Jordan Jarjour

Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template

Therapeutic coding sequences can be targeted to the CCR5 locus of primary human T cells with high efficiency by using megaTAL nuclease and an AAV donor template. Delete and replace Newer gene-editing methods hold promise for correcting human disease but so far have been hampered by low efficiencies when used in primary cells. To address this issue, Sather et al. have devised a more effective way to both disrupt and replace the CCR5 locus in human T cells, a procedure that has already been shown to improve HIV clearance. Serotype 6 of an adeno-associated viral vector worked particularly well for delivery of megaTAL nucleases and homologous donor templates to primary human T cells, achieving efficient gene-editing rates and little toxicity. The megaTALs generate homology-directed repair (rather than previous efforts, which induce nonhomologous end-joining repair) and so was used for both deletion and accurate replacement of the CCR5 locus. The authors demonstrate that chimeric antigen receptors and an HIV fusion inhibitor inserted into the CCR5 locus ameliorate HIV infection in mice and show that their approach also works in CD34+ hematopoietic precursor cells. 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.

Exploitation of binding energy for catalysis and design

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 (KD) and the Michaelis constant under single-turnover conditions (KM*), and those in the (+) half-site primarily decrease the turnover number (kcat*). 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 KM*, whereas redesigns with altered specificities on the (+) side modulate kcat*. Our results illustrate how classical enzymology and modern protein design can each inform the other.

High-resolution profiling of homing endonuclease binding and catalytic specificity using yeast surface display

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.

Engineering domain fusion chimeras from I-OnuI family LAGLIDADG homing endonucleases

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.

LAHEDES: the LAGLIDADG homing endonuclease database and engineering server

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.

Expanding LAGLIDADG endonuclease scaffold diversity by rapidly surveying evolutionary sequence space

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.

Flow cytometric analysis of DNA binding and cleavage by cell surface-displayed homing endonucleases

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.

The design and in vivo evaluation of engineered I-OnuI-based enzymes for HEG gene drive.

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.

Indirect DNA Sequence Recognition and Its Impact on Nuclease Cleavage Activity

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.

Flow Cytometric Assays for Interrogating LAGLIDADG Homing Endonuclease DNA-Binding and Cleavage Properties

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.