%) to deduced human uricase (dHU). Unfortunately, the insoluble expression form of dHU-wPU in E. coli increases the difficulty of production. In this study, we described a more convenient method to efficiently obtain recombinant dHU-wPU protein from E. coli. Combination small ubiquitin-related modifier protein (SUMO) and maltose-binding protein (MBP) was employed to achieve the soluble expression of dHU-wPU. MBP-SUMO-dHU-wPU fusion protein was not only overexpressed in a soluble form, but also showed high purification and cleavage efficiency. Subsequently, we optimized the culture conditions of shake flasks and expanded the production of MBP-SUMO-dHU-wPU fusion protein in a 5 L bioreactor. Finally, about 15 mg of recombinant dHU-wPU was obtained from 1 L M9 fermentation culture by using two-step affinity chromatography, with a SDS-PAGE purity over 90%. In vitro activity analysis showed that dHU-wPU had better ability to catalyze uric acid than wPU.

Erratum: CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy.

An erratum was issued for: Studying TGF-β Signaling and TGF-β-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells. The phrases"surveyor assay"and"SurveyorNuclease" havebeen updated to "T7E1 assay" to " T7endonuclease I" respectively. Step 1.2 inthe Protocol has been updated from: Surveyor nucleaseassay of sgRNA NOTE: The targeting efficiency of the sgRNA used for the knock-in experiment is evaluated by surveyor nuclease assay (also known as T7 endonuclease I (T7EI) assay)17. Select the sgRNA with high DNA cleavage efficiency and a low distance between the sgRNA cutting site and the stop codon. to: T7 endonuclease assay of sgRNA NOTE: The targeting efficiency of the sgRNA used for the knock-in experiment is evaluated by T7 endonuclease (T7EI)assay17. Select the sgRNA with high DNA cleavage efficiency and a low distance between the sgRNA cutting site and the stop codon. Figure 1 in the Representative Results has been updatedfrom: Figure 1: HMEJ-mediated targeted integration in vitro. (A) Experimental scheme for selection of sgRNAs: Six different sgRNAs (Cdx2-sgRNA1~Cdx2-sgRNA6) around the stop codon of the Cdx2 locus with a higher rank and off-target potential were chosen based on online CRISPR design tool. The protospacer adjacent motif (PAM) sequence is in red. (B) Experimental design: The Cas9-CMV-GFP expression plasmids expressing sgRNA, Cas9, and GFP were introduced into N2acells. GFP+ cells were sorted at day 3 for surveyor assay. (C) Surveyor assay for Cdx2 targeting: 6 different sgRNAs were designed for surveyor assay. Normal N2a cell genomic DNA serves as control. *, the sgRNA used for Cdx2-2A-mCherry knock-in experiment. (D) Schematic overview of construction of HMEJ donors using Gibson assembly. (E) Schematic overview of HMEJ-mediated gene targeting strategy at Cdx2 locus. HAL/HAR, left/right homology arm; triangles, sgRNA target sites; OF/OR, outer forward/reverse primer; IF/IR, inner forward/reverse primer. Figure modified from previous report10.Please click here to view a larger version of this figure. to: Figure 1: HMEJ-mediated targeted integration in vitro. (A) Experimental scheme for selection of sgRNAs: Six different sgRNAs (Cdx2-sgRNA1~Cdx2-sgRNA6) around the stop codon of the Cdx2 locus with a higher rank and off-target potential were chosen based on online CRISPR design tool. The protospacer adjacent motif (PAM) sequence is in red. (B) Experimental design: The Cas9-CMV-GFP expression plasmids expressing sgRNA, Cas9, and GFP were introduced into N2acells. GFP+ cells were sorted at day 3 for T7EIassay. (C) T7EI assay for Cdx2 targeting: 6 different sgRNAs were designed for T7EI assay. Normal N2a cell genomic DNA serves as control. *, the sgRNA used for Cdx2-2A-mCherry knock-in experiment. (D) Schematic overview of construction of HMEJ donors using Gibson assembly. (E) Schematic overview of HMEJ-mediated gene targeting strategy at Cdx2 locus. HAL/HAR, left/right homology arm; triangles, sgRNA target sites; OF/OR, outer forward/reverse primer; IF/IR, inner forward/reverse primer. Figure modified from previous report10.Please click here to view a larger version of this figure.

Targeted Integration of a CAR at a Novel Genomic Safe Harbor Directs Potent Therapeutic Outcomes

Chimeric receptor antigen (CAR)-T cell therapy using CARs specific for CD19 have been remarkably successful for treating chemo-refractory/relapsed B cell malignancies. These successes not withstanding, therapeutic outcomes between patients are variable and occasional cases of clonal expansion of the transduced T cells have been observed, albeit without leukemic transformation. These issues are cause for concern and need to be addressed to achieve better and safer therapeutic outcomes. Clonal expansion due to insertional mutagenesis and variegated transgene expression due to position effects are well established to be the consequence of the semi-random integration pattern afforded by gamma-retroviral and lentiviral vectors. We previously established that integration of a CAR cDNA in the TCR alpha locus (TRAC) provides consistent, regulated expression of CD19 CARs and superior CAR T cell efficacy in a mouse model of B-cell acute lymphoblastic leukemia (B-ALL). Here, we identify a novel extragenic site devoid of any known function and remote from endogenous genes, i.e. a 'genomic safe harbor' (GSH), that can be efficiently targeted in human T cells and drives potent CAR T cell therapy in the B-ALL mouse model. To identify GSHs that could be efficiently targeted in T cells by CRISPR-Cas9 and that could also support durable transgene expression, we screened for genomic regions meeting both, GSH criteria and high chromatin accessibility in T cells as measured by ATAC-seq. In human primary T cells, we identified 379 such sites. Ten of the highest accessible sites were investigated. All showed high (>90%) cleavage efficiency and allowed for CAR cDNA targeted integration and expression which also translated into effective cytolytic activity of the CARs within a few days after transduction. However, thereafter CAR expression diminished over the course of a week at most but not all of these sites. In order to prevent possible heterochromatinization, we incorporated chromatin insulator elements with barrier activity flanking the CAR transcription unit. Incorporation of the chromatin insulator element dramatically improved CAR expression and functionality at one site, whereas 3 other GSHs tested were not affected. One of the 10 GSHs maintained long-term CAR expression without requiring an insulator and directed potent anti-leukemic CAR T cell efficacy in a B-ALL 'CAR stress test' mouse model, matching the T cell potency afforded by integrating the CAR cDNA at the TRAC locus. This finding highlights the major effect of the integration site on transgene expression and ensuing therapeutic efficacy. We identified an extragenic GSH site that can be used for effective T cell engineering and sustained expression of a CAR. Through this study, we provide a platform for identifying GSHs that could be reliably targeted for safe and predictable expression of CARs or other immunomodulatory transgenes to potentiate adoptive immunotherapy. Disclosures Sadelain: Fate Therapeutics: Patents & Royalties, Research Funding; Mnemo: Patents & Royalties; Atara: Patents & Royalties, Research Funding; Takeda: Patents & Royalties, Research Funding; Minerva: Other: Biotechnologies, Patents & Royalties.

Knockout of cytidine monophosphate-N-acetylneuraminic acid hydroxylase in Chinese hamster ovary cells by CRISPR/Cas9-based gene-editing technology

Chinese hamster ovary (CHO) cells are mainly used mammalian host to produce therapeutic recombinant glycoproteins which are close to the human-like glycosylation of glycoproteins. However, cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) in CHO cells can produce N-glycolylneuraminic acids (Neu5Gc) which elicits the immune response in the human body. Therefore, it is vital to silence the expression of CMAH in CHO cells using gene-editing technology. CRISPR (clustered regulatory interspaced short palindromic repeat)/Cas9-based gene editing system was performed to knock out the expression of CMAH in CHO cells. Plasmids containing Cas9, single guide (sg) RNA and reporter sequences of CMAH gene were constructed and transformed into HEK293 T cells to screen the sgRNA with high cleavage efficiency. Then the plasmids were co-transfected into the CHO cells. Eight CHO monoclonal cells were obtained by flow cytometry sorting and expanded. Base deletion mutations were identified by PCR and Sanger sequencing. qRT-PCR and MTT assay were confirmed the CMAH gene mutation in the CHO cells. Moreover, the recombinant human erythropoietin (rEPO) protein was also expressed in the mutant CHO cells, and the N-Glycosylation of rEPO showed that G0F, G1F-GN increased and G0F-GN, G0, Man5 were decreased. CRISPR/Cas9 system is a very convenient system for editing CMAH gene in CHO cells. The mutant CHO cells can be used as host to express the recombinant glycoprotein.

A novel thermal Cas12b from a hot spring bacterium with high target mismatch tolerance and robust DNA cleavage efficiency

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas), such as Cas9 and Cpf1, are RNA-guided endonucleases that target and degrade nucleic acids, providing powerful genomic editing and molecular diagnostic tools. Cas12b enzymes are distinct effectors; however, their features and catalytic boundaries require further characterization. We identified BrCas12b from the thermophile bacterium Brevibacillus sp. SYSU G02855 as a novel ortholog of cas12b. Biochemical analyses revealed that BrCas12b is a dual-RNA-guided endonuclease with higher optimum reaction temperature than that of other reported members of Cas12b. The seed sequence of BrCas12b is only 4 nt in length, indicating that it has greater target mismatch tolerance than that of previously reported Cas effectors; however, it contains a compensatory effect at the position of the cleavage site. Using fluorescence-based detection method to evaluate target cleavage efficiency, we showed that BrCas12b has robust enzymatic cleavage activity (Kcat/Km (s−1 M−1) = 8.80 × 1011), which is significantly higher than that of AacCas12b (Kcat/Km (s−1 M−1) = 7.56 × 108) from Alicyclobacillus acidoterrestris. The results increase our understanding of the catalytic mechanism of Cas12b family members and suggest that BrCas12b might be useful in the application of genomic editing and molecular diagnosis.

Effects of different 2A peptides on transgene expression mediated by tricistronic vectors in transfected CHO cells.

Multicistronic vectors can increase transgene expression and decrease the imbalance of gene expression in the Chinese hamster ovary (CHO) cell expression system. Small, self-cleaving 2A peptides have a high cleavage efficiency and are essential for constructing high-expression multicistronic vectors. In this study, we investigated the effects of two different 2A peptides on transgene expression in CHO cells via their mediating action on tricistronic vectors. The enhanced green fluorescent protein (eGFP) and red fluorescent protein (RFP) genes were linked by the porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A) peptides in a multicistronic vector. We transfected CHO cells with these vectors and screened for the presence of blasticidin-resistant colonies. Flow cytometry and real-time quantitative PCR (qPCR) were used to detect the expression levels of eGFP and RFP and the copy numbers of stably transfected cells. The results showed that P2A could enhance eGFP and RFP expression by 1.48- and 1.47-fold, respectively, compared to T2A. The expression levels of the genes were not proportional to their copy numbers. In conclusion, we found that P2A can effectively drive transgene expression in CHO cells and a potent 2A peptide can be used for recombinant protein production in the CHO cell system.

A novel protein purification strategy mediated by the combination of CipA and Ssp DnaB intein

Protein purification is an indispensable step in diverse fields of biological research or production process. Conventional purification methods including the affinity purification or the usage of self-aggregating tags suffered from many drawbacks such as the complicated steps, high cost and low efficiency. Moreover, the fusion tag usually had negative effects on the activity of the target protein. To address the above issues, here we propose a novel protein purification method which needs simple operation steps, and this method is mediated by the combination of CipA protein and a mini-intein (Synechocystis sp. PCC6803 DnaB, Ssp DnaB), depending on the assembly function of CipA and the self-cleavage function of Ssp DnaB. To realize the purification, CipA-DnaB-eGFP protein was expressed and assembled into protein crystalline inclusions (PCIs) in E. coli. Then, only cell lysis, cleavage and centrifugation steps were required to purify eGFP. Purified eGFP was in the supernatant with a purity of over 90%. The cleavage efficiency and the yield of eGFP reached 51.96% and 13.99 ± 0.88 mg/L fermentation broth, respectively. Furthermore, to broaden the application of this approach, three other proteins which were maltose binding protein (MBP), ketoisovalerate decarboxylase (Kivd) and alcohol dehydrogenase (AdhP) were purified with high cleavage efficiency. The purified Kivd and AdhP remained high specific activities. This work demonstrated an effective and convenient protein purification method.

Development of drug-inducible CRISPR-Cas9 systems for large-scale functional screening

BackgroundLarge-scale genetic screening using CRISPR-Cas9 technology has emerged as a powerful approach to uncover and validate gene functions. The ability to control the timing of genetic perturbation during CRISPR screens will facilitate precise dissection of dynamic and complex biological processes. Here, we report the optimization of a drug-inducible CRISPR-Cas9 system that allows high-throughput gene interrogation with a temporal control.ResultsWe designed multiple drug-inducible sgRNA expression vectors and measured their activities using an EGFP gene disruption assay in 11 human and mouse cell lines. The optimal design allows for a tight and inducible control of gene knockout in vitro, and in vivo during a seven-week-long experiment following hematopoietic reconstitution in mice. We next performed parallel genome-wide loss-of-function screens using the inducible and constitutive CRISPR-Cas9 systems. In proliferation-based dropout screens, these two approaches have similar performance in discriminating essential and nonessential genes. In a more challenging phenotypic assay that requires cytokine stimulation and cell staining, we observed similar sensitivity of the constitutive and drug-induced screening approaches in detecting known hits. Importantly, we demonstrate minimal leakiness of our inducible CRISPR screening platforms in the absence of chemical inducers in large-scale settings.ConclusionsIn this study, we have developed a drug-inducible CRISPR-Cas9 system that shows high cleavage efficiency upon induction but low background activity. Using this system, we have achieved inducible gene disruption in a wide range of cell types both in vitro and in vivo. For the first time, we present a systematic side-by-side comparison of constitutive and drug-inducible CRISPR-Cas9 platforms in large-scale functional screens. We demonstrate the tightness and efficiency of our drug-inducible CRISPR-Cas9 system in genome-wide pooled screening. Our design increases the versatility of CRISPR-based genetic screening and represents a significant upgrade on existing functional genomics toolbox.

An internally quenched peptide as a new model substrate for rhomboid intramembrane proteases.

Rhomboids are ubiquitous intramembrane serine proteases that cleave transmembrane substrates. Their functions include growth factor signaling, mitochondrial homeostasis, and parasite invasion. A recent study revealed that the Escherichia coli rhomboid protease EcGlpG is essential for its extraintestinal pathogenic colonization within the gut. Crystal structures of EcGlpG and the Haemophilus influenzae rhomboid protease HiGlpG have deciphered an active site that is buried within the lipid bilayer but exposed to the aqueous environment via a cavity at the periplasmic face. A lack of physiological transmembrane substrates has hampered progression for understanding their catalytic mechanism and screening inhibitor libraries. To identify a soluble substrate for use in the study of rhomboid proteases, an array of internally quenched peptides were assayed with HiGlpG, EcGlpG and PsAarA from Providencia stuartti. One substrate was identified that was cleaved by all three rhomboid proteases, with HiGlpG having the highest cleavage efficiency. Mass spectrometry analysis determined that all enzymes hydrolyze this substrate between norvaline and tryptophan. Kinetic analysis in both detergent and bicellular systems demonstrated that this substrate can be cleaved in solution and in the lipid environment. The substrate was subsequently used to screen a panel of benzoxazin-4-one inhibitors to validate its use in inhibitor discovery.

Screening of 2A peptides for polycistronic gene expression in yeast.

A complexity of pathway expression in yeast compared to prokaryotes is the need for separate promoters and terminators for each gene expressed. Single transcript expression and separated protein production is possible via the use of 2A viral peptides, but detailed characterization to assess their suitability and applications is needed. The present work aimed to characterize multiple 2A peptide sequences to determine suitability for metabolic engineering applications in Saccharomyces cerevisiae. We screened 22 peptides placed between fluorescent protein sequences. Cleaving efficiency was calculated by western blot intensity of bands corresponding to the cleaved and uncleaved forms of the reporter. Three out of the 22 sequences showed high cleavage efficiency: 2A peptide from Equine rhinitis B virus (91%), Porcine teschovirus-1 (85%) and Operophtera brumata cypovirus-18 (83%). Furthermore, expression of the released protein was comparable to its monocistronic expression. As a proof-of-concept, the triterpene friedelin was successfully produced in the same yeast strain by expressing its synthase with the truncated form of HMG1 linked by the 2A peptide of ERBV-1, with production titers comparable to monocistronic expression (via separate promoters). These results suggest that these peptides could be suitable for expression and translation of multiple proteins in metabolic engineering applications in S. cerevisiae.

Exonuclease III-boosted cascade reactions for ultrasensitive SERS detection of nucleic acids

A variety of nucleic acid amplification techniques have been integrated into different detection methods to promote the development of sensitive and convenient analysis of nucleic acids. However, it is still in urgent need to develop amplified nucleic acid biosensors for the analysis of susceptible gene and even distinguishing single-base mismatched DNA in complex biological samples. Benefiting from the achieved detection strategies, here we boost isothermal nucleic acid amplification by resorting to enzyme amplification, and combine this two-stage amplification method with surface-enhanced Raman spectroscopy (SERS) to develop a signal-on nucleic acid detection platform. Due to the high cleavage efficiency of Exonuclease III (Exo III), a large amount of trigger DNA are produced to initiate multiple hybridization chain reaction circles. The product structure tagged with Tamra is then anchored onto the plasmonic SERS substrate and meanwhile enriched. It is demonstrated that this detection platform is sensitive toward the myocardial infarction disease related gene. A detection limit of 1 fM for the gene analysis in a linear relationship in the wide range from 1 fM to 10nM is achieved, better than most of previous counterparts. Meanwhile, our developed detection platform exhibits a high selectivity for the target gene over mismatched analogues. Our strategy provides a robust tool for signal amplification of gene detection even in blood samples.

Nitrodibenzofuran: A One- and Two-Photon Sensitive Protecting Group That Is Superior to Brominated Hydroxycoumarin for Thiol Caging in Peptides.

Photoremovable protecting groups are important for a wide range of applications in peptide chemistry. Using Fmoc-Cys(Bhc-MOM)-OH, peptides containing a Bhc-protected cysteine residue can be easily prepared. However, such protected thiols can undergo isomerization to a dead-end product (a 4-methylcoumarin-3-yl thioether) upon photolysis. To circumvent that photoisomerization problem, we explored the use of nitrodibenzofuran (NDBF) for thiol protection by preparing cysteine-containing peptides where the thiol is masked with an NDBF group. This was accomplished by synthesizing Fmoc-Cys(NDBF)-OH and incorporating that residue into peptides by standard solid-phase peptide synthesis procedures. Irradiation with 365 nm light or two-photon excitation with 800 nm light resulted in efficient deprotection. To probe biological utility, thiol group uncaging was carried out using a peptide derived from the protein K-Ras4B to yield a sequence that is a known substrate for protein farnesyltransferase; irradiation of the NDBF-caged peptide in the presence of the enzyme resulted in the formation of the farnesylated product. Additionally, incubation of human ovarian carcinoma (SKOV3) cells with an NDBF-caged version of a farnesylated peptide followed by UV irradiation resulted in migration of the peptide from the cytosol/Golgi to the plasma membrane due to enzymatic palmitoylation. Overall, the high cleavage efficiency devoid of side reactions and significant two-photon cross-section of NDBF render it superior to Bhc for thiol group caging. This protecting group should be useful for a plethora of applications ranging from the development of light-activatable cysteine-containing peptides to the development of light-sensitive biomaterials.

SpCas9结构域相互作用关键氨基酸的动态网络分析

CRISPR-Cas9系统是一种操作简单、灵活性好、识别和切割效率高的基因编辑技术，具有用于基因治疗的潜能。然而，由于该系统的主要组分Streptococcus pyogenes Cas9 (SpCas9)蛋白过大，难于向靶细胞运输，因而亟需构建截短体蛋白，以提高系统的运输效率、扩展其应用前景。前期结构研究表明，SpCas9结构域分界清晰，各结构域功能相对独立又彼此协同，易于通过对各结构域进行分步改造实现蛋白截短优化。但报道的晶体结构缺乏SpCas9的全长结构，及结构域间相互作用的关键信息。针对此问题，本文运用分子动力学(Molecular Dynamics, MD)模拟和动态网络分析方法研究SpCas9-sgRNA-DNA复合物结构。我们首先对已有晶体结构的缺失片段进行补齐，构建了一个包含全长SpCas9及DNA链的三元复合物原子结构。然后对复合物结构进行长时间尺度MD模拟，通过动态网络分析方法分析了SpCas9蛋白各结构域间的相互作用，找出影响SpCas9结构域间相互作用的关键氨基酸。与已有实验结果比较，所识别的位点是SpCas9蛋白正常工作的关键氨基酸。因此，本研究不仅可以加深人们对SpCas9作用机制的了解，也为SpCas9蛋白的优化改造提供理论指导。 CRISPR-Cas9 system is widely used in gene editing because of its simple operation, flexibility, and high recognition and cleavage efficiency, and it also has the potential for usage in gene therapy. However, Streptococcus pyogenes Cas9 (SpCas9), the main component in the powerful system is too large to be transferred to target cells, which limits its application. So it is necessary to shorten the length of SpCas9. Previous studies revealed that SpCas9 executed its functions by several cooperate but relatively independent domains, so it is possible to optimize this protein section by section. Nevertheless, the integrated structure of SpCas9 and the information on domain interacting is still lacking in reported crystal structures. To solve this problem, this study analyzed the structure of SpCas9-sgRNA-DNA complex using molecular dynamics (MD) simulation and dynamical network analysis. Firstly, the missing fragment of the existing crystal structure was completed to construct a ternary complex atomic structure containing full-length SpCas9 and non-complemen- tary DNA strand. And then, after a long time MD simulation of this new structure, the interactions between domains of SpCas9 were analyzed by dynamical network analysis. Finally, we determined the key residues for SpCas9 domains to interact with each other. Compared with previous experimental results, the identified sites are critical amino acids for proper SpCas9 function. Thus, this study not only deepens the understanding of the working mechanisms of the CRIPSR-Cas9 system, but also offers important guidelines for the optimization and improvement of the system.