Desirazu Narasimha Rao

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

Helicobacter pylori is a carcinogenic bacterium that is responsible for 5.5% of all human gastric cancers. H. pylori codes for an unusually large number of restriction-modification (R-M) systems and several of them are strain specific and phase variable. HpyAII is a novel Type IIs phase variable restriction endonuclease present in 26695 strain of H. pylori. We show that HpyAII prefers two-site substrates over one-site substrates for maximal cleavage activity. HpyAII is less stringent in metal ion requirement and shows higher cleavage activity with Ni²⁺ over Mg²⁺. Mutational analysis of the putative residues of the HNH motif of HpyAII confirms that the protein has an active HNH site for the cleavage of DNA. However, mutation of the first His residue of the HNH motif to Ala does not abolish the enzymatic activity, but instead causes loss of fidelity compared to wild type HpyAII. Previous studies have shown that mutation of the first His residue of the HNH motif of all other known HNH motif motif-containing enzymes completely abolishes enzymatic activity. We found, in the case of HpyAII, mutation of an active site residue leads to the loss of endonuclease fidelity. This study provides further insights into the evolution of restriction enzymes.

Following publication of the original article [1], the authors notified us of an error in the presentation of Fig. 6G.

Nucleic Acids Res (2018) doi: 10.1093/nar/gky126