Target-seq: single workflow for detection of genome integration site, DNA translocation and off-target events.

Designed donor DNA delivery through viral or nonviral systems to target loci in the host genome is a critical step for gene therapy. Adeno-associated virus and lentivirus are leading vehicles for in vivo and ex vivo delivery of therapeutic genes due to their high delivery and editing efficiency. Nonviral editing tools, such as CRISPR/Cas9, are getting more attention for gene modification. However, there are safety concerns; for example, tumorigenesis due to off-target effects and DNA rearrangement. Analysis tools to detect and characterize on-target and off-target genome modification post editing in the host genome are pivotal for evaluating the success and safety of gene therapy. We developed Target-seq combined with different analysis tools to detect the genome integration site, DNA translocation and off-target events.

Click-iT trinucleotide cap analog: Synthesis, mRNA translation, and detection.

The first example of the synthesis of a new trinucleotide cap analog containing propargyl group such as m7,3'-O-propargylG(5')PPP(5')AmpG is reported. The effect of the propargyl group in trinucleotide analog with a standard trinucleotide cap analog (GAG), m7G(5')ppp(5')AmpG was evaluated with respect to their capping efficiency, in vitro T7 RNA polymerase transcription efficiency, and translation activity using cultured A549 lung carcinoma epithelial cells. The new propargyl cap analog is a substrate for T7 RNA polymerase. Notably, the mRNA capped with the propargyl cap is translated âˆ¼ 1.3 times more efficiently than the mRNA capped with the GAG cap. The most characteristic feature of the new propargyl cap analog is that the presence of the propargyl group allows further modification of the mRNA by chemical ligation of an azide-containing fluorescent-labeled substrate to the mRNA via click chemistry.

Trinucleotide Cap Analogue Bearing a Locked Nucleic Acid Moiety: Synthesis, mRNA Modification, and Translation for Therapeutic Applications.

The synthesis of a new trinucleotide cap analogue containing a locked nucleic acid (LNA) moiety such as m7(LNA)G(5')ppp(5')AmpG and its molecular biology applications are described. The most appealing feature is that this new cap analogue outperforms the standard trinucleotide cap m7G(5')ppp(5')AmpG and the anti-reverse cap analogue m27,3'-OG(5')ppp(5')G by a factor of 5 in terms of translational efficiency.

Development of a Proline-Based Selection System for Reliable Genetic Engineering in Chinese Hamster Ovary Cells.

Chinese hamster ovary (CHO) cells are the superior host cell culture models used for the bioproduction of therapeutic proteins. One of the prerequisites for bioproduction using CHO cell lines is the need to generate stable CHO cell lines with optimal expression output. Antibiotic selection is commonly employed to isolate and select CHO cell lines with stable expression, despite its potential negative impact on cellular metabolism and expression level. Herein, we present a novel proline-based selection system for the isolation of stable CHO cell lines. The system exploits a dysfunctional proline metabolism pathway in CHO cells by using a pyrroline-5-carboxylate synthase gene as a selection marker, enabling selection to be made using proline-free media. The selection system was demonstrated by expressing green fluorescent protein (GFP) and a monoclonal antibody. When GFP was expressed, more than 90% of stable transfectants were enriched within 2 weeks of the selection period. When a monoclonal antibody was expressed, we achieved comparable titers (3.35 ± 0.47 µg/mL) with G418 and Zeocin-based selections (1.65 ± 0.46 and 2.25 ± 0.07 µg/mL, respectively). We further developed a proline-based coselection by using S. cerevisiae PRO1 and PRO2 genes as markers, which enables the generation of 99.5% double-transgenic cells. The proline-based selection expands available selection tools and provides an alternative to antibiotic-based selections in CHO cell line development.

Priming Human Repopulating Hematopoietic Stem and Progenitor Cells for Cas9/sgRNA Gene Targeting.

Engineered nuclease-mediated gene targeting through homologous recombination (HR) in hematopoietic stem and progenitor cells (HSPCs) has the potential to treat a variety of genetic hematologic and immunologic disorders. Here, we identify critical parameters to reproducibly achieve high frequencies of RNA-guided (single-guide RNA [sgRNA]; CRISPR)-Cas9 nuclease (Cas9/sgRNA) and rAAV6-mediated HR at the ß-globin (HBB) locus in HSPCs. We identified that by transducing HSPCs with rAAV6 post-electroporation, there was a greater than 2-fold electroporation-aided transduction (EAT) of rAAV6 endocytosis with roughly 70% of the cell population having undergone transduction within 2 hr. When HSPCs are cultured at low densities (1 × 105 cells/mL) prior to HBB targeting, HSPC expansion rates are significantly positively correlated with HR frequencies in vitro as well as in repopulating cells in immunodeficient NSG mice in vivo. We also show that culturing fluorescence-activated cell sorting (FACS)-enriched HBB-targeted HSPCs at low cell densities in the presence of the small molecules, UM171 and SR1, stimulates the expansion of gene-edited HSPCs as measured by higher engraftment levels in immunodeficient mice. This work serves not only as an optimized protocol for genome editing HSPCs at the HBB locus for the treatment of ß-hemoglobinopathies but also as a foundation for editing HSPCs at other loci for both basic and translational research.

TEG-seq: an ion torrent-adapted NGS workflow for in cellulo mapping of CRISPR specificity.

GUIDE-seq was developed to detect CRISPR/Cas9 off-target. However, as originally reported, it was associated with a high level of nonspecific amplification. In an attempt to improve it, we developed target-enriched GUIDE-seq (TEG-seq). The sensitivity level reached 0.1-10 reads-per-million  depending on the NGS platform used, which was equivalent to 0.0002-1% measured by Targeted Amplicon-seq. Application of TEG-seq was demonstrated for the evaluation of various Cas9/gRNA configurations, which suggests delivery of Cas9/gRNA ribonucleoprotein results in significantly fewer off-targets than Cas9/gRNA plasmid. TEG-seq was also applied to 22 gRNAs with relatively high in silico ranking score that targeted the biological relevant SNPs. The result indicated the initial selection of gRNAs with high score is important, although it cannot exclude the possibility of off-target.

Programmable type III-A CRISPR-Cas DNA targeting modules.

The CRISPR-Cas systems provide invader defense in a wide variety of prokaryotes, as well as technologies for many powerful applications. The Type III-A or Csm CRISPR-Cas system is one of the most widely distributed across prokaryotic phyla, and cleaves targeted DNA and RNA molecules. In this work, we have constructed modules of Csm systems from 3 bacterial species and heterologously expressed the functional modules in E. coli. The modules include a Cas6 protein and a CRISPR locus for crRNA production, and Csm effector complex proteins. The expressed modules from L. lactis, S. epidermidis and S. thermophilus specifically eliminate invading plasmids recognized by the crRNAs of the systems. Characteristically, activation of plasmid targeting activity depends on transcription of the plasmid sequence recognized by the crRNA. Activity was not observed when transcription of the crRNA target sequence was blocked, or when the opposite strand or a non-target sequence was transcribed. Moreover, the Csm module can be programmed to recognize plasmids with novel target sequences by addition of appropriate crRNA coding sequences to the module. These systems provide a platform for investigation of Type III-A CRISPR-Cas systems in E. coli, and for introduction of programmable transcription-activated DNA targeting into novel organisms.

Multiuse trail intersection safety analysis: A crowdsourced data perspective.

Real and perceived concerns about cycling safety are a barrier to increased ridership in many cities. Many people prefer to bike on facilities separated from motor vehicles, such as multiuse trails. However, due to underreporting, cities lack data on bike collisions, especially along greenways and multiuse paths. We used a crowdsourced cycling incident dataset (2005-2016) from BikeMaps.org for the Capital Regional District (CRD), BC, Canada. Our goal was to identify design characteristics associated with unsafe intersections between multiuse trails and roads. 92.8% of mapped incidents occurred between 2014 and 2016. We extracted both collision and near miss incidents at intersections from BikeMaps.org. We conducted site observations at 32 intersections where a major multiuse trail intersected with roads. We compared attributes of reported incidents at multiuse trail-road intersections to those at road-road intersections. We then used negative binomial regression to model the relationship between the number of incidents and the infrastructure characteristics at multiuse trail-road intersections. We found a higher proportion of collisions (38%, or 17/45 total reports) at multiuse trail-road intersections compared to road-road intersections (23%, or 62/268 total reports). A higher proportion of incidents resulted in an injury at multiuse trail-road intersections compared to road-road intersections (33% versus 15%). Cycling volumes, vehicle volumes, and trail sight distance were all associated with incident frequency at multiuse trail-road intersections. Supplementing traditional crash records with crowdsourced cycling incident data provides valuable evidence on cycling safety at intersections between multiuse trails and roads, and more generally, when conflicts occur between diverse transportation modes.

Enhanced CRISPR/Cas9-mediated precise genome editing by improved design and delivery of gRNA, Cas9 nuclease, and donor DNA.

While CRISPR-based gene knock out in mammalian cells has proven to be very efficient, precise insertion of genetic elements via the cellular homology directed repair (HDR) pathway remains a rate-limiting step to seamless genome editing. Under the conditions described here, we achieved up to 56% targeted integration efficiency with up to a six-nucleotide insertion in HEK293 cells. In induced pluripotent stem cells (iPSCs), we achieved precise genome editing rates of up to 45% by co-delivering the Cas9 RNP and donor DNA. In addition, the use of a short double stranded DNA oligonucleotide with 3' overhangs allowed integration of a longer FLAG epitope tag along with a restriction site at rates of up to 50%. We propose a model that favors the design of donor DNAs with the change as close to the cleavage site as possible. For small changes such as SNPs or short insertions, asymmetric single stranded donor molecules with 30 base homology arms 3' to the insertion/repair cassette and greater than 40 bases of homology on the 5' end seems to be favored. For larger insertions such as an epitope tag, a dsDNA donor with protruding 3' homology arms of 30 bases is favored. In both cases, protecting the ends of the donor DNA with phosphorothioate modifications improves the editing efficiency.

Improved delivery of Cas9 protein/gRNA complexes using lipofectamine CRISPRMAX.

OBJECTIVES: To identify the best lipid nanoparticles for delivery of purified Cas9 protein and gRNA complexes (Cas9 RNPs) into mammalian cells and to establish the optimal conditions for transfection. RESULTS: Using a systematic approach, we screened 60 transfection reagents using six commonly-used mammalian cell lines and identified a novel transfection reagent (named Lipofectamine CRISPRMAX). Based on statistical analysis, the genome modification efficiencies in Lipofectamine CRISPRMAX-transfected cell lines were 40 or 15 % higher than those in Lipofectamine 3000 or RNAiMAX-transfected cell lines, respectively. Upon optimization of transfection conditions, we observed 85, 75 or 55 % genome editing efficiencies in HEK293FT cells, mouse ES cells, or human iPSCs, respectively. Furthermore, we were able to co-deliver donor DNA with Cas9 RNPs into a disrupted EmGFP stable cell line, resulting in the generation of up to 17 % EmGFP-positive cells. CONCLUSION: Lipofectamine CRISPRMAX was characterized as the best lipid nanoparticles for the delivery of Cas9 RNPs into a variety of mammalian cell lines, including mouse ES cells and iPSCs.

Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection.

CRISPR-Cas9 systems provide a platform for high efficiency genome editing that are enabling innovative applications of mammalian cell engineering. However, the delivery of Cas9 and synthesis of guide RNA (gRNA) remain as steps that can limit overall efficiency and ease of use. Here we describe methods for rapid synthesis of gRNA and for delivery of Cas9 protein/gRNA ribonucleoprotein complexes (Cas9 RNPs) into a variety of mammalian cells through liposome-mediated transfection or electroporation. Using these methods, we report nuclease-mediated indel rates of up to 94% in Jurkat T cells and 87% in induced pluripotent stem cells (iPSC) for a single target. When we used this approach for multigene targeting in Jurkat cells we found that two-locus and three-locus indels were achieved in approximately 93% and 65% of the resulting isolated cell lines, respectively. Further, we found that the off-target cleavage rate is reduced using Cas9 protein when compared to plasmid DNA transfection. Taken together, we present a streamlined cell engineering workflow that enables gRNA design to analysis of edited cells in as little as four days and results in highly efficient genome modulation in hard-to-transfect cells. The reagent preparation and delivery to cells is amenable to high throughput, multiplexed genome-wide cell engineering.

Proximal peroneal perforator in dual-skin paddle configuration of fibula free flap for composite oral reconstruction.

BACKGROUND: Composite defects of the oral cavity are often the result of trauma or advanced-stage tumor extirpation. The resultant deformity frequently requires a three-dimensional reconstruction of bone and soft-tissue. The fibula free flap is the preferred method of reconstruction, with various modifications focused on providing supplemental soft-tissue coverage. The objective of this study was to ascertain both anatomic and clinical data regarding the proximal peroneal perforator and its contribution to the evolution of the fibula free flap. METHODS: Ten cadaver lower extremities were dissected to isolate the most proximal perforator supplying skin over the proximal lateral lower leg. Data were recorded regarding perforator presence, location, and course. Furthermore, review of clinical cases in which the proximal perforator was used in fibula free flap design was performed for operative data collection. RESULTS: Cadaveric dissections revealed the proximal perforator to be present in 90 percent of specimens. Most commonly, the perforator, originating from the peroneal artery, traveled a short intramuscular course through the soleus muscle prior to supplying the overlying skin. In all clinical cases, the perforator was easily located with Doppler prior to incision, and there were no cases of flap failure or skin paddle loss. Flap inset was found to be optimal in all cases, with no tethering or undue tension. CONCLUSIONS: The proximal peroneal perforator was found to be anatomically reliable and clinically useful in composite oral cavity reconstruction following tumor removal. The gained separation between skin paddles allows for greater versatility in flap design and inset. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Biomaterials for reconstruction of the internal orbit.

Orbital floor injuries, alone or combination with other facial fractures, are one of the most commonly encountered midface fractures. Techniques for orbital reconstruction have migrated away from autogenous bone grafts to well-tolerated alloplasts, such as titanium and Medpor. Material for reconstructing the orbit can then be selected based on requirements of the defect matched to the mechanical properties of the material. Material selection is largely and ultimately dependent upon surgeon preference.

Correction of the crooked nose.

Correction of the deviated nose is one of the most difficult tasks in rhinoplasty surgery and should be approached in a systematic manner to ensure a satisfied patient and surgeon. Correction of the deviated nose is unique in that the patient's complaints frequently include aesthetic and functional characteristics. Equal importance should be given to the preoperative, intraoperative, and postoperative aspects of the patient's treatment to ensure a favorable outcome.

Recombination-based DNA assembly and mutagenesis methods for metabolic engineering.

In recent years there has been a growing interest in the precise and concerted assembly of multiple DNA fragments of diverse sizes, including chromosomes, and the fine tuning of gene expression levels and protein activity. Commercial DNA assembly solutions have not been conceived to support the cloning of very large or very small genetic elements or a combination of both. Here we summarize a series of protocols that allow the seamless, simultaneous, flexible, and highly efficient assembly of DNA elements of a wide range of sizes (up to hundred thousand base pairs). The protocols harness the power of homologous recombination and are performed either in vitro or within the living cells. The DNA fragments may or may not share homology at their ends. An efficient site-directed mutagenesis protocol enhanced by homologous recombination is also described.

Genetic assembly tools for synthetic biology.

With the completion of myriad genome sequencing projects, genetic bioengineering has expanded into many applications including the integrated analysis of complex pathways, the construction of new biological parts and the redesign of existing, natural biological systems. All these areas require the precise and concerted assembly of multiple DNA fragments of various sizes, including chromosomes, and the fine-tuning of gene expression levels and protein activity. Current commercial cloning products are not robust enough to support the assembly of very large or very small genetic elements or a combination of both. In addition, current strategies are not flexible enough to allow further modifications to the original design without having to undergo complicated cloning strategies. Here, we present a set of protocols that allow the seamless, simultaneous, flexible, and highly efficient assembly of genetic material, designed for a wide size dynamic range (10s to 100,000s base pairs). The assembly can be performed either in vitro or within the living cells and the DNA fragments may or may not share homology at their ends. A novel site-directed mutagenesis approach enhanced by in vitro recombineering is also presented.

Analysis of microvascular free flaps for reconstruction of advanced mandibular osteoradionecrosis: a retrospective cohort study.

PURPOSE: Previous studies have suggested that radiation therapy does not impact local complication rates after microvascular free flap (MVFF) reconstruction for head and neck cancer. There is little data, however, indicating whether or not the presence of osteoradionecrosis (ORN) affects treatment outcome. The purpose of this retrospective cohort study is to review the outcome of patients undergoing MVFF reconstruction for ORN and to determine if there is a difference in outcome and/or complications when compared to similarly reconstructed patients who received radiation therapy but did not develop ORN, as well as un-radiated controls. PATIENTS AND METHODS: The records of 305 consecutive patients who underwent MVFF reconstruction for a variety of cancer-related therapies or post-traumatic craniofacial defects from 1994 to 2004 were reviewed. Of these, all patients who underwent surgery for Marx stage III ORN involving the mandible were identified (n = 21). For purposes of comparison, patients who received preoperative radiation therapy (XRT) and underwent similar reconstruction but did not have ORN were identified and included in the study group. Similarly matched patients who never received XRT served as controls. Patients were reconstructed with a variety of MVFFs harvested from the fibula (n = 48), radial forearm (n = 11), rectus abdominus (n = 3), latissimus dorsi (n = 3), serratus anterior (n = 1) and iliac crest (n = 1). The study cohort was divided according to XRT status: group 1 (ORN), patients that received XRT and developed ORN (n = 21); group 2 (no ORN), patients that received XRT but did not develop ORN (n = 21); and group 3 (control), patients that never received XRT (n = 25). The following data were collected: age, gender, diagnosis, recipient site, donor site, hyperbaric oxygen therapy (HBO), flap complications, flap survival, patient survival. Outcome measures were defined as flap survival, complications and resolution of ORN. Descriptive statistics were recorded and an analysis of variance was calculated to evaluate differences between the 3 groups. The Fisher's exact test was used to evaluate whether a complication occurred more frequently in any one particular group. RESULTS: The mean age of the 67 patients included in the study was 57 years (SD = 15.4) years (M = 32, F = 35) and there were no significant demographic differences between the 3 groups (P = .8528). All patients were successfully reconstructed although 21% required reoperation for various reasons. Overall flap survival was 88% (ORN = 86%, no ORN = 87%, control = 90%) and there was no difference between the 3 groups studied (P = 1.0). Complications were evenly distributed among the 3 groups (50% overall) and included skin necrosis (P = .824), wound infection (P = .6374), salivary fistula (P = .1178), and partial flap loss (P = 1.0). Carotid blowout occurred in 2 patients in the ORN group, however, this was not statistically significant (P = .1844). Fourteen of the 21 patients in the ORN group had received preoperative HBO. CONCLUSION: Overall MVFF survival and complication rates among patients with ORN versus control groups are the same in this study cohort. Free tissue transfer is a viable option for advanced mandibular ORN.

Preparation of the neck for microvascular reconstruction of the head and neck.

Reconstruction of congenital, developmental, or acquired head and neck defects remains a significant challenge for the oral and maxillofacial surgeon. Microvascular free tissue transfer has several advantages over nonvascularized bone grafts and pedicled soft tissue flaps that currently make it the modality of choice for the reconstruction of extirpative defects of the head and neck. Preoperative planning must include detailed attention to the technical aspects of the microvascular procedure. This includes a thorough understanding of the vascular anatomy of the patient's neck; vascular anatomy of the various flaps including pedicle lengths; and a knowledge of how to facilitate microvascular surgery in the neck and to manage complicating factors in the difficult neck.

Vascularized options for reconstruction of the mandibular condyle.

The temporomandibular joint is elegant in its design, which may make it difficult if not impossible to comprehensively reconstruct. Although a broad range of nonvascularized options exists for reconstruction of degenerative conditions of the temporomandibular joint, vascularized reconstructions such as the fibula or the second metatarsal phalangeal joint are more appropriate for defects resulting from oncologic resection or in patients with compromised soft tissue. An anatomically based classification system for these defects is presented.