Genome Editing Revolution in Life Sciences

Alok Kumar Singh, Sivaprakash Ramalingam, Desirazu N. Rao, Srinivasan Chandrasegaran

Research output: Contribution to journalArticlepeer-review

Abstract

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 nucleases (ZFNs) showing that arbitrary DNA sequences within a mammalian genome, could be addressed by protein engineering, 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 restriction 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 recombination in frog eggs. Transcription activator-like effector 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 multitude of recalcitrant organisms and cell types that were unapproachable previously attesting to the success of protein engineering, long before the arrival of CRISPR. The recent technique 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.

Original languageEnglish (US)
Pages (from-to)971-998
Number of pages28
JournalResonance
Volume26
Issue number7
DOIs
StatePublished - Jul 2021

Keywords

  • CRISPR-Cas9
  • Genome-editing
  • TALENs
  • programmable nucleases
  • zinc-finger nucleases

ASJC Scopus subject areas

  • Education

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