Massively Parallel Biophysical Analysis of CRISPR-Cas Complexes on Next Generation Sequencing Chips.

CRISPR-Cas nucleoproteins target foreign DNA via base pairing with a crRNA. However, a quantitative description of protein binding and nuclease activation at off-target DNA sequences remains elusive. Here, we describe a chip-hybridized association-mapping platform (CHAMP) that repurposes next-generation sequencing chips to simultaneously measure the interactions between proteins and ∼107 unique DNA sequences. Using CHAMP, we provide the first comprehensive survey of DNA recognition by a type I-E CRISPR-Cas (Cascade) complex and Cas3 nuclease. Analysis of mutated target sequences and human genomic DNA reveal that Cascade recognizes an extended protospacer adjacent motif (PAM). Cascade recognizes DNA with a surprising 3-nt periodicity. The identity of the PAM and the PAM-proximal nucleotides control Cas3 recruitment by releasing the Cse1 subunit. These findings are used to develop a model for the biophysical constraints governing off-target DNA binding. CHAMP provides a framework for high-throughput, quantitative analysis of protein-DNA interactions on synthetic and genomic DNA. PAPERCLIP.

HEDGES error-correcting code for DNA storage corrects indels and allows sequence constraints.

Synthetic DNA is rapidly emerging as a durable, high-density information storage platform. A major challenge for DNA-based information encoding strategies is the high rate of errors that arise during DNA synthesis and sequencing. Here, we describe the HEDGES (Hash Encoded, Decoded by Greedy Exhaustive Search) error-correcting code that repairs all three basic types of DNA errors: insertions, deletions, and substitutions. HEDGES also converts unresolved or compound errors into substitutions, restoring synchronization for correction via a standard Reed-Solomon outer code that is interleaved across strands. Moreover, HEDGES can incorporate a broad class of user-defined sequence constraints, such as avoiding excess repeats, or too high or too low windowed guanine-cytosine (GC) content. We test our code both via in silico simulations and with synthesized DNA. From its measured performance, we develop a statistical model applicable to much larger datasets. Predicted performance indicates the possibility of error-free recovery of petabyte- and exabyte-scale data from DNA degraded with as much as 10% errors. As the cost of DNA synthesis and sequencing continues to drop, we anticipate that HEDGES will find applications in large-scale error-free information encoding.

A metaanalysis of bat phylogenetics and positive selection based on genomes and transcriptomes from 18 species.

Historically, the evolution of bats has been analyzed using a small number of genetic loci for many species or many genetic loci for a few species. Here we present a phylogeny of 18 bat species, each of which is represented in 1,107 orthologous gene alignments used to build the tree. We generated a transcriptome sequence of Hypsignathus monstrosus, the African hammer-headed bat, and additional transcriptome sequence for Rousettus aegyptiacus, the Egyptian fruit bat. We then combined these data with existing genomic and transcriptomic data from 16 other bat species. In the analysis of such datasets, there is no clear consensus on the most reliable computational methods for the curation of quality multiple sequence alignments since these public datasets represent multiple investigators and methods, including different source materials (chromosomal DNA or expressed RNA). Here we lay out a systematic analysis of parameters and produce an advanced pipeline for curating orthologous gene alignments from combined transcriptomic and genomic data, including a software package: the Mismatching Isoform eXon Remover (MIXR). Using this method, we created alignments of 11,677 bat genes, 1,107 of which contain orthologs from all 18 species. Using the orthologous gene alignments created, we assessed bat phylogeny and also performed a holistic analysis of positive selection acting in bat genomes. We found that 181 genes have been subject to positive natural selection. This list is dominated by genes involved in immune responses and genes involved in the production of collagens.

Indel-correcting DNA barcodes for high-throughput sequencing.

Many large-scale, high-throughput experiments use DNA barcodes, short DNA sequences prepended to DNA libraries, for identification of individuals in pooled biomolecule populations. However, DNA synthesis and sequencing errors confound the correct interpretation of observed barcodes and can lead to significant data loss or spurious results. Widely used error-correcting codes borrowed from computer science (e.g., Hamming, Levenshtein codes) do not properly account for insertions and deletions (indels) in DNA barcodes, even though deletions are the most common type of synthesis error. Here, we present and experimentally validate filled/truncated right end edit (FREE) barcodes, which correct substitution, insertion, and deletion errors, even when these errors alter the barcode length. FREE barcodes are designed with experimental considerations in mind, including balanced guanine-cytosine (GC) content, minimal homopolymer runs, and reduced internal hairpin propensity. We generate and include lists of barcodes with different lengths and error correction levels that may be useful in diverse high-throughput applications, including >106 single-error-correcting 16-mers that strike a balance between decoding accuracy, barcode length, and library size. Moreover, concatenating two or more FREE codes into a single barcode increases the available barcode space combinatorially, generating lists with >1015 error-correcting barcodes. The included software for creating barcode libraries and decoding sequenced barcodes is efficient and designed to be user-friendly for the general biology community.

A kinetic model predicts SpCas9 activity, improves off-target classification, and reveals the physical basis of targeting fidelity.

The S. pyogenes (Sp) Cas9 endonuclease is an important gene-editing tool. SpCas9 is directed to target sites based on complementarity to a complexed single-guide RNA (sgRNA). However, SpCas9-sgRNA also binds and cleaves genomic off-targets with only partial complementarity. To date, we lack the ability to predict cleavage and binding activity quantitatively, and rely on binary classification schemes to identify strong off-targets. We report a quantitative kinetic model that captures the SpCas9-mediated strand-replacement reaction in free-energy terms. The model predicts binding and cleavage activity as a function of time, target, and experimental conditions. Trained and validated on high-throughput bulk-biochemical data, our model predicts the intermediate R-loop state recently observed in single-molecule experiments, as well as the associated conversion rates. Finally, we show that our quantitative activity predictor can be reduced to a binary off-target classifier that outperforms the established state-of-the-art. Our approach is extensible, and can characterize any CRISPR-Cas nuclease - benchmarking natural and future high-fidelity variants against SpCas9; elucidating determinants of CRISPR fidelity; and revealing pathways to increased specificity and efficiency in engineered systems.

Massively Parallel Selection of NanoCluster Beacons.

NanoCluster Beacons (NCBs) are multicolor silver nanocluster probes whose fluorescence can be activated or tuned by a proximal DNA strand called the activator. While a single-nucleotide difference in a pair of activators can lead to drastically different activation outcomes, termed polar opposite twins (POTs), it is difficult to discover new POT-NCBs using the conventional low-throughput characterization approaches. Here, a high-throughput selection method is reported that takes advantage of repurposed next-generation-sequencing chips to screen the activation fluorescence of ≈40 000 activator sequences. It is found that the nucleobases at positions 7-12 of the 18-nucleotide-long activator are critical to creating bright NCBs and positions 4-6 and 2-4 are hotspots to generate yellow-orange and red POTs, respectively. Based on these findings, a "zipper-bag" model is proposed that can explain how these hotspots facilitate the formation of distinct silver cluster chromophores and alter their chemical yields. Combining high-throughput screening with machine-learning algorithms, a pipeline is established to design bright and multicolor NCBs in silico.

Massively parallel kinetic profiling of natural and engineered CRISPR nucleases.

Engineered SpCas9s and AsCas12a cleave fewer off-target genomic sites than wild-type (wt) Cas9. However, understanding their fidelity, mechanisms and cleavage outcomes requires systematic profiling across mispaired target DNAs. Here we describe NucleaSeq-nuclease digestion and deep sequencing-a massively parallel platform that measures the cleavage kinetics and time-resolved cleavage products for over 10,000 targets containing mismatches, insertions and deletions relative to the guide RNA. Combining cleavage rates and binding specificities on the same target libraries, we benchmarked five SpCas9 variants and AsCas12a. A biophysical model built from these data sets revealed mechanistic insights into off-target cleavage. Engineered Cas9s, especially Cas9-HF1, dramatically increased cleavage specificity but not binding specificity compared to wtCas9. Surprisingly, AsCas12a cleavage specificity differed little from that of wtCas9. Initial DNA cleavage sites and end trimming varied by nuclease, guide RNA and the positions of mispaired nucleotides. More broadly, NucleaSeq enables rapid, quantitative and systematic comparisons of specificity and cleavage outcomes across engineered and natural nucleases.

Towards the development of a pediatric ventricular assist device.

The very limited options available to treat ventricular failure in children with congenital and acquired heart diseases have motivated the development of a pediatric ventricular assist device at the University of Pittsburgh (UoP) and University of Pittsburgh Medical Center (UPMC). Our effort involves a consortium consisting of UoP, Children's Hospital of Pittsburgh (CHP), Carnegie Mellon University, World Heart Corporation, and LaunchPoint Technologies, Inc. The overall aim of our program is to develop a highly reliable, biocompatible ventricular assist device (VAD) for chronic support (6 months) of the unique and high-risk population of children between 3 and 15 kg (patients from birth to 2 years of age). The innovative pediatric ventricular assist device we are developing is based on a miniature mixed flow turbodynamic pump featuring magnetic levitation, to assure minimal blood trauma and risk of thrombosis. This review article discusses the limitations of current pediatric cardiac assist treatment options and the work to date by our consortium toward the development of a pediatric VAD.

Single ventricle palliation: greater risk of complications with the Fontan procedure than with the bidirectional Glenn procedure alone.

BACKGROUND: This study was performed to evaluate and compare the early, intermediate, and long-term outcomes of the bidirectional Glenn procedure and Fontan procedure in patients who live at moderately high altitude. METHODS: The outcome of each method of palliation for patients with a functionally single ventricle was retrospectively evaluated from a review of medical records. RESULTS: The bidirectional Glenn procedure was performed in 177 patients from October 1984 to June 2004. The Fontan procedure was performed in 149 patients from June 1978 to June 2004. Cardiovascular death or heart transplantation occurred in 8% of patients after the bidirectional Glenn procedure and 17% of patients after the Fontan procedure. Complications of systemic thromboembolic events, bleeding associated with anticoagulation therapy, protein losing enteropathy, and arrhythmias requiring implantation of a pacemaker, cardioversion, or radiofrequency ablation occurred in 7% of patients after the bidirectional Glenn procedure and 47% of patients after the Fontan procedure. Cardiovascular deaths and heart transplantation occurred less frequently when the Fontan procedure was performed in patients with a previous bidirectional Glenn procedure. However, the actuarial transplant-free survival and freedom from complications was not superior for a subgroup of patients who had a Fontan procedure after a bidirectional Glenn procedure in comparison to a subgroup of patients who had a bidirectional Glenn procedure alone. CONCLUSIONS: The bidirectional Glenn procedure can be used for long-term palliation of patients with a functionally single ventricle. Additional palliation with a Fontan procedure may increase the risk of stroke, protein losing enteropathy and arrhythmias without improving survival.

The PediaFlow pediatric ventricular assist device.

The very limited options available to treat ventricular failure in patients with congenital and acquired heart diseases have motivated the development of a pediatric ventricular assist device (VAD). Our effort involves a consortium consisting of the University of Pittsburgh, Carnegie Mellon University, Children's Hospital of Pittsburgh, World Heart Corporation, and LaunchPoint Technologies, LLC. The overall aim of our program is to develop a highly reliable, biocompatible VAD for chronic support (6 months) of the unique and high-risk population of children between 3 kg and 15 kg (patients from birth to 2 years of age). The innovative pediatric VAD we are developing (PediaFlow) is based on a miniature mixed-flow turbodynamic pump featuring magnetic levitation, with the design goal being to assure minimal blood trauma and risk of thrombosis. This article discusses the limitations of current pediatric cardiac assist treatment options and the work to date by our consortium toward the development of a pediatric VAD.

Follow-up of aortic coarctation repair in neonates.

OBJECTIVES: The purposes of this study were to assess the growth of left heart (LH) structures, to evaluate midterm outcomes, and to define echocardiographic parameters predictive of increased risk of re-intervention in patients born with aortic coarctation and hypoplasia of LH structures that underwent neonatal coarctation repair. BACKGROUND: Neonatal coarctation is often associated with hypoplasia of LH structures. Although previous studies have shown that coarctation repair can be performed with good results in these neonates, there are little data regarding growth of the LH structures or outcomes in these patients. METHODS: Patients with isolated coarctation and at least one hypoplastic LH valve (mitral or aortic Z-score <-2) who underwent a neonatal coarctation repair were identified. Clinic charts and the latest echocardiograms were reviewed. RESULTS: All 55 patients were alive and well, and no patient had clinical evidence of mitral stenosis. Three patients (5%) required re-intervention. Thirty-eight patients had echocardiograms that demonstrated normal left ventricular (LV) size and function with a follow-up duration of 73 +/- 19 months (range 3 to 9 years). Both mitral and aortic annulus Z-scores increased significantly: -3.1 +/- 1.5 to -0.5 +/- 1.6 (p < 0.001) and -3.5 +/- 1.9 to 0.7 +/- 1.6 (p < 0.001), respectively. Nine patients (24%) developed LV outflow tract obstruction by echocardiographic criteria. CONCLUSIONS: After neonatal coarctation repair with associated LH hypoplasia, LH structures increase substantially in size, and clinical outcomes are excellent at midterm follow-up. Despite initial annular hypoplasia, the need for intervention for mitral or aortic/subaortic stenosis is uncommon.

Two-year reduction of panel reactive human leukocyte antigen antibodies in children receiving mycophenolate mofetil after valved allograft placement.

Allografts used in the repair of congenital heart defects in children induce a persistent broad HLA antibody response. We have previously shown that a 3-month course of mycophenolic mofetil (MMF) significantly reduces the HLA class I antibody response to valved allograft implantation in children. The purpose of this study was to determine if this reduction in HLA antibody persists after discontinuation of MMF. We conducted follow-up (mean 2 +/- 0.5 years) of seven patients who had received allograft placement for repair of congenital heart defects. These patients received 3 months of immunosuppression with MMF following allograft implantation. When compared to historical controls, patients who received MMF following surgery showed a significantly decreased HLA class I antibody response at 2 years postimplantation. This study demonstrates the ability to persistently alter the HLA class I antibody response using 3 months of MMF following allograft implantation in children.

Filovirus receptor NPC1 contributes to species-specific patterns of ebolavirus susceptibility in bats.

Biological factors that influence the host range and spillover of Ebola virus (EBOV) and other filoviruses remain enigmatic. While filoviruses infect diverse mammalian cell lines, we report that cells from African straw-colored fruit bats (Eidolon helvum) are refractory to EBOV infection. This could be explained by a single amino acid change in the filovirus receptor, NPC1, which greatly reduces the affinity of EBOV-NPC1 interaction. We found signatures of positive selection in bat NPC1 concentrated at the virus-receptor interface, with the strongest signal at the same residue that controls EBOV infection in Eidolon helvum cells. Our work identifies NPC1 as a genetic determinant of filovirus susceptibility in bats, and suggests that some NPC1 variations reflect host adaptations to reduce filovirus replication and virulence. A single viral mutation afforded escape from receptor control, revealing a pathway for compensatory viral evolution and a potential avenue for expansion of filovirus host range in nature.

Immunogenicity of decellularized cryopreserved allografts in pediatric cardiac surgery: comparison with standard cryopreserved allografts.

BACKGROUND: Recognition of the immunogenicity of standard cryopreserved allografts has led to the development of new decellularized allografts (CryoValve SG; CryoLife, Inc, Kennesaw, Ga). This preliminary study examined the HLA antibody response to these decellularized allografts and compared it with the response to standard allograft material. METHODS: We prospectively measured the frequency of panel-reactive HLA class I (HLA-A, HLA-B, and HLA-C) and class II (HLA-DR/DQ) alloantibodies in 14 children (age 8.5 +/- 7.9 years) receiving decellularized, cryopreserved allografts, including 6 undergoing allograft patch insertion and 8 with a valved pulmonary allograft. We compared them with 20 historical control subjects (age 1.7 +/- 2.4 years) undergoing implantation of standard cryopreserved allografts, 8 with valves and 12 with allograft patch. All patients had panel-reactive antibody levels measured before and at 1, 3, and 12 months after the operation. HLA class I and class II panel-reactive antibody levels were determined with a sensitive flow cytometry technique. RESULTS: We found panel-reactive antibody levels in decellularized allografts to be elevated slightly from preoperative levels for both class I and class II antibodies at 1, 3, and 12 months (P >.05). The panel-reactive antibody level for both class I and class II antibodies were significantly lower for decellularized allografts as compared to standard allografts. Functionally, the allografts were similar with decellularized valved grafts showing a peak echo-determined systolic gradient of 13 +/- 15 mm Hg at 8 +/- 2.6 months postoperatively as compared to a gradient of 24 +/- 18 mm Hg measured 12 +/- 6 months postoperatively in standard allografts (P =.11). CONCLUSIONS: Decellularized grafts elicited significantly lower levels of class I and class II HLA antibody formation at 1, 3, and 12 months after implantation than did standard cryopreserved allografts. Early hemodynamic function of decellularized grafts was similar to that of standard cryopreserved allograft valves. Further experience is necessary to determine whether the reduced immunogenicity of decellularized allografts will truly allow tissue ingrowth and improved long-term durability in patients.

Immunology and failure of valved allografts in children.

Valved allografts are frequently used in the repair of congenital heart defects in children. Although the longevity of these grafts is generally very good, there continue to be ongoing problems with allograft stenosis, allograft valve insufficiency, and subsequent allograft failure, particularly in younger children. This review presents data on the immunologic and nonimmunologic risk factors implicated in valved allograft failure, in addition to ongoing investigation into the improvement of allograft function.

Mycophenolic mofetil reduces the HLA antibody response of children to valved allograft implantation.

BACKGROUND: Valved allografts induce a brisk, broadly reactive human leukocyte antigen (HLA) antibody response in children after implantation. Mycophenolic mofetil (MMF) is a powerful immunosuppressant that inhibits the proliferation of both T cells and B cells and has been reported to possibly reduce HLA panel reactive antibody (PRA) in sensitized transplant recipients. METHODS: The purpose of this study was to determine whether MMF can blunt the HLA antibody response to valved allografts in children. Eight patients completed (of 28 approached) a pilot study to determine the effects of 3 months of twice daily MMF (600 mg/m(2)/dose) on the HLA antibody response measured before surgery, at 1 month, and at 3 months after implantation. Patients were 7.5 +/- 4 yrs old (mean +/- standard deviation [SD]), with 5 patients undergoing repair of tetralogy of Fallot, 2 Ross procedures, and 1 aortic valve replacement. RESULTS: In contrast to historical controls with a virtual 100% HLA class I PRA response to valved allograft implantation, MMF markedly decreased the HLA class I antibody response at 1 and 3 months postimplantation. In 6 cases where the HLA type of the donor was defined, PRA specificity correlated with incompatible antigens on the allograft. One patient withdrew after 2 weeks due to a sinus infection that was successfully treated with oral antibiotics, and 3 patients had a transient adverse effect of postoperative vomiting. CONCLUSIONS: This study demonstrates the ability to pharmacologically abrogate the HLA class I antibody response to valved allograft implantation in children using MMF.

Current results with intraaortic balloon pumping in infants and children.

BACKGROUND: Intraaortic balloon pumping (IABP) is useful for support in patients with moderate left ventricular dysfunction. IABP is usually timed with the R wave of the electrocardiogram. We have utilized M-mode echocardiography timed IABP in children with left-side heart failure since 1994. Electrocardiogram timing seems inappropriate for children, who have much higher heart rates. We describe our experience with children who underwent IABP therapy before and after 1994, when echocardiographic timing was instituted. METHODS: We reviewed records of 29 children who underwent IABP for all indications at Primary Children's Medical Center since 1988. RESULTS: Overall survival was 62.1% (18 of 29) in this series. Survival was similar for infants (odds ratio = 2.0, 95% confidence interval = 0.29 to 14.31, p = 0.43) and older children. Survival was similar in the echocardiography era when compared with the electrocardiogram era (odds ratio = 2.4, 95% confidence interval = 0.56 to 10.4, p = 0.44). CONCLUSIONS: IABP is a useful means of support in children with left ventricular dysfunction. M-mode echocardiography is effective in triggering IABP. The sample size in this study is too small to detect a mortality rate difference.

Current treatment of pediatric empyema.

Pneumonia with complicated parapneumonic effusion and empyema is increasing in incidence and continues to be a source of morbidity in children seen in our institution. Current diagnostic modalities include chest radiographs and CT scanning with ultrasound being helpful in some situations. Exact management of empyema remains controversial. Although open thoracotomy drainage is well accepted in children, video-assisted thoracoscopic surgery (VATS) drainage has become more prevalent in the current era. Over the last 4 years, we have treated 58 children with intrapleural placement of pigtail catheters and administration fibrinolytics consisting of tissue plasminogen activator (tPA). Successful drainage and resolution of 54 of the 58 effusions was achieved with percutaneous methods alone. There was no mortality or 30-day recurrence. Mean hospital stay was 9.1 days (range 5 to 21) and mean chest catheter removal was 6 days post placement (range 1.5 to 20). Of the four patients that failed percutaneous tube therapy, 3 underwent video assisted thoracic surgery (VATS), and one had open thoracotomy with decortication. Based on our experience, tPA administered through a small bore chest tube for drainage of complicated parapneumonic effusions has become our standard practice. We reserve VATS for treatment failures and open thoracotomy and decortication for patients with VATS failure.

Regional myocardial dysfunction following Norwood with right ventricle to pulmonary artery conduit in patients with hypoplastic left heart syndrome.

BACKGROUND: Improved early survival has led many centers to use the right ventricle-to-pulmonary artery (RVPA) conduit instead of the modified Blalock-Taussig shunt for Norwood palliation of hypoplastic left-heart syndrome. However, there is concern regarding the potential deleterious effects of the required right ventriculotomy for placement of the RVPA conduit on global and regional right ventricular (RV) function. The purpose of this study was to investigate global and regional RV wall motion abnormalities after Norwood palliation with RVPA conduit using Velocity Vector Imaging (VVI). METHODS: Thirty consecutive patients with hypoplastic left-heart syndrome who underwent stage 2 palliation between January 2007 and December 2009 were identified from the surgical database. VVI was performed on two-dimensional echocardiographic images obtained before second-stage palliation. Peak systolic circumferential and radial velocity, strain, and strain rate were measured from parasternal short-axis and apical four-chamber views. RV ejection fraction was measured using the biplane modified Simpson's rule. Regional RV systolic deformations were compared between different RV segments. VVI measures were also compared with RV systolic function. In a subgroup (n = 14), VVI was repeated on follow-up after stage 2 palliation to evaluate changes in regional and global RV deformation. RESULTS: A total of 30 patients (20 males) were studied. The median age at the time of interstage echocardiography was 12 weeks (range, 8-18 weeks). In the short axis, average peak systolic circumferential strain values for the anterior, posterior, septal, and RV free wall segments were 3.79 ± 2.52%, 11.4 ± 5.2%, 13.3 ± 6.5%, and 11.1 ± 5.0%, respectively. From the short-axis view, the anterior RV segment (ventriculotomy site) exhibited significantly reduced circumferential velocity, peak systolic strain, and strain rate (P < .0001). Mean global VVI measurements were correlated with RV ejection fraction. On follow-up after stage 2 palliation, the ventriculotomy region showed persistently reduced velocity, peak systolic strain, and strain rate compared with all other segments. CONCLUSIONS: In patients with hypoplastic left-heart syndrome after Norwood palliation with RVPA conduit, RV myocardial deformation was significantly reduced at the ventriculotomy site, which persisted after stage 2 palliation. VVI-derived measures demonstrating impairment of global systolic myocardial deformation were correlated with RV systolic function. Long-term multicenter studies to evaluate the effects of ventriculotomy scar on single systemic right ventricle are required.