Evaluating the Mutagenic Activity of Targeted Endonucleases Containing aSharkeyFokI Cleavage Domain Variant in Zebrafish

Abstract

Synthetic targeted endonucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have recently emerged as powerful tools for targeted mutagenesis, especially in organisms that are not amenable to embryonic stem cell manipulation. Both ZFNs and TALENs consist of DNA-binding arrays that are fused to the nonspecific FokI nuclease domain. In an effort to improve targeted endonuclease mutagenesis efficiency, we enhanced their catalytic activity using the Sharkey FokI nuclease domain variant. All constructs tested display increased DNA cleavage activity in vitro. We demonstrate that one out of four ZFN arrays containing the Sharkey FokI variant exhibits a dramatic increase in mutagenesis frequency in vivo in zebrafish. The other three ZFNs exhibit no significant alteration of activity in vivo. Conversely, we demonstrate that TALENs containing the Sharkey FokI variant exhibit absent or severely reduced in vivo mutagenic activity in zebrafish. Notably, Sharkey ZFNs and TALENs do not generate increased toxicity-related defects or mortality. Our results present Sharkey ZFNs as an effective alternative to conventional ZFNs, but advise against the use of Sharkey TALENs.

Improved Specificity and Safety of Anti-Hepatitis B Virus TALENs Using Obligate Heterodimeric FokI Nuclease Domains

“…In contrast the third-generation TALENs containing Sharkey mutations exhibited reduced silencing activity against HBV. Similar results have been reported before [ 28 , 29 ]. This suggests that incorporating Sharkey mutations into the TALEN architecture, and more specifically into third-generation Fok I nuclease domains, is deleterious to silencing activity [ 28 , 29 ].…”

“…Similar results have been reported before [ 28 , 29 ]. This suggests that incorporating Sharkey mutations into the TALEN architecture, and more specifically into third-generation Fok I nuclease domains, is deleterious to silencing activity [ 28 , 29 ]. The Sharkey mutations were generated by directed evolution of ZFNs and possibly provides the reason for reduced silencing activity within TALENs.…”

Nuclease‐mediated genome editing: At the front‐line of functional genomics technology

“…), yet Sharkey‐TALENs appear not to recapitulate increased activity (Pillay et al . ). In order to decrease genotoxicity by off‐target activity resulting from self‐dimerization, obligate heterodimers of FokI have been used for both ZFNs (Miller et al .…”

Nrl is dispensable for specification of rod photoreceptors in adult zebrafish contrasting a deeply conserved requirement earlier in ontogeny

“…The gRNAs were mixed with protein Cas9 (NEB), allowed minutes at 37°C to assemble into ribonucleoprotein complexes, and the mixture was microinjected into the cell of zebrafish embryos at the cell-stage. Animals at 2dpf were sacrificed for high-resolution meltcurve (HRM) analysis of nrl cutting (STAR methods for primers; methods as previously described [55,56]. For nrl ua5009 , the 5'-most guide RNA used above, and a second guide RNA targeting eGFP (CR.GFP.GA5'.break, STAR methods) were mixed together and allowed to complex with protein Cas9.…”