publications

2021

1. Biosens. Bioelect.

   Rapid and accurate nucleobase detection using FnCas9 and its application in COVID-19 diagnosis

   Mohd Azhar , Rhythm Phutela , Manoj Kumar , and 24 more authors

   Biosensors and Bioelectronics, 2021

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   Rapid detection of DNA/RNA pathogenic sequences or variants through point-of-care diagnostics is valuable for accelerated clinical prognosis, as witnessed during the recent COVID-19 outbreak. Traditional methods relying on qPCR or sequencing are tough to implement with limited resources, necessitating the development of accurate and robust alternative strategies. Here, we report FnCas9 Editor Linked Uniform Detection Assay (FELUDA) that utilizes a direct Cas9 based enzymatic readout for detecting nucleobase and nucleotide sequences without trans-cleavage of reporter molecules. We also demonstrate that FELUDA is 100% accurate in detecting single nucleotide variants (SNVs), including heterozygous carriers, and present a simple web-tool JATAYU to aid end-users. FELUDA is semi-quantitative, can adapt to multiple signal detection platforms, and deploy for versatile applications such as molecular diagnosis during infectious disease outbreaks like COVID-19. Employing a lateral flow readout, FELUDA shows 100% sensitivity and 97% specificity across all ranges of viral loads in clinical samples within 1hr. In combination with RT-RPA and a smartphone application True Outcome Predicted via Strip Evaluation (TOPSE), we present a prototype for FELUDA for CoV-2 detection closer to home.

2019

1. PNAS

   Francisella novicida Cas9 interrogates genomic DNA with very high specificity and can be used for mammalian genome editing

   Sundaram Acharya , Arpit Mishra , Deepanjan Paul , and 14 more authors

   Proceedings of the National Academy of Sciences, 2019

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   Genome editing using the CRISPR/Cas9 system has been used to make precise heritable changes in the DNA of organisms. Although the widely used Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants have been efficiently harnessed for numerous gene-editing applications across different platforms, concerns remain regarding their putative off-targeting at multiple loci across the genome. Here we report that Francisella novicida Cas9 (FnCas9) shows a very high specificity of binding to its intended targets and negligible binding to off-target loci. The specificity is determined by its minimal binding affinity with DNA when mismatches to the target single-guide RNA (sgRNA) are present in the sgRNA:DNA heteroduplex. FnCas9 produces staggered cleavage, higher homology-directed repair rates, and very low nonspecific genome editing compared to SpCas9. We demonstrate FnCas9-mediated correction of the sickle cell mutation in patient-derived induced pluripotent stem cells and propose that it can be used for precise therapeutic genome editing for a wide variety of genetic disorders.

2. US Patent

   CRISPR Effector System Mediated Detection of Nucleic Acid Variants in DNA

   Debojyoti Chakraborty , Souvik Maiti , Mohd Azhar , and 6 more authors

   2019

   Provisional US Patent 201911049432

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   The invention describes a method to detect Single nucleotide variants (SNVs) in DNA using a CRISPR effector system that compromise of Cas9 from Francisella novicida (FnCas9), a synthetic sgRNA harboring a sequence that consist of mismatches specific to the DNA substrate being analyzed and a detection scheme based on enzymatic cleavage of the substrate. The method can be applied to a large number of SNVs both pathogenic and non-pathogenic and can be used to distinguish DNA substrates different by a single mismatch without the need for sequencing. The method is economical, easy to assemble and provides a robust and rapid readout that can be appropriately adapted for point of care applications.

2017

1. IIC

   Influence of plant extracts on reproductive physiology and behavior of Aedes aegypti

   Namrata Sharma

   In Proceedings of the INSCR International Conference , 2017

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   Due to development of resistance against a large number of insecticides, the control of dengue fever mosquito, Aedes aegypti has now becomes a challenge for the researchers. Aedes aegypti is ubiquitous and causes many deadly diseases like, dengue fever, dengue haemorrhagic fever, chikungunya and yellow fever. Use of phytochemicals to control insect pests/vectors is known since ages as a part of indigenous and traditional knowledge. Many plant products decrease insect longevity, and interfere with their reproductive bioactivities. In view of this ethanolic and hexane plant extracts of three plants Ocimum, Nerium and Citrus peel were evaluated at different doses, viz., 100ppm, 200ppm and 400ppm with respect to reproductive behaviour and development of Aedes aegypti.All tested concentrations of plant extracts were observed to act as ovipositional deterrent, showing negative values of oviposition activity index. The hexane extract of Ocimum at 400ppm showed maximum (98.7%) ovipositional deterrence. The studies on larvicidal activity of these plant extracts revealed that at 400ppm, Citrus (orange peel) had maximum larvicidal activity (72.5%) followed by Nerium (52.5%) and Ocimum (40%). The plant extracts impaired behavioural responses of Aedes larvae too. The treated larvae deviated from their normal wriggling, swimming and resting pattern. The extract of orange peel exhibited maximum metamorphic inhibition at 400ppm, only 26.7% fourth instar treated larvae could metamorphose into pupae. These results suggest that the phytochemicals extracted from common Indian plants and plant waste can be employed in an effective vector control program against A. aegypti. Further detailed research is needed to identify the active ingredients present in these extracts and to get more conclusive results.