Effector DNA-binding Domain Research Articles

The Evolution of Meganucleases Genome Engineering Tools: an overview

Human beings today have been able to pursue goals with non-random changes in the genome of living organisms. This is the process of genetic engineering or genome editing, which involves replacing, inserting, or deleting an arbitrary genomic sequence using an artificial restriction enzyme capable of cleaving a specific part of the genome. The ability to manipulate and modify genes and functional studies as well as awareness of the molecular basis of diseases and the development of new and targeted therapies with these techniques have been created. To date, different techniques have now been developed with the ability to edit targeted genomics including Zinc-finger nucleases (ZFNs), Transcription activator-like effector nucleases (TALEN) and Clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR associated protein 9 (Cas9) (CRISPR/Cas9). The fusing of a zinc-finger DNA-binding domain to a DNA-cleavage domain produces artificial restriction enzymes known as Zinc-finger nucleases (ZFNs). Zinc finger domains can be designed to target a specific DNA sequence which causes ZFNs to target unique sequences in complex genomes. TALENs are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain to cut DNA strands at specific locations. CRISPR/Cas9 is a bacterial immune system against viruses in which the Cas9 nucleus connects with a single-strand guide to a complementary target sequence and makes changes. In this study, we will review the evolution and various aspects of each one over time.

Genome Editing: Revolutionizing the Crop Improvement

Modern biotechnology is progressed to sequence-specific, site-directed, precise, and safe strategies for genetic manipulation. The genome-editing strategy is being proposed as one of the most promising tools for genomic study and crop improvement. The basic mechanism involved in genetic manipulations through programmable nucleases is recognition of target genomic loci and binding of effector DNA-binding domain (DBD), double-strand breaks (DSBs) in target DNA by the restriction endonucleases (FokI and Cas) and the repair of DSBs through homology-directed recombination (HDR) or non-homologous end joining (NHEJ). The less efficient and more precise HDR results in the replacement of nucleotides, whereas the more efficient and error-prone NHEJ results in the insertion or deletion of nucleotides. The genome-editing strategies like ZFN, TALEN, and CRISPR/Cas involved in genome editing are being employed to add the desirable trait(s) and remove the undesirable. The impact of gene editing application in crop improvement has proven it the next-generation tool to enhance agricultural productivity. The modifications in crop plants include biotic and abiotic stresses, nutritional improvements, alteration in phenotype, and other characters such as crafting male sterility, haploid production, and increase in yield. The repurposing of CRISPR applications by catalytically inactive Cas9 marks CRISPR as an indispensable tool for genome editing and beyond in biological research. However, the genome-edited plants may have unintended alteration(s) that create biosafety risks. The tools are eventually progressing for more accuracy and efficiency to ensure the safety of consumers and the environment. This article is an attempt to review these genome-editing strategies, the biochemistry attributed to application in crop improvement, and the associated challenges.

EcoRII Restriction Endonuclease Forms Specific Contacts to the Bases of Its Target Sequence Flipped from DNA in a Transition Complex with Photoactivatable Substrates

The photoactivatable modified oligonucleotides were used to investigate direct contacts formed by the type IIE EcoRII restriction endonuclease and the T/A bases of its recognition site (5'-CCT/AGG). EcoRII dimer consists of a central catalytic core, made of two C-terminal endonuclease-like domains (EcoRII-C) from different subunits, and two N-terminal effector DNA binding domains (EcoRII-N). According to co-crystal structure of isolated EcoRII-C with DNA catalytic dimer EcoRII-C flips nucleotides of the central T/A pair into the enzyme binding pockets. Нere, photocross-linking technique was used to investigate the direct contacts formed by extrahelical T/A bases in the protein pockets of full-length EcoRII within the pre-reactive EcoRII–DNA complex obtained in the presence of Ca2+ in solution. Photoreactive zero-length agent 5-iodo-2'-deoxyuridine (IdU) was introduced as single substituent into the central T/A position of EcoRII recognition site or into the flanking nucleotide sequences of 14-mer DNA substrate. The substitution of only dT or dA residues in EcoRII recognition site resulted in formation of photocross-links upon irradiation only in the presence of Ca2+. Proteolytic digestion of the enzyme-oligonucleotide conjugates followed by MALDI-MS analysis have allowed to identify the 224VEYD227 EcoRII region involved in the formation of the cross-links. This region belongs to the central part of H-10 α-helix. Y226 residue was suggested to form cross-link with T or A bases of EcoRII site replaced by IdU within the pre-reactive complex. The flipped base pair protein pockets of EcoRII seem to accommodate equally well both A and T bases of the DNA substrate. Altogether, IdU-containing photoactivatable DNA substrates have allowed to trap the flipped bases in complex with full-length EcoRII before DNA cleavage in the solution and to identify direct enzyme–DNA contacts important for high specificity of EcoRII for the Т/A nucleotides providing a highly specific cleavage reaction.

Assembly and characterization of megaTALs for hyperspecific genome engineering applications.

Rare-cleaving nucleases have emerged as valuable tools for creating targeted genomic modification for both therapeutic and research applications. MegaTALs are novel monomeric nucleases composed of a site-specific meganuclease cleavage head with additional affinity and specificity provided by a TAL effector DNA binding domain. This fusion product facilitates the transformation of meganucleases into hyperspecific and highly active genome engineering tools that are amenable to multiplexing and compatible with multiple cellular delivery methods. In this chapter, we describe the process of assembling a megaTAL from a meganuclease, as well as a method for characterization of nuclease cleavage activity in vivo using a fluorescence reporter assay.

Enhancing the Specificity of Recombinase-Mediated Genome Engineering through Dimer Interface Redesign

Despite recent advances in genome engineering made possible by the emergence of site-specific endonucleases, there remains a need for tools capable of specifically delivering genetic payloads into the human genome. Hybrid recombinases based on activated catalytic domains derived from the resolvase/invertase family of serine recombinases fused to Cys2-His2 zinc-finger or TAL effector DNA-binding domains are a class of reagents capable of achieving this. The utility of these enzymes, however, has been constrained by their low overall targeting specificity, largely due to the formation of side-product homodimers capable of inducing off-target modifications. Here, we combine rational design and directed evolution to re-engineer the serine recombinase dimerization interface and generate a recombinase architecture that reduces formation of these undesirable homodimers by >500-fold. We show that these enhanced recombinases demonstrate substantially improved targeting specificity in mammalian cells and achieve rates of site-specific integration similar to those previously reported for site-specific nucleases. Additionally, we show that enhanced recombinases exhibit low toxicity and promote the delivery of the human coagulation factor IX and α-galactosidase genes into endogenous genomic loci with high specificity. These results provide a general means for improving hybrid recombinase specificity by protein engineering and illustrate the potential of these enzymes for basic research and therapeutic applications.

Cell-Penetrating Peptide-Mediated Delivery of TALEN Proteins via Bioconjugation for Genome Engineering

Transcription activator-like (TAL) effector nucleases (TALENs) have enabled the introduction of targeted genetic alterations into a broad range of cell lines and organisms. These customizable nucleases are comprised of programmable sequence-specific DNA-binding modules derived from TAL effector proteins fused to the non-specific FokI cleavage domain. Delivery of these nucleases into cells has proven challenging as the large size and highly repetitive nature of the TAL effector DNA-binding domain precludes their incorporation into many types of viral vectors. Furthermore, viral and non-viral gene delivery methods carry the risk of insertional mutagenesis and have been shown to increase the off-target activity of site-specific nucleases. We previously demonstrated that direct delivery of zinc-finger nuclease proteins enables highly efficient gene knockout in a variety of mammalian cell types with reduced off-target effects. Here we show that conjugation of cell-penetrating poly-Arg peptides to a surface-exposed Cys residue present on each TAL effector repeat imparted cell-penetrating activity to purified TALEN proteins. These modifications are reversible under reducing conditions and enabled TALEN-mediated gene knockout of the human CCR5 and BMPR1A genes at rates comparable to those achieved with transient transfection of TALEN expression vectors. These findings demonstrate that direct protein delivery, facilitated by conjugation of chemical functionalities onto the TALEN protein surface, is a promising alternative to current non-viral and viral-based methods for TALEN delivery into mammalian cells.

Genome engineering with custom recombinases.

Site-specific recombinases are valuable tools for myriad basic research and genome engineering applications. In particular, hybrid recombinases consisting of catalytic domains from the resolvase/invertase family of serine recombinases fused to Cys2-His2 zinc-finger or TAL effector DNA-binding domains are capable of introducing targeted modifications into mammalian cells. Due to their inherent modularity, new recombinases with distinct targeting specificities can readily be generated and utilized in a "plug-and-play" manner. In this protocol, we provide detailed, step-by-step instructions for generating new hybrid recombinases with user-defined specificity, as well as methods for achieving site-specific integration into targeted genomic loci using these systems.

Genome Engineering with TAL-Effector Nucleases and Alternative Modular Nuclease Technologies

Over three years following the discovery of the TAL code, artificial TAL effector DNA binding domains have emerged as the premier platform for building site-specific DNA binding polypeptides for use in biological research. Here, we provide an overview of TAL effector and alternative modular DNA binding domain (mDBD) technologies, focusing on their use in established and emerging architectures for building site-specific endonucleases for genome engineering applications. We also discuss considerations for choosing TAL effector/mDBD or alternative nuclease technologies for genome engineering projects ranging from basic laboratory gene editing of cultured cell lines to therapeutics. Finally, we highlight how the rapid pace of development of mDBD-based, such as monomeric TALENs (I-TevI-TAL), and more recently RNA-guided nucleases (CRISPR-Cas9) has led to a transition in the field of genome engineering towards development of the next generation of technologies aimed at controlling events that occur after targeted DNA breaks are made.

Transcription activator like effector (TALE)-directed piggyBac transposition in human cells

Insertional therapies have shown great potential for combating genetic disease and safer methods would undoubtedly broaden the variety of possible illness that can be treated. A major challenge that remains is reducing the risk of insertional mutagenesis due to random insertion by both viral and non-viral vectors. Targetable nucleases are capable of inducing double-stranded breaks to enhance homologous recombination for the introduction of transgenes at specific sequences. However, off-target DNA cleavages at unknown sites can lead to mutations that are difficult to detect. Alternatively, the piggyBac transposase is able perform all of the steps required for integration; therefore, cells confirmed to contain a single copy of a targeted transposon, for which its location is known, are likely to be devoid of aberrant genomic modifications. We aimed to retarget transposon insertions by comparing a series of novel hyperactive piggyBac constructs tethered to a custom transcription activator like effector DNA-binding domain designed to bind the first intron of the human CCR5 gene. Multiple targeting strategies were evaluated using combinations of both plasmid-DNA and transposase-protein relocalization to the target sequence. We demonstrated user-defined directed transposition to the CCR5 genomic safe harbor and isolated single-copy clones harboring targeted integrations.

Structural mechanisms for the 5′-CCWGG sequence recognition by the N- and C-terminal domains of EcoRII

EcoRII restriction endonuclease is specific for the 5′-CCWGG sequence (W stands for A or T); however, it shows no activity on a single recognition site. To activate cleavage it requires binding of an additional target site as an allosteric effector. EcoRII dimer consists of three structural units: a central catalytic core, made from two copies of the C-terminal domain (EcoRII-C), and two N-terminal effector DNA binding domains (EcoRII-N). Here, we report DNA-bound EcoRII-N and EcoRII-C structures, which show that EcoRII combines two radically different structural mechanisms to interact with the effector and substrate DNA. The catalytic EcoRII-C dimer flips out the central T:A base pair and makes symmetric interactions with the CC:GG half-sites. The EcoRII-N effector domain monomer binds to the target site asymmetrically in a single defined orientation which is determined by specific hydrogen bonding and van der Waals interactions with the central T:A pair in the major groove. The EcoRII-N mode of the target site recognition is shared by the large class of higher plant transcription factors of the B3 superfamily.

Structure of the metal-independent restriction enzyme BfiI reveals fusion of a specific DNA-binding domain with a nonspecific nuclease.

Among all restriction endonucleases known to date, BfiI is unique in cleaving DNA in the absence of metal ions. BfiI represents a different evolutionary lineage of restriction enzymes, as shown by its crystal structure at 1.9-A resolution. The protein consists of two structural domains. The N-terminal catalytic domain is similar to Nuc, an EDTA-resistant nuclease from the phospholipase D superfamily. The C-terminal DNA-binding domain of BfiI exhibits a beta-barrel-like structure very similar to the effector DNA-binding domain of the Mg(2+)-dependent restriction enzyme EcoRII and to the B3-like DNA-binding domain of plant transcription factors. BfiI presumably evolved through domain fusion of a DNA-recognition element to a nonspecific nuclease akin to Nuc and elaborated a mechanism to limit DNA cleavage to a single double-strand break near the specific recognition sequence. The crystal structure suggests that the interdomain linker may act as an autoinhibitor controlling BfiI catalytic activity in the absence of a specific DNA sequence. A psi-blast search identified a BfiI homologue in a Mesorhizobium sp. BNC1 bacteria strain, a plant symbiont isolated from an EDTA-rich environment.