ABSTRACT
Chimeric antigen receptor (CAR) designs that incorporate pharmacologic control are desirable; however, designs suitable for clinical translation are needed. We designed a fully human, rapamycin-regulated drug product for targeting CD33+ tumors called dimerizaing agent-regulated immunoreceptor complex (DARIC33). T cell products demonstrated target-specific and rapamycin-dependent cytokine release, transcriptional responses, cytotoxicity, and in vivo antileukemic activity in the presence of as little as 1 nM rapamycin. Rapamycin withdrawal paused DARIC33-stimulated T cell effector functions, which were restored following reexposure to rapamycin, demonstrating reversible effector function control. While rapamycin-regulated DARIC33 T cells were highly sensitive to target antigen, CD34+ stem cell colony-forming capacity was not impacted. We benchmarked DARIC33 potency relative to CD19 CAR T cells to estimate a T cell dose for clinical testing. In addition, we integrated in vitro and preclinical in vivo drug concentration thresholds for off-on state transitions, as well as murine and human rapamycin pharmacokinetics, to estimate a clinically applicable rapamycin dosing schedule. A phase I DARIC33 trial has been initiated (PLAT-08, NCT05105152), with initial evidence of rapamycin-regulated T cell activation and antitumor impact. Our findings provide evidence that the DARIC platform exhibits sensitive regulation and potency needed for clinical application to other important immunotherapy targets.
Subject(s)
Leukemia, Myeloid, Acute , Sialic Acid Binding Ig-like Lectin 3 , Sirolimus , T-Lymphocytes , Animals , Female , Humans , Male , Mice , Immunotherapy, Adoptive , Leukemia, Myeloid, Acute/immunology , Leukemia, Myeloid, Acute/therapy , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/pathology , Receptors, Chimeric Antigen/immunology , Sialic Acid Binding Ig-like Lectin 3/immunology , Sialic Acid Binding Ig-like Lectin 3/metabolism , Sirolimus/pharmacology , Sirolimus/administration & dosage , T-Lymphocytes/immunology , T-Lymphocytes/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Chimeric antigen receptor (CAR) T cell therapies have achieved promising outcomes in several cancers, however more challenging oncology indications may necessitate advanced antigen receptor designs and functions. Here we describe a bipartite receptor system comprised of separate antigen targeting and signal transduction polypeptides, each containing an extracellular dimerization domain. We demonstrate that T cell activation remains antigen dependent but can only be achieved in the presence of a dimerizing drug, rapamycin. Studies performed in vitro and in xenograft mouse models illustrate equivalent to superior anti-tumor potency compared to currently used CAR designs, and at rapamycin concentrations well below immunosuppressive levels. We further show that the extracellular positioning of the dimerization domains enables the administration of recombinant re-targeting modules, potentially extending antigen targeting. Overall, this novel regulatable CAR design has exquisite drug sensitivity, provides robust anti-tumor responses, and is uniquely flexible for multiplex antigen targeting or retargeting, which may further assist the development of safe, potent and durable T cell therapeutics.
Subject(s)
Antigens, CD19/immunology , Immunotherapy, Adoptive/methods , Neoplasms/therapy , Receptors, Chimeric Antigen/genetics , Recombinant Fusion Proteins/genetics , Animals , Antigens, CD19/metabolism , Cell Line, Tumor , Female , Humans , Lymphocyte Activation , Mice , Neoplasms/immunology , Neoplasms/pathology , Promoter Regions, Genetic/drug effects , Promoter Regions, Genetic/genetics , Protein Domains/genetics , Protein Multimerization/drug effects , Protein Multimerization/genetics , Receptors, Chimeric Antigen/immunology , Receptors, Chimeric Antigen/metabolism , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/metabolism , Single-Chain Antibodies/genetics , Single-Chain Antibodies/immunology , Single-Chain Antibodies/metabolism , Sirolimus/administration & dosage , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , T-Lymphocytes/transplantation , Xenograft Model Antitumor AssaysABSTRACT
The retargeting of protein-DNA specificity, outside of extremely modular DNA binding proteins such as TAL effectors, has generally proved to be quite challenging. Here, we describe structural analyses of five different extensively retargeted variants of a single homing endonuclease, that have been shown to function efficiently in ex vivo and in vivo applications. The redesigned proteins harbor mutations at up to 53 residues (18%) of their amino acid sequence, primarily distributed across the DNA binding surface, making them among the most significantly reengineered ligand-binding proteins to date. Specificity is derived from the combined contributions of DNA-contacting residues and of neighboring residues that influence local structural organization. Changes in specificity are facilitated by the ability of all those residues to readily exchange both form and function. The fidelity of recognition is not precisely correlated with the fraction or total number of residues in the protein-DNA interface that are actually involved in DNA contacts, including directional hydrogen bonds. The plasticity of the DNA-recognition surface of this protein, which allows substantial retargeting of recognition specificity without requiring significant alteration of the surrounding protein architecture, reflects the ability of the corresponding genetic elements to maintain mobility and persistence in the face of genetic drift within potential host target sites.
Subject(s)
DNA/chemistry , DNA/metabolism , Endodeoxyribonucleases/chemistry , Endodeoxyribonucleases/metabolism , Amino Acid Sequence , Animals , Animals, Genetically Modified , Base Sequence , Binding Sites/genetics , Crystallography , Culicidae/enzymology , Culicidae/genetics , DNA/genetics , Endodeoxyribonucleases/genetics , Models, Molecular , Mutation , Nucleic Acid Conformation , Protein Binding , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid , Substrate SpecificityABSTRACT
A naturally occurring 32-base pair deletion of the HIV-1 co-receptor CCR5 has demonstrated protection against HIV infection of human CD4+ T cells. Recent genetic engineering approaches using engineered nucleases to disrupt the gene and mimic this mutation show promise for HIV therapy. We developed a megaTAL nuclease targeting the third extracellular loop of CCR5 that we delivered to primary human T cells by mRNA transfection. The CCR5 megaTAL nuclease established resistance to HIV in cell lines and disrupted the expression of CCR5 on primary human CD4+ T cells with a high efficiency, achieving up to 80% modification of the locus in primary cells as measured by molecular analysis. Gene-modified cells engrafted at levels equivalent to unmodified cells when transplanted into immunodeficient mice. Furthermore, genetically modified CD4+ cells were preferentially expanded during HIV-1 infection in vivo in an immunodeficient mouse model. Our results demonstrate the feasibility of targeting CCR5 in primary T cells using an engineered megaTAL nuclease, and the potential to use gene-modified cells to reconstitute a patient's immune system and provide protection from HIV infection.
ABSTRACT
LAGLIDADG meganucleases are DNA cleaving enzymes used for genome engineering. While their cleavage specificity can be altered using several protein engineering and selection strategies, their overall targetability is limited by highly specific indirect recognition of the central four base pairs within their recognition sites. In order to examine the physical basis of indirect sequence recognition and to expand the number of such nucleases available for genome engineering, we have determined the target sites, DNA-bound structures, and central four cleavage fidelities of nine related enzymes. Subsequent crystallographic analyses of a meganuclease bound to two noncleavable target sites, each containing a single inactivating base pair substitution at its center, indicates that a localized slip of the mutated base pair causes a small change in the DNA backbone conformation that results in a loss of metal occupancy at one binding site, eliminating cleavage activity.
Subject(s)
DNA/chemistry , DNA/metabolism , Deoxyribonucleases/chemistry , Deoxyribonucleases/metabolism , Base Sequence , Binding Sites , DNA Cleavage , Models, Molecular , Nucleic Acid Conformation , Protein Conformation , Substrate SpecificityABSTRACT
Genetic mutations or engineered nucleases that disrupt the HIV co-receptor CCR5 block HIV infection of CD4(+) T cells. These findings have motivated the engineering of CCR5-specific nucleases for application as HIV therapies. The efficacy of this approach relies on efficient biallelic disruption of CCR5, and the ability to efficiently target sequences that confer HIV resistance to the CCR5 locus has the potential to further improve clinical outcomes. We used RNA-based nuclease expression paired with adeno-associated virus (AAV)-mediated delivery of a CCR5-targeting donor template to achieve highly efficient targeted recombination in primary human T cells. This method consistently achieved 8 to 60% rates of homology-directed recombination into the CCR5 locus in T cells, with over 80% of cells modified with an MND-GFP expression cassette exhibiting biallelic modification. MND-GFP-modified T cells maintained a diverse repertoire and engrafted in immune-deficient mice as efficiently as unmodified cells. Using this method, we integrated sequences coding chimeric antigen receptors (CARs) into the CCR5 locus, and the resulting targeted CAR T cells exhibited antitumor or anti-HIV activity. Alternatively, we introduced the C46 HIV fusion inhibitor, generating T cell populations with high rates of biallelic CCR5 disruption paired with potential protection from HIV with CXCR4 co-receptor tropism. Finally, this protocol was applied to adult human mobilized CD34(+) cells, resulting in 15 to 20% homologous gene targeting. Our results demonstrate that high-efficiency targeted integration is feasible in primary human hematopoietic cells and highlight the potential of gene editing to engineer T cell products with myriad functional properties.
Subject(s)
Deoxyribonucleases/metabolism , Dependovirus/metabolism , Hematopoietic Stem Cells/metabolism , Receptors, CCR5/metabolism , Adult , Antigens, CD34/metabolism , CD3 Complex/metabolism , Cells, Cultured , DNA Repair , Genetic Loci , Genetic Therapy , Green Fluorescent Proteins/metabolism , Humans , RNA Editing/genetics , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/metabolismABSTRACT
Genome engineering with designer nucleases is a rapidly progressing field, and the ability to correct human gene mutations in situ is highly desirable. We employed fibroblasts derived from a patient with Fanconi anemia as a model to test the ability of the clustered regularly interspaced short palindromic repeats/Cas9 nuclease system to mediate gene correction. We show that the Cas9 nuclease and nickase each resulted in gene correction, but the nickase, because of its ability to preferentially mediate homology-directed repair, resulted in a higher frequency of corrected clonal isolates. To assess the off-target effects, we used both a predictive software platform to identify intragenic sequences of homology as well as a genome-wide screen utilizing linear amplification-mediated PCR. We observed no off-target activity and show RNA-guided endonuclease candidate sites that do not possess low sequence complexity function in a highly specific manner. Collectively, we provide proof of principle for precision genome editing in Fanconi anemia, a DNA repair-deficient human disorder.
Subject(s)
Bacterial Proteins/metabolism , Clustered Regularly Interspaced Short Palindromic Repeats , Deoxyribonuclease I/metabolism , Fanconi Anemia Complementation Group C Protein/metabolism , Fibroblasts/metabolism , Genome, Human , Bacterial Proteins/genetics , Base Sequence , Deoxyribonuclease I/genetics , Electroporation , Fanconi Anemia/genetics , Fanconi Anemia/metabolism , Fanconi Anemia/pathology , Fanconi Anemia/therapy , Fanconi Anemia Complementation Group C Protein/genetics , Fibroblasts/pathology , Gene Expression , Genetic Engineering , Genetic Loci , Humans , Lipids , Molecular Sequence Data , Molecular Targeted Therapy , Plasmids/chemistry , Plasmids/metabolism , Polymerase Chain Reaction/methods , Primary Cell Culture , RNA, Guide, Kinetoplastida/genetics , RNA, Guide, Kinetoplastida/metabolism , Streptococcus pyogenes/chemistry , Streptococcus pyogenes/enzymology , TransfectionABSTRACT
LAGLIDADG homing endonucleases (LHEs) are compact endonucleases with 20-22 bp recognition sites, and thus are ideal scaffolds for engineering site-specific DNA cleavage enzymes for genome editing applications. Here, we describe a general approach to LHE engineering that combines rational design with directed evolution, using a yeast surface display high-throughput cleavage selection. This approach was employed to alter the binding and cleavage specificity of the I-Anil LHE to recognize a mutation in the mouse Bruton tyrosine kinase (Btk) gene causative for mouse X-linked immunodeficiency (XID)-a model of human X-linked agammaglobulinemia (XLA). The required re-targeting of I-AniI involved progressive resculpting of the DNA contact interface to accommodate nine base differences from the native cleavage sequence. The enzyme emerging from the progressive engineering process was specific for the XID mutant allele versus the wild-type (WT) allele, and exhibited activity equivalent to WT I-AniI in vitro and in cellulo reporter assays. Fusion of the enzyme to a site-specific DNA binding domain of transcription activator-like effector (TALE) resulted in a further enhancement of gene editing efficiency. These results illustrate the potential of LHE enzymes as specific and efficient tools for therapeutic genome engineering.
Subject(s)
Endodeoxyribonucleases/chemistry , Protein-Tyrosine Kinases/genetics , Agammaglobulinaemia Tyrosine Kinase , Animals , Cells, Cultured , DNA Cleavage , DNA-Binding Proteins/chemistry , Directed Molecular Evolution , Endodeoxyribonucleases/metabolism , Genetic Loci , Genomics , HEK293 Cells , Homologous Recombination , Humans , Indicators and Reagents , Mice , Mutation , Protein Structure, TertiaryABSTRACT
Rare-cleaving endonucleases have emerged as important tools for making targeted genome modifications. While multiple platforms are now available to generate reagents for research applications, each existing platform has significant limitations in one or more of three key properties necessary for therapeutic application: efficiency of cleavage at the desired target site, specificity of cleavage (i.e. rate of cleavage at 'off-target' sites), and efficient/facile means for delivery to desired target cells. Here, we describe the development of a single-chain rare-cleaving nuclease architecture, which we designate 'megaTAL', in which the DNA binding region of a transcription activator-like (TAL) effector is used to 'address' a site-specific meganuclease adjacent to a single desired genomic target site. This architecture allows the generation of extremely active and hyper-specific compact nucleases that are compatible with all current viral and nonviral cell delivery methods.
Subject(s)
DNA Cleavage , Endodeoxyribonucleases/chemistry , Endodeoxyribonucleases/metabolism , Cells, Cultured , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Endodeoxyribonucleases/genetics , Gene Knockout Techniques , Genes, T-Cell Receptor alpha , Genetic Engineering , Genomics/methods , HEK293 Cells , Humans , Protein Structure, Tertiary/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolismABSTRACT
The homing endonuclease gene (HEG) drive system, a promising genetic approach for controlling arthropod populations, utilises engineered nucleases to spread deleterious mutations that inactivate individual genes throughout a target population. Previous work with a naturally occurring LAGLIDADG homing endonuclease (I-SceI) demonstrated its feasibility in both Drosophila and Anopheles. Here we report on the next stage of this strategy: the redesign of HEGs with customized specificity in order to drive HEG-induced 'homing' in vivo via break-induced homologous recombination. Variants targeting a sequence within the Anopheles AGAP004734 gene were created from the recently characterized I-OnuI endonuclease, and tested for cleavage activity and frequency of homing using a model Drosophila HEG drive system. We observed cleavage and homing at an integrated reporter for all endonuclease variants tested, demonstrating for the first time that engineered HEGs can cleave their target site in insect germline cells, promoting targeted mutagenesis and homing. However, in comparison to our previously reported work with I-SceI, the engineered I-OnuI variants mediated homing with a reduced frequency, suggesting that site-specific cleavage activity is insufficient by itself to ensure efficient homing. Taken together, our experiments take a further step towards the development of a viable HEG-based population control strategy for insects.
Subject(s)
Anopheles/genetics , Drosophila melanogaster/genetics , Endodeoxyribonucleases/genetics , Gene Targeting/methods , Insect Proteins/genetics , Pest Control, Biological/methods , Animals , Deoxyribonucleases, Type II Site-Specific/genetics , Genes, Reporter , Genetic Engineering , Germ-Line Mutation , Homologous Recombination , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
A fast, easy, and scalable method to assess the properties of site-specific nucleases is crucial to -understanding their in cellulo behavior in genome engineering or population-level gene drive applications. Here we describe an analytical platform that enables high-throughput, semiquantitative interrogation of the DNA-binding and catalytic properties of LAGLIDADG homing endonucleases (LHEs). Using this platform, natural or engineered LHEs are expressed on the surface of Saccharomyces cerevisiae yeast where they can be rapidly evaluated against synthetic DNA target sequences using flow cytometry.
Subject(s)
DNA/metabolism , Endonucleases/metabolism , Flow Cytometry/methods , Protein Binding , Saccharomyces cerevisiaeABSTRACT
Although engineered LAGLIDADG homing endonucleases (LHEs) are finding increasing applications in biotechnology, their generation remains a challenging, industrial-scale process. As new single-chain LAGLIDADG nuclease scaffolds are identified, however, an alternative paradigm is emerging: identification of an LHE scaffold whose native cleavage site is a close match to a desired target sequence, followed by small-scale engineering to modestly refine recognition specificity. The application of this paradigm could be accelerated if methods were available for fusing N- and C-terminal domains from newly identified LHEs into chimeric enzymes with hybrid cleavage sites. Here we have analyzed the structural requirements for fusion of domains extracted from six single-chain I-OnuI family LHEs, spanning 40-70% amino acid identity. Our analyses demonstrate that both the LAGLIDADG helical interface residues and the linker peptide composition have important effects on the stability and activity of chimeric enzymes. Using a simple domain fusion method in which linker peptide residues predicted to contact their respective domains are retained, and in which limited variation is introduced into the LAGLIDADG helix and nearby interface residues, catalytically active enzymes were recoverable for ≈ 70% of domain chimeras. This method will be useful for creating large numbers of chimeric LHEs for genome engineering applications.
Subject(s)
Endodeoxyribonucleases/chemistry , Protein Engineering/methods , Amino Acid Motifs , Amino Acid Sequence , Base Pairing , DNA/chemistry , Endodeoxyribonucleases/genetics , Endodeoxyribonucleases/metabolism , Enzyme Stability , HEK293 Cells , Humans , Molecular Sequence Data , Peptides/chemistry , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolismABSTRACT
LAGLIDADG homing endonucleases (LHEs) are DNA cleaving enzymes, also termed 'meganucleases' that are employed as gene-targeting reagents. This use of LHEs requires that their DNA specificity be altered to match sequences in genomic targets. The choice of the most appropriate LHE to target a particular gene is facilitated by the growing number of such enzymes with well-characterized activities and structures. 'LAHEDES' (The LAGLIDADG Homing Endonuclease Database and Engineering Server) provides both an online archive of LHEs with validated DNA cleavage specificities and DNA-binding interactions, as well as a tool for the identification of DNA sequences that might be targeted by various LHEs. Searches can be performed using four separate scoring algorithms and user-defined choices of LHE scaffolds. The webserver subsequently provides information regarding clusters of amino acids that should be interrogated during engineering and selection experiments. The webserver is fully open access and can be found at http://homingendonuclease.net.
Subject(s)
Databases, Genetic , Endodeoxyribonucleases/chemistry , Endodeoxyribonucleases/metabolism , Software , DNA/chemistry , DNA/metabolism , DNA Cleavage , Endodeoxyribonucleases/genetics , Gene Targeting , Internet , Mutation , Protein Engineering , Protein Structure, Tertiary , Substrate SpecificityABSTRACT
LAGLIDADG homing endonucleases (LHEs) are a family of highly specific DNA endonucleases capable of recognizing target sequences ≈ 20 bp in length, thus drawing intense interest for their potential academic, biotechnological and clinical applications. Methods for rational design of LHEs to cleave desired target sites are presently limited by a small number of high-quality native LHEs to serve as scaffolds for protein engineering-many are unsatisfactory for gene targeting applications. One strategy to address such limitations is to identify close homologs of existing LHEs possessing superior biophysical or catalytic properties. To test this concept, we searched public sequence databases to identify putative LHE open reading frames homologous to the LHE I-AniI and used a DNA binding and cleavage assay using yeast surface display to rapidly survey a subset of the predicted proteins. These proteins exhibited a range of capacities for surface expression and also displayed locally altered binding and cleavage specificities with a range of in vivo cleavage activities. Of these enzymes, I-HjeMI demonstrated the greatest activity in vivo and was readily crystallizable, allowing a comparative structural analysis. Taken together, our results suggest that even highly homologous LHEs offer a readily accessible resource of related scaffolds that display diverse biochemical properties for biotechnological applications.
Subject(s)
Endodeoxyribonucleases/chemistry , Amino Acid Sequence , Crystallography , DNA/metabolism , Endodeoxyribonucleases/genetics , Endodeoxyribonucleases/metabolism , Evolution, Molecular , HEK293 Cells , Humans , Models, Molecular , Molecular Sequence Data , Open Reading Frames , Sequence Homology, Amino Acid , Substrate SpecificityABSTRACT
Site-specific genome engineering technologies are increasingly important tools in the postgenomic era, where biotechnological objectives often require organisms with precisely modified genomes. Rare-cutting endonucleases, through their capacity to create a targeted DNA strand break, are one of the most promising of these technologies. However, realizing the full potential of nuclease-induced genome engineering requires a detailed understanding of the variables that influence resolution of nuclease-induced DNA breaks. Here we present a genome engineering reporter system, designated 'traffic light', that supports rapid flow-cytometric analysis of repair pathway choice at individual DNA breaks, quantitative tracking of nuclease expression and donor template delivery, and high-throughput screens for factors that bias the engineering outcome. We applied the traffic light system to evaluate the efficiency and outcome of nuclease-induced genome engineering in human cell lines and identified strategies to facilitate isolation of cells in which a desired engineering outcome has occurred.
Subject(s)
DNA Damage/genetics , DNA Repair/genetics , Genes, Reporter/genetics , Genetic Engineering/methods , Genome/geneticsABSTRACT
Enzymes use substrate-binding energy both to promote ground-state association and to stabilize the reaction transition state selectively. The monomeric homing endonuclease I-AniI cleaves with high sequence specificity in the centre of a 20-base-pair (bp) DNA target site, with the amino (N)-terminal domain of the enzyme making extensive binding interactions with the left (-) side of the target site and the similarly structured carboxy (C)-terminal domain interacting with the right (+) side. Here we show that, despite the approximate twofold symmetry of the enzyme-DNA complex, there is almost complete segregation of interactions responsible for substrate binding to the (-) side of the interface and interactions responsible for transition-state stabilization to the (+) side. Although single base-pair substitutions throughout the entire DNA target site reduce catalytic efficiency, mutations in the (-) DNA half-site almost exclusively increase the dissociation constant (K(D)) and the Michaelis constant under single-turnover conditions (K(M)*), and those in the (+) half-site primarily decrease the turnover number (k(cat)*). The reduction of activity produced by mutations on the (-) side, but not mutations on the (+) side, can be suppressed by tethering the substrate to the endonuclease displayed on the surface of yeast. This dramatic asymmetry in the use of enzyme-substrate binding energy for catalysis has direct relevance to the redesign of endonucleases to cleave genomic target sites for gene therapy and other applications. Computationally redesigned enzymes that achieve new specificities on the (-) side do so by modulating K(M)*, whereas redesigns with altered specificities on the (+) side modulate k(cat)*. Our results illustrate how classical enzymology and modern protein design can each inform the other.
Subject(s)
Biocatalysis , Computer Simulation , Endonucleases/metabolism , RNA-Directed DNA Polymerase/metabolism , Thermodynamics , Binding Sites , Computational Biology , DNA/chemistry , DNA/metabolism , Endonucleases/chemistry , Kinetics , Models, Molecular , Protein Binding , Protein Conformation , RNA-Directed DNA Polymerase/chemistry , Saccharomyces cerevisiae/metabolism , Substrate SpecificityABSTRACT
Experimental analysis and manipulation of protein-DNA interactions pose unique biophysical challenges arising from the structural and chemical homogeneity of DNA polymers. We report the use of yeast surface display for analytical and selection-based applications for the interaction between a LAGLIDADG homing endonuclease and its DNA target. Quantitative flow cytometry using oligonucleotide substrates facilitated a complete profiling of specificity, both for DNA-binding and catalysis, with single base pair resolution. These analyses revealed a comprehensive segregation of binding specificity and affinity to one half of the pseudo-dimeric interaction, while the entire interface contributed specificity at the level of catalysis. A single round of targeted mutagenesis with tandem affinity and catalytic selection steps provided mechanistic insights to the origins of binding and catalytic specificity. These methods represent a dynamic new approach for interrogating specificity in protein-DNA interactions.
Subject(s)
Deoxyribonucleases, Type II Site-Specific/metabolism , Binding Sites , Catalysis , DNA/metabolism , DNA Restriction Enzymes/metabolism , Endonucleases/metabolism , Flow Cytometry , Models, Molecular , Saccharomyces cerevisiae/metabolism , Substrate SpecificityABSTRACT
LAGLIDADG homing endonucleases (LHEs) cleave 18-24 bp DNA sequences and are promising enzymes for applications requiring sequence-specific DNA cleavage amongst genome-sized DNA backgrounds. Here, we report a method for cell surface display of LHEs, which facilitates analysis of their DNA binding and cleavage properties by flow cytometry. Cells expressing surface LHEs can be stained with fluorescently conjugated double-stranded oligonucleotides (dsOligos) containing their respective target sequences. The signal is absolutely sequence specific and undetectable with dsOligos carrying single base-pair substitutions. LHE-dsOligo interactions facilitate rapid enrichment and viable recovery of rare LHE expressing cells by both fluorescence-activated cell sorting (FACS) and magnetic cell sorting (MACS). Additionally, dsOligos conjugated with unique fluorophores at opposite termini can be tethered to the cell surface and used to detect DNA cleavage. Recapitulation of DNA binding and cleavage by surface-displayed LHEs provides a high-throughput approach to library screening that should facilitate rapid identification and analysis of enzymes with novel sequence specificities.