Multi-reporter selection for the design of active and more specific zinc-finger nucleases for genome editing

Abstract

Engineered nucleases have transformed biological research and offer great therapeutic potential by enabling the straightforward modification of desired genomic sequences. While many nuclease platforms have proven functional, all can produce unanticipated off-target lesions and have difficulty discriminating between homologous sequences, limiting their therapeutic application. Here we describe a multi-reporter selection system that allows the screening of large protein libraries to uncover variants able to discriminate between sequences with substantial homology. We have used this system to identify zinc-finger nucleases that exhibit high cleavage activity (up to 60% indels) at their targets within the CCR5 and HBB genes and strong discrimination against homologous sequences within CCR2 and HBD. An unbiased screen for off-target lesions using a novel set of CCR5-targeting nucleases confirms negligible CCR2 activity and demonstrates minimal off-target activity genome wide. This system offers a straightforward approach to generate nucleases that discriminate between similar targets and provide exceptional genome-wide specificity.

Engineered dual selection for directed evolution of SpCas9 PAM specificity

“…We describe here the engineering of two in vivo reporter systems that provide simultaneous positive and negative selection, or positive selection alone, to facilitate directed evolution of Cas9’s PAM specificity. Our ω-dCas9 system was derived from a plasmid-based B1H system that was previously used to evolve synthetic zinc fingers via HIS3 -dependent positive selection and URA3 -dependent negative selection with 5-fluoroorotic acid (5-FOA) 34 . Instead of the dual-promoter design used for zinc finger evolution, the ω-dCas9 system exploits a single-promoter architecture that allows positive selection stringency to be tuned with dCas9-dependent repression at a downstream secondary target (Fig.…”

Linking the environment, DAF-7/TGFβ signaling and LAG-2/DSL ligand expression in the germline stem cell niche

“…Mutant and wild-type versions the lag-2 upstream sequence (600 bp) were cloned into MRB1H-reporter vector (also known as 'GHUC', see Table S1) (Oakes et al, 2016) between NotI and EcoRI upstream of the HIS3-GFP cassette, generating GHUC-1, GHUC-2, GHUC-3, GHUC-4, GHUC-5, GHUC-6 and GHUC-7. DNA encoding amino acids 1-250 of DAF-3 (DAF-3-N') was cloned into the pB1Hw2-omega vector (Noyes et al, 2008) between KpnI and XbaI, creating an omega-DAF-3-N' fusion in pB1Hw2-Daf3250 (see Table S1).…”

A multi-reporter bacterial 2-hybrid assay for the fast, simple, and dynamic assay of PDZ domain – peptide interactions

“…Adapting the multi-reporter bacterial 1-hybrid (B1H) to a B2H. The omega (ω)-based B1H is a well-established, robust, and rapid method for characterizing protein-DNA interactions [28][29][30] . The strength of this system is that assays can be done in a matter of days and multiple domains can be investigated in parallel.…”

“…As compared to phage display, the nature of our method sidesteps the laborious and often difficult need to generate a functional purified protein in vitro. This approach has been applied to many DNA-binding domains [28][29][30]44,45 and the sensitivity of the MR-B2H system may enable a similar approach to understand SLiD function. Finally, the simplicity and throughput of this technique allows the seamless application of multiple libraries.…”