Programmable DNA-binding Proteins Research Articles

Imaging Method Using CRISPR/dCas9 and Engineered gRNA Scaffolds Can Perturb Replication Timing at the HSPA1 Locus.

Fluorescence microscopy imaging of specific chromosomal sites is essential for genome architecture research. To enable visualization of endogenous loci in mammalian cells, programmable DNA-binding proteins such as TAL effectors and CRISPR/dCas9 are commonly utilized. In addition, site-specific insertion of a TetO repeat array, coupled with TetR-enhanced green fluorescent protein fusion protein expression, can be used for labeling nonrepetitive endogenous loci. Here, we performed a comparison of several live-cell chromosome tagging methods, including their effect on subnuclear positioning, expression of adjacent genes, and DNA replication timing. Our results showed that the CRISPR-based imaging method can delay DNA replication timing and sister chromatid resolution at certain region. However, subnuclear localization of the labeled locus and gene expression from adjacent loci were unaffected by either TetO/TetR or CRISPR-based methods, suggesting that CRISPR-based imaging could be used for applications that do not require DNA replication analysis.

Imaging-Based In Situ Analysis of 5-Methylcytosine at Low Repetitive Single Gene Loci with Transcription-Activator-Like Effector Probes.

Transcription-activator-like effectors (TALEs) are programmable DNA binding proteins that can be used for sequence-specific, imaging-based analysis of cellular 5-methylcytosine. However, this has so far been limited to highly repetitive satellite DNA. To expand this approach to the analysis of coding single gene loci, we here explore a number of signal amplification strategies for increasing imaging sensitivity with TALEs. We develop a straightforward amplification protocol and employ it to target the MUC4 gene, which features only a small cluster of repeat sequences. This offers high sensitivity imaging of MUC4, and in costaining experiments with pairs of one TALE selective for unmethylated cytosine and one universal control TALE enables analyzing methylation changes in the target independently of changes in target accessibility. These advancements offer prospects for 5-methylcytosine analysis at coding, nonrepetitive gene loci by the use of designed TALE probe collections.

Base Editing of Human Hematopoietic Stem Cells.

Base editing by nucleotide deaminases linked to programmable DNA-binding proteins represents a promising approach to remedy blood disorders. Here we describe the ex vivo base editing of human CD34+ hematopoietic stem and progenitor cells (HSPCs) by electroporation of base editor mRNA or protein.

Cell-Based Delivery Approaches for DNA-Binding Domains to the Central Nervous System.

Advancements in programmable DNA-Binding Proteins (DBDs) that target the genome, such as zinc fingers, transcription activator-like effectors, and Cas9, have broadened drug target design beyond traditional protein substrates. Effective delivery methodologies remain a major barrier in targeting the central nervous system. Currently, adeno-associated virus is the most well-validated delivery system for the delivery of DBDs towards the central nervous with multiple, on-going clinical trials. While effective in transducing neuronal cells, viral delivery systems for DBDs remain problematic due to inherent viral packaging limits or immune responses that hinder translational potential. Direct administration of DBDs or encapsulation in lipid nanoparticles may provide alternative means towards delivering gene therapies into the central nervous system. This review will evaluate the strengths and limitations of current DBD delivery strategies in vivo. Furthermore, this review will discuss the use of adult stem cells as a putative delivery vehicle for DBDs and the potential advantages that these systems have over previous methodologies.

Engineered TALE Repeats for Enhanced Imaging-Based Analysis of Cellular 5-Methylcytosine.

Transcription‐activator‐like effectors (TALEs) are repeat‐based, programmable DNA‐binding proteins that can be engineered to recognize sequences of canonical and epigenetically modified nucleobases. Fluorescent TALEs can be used for the imaging‐based analysis of cellular 5‐methylcytosine (5 mC) in repetitive DNA sequences. This is based on recording fluorescence ratios from cell co‐stains with two TALEs: an analytical TALE targeting the cytosine (C) position of interest through a C‐selective repeat that is blocked by 5 mC, and a control TALE targeting the position with a universal repeat that binds both C and 5 mC. To enhance this approach, we report herein the development of novel 5 mC‐selective repeats and their integration into TALEs that can replace universal TALEs in imaging‐based 5 mC analysis, resulting in a methylation‐dependent response of both TALEs. We screened a library of size‐reduced repeats and identified several 5 mC binders. Compared to the 5 mC‐binding repeat of natural TALEs and to the universal repeat, two repeats containing aromatic residues showed enhancement of 5 mC binding and selectivity in cellular transcription activation and electromobility shift assays, respectively. In co‐stains of cellular SATIII DNA with a corresponding C‐selective TALE, this selectivity results in a positive methylation response of the new TALE, offering perspectives for studying 5 mC functions in chromatin regulation by in situ imaging with increased dynamic range.

A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing.

Bacterial toxins represent a vast reservoir of biochemical diversity that can be repurposed for biomedical applications. Such proteins include a group of predicted interbacterial toxins of the deaminase superfamily, members of which have found application in gene-editing techniques1,2. Since previously described cytidine deaminases operate on single-stranded nucleic acids3, their use in base editing requires the unwinding of double-stranded DNA (dsDNA), for example, by a CRISPR–Cas9 system. Base editing within mitochondrial DNA (mtDNA), however, has thus far been hindered by challenges associated with the delivery of guide RNA into the mitochondria4. Here we describe an interbacterial toxin, which we named DddA, that catalyses the deamination of cytidines within dsDNA. We engineered split-DddA halves that are non-toxic and inactive until brought together on target DNA by adjacently bound programmable DNA-binding proteins. Fusions of the split-DddA halves, transcription activator-like effector array proteins, and a uracil glycosylase inhibitor resulted in RNA-free DddA-derived cytosine base editors (DdCBEs) that catalyse C•G-to-T•A conversions in human mtDNA with high target specificity and product purity. We used DdCBEs to model a disease-associated mtDNA mutation in human cells, resulting in changes in respiration rates and oxidative phosphorylation. CRISPR-free DdCBEs enable the precise manipulation of mtDNA, rather than the elimination of mtDNA copies that results from its cleavage by targeted nucleases, with broad implications for the study and potential treatment of mitochondrial disorders.

DNA methylation in plants: mechanisms and tools for targeted manipulation.

DNA methylation is an epigenetic mark that regulates multiple processes, such as gene expression and genome stability. Mutants and pharmacological treatments have been instrumental in the study of this mark in plants, although their genome-wide effect complicates the direct association between changes in methylation and a particular phenotype. A variety of tools that allow locus-specific manipulation of DNA methylation can be used to assess its direct role in specific processes, as well as to create novel epialleles. Recently, new tools that recruit the methylation machinery directly to target loci through programmable DNA-binding proteins have expanded the tool kit available to researchers. This review provides an overview of DNA methylation in plants and discusses the tools that have recently been developed for its manipulation.

Application and prospects of CRISPR/Cas9-based methods to trace defined genomic sequences in living and fixed plant cells.

The 3D organization of chromatin plays an important role in genome stability and many other pivotal biological programs. Therefore, the establishment of imaging methods, which enable us to study the dynamics of chromatin in living cells, is necessary. Although primary live cell imaging methods were a breakthrough, there is a need to develop more specific labeling techniques. With the discovery of programmable DNA binding proteins, such zinc finger proteins (ZFP), transcription activator-like effectors (TALE), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), a major leap forward was made. Here, we review the applications and potential of fluorescent repressor-operator systems, programmable DNA binding proteins with an emphasis on CRISPR-based chromatin imaging in living and fixed cells, and their potential application in plant science.

The N6-Position of Adenine Is a Blind Spot for TAL-Effectors That Enables Effective Binding of Methylated and Fluorophore-Labeled DNA.

Transcription-activator-like effectors (TALEs) are programmable DNA binding proteins widely used for genome targeting. TALEs consist of multiple concatenated repeats, each selectively recognizing one nucleobase via a defined repeat variable diresidue (RVD). Effective use of TALEs requires knowledge about their binding ability to epigenetic and other modified nucleobases occurring in target DNA. However, aside from epigenetic cytosine-5 modifications, the binding ability of TALEs to modified DNA is unknown. We here study the binding of TALEs to the epigenetic nucleobase N6-methyladenine (6mA) found in prokaryotic and recently also eukaryotic genomes. We find that the natural, adenine (A)-binding RVD NI is insensitive to 6mA. Model-assisted structure-function studies reveal accommodation of 6mA by RVDs with altered hydrophobic surfaces and abilities of hydrogen bonding to the N6-amino group or N7 atom of A. Surprisingly, this tolerance of N6 substitution was transferrable to bulky N6-alkynyl substituents usable for click chemistry and even to a large rhodamine dye, establishing the N6 position of A as the first site of DNA that offers label introduction within TALE target sites without interference. These findings will guide future in vivo studies with TALEs and expand their applicability as DNA capture probes for analytical applications in vitro.

Genomic targeting of epigenetic probes using a chemically tailored Cas9 system

Recent advances in the field of programmable DNA-binding proteins have led to the development of facile methods for genomic localization of genetically encodable entities. Despite the extensive utility of these tools, locus-specific delivery of synthetic molecules remains limited by a lack of adequate technologies. Here we combine the flexibility of chemical synthesis with the specificity of a programmable DNA-binding protein by using protein trans-splicing to ligate synthetic elements to a nuclease-deficient Cas9 (dCas9) in vitro and subsequently deliver the dCas9 cargo to live cells. The versatility of this technology is demonstrated by delivering dCas9 fusions that include either the small-molecule bromodomain and extra-terminal family bromodomain inhibitor JQ1 or a peptide-based PRC1 chromodomain ligand, which are capable of recruiting endogenous copies of their cognate binding partners to targeted genomic binding sites. We expect that this technology will allow for the genomic localization of a wide array of small molecules and modified proteinaceous materials.

TALE proteins search DNA using a rotationally decoupled mechanism.

Transcription activator-like effector (TALE) proteins are a class of programmable DNA-binding proteins used extensively for gene editing. Despite recent progress, however, little is known about their sequence search mechanism. Here, we use single-molecule experiments to study TALE search along DNA. Our results show that TALEs utilize a rotationally decoupled mechanism for nonspecific search, despite remaining associated with DNA templates during the search process. Our results suggest that the protein helical structure enables TALEs to adopt a loosely wrapped conformation around DNA templates during nonspecific search, facilitating rapid one-dimensional (1D) diffusion under a range of solution conditions. Furthermore, this model is consistent with a previously reported two-state mechanism for TALE search that allows these proteins to overcome the search speed-stability paradox. Taken together, our results suggest that TALE search is unique among the broad class of sequence-specific DNA-binding proteins and supports efficient 1D search along DNA.

DNA-binding proteins from marine bacteria expand the known sequence diversity of TALE-like repeats

Transcription Activator-Like Effectors (TALEs) of Xanthomonas bacteria are programmable DNA binding proteins with unprecedented target specificity. Comparative studies into TALE repeat structure and function are hindered by the limited sequence variation among TALE repeats. More sequence-diverse TALE-like proteins are known from Ralstonia solanacearum (RipTALs) and Burkholderia rhizoxinica (Bats), but RipTAL and Bat repeats are conserved with those of TALEs around the DNA-binding residue. We study two novel marine-organism TALE-like proteins (MOrTL1 and MOrTL2), the first to date of non-terrestrial origin. We have assessed their DNA-binding properties and modelled repeat structures. We found that repeats from these proteins mediate sequence specific DNA binding conforming to the TALE code, despite low sequence similarity to TALE repeats, and with novel residues around the BSR. However, MOrTL1 repeats show greater sequence discriminating power than MOrTL2 repeats. Sequence alignments show that there are only three residues conserved between repeats of all TALE-like proteins including the two new additions. This conserved motif could prove useful as an identifier for future TALE-likes. Additionally, comparing MOrTL repeats with those of other TALE-likes suggests a common evolutionary origin for the TALEs, RipTALs and Bats.

Direct observation of TALE protein dynamics reveals a two-state search mechanism.

Transcription activator-like effector (TALE) proteins are a class of programmable DNA-binding proteins for which the fundamental mechanisms governing the search process are not fully understood. Here we use single-molecule techniques to directly observe TALE search dynamics along DNA templates. We find that TALE proteins are capable of rapid diffusion along DNA using a combination of sliding and hopping behaviour, which suggests that the TALE search process is governed in part by facilitated diffusion. We also observe that TALE proteins exhibit two distinct modes of action during the search process—a search state and a recognition state—facilitated by different subdomains in monomeric TALE proteins. Using TALE truncation mutants, we further demonstrate that the N-terminal region of TALEs is required for the initial non-specific binding and subsequent rapid search along DNA, whereas the central repeat domain is required for transitioning into the site-specific recognition state.

Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers

Technologies that facilitate the targeted manipulation of epigenetic marks could be used to precisely control cell phenotype or interrogate the relationship between the epigenome and transcriptional control. Here we have generated a programmable acetyltransferase based on the CRISPR/Cas9 gene regulation system, consisting of the nuclease-null dCas9 protein fused to the catalytic core of the human acetyltransferase p300. This fusion protein catalyzes acetylation of histone H3 lysine 27 at its target sites, corresponding with robust transcriptional activation of target genes from promoters, proximal enhancers, and distal enhancers. Gene activation by the targeted acetyltransferase is highly specific across the genome. In contrast to conventional dCas9-based activators, the acetyltransferase effectively activates genes from enhancer regions and with individual guide RNAs. The core p300 domain is also portable to other programmable DNA-binding proteins. These results support targeted acetylation as a causal mechanism of transactivation and provide a new robust tool for manipulating gene regulation.

Direct Observation of TALE Protein Search Dynamics along DNA

In this work, we discuss the direct observation of transcription activator-like effector (TALE) protein dynamics along DNA templates using single molecule techniques. TALE proteins are robust, programmable DNA-binding proteins, often fused to a nuclease domain to generate the TALEN system, a leading technology in genetic engineering. Recently, powerful methods for gene editing have been developed, including zinc finger nucleases, the CRISPR/Cas9 system, and TALENs. Despite great promise for treating human disease, however, we still lack a complete understanding of the mechanisms governing TALE search dynamics and the role of off-target binding events threatening to inhibit clinical implementation. Our work aims to develop a molecular-level understanding of TALE binding and target sequence search, which will facilitate the design of new and efficient TALEN systems. To this end we have developed a single molecule assay to directly visualize the binding and 1-D search of TALE proteins along stretched, dual-tethered DNA templates. We implemented an efficient method for specific labeling of TALE proteins using an aldehyde-based bioorthogonal labeling scheme relying on formylglycine generating enzyme. Single molecule data on TALE search dynamics reveal a previously unknown two-state “search and check” model, wherein periods of rapid DNA search are interspersed with stationary local binding. This model reconciles the ability for TALE proteins to locate their target sequence amongst thousands of potential binding sites. We further generated a series of truncated TALE variants and observed the dynamics of these proteins at the single molecule level. In this way, we are able to identify each subdomain's role in sequence search, thereby further advancing the understanding of TALE search. Overall, our work provides a ‘first-of-its-kind’ view of the 1-D diffusion of TALE proteins on DNA, which will be critically important for engineering improved TALE proteins for precise genomic editing.

Programmable DNA-binding proteins from Burkholderia provide a fresh perspective on the TALE-like repeat domain.

The tandem repeats of transcription activator like effectors (TALEs) mediate sequence-specific DNA binding using a simple code. Naturally, TALEs are injected by Xanthomonas bacteria into plant cells to manipulate the host transcriptome. In the laboratory TALE DNA binding domains are reprogrammed and used to target a fused functional domain to a genomic locus of choice. Research into the natural diversity of TALE-like proteins may provide resources for the further improvement of current TALE technology. Here we describe TALE-like proteins from the endosymbiotic bacterium Burkholderia rhizoxinica, termed Bat proteins. Bat repeat domains mediate sequence-specific DNA binding with the same code as TALEs, despite less than 40% sequence identity. We show that Bat proteins can be adapted for use as transcription factors and nucleases and that sequence preferences can be reprogrammed. Unlike TALEs, the core repeats of each Bat protein are highly polymorphic. This feature allowed us to explore alternative strategies for the design of custom Bat repeat arrays, providing novel insights into the functional relevance of non-RVD residues. The Bat proteins offer fertile grounds for research into the creation of improved programmable DNA-binding proteins and comparative insights into TALE-like evolution.

Precision genetic modifications: a new era in molecular biology and crop improvement

Recently, the use of programmable DNA-binding proteins such as ZFP/ZFNs, TALE/TALENs and CRISPR/Cas has produced unprecedented advances in gene targeting and genome editing in prokaryotes and eukaryotes. These advances allow researchers to specifically alter genes, reprogram epigenetic marks, generate site-specific deletions and potentially cure diseases. Unlike previous methods, these precision genetic modification techniques (PGMs) are specific, efficient, easy to use and economical. Here we discuss the capabilities and pitfalls of PGMs and highlight the recent, exciting applications of PGMs in molecular biology and crop genetic engineering. Further improvement of the efficiency and precision of PGM techniques will enable researchers to precisely alter gene expression and biological/chemical pathways, probe gene function, modify epigenetic marks and improve crops by increasing yield, quality and tolerance to limiting biotic and abiotic stress conditions.

Zinc Fingers, TAL Effectors, or Cas9-Based DNA Binding Proteins: What’s Best for Targeting Desired Genome Loci?

Programmable DNA binding proteins (PDPs) that selectively bind user-defined nucleotide sequences can navigate protein domains to any desired target within the genome. Different functional domains can be fused to PDPs to achieve (1) activation or repression of transcription, (2) nicking or cleavage of DNA, or (3) modification or removal of epigenetic marks at the targeted genome spot. Given their broad applicability, the development of highly specific, easy-to-use PDPs has been a holy grail of biotechnology since the determination of the 3-D structure of zinc fingers (ZFs) bound to DNA in 1991 (Pavletich and Pabo, 1991).