Mithlesh Kumar سينغ

• Led CRISPR/Cas9-mediated genome editing for trait improvement in cotton, maize, and
tomato.
• Led next-generation sequencing (Nanopore) and regulatory molecular characterization for
event selection, event characterization, and regulatory compliance of BGII cotton and novel
transgenic events.
• Performed transgene integration analysis, insertion-site mapping, copy number analysis
(Southern blot), and vector backbone confirmation.
• Conducted spatio-temporal transgene expression and data analysis (qRT-PCR, ELISA).
• Designed and validated high-throughput KASP and TaqMan assays for genotyping and
zygosity determination.
• Generated regulatory biosafety datasets covering ORF analysis, allergenicity, toxicity, and
antinutrient assessment.
• Supported BRL-I and BRL-II confined field trials, trait validation, regulatory documentation,
and regulatory affairs activities for product advancement.

• Led CSIR-funded cotton mission project on next-generation insect-resistant cotton.
• Evaluated recombinant toxic proteins against major pests including Bemisia tabaci,
Spodoptera litura, and Pectinophora gossypiella.
• Performed Nanopore genome sequencing for regulatory molecular characterization.
• Engineered, expressed, and evaluated recombinant Cry protein variants against key insect
pests.
• Conducted in silico allergenicity, toxicity, antinutrient and biosafety risk assessments.
• Prepared regulatory dossiers and biosafety assessments for trait deregulation in BRL trials.

• Developed gene pyramiding strategies for enhanced insect resistance in crops.
• Characterized more than 20 transgenic events by insertion site and flanking sequence
analysis and assessed transgene expression using qRT-PCR, ELISA, and Western blot.
• Established event and zygosity-specific PCR assays with 0.01% detection sensitivity.
• Conducted in vitro and in planta bioassays for protein efficacy validation.

• Processed clinical samples for RNA isolation and RT-PCR diagnostics in a BSL-3 laboratory.
• Maintained biosafety compliance, laboratory documentation, and data reporting.

• Characterized more than 50 transgenic cotton lines expressing insecticidal proteins.
• Performed plant protein purification and mass spectrometry analysis.
• Conducted confined field trials for evaluation and selection of GM events.
• Studied insect biology and resistance mechanisms.
• Designed trap-crop strategies for whitefly management.

• Conducted phenotyping and molecular characterization of insect resistant GM cotton lines.
• Performed gene expression analysis using qRT-PCR and Western blot.
• Conducted insect bioassays and beneficial insect safety studies.
• Determined transgene copy number (Southern blot), and integration profiles.