Monomeric site-specific nucleases for genome editing

Abstract

Targeted manipulation of complex genomes often requires the introduction of a double-strand break at defined locations by site-specific DNA endonucleases. Here, we describe a monomeric nuclease domain derived from GIY-YIG homing endonucleases for genomeediting applications. Fusion of the GIY-YIG nuclease domain to threemember zinc-finger DNA binding domains generated chimeric GIYzinc finger endonucleases (GIY-ZFEs). Significantly, the I-TevI-derived fusions (Tev-ZFEs) function in vitro as monomers to introduce a double-strand break, and discriminate in vitro and in bacterial and yeast assays against substrates lacking a preferred 5′-CNNNG-3′ cleavage motif. The Tev-ZFEs function to induce recombination in a yeastbased assay with activity on par with a homodimeric Zif268 zincfinger nuclease. We also fused the I-TevI nuclease domain to a catalytically inactive LADGLIDADG homing endonuclease (LHE) scaffold. The monomeric Tev-LHEs are active in vivo and similarly discriminate against substrates lacking the 5′-CNNNG-3′ motif. The monomeric Tev-ZFEs and Tev-LHEs are distinct from the FokI-derived zinc-finger nuclease and TAL effector nuclease platforms as the GIY-YIG domain alleviates the requirement to design two nuclease fusions to target a given sequence, highlighting the diversity of nuclease domains with distinctive biochemical properties suitable for genome-editing applications.

Biasing genome-editing events toward precise length deletions with an RNA-guided TevCas9 dual nuclease

“…The I-TevI nuclease and linker domains have successfully been fused to four different DNA-binding architectures that are used in genome editing: zinc fingers (27), TALEs (29), meganucleases (30), and, as reported here, Cas9. An on-going debate in the genomeediting field centers on the ease of use (targeting range) versus specificity (off-target effects) of the various reagents, particularly for common laboratory manipulations of cell lines or model organisms.…”

“…Subsequent repair would effectively delete the majority of the target site, preventing further cleavage. We previously created a dual nuclease termed MegaTev, a fusion of I-TevI to LAGLIDADG homing endonucleases (or meganuclease) (27,34,35). We showed that MegaTevs efficiently excises a 30-bp fragment from model DNA substrates in HEK293 cells (30).…”

“…Previous studies revealed that the length of the DNA spacer is crucial for I-TevI cleavage function, with lengths of 14-19 bp supporting activity in various chimeric contexts (27,29,30). We coexpressed TevCas9 with a C-terminal histidine tag and a gRNA targeting a site in the retinoic acid receptor alpha gene (RARA.233, numbered according to start of target site in the RARA cDNA) from the same plasmid in Escherichia coli.…”

“…To create a TevCas9 RNA-guided dual nuclease, we fused the I-TevI nuclease domain (residues 1-92) and a portion of the linker domain (residues 92-169) to the N terminus of SpCas9 nuclease derived from the Streptococcus pyogenes type II CRISPR system (27,28) (Fig. 1A).…”

“…We created a dual nuclease that introduces two noncompatible DNA breaks at a target site such that the majority of the target site is deleted, preventing regeneration of the target site and continued cleavage. We fused the monomeric nuclease and linker domains from the I-TevI homing endonuclease to Cas9 (27,28), creating an RNA-guided TevCas9 dual nuclease that functions robustly in HEK293 cells at endogenous target sites. TevCas9 generates defined length deletions of 33-36 bp at high frequencies with minimal DNA end processing compared with Cas9.…”