Precise therapeutic gene correction by a simple nuclease-induced double-stranded break.

Current programmable nuclease-based methods (for example, CRISPR-Cas9) for the precise correction of a disease-causing genetic mutation harness the homology-directed repair pathway. However, this repair process requires the co-delivery of an exogenous DNA donor to recode the sequence and can be inefficient in many cell types. Here we show that disease-causing frameshift mutations that result from microduplications can be efficiently reverted to the wild-type sequence simply by generating a DNA double-stranded break near the centre of the duplication. We demonstrate this in patient-derived cell lines for two diseases: limb-girdle muscular dystrophy type 2G (LGMD2G)1 and Hermansky-Pudlak syndrome type 1 (HPS1)2. Clonal analysis of inducible pluripotent stem (iPS) cells from the LGMD2G cell line, which contains a mutation in TCAP, treated with the Streptococcus pyogenes Cas9 (SpCas9) nuclease revealed that about 80% contained at least one wild-type TCAP allele; this correction also restored TCAP expression in LGMD2G iPS cell-derived myotubes. SpCas9 also efficiently corrected the genotype of an HPS1 patient-derived B-lymphoblastoid cell line. Inhibition of polyADP-ribose polymerase 1 (PARP-1) suppressed the nuclease-mediated collapse of the microduplication to the wild-type sequence, confirming that precise correction is mediated by the microhomology-mediated end joining (MMEJ) pathway. Analysis of editing by SpCas9 and Lachnospiraceae bacterium ND2006 Cas12a (LbCas12a) at non-pathogenic 4-36-base-pair microduplications within the genome indicates that the correction strategy is broadly applicable to a wide range of microduplication lengths and can be initiated by a variety of nucleases. The simplicity, reliability and efficacy of this MMEJ-based therapeutic strategy should permit the development of nuclease-based gene correction therapies for a variety of diseases that are associated with microduplications.

Functional restoration of mouse Nf1 nonsense alleles in differentiated cultured neurons.

Neurofibromatosis type 1 (NF1), one of the most common autosomal dominant genetic disorders, is caused by mutations in the NF1 gene. NF1 patients have a wide variety of manifestations with a subset at high risk for the development of tumors in the central nervous system (CNS). Nonsense mutations that result in the synthesis of truncated NF1 protein (neurofibromin) are strongly associated with CNS tumors. Therapeutic nonsense suppression with small molecule drugs is a potentially powerful approach to restore the expression of genes harboring nonsense mutations. Ataluren is one such drug that has been shown to restore full-length functional protein in several models of nonsense mutation diseases, as well as in patients with nonsense mutation Duchenne muscular dystrophy. To test ataluren's potential applicability to NF1 nonsense mutations associated with CNS tumors, we generated a homozygous Nf1R683X/R683X-3X-FLAG mouse embryonic stem (mES) cell line which recapitulates an NF1 patient nonsense mutation (c.2041 C > T; p.Arg681X). We differentiated Nf1R683X/R683X-3X-FLAG mES cells into cortical neurons in vitro, treated the cells with ataluren, and demonstrated that ataluren can promote readthrough of the nonsense mutation at codon 683 of Nf1 mRNA in neural cells. The resulting full-length protein is able to reduce the cellular level of hyperactive phosphorylated ERK (pERK), a RAS effector normally suppressed by the NF1 protein.

Absolute quantitative measurement of transcriptional kinetic parameters in vivo.

mRNA expression involves transcription initiation, elongation and degradation. In cells, these dynamic processes are highly regulated. However, experimental characterization of the dynamic processes in vivo is difficult due to the paucity of methods capable of direct measurements. We present a highly sensitive and versatile method enabling direct characterization of the dynamic processes. Our method is based on single-molecule fluorescence in situ hybridization (smFISH) and quantitative analyses of hybridization signals. We hybridized multiple probes labelled with spectrally distinct fluorophores to multiple sub-regions of single mRNAs, and visualized the kinetics of synthesis and degradation of the sub-regions. Quantitative analyses of the data lead to absolute quantification of the lag time of mRNA induction (the time it takes for external signals to activate transcription initiation), transcription initiation rate, transcription elongation speed (i.e. mRNA chain-growth speed), the rate of premature termination of transcripts and degradation rates. Applying our method to three different biological problems, we demonstrated how our method may be applicable to reveal dynamics of mRNA expression that was difficult to study previously. We expect such absolute quantification can greatly facilitate understanding of gene expression and its regulation working at the levels of transcriptional initiation, elongation and degradation.

Expression optimization and synthetic gene networks in cell-free systems.

Synthetic biology offers great promise to a variety of applications through the forward engineering of biological function. Most efforts in this field have focused on employing living cells, yet cell-free approaches offer simpler and more flexible contexts. Here, we evaluate cell-free regulatory systems based on T7 promoter-driven expression by characterizing variants of TetR and LacI repressible T7 promoters in a cell-free context and examining sequence elements that determine expression efficiency. Using the resulting constructs, we then explore different approaches for composing regulatory systems, leading to the implementation of inducible negative feedback in Escherichia coli extracts and in the minimal PURE system, which consists of purified proteins necessary for transcription and translation. Despite the fact that negative feedback motifs are common and essential to many natural and engineered systems, this simple building block has not previously been implemented in a cell-free context. As a final step, we then demonstrate that the feedback systems developed using our cell-free approach can be implemented in live E. coli as well, illustrating the potential for using cell-free expression to fast track the development of live cell systems in synthetic biology. Our quantitative cell-free component characterizations and demonstration of negative feedback embody important steps on the path to harnessing biological function in a bottom-up fashion.

Optimization of Nuclear Localization Signal Composition Improves CRISPR-Cas12a Editing Rates in Human Primary Cells.

Type V CRISPR-Cas12a systems are an attractive Cas9-alternative nuclease platform for specific genome editing applications. However, previous studies demonstrate that there is a gap in overall activity between Cas12a and Cas9 in primary cells.1 Here we describe optimization to the NLS composition and architecture of Cas12a to facilitate highly efficient targeted mutagenesis in human transformed cell lines (HEK293T, Jurkat, and K562 cells) and primary cells (NK cells and CD34+ HSPCs), regardless of Cas12a ortholog. Our 3xNLS Cas12a architecture resulted in the most robust editing platform. The improved editing activity of Cas12a in both NK cells and CD34+ HSPCs resulted in pronounced phenotypic changes associated with target gene editing. Lastly, we demonstrated that optimization of the NLS composition and architecture of Cas12a did not increase editing at potential off-target sites in HEK293T or CD34+ HSPCs. Our new Cas12a NLS variant provides an improved nuclease platform for therapeutic genome editing.

Efficient Homology-Directed Repair with Circular Single-Stranded DNA Donors.

While genome editing has been revolutionized by the advent of CRISPR-based nucleases, difficulties in achieving efficient, nuclease-mediated, homology-directed repair (HDR) still limit many applications. Commonly used DNA donors such as plasmids suffer from low HDR efficiencies in many cell types, as well as integration at unintended sites. In contrast, single-stranded DNA (ssDNA) donors can produce efficient HDR with minimal off-target integration. In this study, we describe the use of ssDNA phage to efficiently and inexpensively produce long circular ssDNA (cssDNA) donors. These cssDNA donors serve as efficient HDR templates when used with Cas9 or Cas12a, with integration frequencies superior to linear ssDNA (lssDNA) donors. To evaluate the relative efficiencies of imprecise and precise repair for a suite of different Cas9 or Cas12a nucleases, we have developed a modified traffic light reporter (TLR) system (TLR-multi-Cas variant 1 [MCV1]) that permits side-by-side comparisons of different nuclease systems. We used this system to assess editing and HDR efficiencies of different nuclease platforms with distinct DNA donor types. We then extended the analysis of DNA donor types to evaluate efficiencies of fluorescent tag knockins at endogenous sites in HEK293T and K562 cells. Our results show that cssDNA templates produce efficient and robust insertion of reporter tags. Targeting efficiency is high, allowing production of biallelic integrants using cssDNA donors. cssDNA donors also outcompete lssDNA donors in template-driven repair at the target site. These data demonstrate that circular donors provide an efficient, cost-effective method to achieve knockins in mammalian cell lines.

Novel MRI deformation-heterogeneity radiomic features are associated with molecular subgroups and overall survival in pediatric medulloblastoma: Preliminary findings from a multi-institutional study.

Introduction: Medulloblastoma (MB) is a malignant, heterogenous brain tumor. Advances in molecular profiling have led to identifying four molecular subgroups of MB (WNT, SHH, Group 3, Group 4), each with distinct clinical behaviors. We hypothesize that (1) aggressive MB tumors, growing heterogeneously, induce pronounced local structural deformations in the surrounding parenchyma, and (b) these local deformations as captured on Gadolinium (Gd)-enhanced-T1w MRI are independently associated with molecular subgroups, as well as overall survival in MB patients. Methods: In this work, a total of 88 MB studies from 2 institutions were analyzed. Following tumor delineation, Gd-T1w scan for every patient was registered to a normal age-specific T1w-MRI template via deformable registration. Following patient-atlas registration, local structural deformations in the brain parenchyma were obtained for every patient by computing statistics from deformation magnitudes obtained from every 5mm annular region, 0 < d < 60 mm, where d is the distance from the tumor infiltrating edge. Results: Multi-class comparison via ANOVA yielded significant differences between deformation magnitudes obtained for Group 3, Group 4, and SHH molecular subgroups, observed up to 60-mm outside the tumor edge. Additionally, Kaplan-Meier survival analysis showed that the local deformation statistics, combined with the current clinical risk-stratification approaches (molecular subgroup information and Chang's classification), could identify significant differences between high-risk and low-risk survival groups, achieving better performance results than using any of these approaches individually. Discussion: These preliminary findings suggest there exists significant association of our tumor-induced deformation descriptor with overall survival in MB, and that there could be an added value in using the proposed radiomic descriptor along with the current risk classification approaches, towards more reliable risk assessment in pediatric MB.

A Protease-Activated Fluorescent Probe Allows Rapid Visualization of Keratinocyte Carcinoma during Excision.

Keratinocyte carcinomas, including basal and squamous cell carcinomas, are the most common human cancers worldwide. While 75% of all keratinocyte carcinoma (4 million annual cases in the United States) are treated with conventional excision, this surgical modality has much lower cure rates than Mohs micrographic surgery, likely due to the bread-loaf histopathologic assessment that visualizes <1% of the tissue margins. A quenched protease-activated fluorescent probe 6qcNIR, which produces a signal only in the protease-rich tumor microenvironment, was topically applied to 90 specimens ex vivo immediately following excision. "Puzzle-fit" analysis was used to correlate the fluorescent images with histology. Probe-dependent fluorescent images correlated with cancer determined by conventional histology. Point-of-care fluorescent detection of skin cancer had a clinically relevant sensitivity of 0.73 and corresponding specificity of 0.88. Importantly, clinicians were effectively trained to read fluorescent images within 15 minutes with reliability and confidence, resulting in sensitivities of 62%-78% and specificities of 92%-97%. Fluorescent imaging using 6qcNIR allows 100% tumor margin assessment by generating en face images that correlate with histology and may be used to overcome the limitations of conventional bread-loaf histology. The utility of 6qcNIR was validated in a busy real-world clinical setting, and clinicians were trained to effectively read fluorescent margins with a short guided instruction, highlighting clinical adaptability. When used in conventional excision, this approach may result in higher cure rates at a lower cost by allowing same-day reexcision when needed, reducing patient anxiety and improving compliance by expediting postsurgical specimen assessment. SIGNIFICANCE: A fluorescent-probe-tumor-visualization platform was developed and validated in human keratinocyte carcinoma excision specimens that may provide simple, rapid, and global assessment of margins during skin cancer excision, allowing same-day reexcision when needed.

Molecular imaging and validation of margins in surgically excised nonmelanoma skin cancer specimens.

In an effort to increase the efficiency and cure rate of nonmelanoma skin cancer (NMSC) excisions, we have developed a point-of-care method of imaging and evaluation of skin cancer margins. We evaluate the skin surgical specimens using a smart, near-infrared probe (6qcNIR) that fluoresces in the presence of cathepsin proteases overexpressed in NMSC. Imaging is done with an inverted, flying-spot fluorescence scanner that reduces scatter, giving a 70% improved step response as compared to a conventional imaging system. We develop a scheme for careful comparison of fluorescent signals to histological annotation, which involves image segmentation, fiducial-based registration, and nonrigid free-form deformation on fluorescence images, corresponding color images, "bread-loafed" tissue images, hematoxylin and eosin (H&E)-stained slides, and pathological annotations. From epidermal landmarks, spatial accuracy in the bulk of the sample is ∼ 500 µ m , which when extrapolated with a linear stretch model, suggests an error at the margin of ∼ 100 µ m , within clinical reporting standards. Cancer annotations on H&E slides are transformed and superimposed on the fluorescence images to generate the final results. Using this methodology, fluorescence cancer signals are generally found to correspond spatially with histological annotations. This method will allow us to accurately analyze molecular probes for imaging skin cancer margins.

Distinct mechanisms coordinate transcription and translation under carbon and nitrogen starvation in Escherichia coli.

Bacteria adapt to environmental stress by producing proteins that provide stress protection. However, stress can severely perturb the kinetics of gene expression, disrupting protein production. Here, we characterized how Escherichia coli mitigates such perturbations under nutrient stress through the kinetic coordination of transcription and translation. We observed that, when translation became limiting under nitrogen starvation, transcription elongation slowed accordingly. This slowdown was mediated by (p)ppGpp, the alarmone whose primary role is thought to be promoter regulation. This kinetic coordination by (p)ppGpp was critical for the robust synthesis of gene products. Surprisingly, under carbon starvation, (p)ppGpp was dispensable for robust synthesis. Characterization of the underlying kinetics revealed that under carbon starvation, transcription became limiting, and translation aided transcription elongation. This mechanism naturally coordinated transcription with translation, alleviating the need for (p)ppGpp as a mediator. These contrasting mechanisms for coordination resulted in the condition-dependent effects of (p)ppGpp on global protein synthesis and starvation survival. Our findings reveal a kinetic aspect of gene expression plasticity, establishing (p)ppGpp as a condition-dependent global effector of gene expression.

Thrombin-mediated transcriptional regulation using DNA aptamers in DNA-based cell-free protein synthesis.

Realizing the potential of cell-free systems will require development of ligand-sensitive gene promoters that control gene expression in response to a ligand of interest. Here, we describe an approach to designing ligand-sensitive transcriptional control in cell-free systems that is based on the combination of a DNA aptamer that binds thrombin and the T7 bacteriophage promoter. Placement of the aptamer near the T7 promoter, and using a primarily single-stranded template, results in up to a 6-fold change in gene expression in a ligand concentration-dependent manner. We further demonstrate that the sensitivity to thrombin concentration and the fold change in expression can be tuned by altering the position of the aptamer. The results described here pave the way for the use of DNA aptamers to achieve modular regulation of transcription in response to a wide variety of ligands in cell-free systems.

Multi-input regulation and logic with T7 promoters in cells and cell-free systems.

Engineered gene circuits offer an opportunity to harness biological systems for biotechnological and biomedical applications. However, reliance on native host promoters for the construction of circuit elements, such as logic gates, can make the implementation of predictable, independently functioning circuits difficult. In contrast, T7 promoters offer a simple orthogonal expression system for use in a variety of cellular backgrounds and even in cell-free systems. Here we develop a T7 promoter system that can be regulated by two different transcriptional repressors for the construction of a logic gate that functions in cells and in cell-free systems. We first present LacI repressible T7lacO promoters that are regulated from a distal lac operator site for repression. We next explore the positioning of a tet operator site within the T7lacO framework to create T7 promoters that respond to tet and lac repressors and realize an IMPLIES gate. Finally, we demonstrate that these dual input sensitive promoters function in an E. coli cell-free protein expression system. Our results expand the utility of T7 promoters in cell based as well as cell-free synthetic biology applications.

Surface charge- and space-dependent transport of proteins in crowded environments of nanotailored posts.

The reaction and diffusion of molecules across barriers and through crowded environments is integral to biological system function and to separation technologies. Ordered, microfabricated post arrays are a promising route to creating synthetic barriers with controlled chemical and physical characteristics. They can be used to create crowded environments, to mimic aspects of cellular membranes, and to serve as engineered replacements of polymer-based separation media. Here, the translational diffusion of fluorescein isothiocyante and various forms of green fluorescent protein (GFP), including "supercharged" variants, are examined in a silicon-based post array environment. The technique of fluorescence recovery after photobleaching (FRAP) is combined with analytical approximations and numerical simulations to assess the relative effects of reaction and diffusion on molecular transport, respectively. FRAP experiments were conducted for 64 different cases where the molecular species, the density of the posts, and the chemical surface charge of the posts were varied. In all cases, the dense packing of the posts hindered the diffusive transport of the fluorescent species. The supercharged GFPs strongly interacted with oppositely charged surfaces. With similar molecular and surface charges, transport is primarily limited by hindered diffusion. For conventional, enhanced GFP in a positively charged surface environment, transport was limited by the coupled action of hindered diffusion and surface interaction with the posts. Quantification of the size-, space-, time-, and charge-dependent translational diffusion in the post array environments can provide insight into natural processes and guide the design and development of selective membrane systems.