DNA hydrolysis and genome editing applications of GIY-YIG family homing endonucleases

The ability to manipulate complex genomes in a precise manner is essential for studying biological processes in model systems, engineering plant strains for agriculture, or advancing human cellular therapies to treat diseases. Genomic alterations are most efficient when a double-strand DNA break is introduced at the loci where the modification is desired. Different classes of naturally occurring DNA endonucleases, including homing endonucleases, have therefore been explored as candidates for genome modification studies as they target long stretches of DNA. Homing endonucleases are mobile genetic elements whose biological role is to introduce site-specific double-strand breaks into naïve genomes, ultimately resulting in the selfish propagation of their own genes. Consequently, homing endonucleases are an ideal enzymatic system whose natural properties can be exploited to manipulate genes. In the present studies, I examine the cleavage mechanism of GIY-YIG family homing endonucleases, as until now the method by which they hydrolyze DNA has remained poorly understood. Using the GIY-YIG homing endonuclease I-BmoI as a model system, I investigate the amino acid, nucleotide, and divalent metal ion requirements of the GIY-YIG nuclease domain to generate a double-strand break. I specifically test models of hydrolysis by which enzymes with a single active site could nick both strands of DNA, and determine that I-BmoI functions as a monomer throughout the reaction pathway. Furthermore, I demonstrate that the nuclease domain itself has weak binding affinity, is tethered to DNA by a high affinity binding domain, and must reposition across each strand through a series of protein and substrate conformational changes to facilitate DNA hydrolysis. To explore the relevance of GIY-YIG homing endonucleases as genome editing reagents, I fused the nuclease domain of I-TevI to three different re-targetable DNA-binding platforms utilized in the field. The engineered nucleases developed within the present studies are mechanistically distinct from established technologies, as they function as monomers and cleave DNA at a preferred sequence motif. I therefore envision that the engineered GIY-YIG nucleases may circumvent complications associated with established technologies, and provide an alternative and potentially safer set of genome editing reagents

Alteration of Sequence Specificity of the Type IIS Restriction Endonuclease BtsI

The Type IIS restriction endonuclease BtsI recognizes and digests at GCAGTG(2/0). It comprises two subunits: BtsIA and BtsIB. The BtsIB subunit contains the recognition domain, one catalytic domain for bottom strand nicking and part of the catalytic domain for the top strand nicking. BtsIA has the rest of the catalytic domain that is responsible for the DNA top strand nicking. BtsIA alone has no activity unless it mixes with BtsIB to reconstitute the BtsI activity. During characterization of the enzyme, we identified a BtsIB mutant R119A found to have a different digestion pattern from the wild type BtsI. After characterization, we found that BtsIB(R119A) is a novel restriction enzyme with a previously unreported recognition sequence CAGTG(2/0), which is named as BtsI-1. Compared with wild type BtsI, BtsI-1 showed different relative activities in NEB restriction enzyme reaction buffers NEB1, NEB2, NEB3 and NEB4 and less star activity. Similar to the wild type BtsIB subunit, the BtsI-1 B subunit alone can act as a bottom nicking enzyme recognizing CAGTG(-/0). This is the first successful case of a specificity change among this restriction endonuclease type

Strand Displacement Amplification for Multiplex Detection of Nucleic Acids

The identification of various targets such as bacteria, viruses, and other cells remains a prerequisite for point-of-care diagnostics and biotechnological applications. Nucleic acids, as encoding information for all forms of life, are excellent biomarkers for detecting pathogens, hereditary diseases, and cancers. To date, many techniques have been developed to detect nucleic acids. However, most of them are based on polymerase chain reaction (PCR) technology. These methods are sensitive and robust, but they require expensive instruments and trained personnel. DNA strand displacement amplification is carried out under isothermal conditions and therefore does not need expensive instruments. It is simple, fast, sensitive, specific, and inexpensive. In this chapter, we introduce the principles, methods, and updated applications of DNA strand displacement technology in the detection of infectious diseases. We also discuss how robust, sensitive, and specific nucleic acid detection could be obtained when combined with the novel CRISPR/Cas system

Isolation and Characterisation of Relaxed Specificity I-TevI Nuclease Domain Mutants

Engineering nucleases is important to the advancement of genetic engineering and gene therapy approaches. Engineering requires a knowledge of which residues are contributing to each function of the nuclease. The residues which contribute to cleavage specificity of the I-TevI nuclease domain (ND) are unknown. I suspect that some of these contributions derive from the ND, thus my null hypothesis is that mutation of the ND will not alter the substrates this enzyme can cut. I have mutagenised the I-TevI nuclease domain and using directed evolution I have isolated mutations which were characterised in vivo and in vitro. These mutations permit cleavage of otherwise cleavage resistant substrates, indicating that the ND does contribute to cleavage specificity. Mutations which provided the greatest increase in activity against cleavage resistant substrates (K26R, T95S, and Q158R) were combined into a single relaxed specificity nuclease domain which exhibits 1.2-5-fold improved cleavage of resistant substrates

Dual-active genome-editing reagents

Manipulation of complex genomes has many beneficial downstream applications in agriculture and human gene therapy. Precise genome-editing requires the introduction of a specific DNA double-stand break at a locus of interest, in turn inducing host DNA repair pathways to cause gene knockout through non-homologous end-joining or gene repair using homologous recombination and donor template. No matter the application, the field has depended on a few reagents to introduce precise double-strand breaks in host genomes. LAGLIDADG homing endonucleases or meganucleases harness the natural properties of these rare-cutting enzymes to target precise sequences in a complex genome. Other successful reagents are derived from a type IIS restriction endonuclease, FokI, fused to various DNA-binding architectures such as zinc finger domains and transcription activator-like effector domains. However, the discovery of clustered regularly interspaced short palindromic repeat-associated protein, CRISPR-Cas9, has dominated the field with its ease of design requiring a single RNA molecule to target the sequence of interest. Even with a handful of reagents to choose from, no one reagent is suitable for every application as every reagent has its own set of limitations and advantages. Here we present another potential genome-editing reagent derived from a GIY-YIG homing endonuclease, I-TevI, fused to all four DNA-targeting proteins described above. First, we demonstrate that I-TevI is a portable nuclease domain that can be targeted using Zinc-Fingers and LAGLIDADG proteins. Using these new reagents, we were able to further characterize I-TevI specificity using high throughput in vitro and in vivo screens to highlight important sequence requirements for targeting. Using this knowledge, we systematically engineered new I-TevI variants with altered specificity to broaden the number of targets available for I-TevI-derived reagents. We incorporated these new I-TevI variants into a more versatile dual-active nuclease, TevCas9, capable of introducing two double-strand breaks at a single target site. This dual cleavage event is capable of excising a short DNA fragment from the target site and is unique to I-TevI derived fusions. We envisioned that the monomeric, sequence-specific I-TevI catalytic domain would improve current tools by providing additional specificity and the ability to introduce dual-cleavage event would present unique applications for genome engineering

Catalytic domain of restriction endonuclease BmrI as a cleavage module for engineering endonucleases with novel substrate specificities

Creating endonucleases with novel sequence specificities provides more possibilities to manipulate DNA. We have created a chimeric endonuclease (CH-endonuclease) consisting of the DNA cleavage domain of BmrI restriction endonuclease and C.BclI, a controller protein of the BclI restriction-modification system. The purified chimeric endonuclease, BmrI198-C.BclI, cleaves DNA at specific sites in the vicinity of the recognition sequence of C.BclI. Double-strand (ds) breaks were observed at two sites: 8 bp upstream and 18 bp within the C-box sequence. Using DNA substrates with deletions of C-box sequence, we show that the chimeric endonuclease requires the 5′ half of the C box only for specific cleavage. A schematic model is proposed for the mode of protein–DNA binding and DNA cleavage. The present study demonstrates that the BmrI cleavage domain can be used to create combinatorial endonucleases that cleave DNA at specific sequences dictated by the DNA-binding partner. The resulting endonucleases will be useful in vitro and in vivo to create ds breaks at specific sites and generate deletions

The Type II restriction endonuclease MvaI has dual specificity

The MvaI restriction endonuclease cuts 5′-CC↓AGG-3′/5′-CC↑TGG-3′ sites as indicated by the arrows. N4-methylation of the inner cytosines (Cm4CAGG/Cm4CTGG) protects the site against MvaI cleavage. Here, we show that MvaI nicks the G-strand of the related sequence (CCGGG/CCCGG, BcnI site) if the inner cytosines are C5-methylated: Cm5C↓GGG/CCm5CGG. At M.SssI-methylated SmaI sites, where two oppositely oriented methylated BcnI sites partially overlap, double-nicking leads to double-strand cleavage (CCm5C↓GGG/CCm5C↑GGG) generating fragments with blunt ends. The double-strand cleavage rate and the stringency of substrate site recognition is lower at the methylation-dependent site than at the canonical target site. MvaI is the first restriction endonuclease shown to possess, besides the ‘normal’ activity on its unmethylated recognition site, also a methylation-directed activity on a different sequence

MegaTevs: Single-Chain Dual Nucleases for Efficient Gene Disruption

Targeting gene disruptions in complex genomes relies on imprecise repair by the non-homologous end-joining DNA pathway, creating mutagenic insertions or deletions (indels) at the break point. DNA end-processing enzymes are often co-expressed with genome-editing nucleases to enhance the frequency of indels, as the compatible cohesive ends generated by the nucleases can be precisely repaired, leading to a cycle of cleavage and non-mutagenic repair. Here, we present an alternative strategy to bias repair toward gene disruption by fusing two different nuclease active sites from I-TevI (a GIY-YIG enzyme) and I-OnuI E2 (an engineered meganuclease) into a single polypeptide chain. In vitro, the MegaTev enzyme generates two double-strand breaks to excise an intervening 30-bp fragment. In HEK 293 cells, we observe a high frequency of gene disruption without co-expression of DNA end-processing enzymes. Deep sequencing of disrupted target sites revealed minimal processing, consistent with the MegaTev sequestering the double-strand breaks from the DNA repair machinery. Off-target profiling revealed no detectable cleavage at sites where the I-TevI CNNNG cleavage motif is not appropriately spaced from the I-OnuI binding site. The MegaTev enzyme represents a small, programmable nuclease platform for extremely specific genome-engineering applications