• Alok Kumar

      Articles written in Resonance – Journal of Science Education

    • What can we Learn from the Electromagnetic Spectrum?

      A W Joshi Alok Kumar

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    • Scalars and Vectors in Physics – I.

      A W Joshi Alok Kumar

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    • Scalars and Vectors in Physics – II

      A W Joshi Alok Kumar

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    • Nature Watch - Sarus Crane: On its Way to Extinction

      Alok Kumar Mishra

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    • Genome Editing: Revolution in Life Sciences

      Alok Kumar Singh Sivaprakash Ramalingam Desirazu N Rao Srinivasan Chandrasegaran

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      Programmable nucleases—ZFNs, TALENs and CRISPR-Cas9—have equipped scientists with an unprecedented ability to modify cells and organisms almost at will, with great implications across life sciences: biology, agriculture, ecology and medicine. Nucleases-based genome editing (aka gene editing) depends on cellular responses to a targeted double-strand break(DSB). The first truly targetable reagents were zinc finger nu-cleases (ZFNs) showing that arbitrary DNA sequences within a mammalian genome, could be addressed by protein engi-neering, ushering in the era of genome editing. ZFNs that are fusions of zinc finger proteins (ZFPs) and FokI cleavage domain, resulted from the basic research on Type IIs FokI re-striction enzyme, which showed a bipartite structure with a separable DNA-binding domain and a non-specific cleavage domain. Studies on 3-finger ZFNs established that the preferred substrates were paired binding sites, which doubled the size of the target recognition sequence from 9 to 18 bp that is large enough to specify a unique genomic locus in plant and mammalian cells, including human cells. Subsequently, a ZFN-induced DSB was shown to stimulate homologous re-combination in frog eggs. Transcription activator-like effec-tor nucleases (TALENs) that are based on bacterial TALEs fused to FokI cleavage domain expanded the capability. ZFNs and TALENs have been successfully used to modify a multi-tude of recalcitrant organisms and cell types that were unap-proachable previously attesting to the success of protein engi-neering, long before the arrival of CRISPR. The recent tech-nique to deliver a targeted DSB to cellular genomes are RNA-guided nucleases as exemplified by the Type II prokaryotic CRISPR-Cas9 system. Unlike ZFNs and TALENs that use protein motifs for DNA sequence recognition, CRISPR-Cas9 depends on RNA-DNA recognition. The advantages of the CRISPR-Cas9 system, which include ease of RNA design for new targets and dependence on a single constant Cas9 protein, have led to its wide adoption by research labs around the world. The 2020 Nobel Prize for Chemistry was awarded to Jennifer Doudna and Emmanualle Charpentier for harnessing CRISPR-Cas9 system to provide a simplified technique for genome editing. The programmable nucleases have also been shown to cut at off-target sites with mutagenic consequences, which is a serious concern for human therapeutic applications. Therefore, applications of genome editing technologies to human therapeutics will ultimately depend on risk versus benefit analysis and informed consent

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