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

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

Mohd Azhar, Rhythm Phutela, Manoj Kumar, Asgar Hussain Ansari, Riya Rauthan, Sneha Gulati, Namrata Sharma, Dipanjali Sinha, Saumya Sharma, Sunaina Singh, Sundaram Acharya, Sajal Sarkar, Deepanjan Paul, Poorti Kathpalia, Meghali Aich, Paras Sehgal, Gyan Ranjan, Rahul C Bhoyar, Khushboo Singhal, Harsha Lad, Pradeep Kumar Patra, Govind Makharia, Giriraj Ratan Chandak, Bala Pesala, Debojyoti Chakraborty, Souvik Maiti

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.

2021

SARS-CoV-2, LFA, fncas 9, CRISPR Dx, FELUDA, SNV detection

10.1016/j.bios.2021.113207

Biosensors & bioelectronics