Biology subjects

Sang, R.

Publications and source records attributed to Sang, R..

10 recordsLinked to original sources

bioRxiv2026

A light-responsive lipid nanoparticle platform delivering Cas9 RNP for VEGFA editing suppresses choroidal neovascularisation

Choroidal neovascularisation (CNV) is a major cause of irreversible vision loss. Conventional anti-VEGF therapies for CNV often require repeated intravitreal injections, imposing substantial treatment burden and cumulative injection-related risk. Genome editing to disrupt VEGFA could provide durable suppression, potentially from a single administration. However, durability depends on permanently disrupting the target in a well-controlled way, as unrestricted VEGFA loss can cause choriocapillaris atrophy and compromise retinal function. This represents a major safety barrier to clinical translation. In this study, we developed lipid-based nanoparticles to release Cas9/sgRNA ribonucleoprotein (RNP) complexes under light illumination, enabling light-controlled editing of the VEGFA gene. The nanoparticles had a mean hydrodynamic diameter of approximately 130 nm with low polydispersity (0.296) and high sgRNA encapsulation efficiency (90.33%). In human retinal pigment epithelial cells, light exposure increased the extent of VEGFA disruption relative to the non-illuminated condition, as confirmed by targeted next-generation sequencing, with corresponding reductions in VEGFA mRNA and protein expression. Following a single intravitreal injection in a laser-induced rat CNV model, the nanoparticles combined with light illumination significantly reduced vascular leakage and CNV-associated structural abnormalities compared with both vehicle and non-illuminated controls, and ocular safety assessment revealed no apparent nanoparticle-related structural abnormalities in the retina. This study provides a foundation for spatially controlled VEGFA genome editing as a potential therapeutic strategy for VEGFA-driven macular neovascular disease.

bioengineering↗

bioRxiv2026

Lipid nanoparticle-mediated RNAi targeting of KRAS mutants sensitises colorectal cancer cells to chemotherapy

KRAS mutations, including G12D, G12V and G13D, are major oncogenic drivers in colorectal cancer (CRC) and are associated with therapeutic resistance. However, approved KRAS inhibitors remain focused on G12C subtype, leaving these prevalent variants without approved targeted therapies. Here, we developed a lipid nanoparticle (LNP)-based RNA interference (RNAi) strategy to target these three KRAS variants using a single small interfering RNA (siRNA) sequence. The developed siRNA-LNPs reduced KRAS mRNA and protein expression in HCT116 (G13D), SW620 (G12V) and LS180 (G12D) cells, but not in KRAS wild-type HT29 cells, suppressing the cellular proliferation, clonogenic survival and migration. Transcriptomic profiling revealed changes in KRAS associated signalling, including suppression of cell-cycle and DNA repair programmes, providing a rationale for combining siRNA silencing with the fluoropyrimidine chemotherapeutic agent 5-fluorouracil (5-FU). The combination produced synergistic effects across all three KRAS-mutant CRC cell models and enhanced the apoptosis in both monolayer cultures and 3D spheroids. Collectively, these findings provide proof of concept for this combination strategy and support its further investigation as a potential therapeutic approach for KRAS-mutant CRC.

molecular biology↗

bioRxiv2026

Modular split-trigger based cooperative activation platform for Cas13-12 tandem biosensing system

Achieving precise control of CRISPR/Cas trans-cleavage depends on understanding how nucleic acid activators engage Cas effectors, yet the fundamental principles of split-trigger activation of Cas12a remain unclear. Here, we uncover the mechanistic determinants that enable fragmented nucleic acids to collectively initiate Cas12a activity. We show that split triggers bearing external extensions fully support the R-loop formation, whereas internal extensions which disrupt the spacer complementarity abolish Csa12a activation. We further demonstrate that covalent linkage of split-trigger fragments prevents R-loop propagation, revealing that Cas12as activation strictly requires two physically independent split fragments. Together, these findings establish a synergistic split-trigger activation mechanism in which cooperative hybridization of two individually fragments nucleates and extends the Cas12a R-loop with high efficiency. Conceptually, this mechanism enables a cascade architecture that transforms CRISPR diagnostics from a one-target one-Cas ribonucleoprotein (RNP) paradigm into a highly amplifying process in which a single target molecule activates numerous downstream Cas RNPs. Building on this principle, we show that the cleavage of a rationally designed linear DNA-RNA-DNA mediator by LbuCas13a generates optimally configured split triggers for Cas12a activation, thereby coupling RNA recognition to large-scale Cas12a activation without enzymatic preamplification. The resulting Split Trigger Activated Cas13-Cas12 Cascade System (STACS) achieves amplification-free detection down to 1 copy/{micro}L within 15 minutes and maintains robust performance in complex biological (serum, saliva) and environmental (mud) matrices. This work establishes a generalizable strategy for engineering programmable CRISPR cascades with high Cas RNP activation multiplicity for ultrasensitive molecular diagnostics. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/734747v2_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@104a609org.highwire.dtl.DTLVardef@30cebcorg.highwire.dtl.DTLVardef@54b15borg.highwire.dtl.DTLVardef@47f89e_HPS_FORMAT_FIGEXP M_FIG C_FIG Mechanism and detection workflow of the Split Trigger Activated Cas13-12 Cascade System (STACS).

Molecular Biology↗

bioRxiv2024

Hairpin-locker mediated CRISPR/Cas tandem system for ultrasensitive detection of DNA without pre-amplification

Achieving ultra-sensitive detection of DNA is of paramount importance in the field of molecular analytics. Conventional amplification technologies such as polymerase chain reaction (PCR) currently play a leading role in ultrasensitive DNA detection. However, amplicon contamination common in these techniques may lead to false positives. To date, CRISPR-associated nucleases (type V & VI) with their programmable cleavage have been utilised for sensitive detection of unamplified nucleic acids in complex real samples. Nevertheless, without additional amplification strategies, the pM range sensitivity of such CRISPR/Cas sensors is not sufficient for clinical applications. Here, we established a hairpin-locker (H-locker) mediated Cas12-Cas13 tandem biosensing system (Cas12-13 tandem-sensor) for ultrasensitive detection of DNA targets. Without the need for any additional amplification reaction or device, this system is capable of detecting DNA at a notable 1 aM level (<1 copy/uL) sensitivity. In addition, the system was able to distinguish cancer mutations in colorectal cancer (CRC) mice. This is a significant advance for CRISPR/Cas biosensing technology offering simple, highly sensitive, and user-friendly diagnostics for next-generation nucleic acid detection. O_TBL View this table: org.highwire.dtl.DTLVardef@7fc32eorg.highwire.dtl.DTLVardef@1e716ceorg.highwire.dtl.DTLVardef@8a43e0org.highwire.dtl.DTLVardef@16e286dorg.highwire.dtl.DTLVardef@1202eb0_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 1.C_FLOATNO O_TABLECAPTIONDNA and RNA oligos used in this study. C_TABLECAPTION C_TBL

molecular biology↗

bioRxiv2024

Modulating Cas13a trans-cleavage by double strand RNA: Application to the development of an autocatalytic sensor

Cas13a-based diagnostic systems have been widely utilized for the detection of RNA targets. However, without preamplification such systems have sensitivity in the picomolar range only. Here, we found that double strand RNA (dsRNA) over 20nt is able to effectively activate the trans-cleavage activity of Cas13a RNP, while the cleavage rates of dsRNA by activated Cas13a RNP are very low. In addition, specially designed small circular RNA constructs (Cir-mediators comprising a 20nt dsRNA trigger with a 5nt ssRNA linker) have limited ability to activate Cas13a RNP, but this activation is restored once the circular structures are cleaved and become linear. Based on this new method to control trans-cleavage activity of Cas13 RNP, we developed a Cas13a autocatalytic biosensing system assisted by Cir-mediators, which allows one target RNA to activate numerous Cas13a RNPs. With this approach we show ultrasensitive detection of 1aM of synthetic RNA targets without preamplification within 15min. The sensor was successfully applied to monitor miRNA-21 concentration in clinical plasma samples in colorectal cancer. This investigation yields novel insights into the properties of Cas13a RNPs, and the Cir-mediator-based autosensor introduces a novel method for detecting RNA targets with exceptional sensitivity. HighlightsO_LIdsRNA is able to trigger activation of Cas13a RNP. C_LIO_LIActivated Cas13a RNP do not cleave dsRNA. C_LIO_LICir-mediator induces low levels of Cas13a RNP activation. C_LIO_LICir-mediator based Cas13a auto-catalysis biosensor can detect 1aM RNA targets. C_LI

molecular biology↗

bioRxiv2023

Extensive variation and strain-specificity in dengue virus susceptibility among African Aedes aegypti populations

African populations of the mosquito Aedes aegypti are usually considered less susceptible to infection by human-pathogenic flaviviruses than globally invasive populations found outside Africa. Although this contrast has been well documented for Zika virus (ZIKV), it is unclear to what extent it is true for dengue virus (DENV), the most prevalent flavivirus of humans. Addressing this question is complicated by substantial genetic diversity among DENV strains, most notably in the form of four genetic types (DENV1 to DENV4), that can lead to genetically specific interactions with mosquito populations. Here, we carried out a continent-wide survey of DENV susceptibility using a panel of field-derived Ae. aegypti colonies from across the African range of the species and a colony from Guadeloupe, French West Indies as non-African reference. We found considerable variation in the ability of African Ae. aegypti populations to acquire and replicate a panel of six DENV strains spanning the four DENV types. Although African Ae. aegypti populations were generally less susceptible than the reference non-African population from Guadeloupe, in several instances some African populations were equally or more susceptible than the Guadeloupe population. Moreover, the relative level of susceptibility between African mosquito populations depended on the DENV strain, indicating genetically specific interactions. We conclude that unlike ZIKV susceptibility, there is no clear-cut dichotomy in DENV susceptibility between African and non-African Ae. aegypti. DENV susceptibility of African Ae. aegypti populations is highly heterogeneous and largely governed by the specific pairing of mosquito population and DENV strain. Author summaryAfrican populations of the mosquito Aedes aegypti are usually thought to be less likely to get infected by flaviviruses compared to Ae. aegypti mosquitoes found outside Africa. While this has been well-demonstrated for Zika virus, it is not clear if the same is true for dengue virus, which is the most common flavivirus in humans. Studying this is complicated by the strain diversity of dengue virus, including four main genetic types, potentially causing different interactions. In this study, we compared several mosquito populations and found that, in general, African mosquitoes were less likely to get infected by dengue virus compared to mosquitoes from outside Africa. However, in some cases, African mosquitoes were just as or even more likely to get infected. The specific strain of dengue virus also influenced how likely African mosquitoes were to get infected, showing that the relationship between African mosquitoes and dengue virus is complex.

microbiology↗

bioRxiv2023

Genetic variability and vector competence of Aedes aegypti populations from Kisumu and Busia Counties, Western Kenya, for Chikungunya and Zika viruses

Aedes aegypti is the primary vector of several arboviruses, including dengue virus (DENV), chikungunya virus (CHIKV), yellow fever virus (YFV), and Zika virus (ZIKV). This vector is widespread globally in tropical and subtropical areas, but also found in temperate areas. Kenya experienced its first chikungunya outbreaks in Lamu County in 2004 and later in Mandera: 2016, and Mombasa: 2017. While there is yet to be a report of Zika outbreaks in Kenya, sero-surveillance studies indicate low-level transmission of this virus in coastal and northern parts of the country. Despite the presence of Ae. aegypti in Kisumu and Busia counties in sufficient densities, and free movement of people between the coast and the two western Kenya counties, no outbreaks of either disease have been reported in these regions. To investigate this phenomenon, we collected Ae. aegypti mosquitoes from county headquarter towns near railway stations connecting the coast and western Kenya and reared them under controlled laboratory conditions. The mosquitoes were then assessed for genetic variability using CO1 genes as well as their efficiency to transmit viruses using Laboratory colonies (F1) of the field mosquitoes challenged with an infectious blood meal containing CHIKV and ZIKV. Genetic analysis revealed the presence of both Ae. aegypti subspecies, (Ae. aegypti aegypti [Aaa] and Ae. aegypti formosus [Aaf]) in the two western Kenya counties, with Aaf being dominant (19:8 for Kisumu samples and 25:6 for Busia samples). Additionally, pairwise comparison revealed minimal genetic differentiation (0.62%) between the study populations, with a high genetic variation (99.38%) observed within each population, indicating significant diversity within individual populations. Ae. aegypti populations from Kisumu and Busia counties exhibited competence for CHIKV, with infection, dissemination, and transmission rates of 55.2%, 85.5%, and 27.1% for Kisumu; and 57.8%, 71.8%, and 25% for Busia populations, respectively. There was no significant difference in vector competence between these two populations. Interestingly, neither population was competent for ZIKV. In conclusion, the data shows that the Ae. aegypti populations in the two cities were homogeneous. This could explain the observed similarity in vector competence for CHIKV and ZIKV. Author SummaryOur study investigated the genetic variability and vector competence of Ae. aegypti mosquito populations in Kisumu and Busia Counties to CHIKV and ZIKV; revealing the presence and even distribution of both Aaa and Aaf subspecies. We also found that the Ae. aegypti populations from the two counties were not genetically differentiated. Furthermore, our study revealed that the Ae. aegypti mosquitoes from Kisumu and Busia counties were competent for CHIKV but may be refractory to ZIKV infection. These findings highlight the importance of continued monitoring of Ae. aegypti populations and their potential for arboviral disease transmission in the region.

microbiology↗

bioRxiv2020

Climate and urbanization drive mosquito preference for humans

The majority of mosquito-borne illness is spread by a few mosquito species that have evolved to specialize in biting humans, yet the precise causes of this behavioral shift are poorly understood. We address this gap in the arboviral vector Aedes aegypti. We first characterize the behaviour of mosquitoes from 27 sites scattered across the species ancestral range in sub-Saharan Africa, revealing previously unrecognized diversity in female preference for human versus animal odor. We then use modelling to show that this diversity can be almost fully predicted by two ecological factors - dry season intensity and human population density. Finally we integrate this information with whole genome sequence data from 345 individual mosquitoes to identify a single underlying ancestry component linked to human preference, with genetic changes concentrated in a few key chromosomal regions. Our findings strongly suggest that human-biting in this important disease vector originally evolved as a by-product of breeding in human-stored water in areas where doing so provided the only means to survive the long, hot dry season. Our model also predicts that changes in human population density are likely to drive future mosquito evolution. Rapid urbanization may drive a shift to human-biting in many cities across Africa by 2050.

evolutionary biology↗

bioRxiv2019

Positive association between Brucella spp seroprevalences in livestock and humans from a cross-sectional study in Garissa and Tana River Counties, Kenya

BackgroundBrucella spp. is a zoonotic bacterial agent of high public health and socio-economic importance. It infects many species of animals including wildlife, and people may get exposed through direct contact with an infected animal or consumption of raw or undercooked animal products. We implemented a linked livestock-human cross-sectional study to determine seroprevalences and risk factors of Brucella spp. in livestock and humans. We also estimated intra-cluster correlation coefficients (ICCs) for these observations at the household and village levels.\n\nMethodologyThe study was implemented in Garissa County (specifically Ijara and Sangailu areas) and Tana River (Bura and Hola) counties. A household was the unit of analysis and the sample size was derived using the standard procedures. Serum samples were obtained from selected livestock and people from randomly selected households. Humans were sampled in both counties while livestock could be sampled only in Tana River County. Samples obtained were screened for anti-Brucella IgG antibodies using ELISA kits. Data were analyzed using generalized linear mixed effects logistic regression models with the household (herd) and village being used as random effects.\n\nResultsThe overall seroprevalences were 3.47% (95% confidence interval [CI]: 2.72 - 4.36%) and 35.81% (95% CI: 32.87 - 38.84) in livestock and humans, respectively. In livestock, older animals and those sampled in Hola had significantly higher seroprevalences that younger ones or those sampled in Bura. Herd and village random effects were significant and ICC estimates associated with these variables were 0.40 (95% CI: 0.22 - 0.60) and 0.24 (95% CI: 0.08 - 0.52), respectively. For human data, older people, males, and people who lived in pastoral areas had significantly higher Brucella spp. seroprevalences than younger ones, females or those who lived in irrigated or riverine areas. People from households that had at least one seropositive animal were 3.35 (95% CI: 1.51 - 7.41) times more likely to be seropositive compared to those that did not. Human exposures significantly clustered at the household level; the ICC estimate obtained was 0.21 (95% CI: 0.06 - 0.52).\n\nConclusionThe presence of a seropositive animal in a household significantly increased the risk of exposure in people in that household. Brucella spp. exposures in both livestock and humans clustered significantly at the household level. This suggests that risk-based surveillance measures, guided by locations of primary cases reported, either in humans or livestock, can be used to detect Brucella spp. infections livestock or humans, respectively.\n\nAuthor summaryBrucellosis is an important zoonotic disease that primarily affects livestock and wildlife. In humans, the disease is characterized by prolonged fever, body aches, joint pains and weakness while in livestock, the disease causes abortions and infertility. We carried out a study in northeastern Kenya (Garissa and Tana River Counties) to identify factors that affect the distribution of the disease in people and livestock. Livestock and people from randomly selected households were recruited and serum samples obtained for screening using anti-Brucella IgG ELISA kits to determine their Brucella spp. exposure. Data obtained were analyzed using mixed effects logistic regression models. Results obtained show that human and animal Brucella spp. seroprevalences cluster at the household level. The odds of exposure in humans were at least three times higher in households that had at least one seropositive animal compared to those that did not. These results can be used to design risk-based surveillance systems where each Brucella infection identified in livestock or humans could signal potential locations of other secondary infections.

epidemiology↗

bioRxiv2018

Recent outbreaks of chikungunya virus (CHIKV) in Africa and Asia are driven by a variant carrying mutations associated with increased fitness for Aedes aegypti

BackgroundIn 2016, a chikungunya virus (CHIKV) outbreak was reported in Mandera, Kenya. This was the first major CHIKV outbreak in the country since the global re-emergence of this virus, which arose as an initial outbreak in Kenya in 2004. Therefore, we collected samples and sequenced viral genomes from the 2016 Mandera outbreak.\n\nMethodology/Principal FindingsAll Kenyan genomes contained two mutations, E1:K211E and E2:V264A, recently reported to have an association with increased infectivity, dissemination and transmission in the Aedes aegypti (Ae. aegypti) vector. Phylogeographic inference of temporal and spatial virus relationships using Bayesian approaches showed that this Ae. aegypti adapted strain emerged within the East, Central, and South African (ECSA) lineage of CHIKV between 2005 and 2008, most probably in India. It was also in India where the first large outbreak caused by this strain appeared, in New Delhi, 2010. More importantly, our results also showed that this strain is no longer contained to India, and that it has more recently caused several major outbreaks of CHIKV, including the 2016 outbreaks in India, Pakistan and Kenya, and the 2017 outbreak in Bangladesh. In addition to its capability to cause large outbreaks in different regions of the world, this CHIKV strain has the capacity to replace less adapted wild type strains in Ae. aegypti-rich regions. Indeed, all the latest full CHIKV genomes of the ECSA Indian Ocean Lineage (IOL), from the regions of high Ae. aegypti prevalence, carry these two mutations, including samples collected in Japan, Australia, and China.\n\nConclusions/SignificanceOur results point to the importance of continued genomic-based surveillance of this strains global spread, and they prompt urgent vector competence studies in Asian and African countries, in order to assess the level of vector receptiveness, virus transmission, and the impact this might have on this strains ability to cause major outbreaks.\n\nAuthor summaryChikungunya virus (CHIKV) causes a debilitating infection with high fever, intense muscle and bone pain, rash, nausea, vomiting and headaches, and persistent and/or recurrent joint pains for months or years after contracting the virus. CHIKV is spread by two mosquito vectors, Aedes albopictus and Aedes aegypti, with increased presence around the globe. In this study, we report global spread of a CHIKV strain that carries two mutations that have been suggested to increase this virus ability to infect the Aedes aegypti mosquito, as well as to increase CHIKVs ability to be transmitted by this vector. We show that this strain appeared sometime between 2005 and 2008, most probably in India, and has now spread to Africa, Asia, and Australia. We show that this strain is capable of driving large outbreaks of CHIKV in the human population, causing recent major outbreaks in Kenya, Pakistan, India and Bangladesh. Thus, our results stress the importance of monitoring this strains global spread, as well as the need of improved vector control strategies in the areas of Aedes aegypti prevalence.

evolutionary biology↗