CRISPR/Cas9 Editing Induces High Rates of Unintended Large Gene Modifications in HSPCs from Patients with Sickle Cell Disease

Introduction: Several gene editing strategies have been developed to cure sickle cell disease (SCD), including the use of CRISPR/Cas9 to edit beta-globin (HBB), gamma-globin (HBG), or B-cell lymphoma/leukemia 11A (BCL11A) in hematopoietic stem and progenitor cells (HSPCs) from patients with SCD. Although high gene-editing rates can be achieved and off-target effects reduced, new challenges in applying the gene-editing strategies, including unintended gene modifications, need to be addressed in order to cure SCD with high efficacy and safety. To date, due to limitations in sequencing methods, studies on CRISPR/Cas9 genome editing for treating SCD only identified small insertions/deletions (INDELs); the extent and consequences of unintended large gene modifications are generally unknown. Here we provide accurate quantification and profiling of unintended gene modifications due to Cas9 induced double-stranded breaks (DSBs) in SCD HSPCs, including large deletions, insertions, and complex chromosomal arrangements, and the comparison of different approaches. Methods: R-66S gRNA targets the sickle mutation on the HBB. R-02 gRNA generates a DSB 16 bp away from the sickle mutation site. SD-02 gRNA introduces a 13-bp Hereditary Persistence of Fetal Hemoglobin (HPFH) deletion as a major INDEL in the HBG1/HBG2 promoter to reactivate fetal hemoglobin (HbF). BCL11A gRNA targets the GATA1 site at the BCL11A erythroid enhancer to induce HbF. R-66S, R-02, SD-02, and BCL11A gRNAs were respectively complexed with SpyCas9 and delivered as ribonucleoprotein (RNP) to SCD HSPCs. To accurately quantify CRISPR/Cas9 induced large modifications in gene-edited SCD HSPCs, we used PacBio Single Molecule, Real-Time (SMRT) Sequencing with Unique Molecular Identifiers (UMI). The 5-6 kb region around the Cas9 cut-site was dual-UMI tagged using two PCR cycles. The second and third PCR was performed with minimal cycle numbers to enrich the UMI-tagged template molecules. The SMRTbell library composed of edited and unedited SCD HSPCs samples was sequenced on a PacBio Sequel II 8M flowcell using the circular consensus sequencing (CCS) mode. The PacBio subreads were converted to HiFi reads and subjected to UMI consensus read generation and variant calling. Results: SMRT-seq with UMI revealed high rates and broad spectra of unintended large deletions (> 200 bp) induced by Cas9 cutting at HBB, HBG1, and BCL11A genes in RNP treated samples, with respectively R-66S RNP, 31.7%; R-02 RNP, 17.4%; SD-02 RNP, 13.3%; BCL11A RNP, 40%. The large deletions have a very broad distribution of sizes and locations. In addition, we found large insertions (> 50 bp) and local complex chromosomal rearrangements at the Cas9 cut-sites. Therefore, the current assessment of gene-editing rates using short-read Next Generation Sequencing (NGS) misses a substantial proportion of Cas9-cutting induced large gene modifications, resulting in an inaccurate measure of both allele and genotype frequencies. Discussions: We found that unintended on-target large deletions occur at high rates at HBB, HBG1, and BCL11A in gene-edited SCD HSPCs. These results raise significant safety concerns regarding gene-editing of HSPCs to treat SCD. Our results demonstrate the importance of detecting and quantifying all possible CRISPR/Cas9 gene-editing outcomes to ensure the efficient and safe translation of gene-editing-based strategies to cure SCD and other human diseases. Additional work is required to determine the functional consequences of the unintended gene modifications and the persistence of the unintended large gene modifications at the on-target cut-sites. Disclosures Sheehan: Forma Therapeutics: Research Funding; Beam Therapeutics: Research Funding; Novartis: Research Funding.

Minimal Cycle Representatives in Persistent Homology Using Linear Programming: An Empirical Study With User’s Guide

Cycle representatives of persistent homology classes can be used to provide descriptions of topological features in data. However, the non-uniqueness of these representatives creates ambiguity and can lead to many different interpretations of the same set of classes. One approach to solving this problem is to optimize the choice of representative against some measure that is meaningful in the context of the data. In this work, we provide a study of the effectiveness and computational cost of several ℓ1 minimization optimization procedures for constructing homological cycle bases for persistent homology with rational coefficients in dimension one, including uniform-weighted and length-weighted edge-loss algorithms as well as uniform-weighted and area-weighted triangle-loss algorithms. We conduct these optimizations via standard linear programming methods, applying general-purpose solvers to optimize over column bases of simplicial boundary matrices. Our key findings are: 1) optimization is effective in reducing the size of cycle representatives, though the extent of the reduction varies according to the dimension and distribution of the underlying data, 2) the computational cost of optimizing a basis of cycle representatives exceeds the cost of computing such a basis, in most data sets we consider, 3) the choice of linear solvers matters a lot to the computation time of optimizing cycles, 4) the computation time of solving an integer program is not significantly longer than the computation time of solving a linear program for most of the cycle representatives, using the Gurobi linear solver, 5) strikingly, whether requiring integer solutions or not, we almost always obtain a solution with the same cost and almost all solutions found have entries in {‐1,0,1} and therefore, are also solutions to a restricted ℓ0 optimization problem, and 6) we obtain qualitatively different results for generators in Erdős-Rényi random clique complexes than in real-world and synthetic point cloud data.

Detecting Determinism in Time Series with Complex Networks Constructed Using a Compression Algorithm

The properties of complex networks derived from applying a compression algorithm to time series subject to symbolic ordinal-based encoding is explored. The information content of compression codewords can be used to detect forbidden symbolic patterns indicative of nonlinear determinism. The connectivity structure of ordinal-based compression networks summarized by their minimal cycle basis structure can also be used in tests for nonlinear determinism, in particular, detection of time irreversibility in a signal.

Average Reward Dynamic Programming Applied to a Persistent Visitation and Data Delivery Problem

We are interested in the persistent surveillance of an area of interest comprised of stations/ data nodes that need to be visited in a cyclic manner. The data collection task is undertaken by a UAV which autonomously executes the mission. In addition to geographically distributed stations, the scenario also includes a central depot, where data collected from the different nodes must be delivered. In this context, the performance criteria, in addition to a desired minimal cycle time, also entails minimizing the delay in delivering the data collected from each node to the depot. Each node has a priority/ weight associated with it that characterizes the relative importance between timely delivery of data from the nodes. We pose the problem as an average/ cycle reward maximization problem; where the UAV gains a reward that is a decreasing function of weighted delay in data delivery from the nodes. Since we aim to maximize the average reward, the solution also favors shorter overall cycle time. In a cycle, each station is visited exactly once; however, we allow the UAV to visit the depot more than once in a cycle. Evidently, this allows for quicker delivery of data from a higher priority node. We apply results from average reward maximization stochastic dynamic programming to our deterministic case and solve the problem using Linear Programming. We also discuss the special case of no penalty on delivery delay, whence the problem collapses to the well known metric Traveling Salesman Problem.

Deadlock Prevention with Wormhole Routing

In this chapter, the problem of constructing minimal cycle-breaking connectivity preserving sets of turns for graphs that model regular or near regular multiprocessor systems, as a method to prevent deadlocks is investigated. Cycle-breaking provides for deadlock-free wormhole routing defined by turns prohibited at some nodes. The lower and upper bounds for minimal cardinalities of cycle-breaking connectivity preserving sets for several classes of graphs such as homogeneous meshes, p-ary n-cubes, cube-connected cycles, hexagonal and honeycomb meshes and tori, Hamiltonian graphs and others are obtained and presented along with some preliminary experimental results.

Minimizing the cycle time in multiple-dwell cams

Presented in this article are closed-form kinematic equations that give the minimum cycle time for multiple-dwell cam-follower systems subject to acceleration and jerk constraints set by the user. Cam-driven machines are used extensively in manufacturing because of their low cost, great precision and high production rates when compared with alternatives. Since they are frequently used in mass production operations, there is a need for minimizing their cycle time to increase manufacturing throughput and reduce capital costs for machines and facilities. Their widespread use means that there is the potential for significant improvement in overall manufacturing efficiency. The incorporation of acceleration and jerk limits in the kinematic formulation ensures minimal cycle time without compromising the operational limits of the manufacturing machines. The equations are given in a form suitable for spreadsheet or equation-solver programs, making it easy for the cam designer to generate an arbitrary number of data points for cam manufacturing. Multiple-dwell cams are particularly well suited for manufacturing operations; therefore, the intended audience of this paper is cam designers in the manufacturing sector.

Multimode Flex-Interleaver Core for Baseband Processor Platform

This paper presents a flexible interleaver architecture supporting multiple standards like WLAN, WiMAX, HSPA+, 3GPP-LTE, and DVB. Algorithmic level optimizations like 2D transformation and realization of recursive computation are applied, which appear to be the key to reach to an efficient hardware multiplexing among different interleaver implementations. The presented hardware enables the mapping of vital types of interleavers including multiple block interleavers and convolutional interleaver onto a single architecture. By exploiting the hardware reuse methodology the silicon cost is reduced, and it consumes 0.126 mm2area in total in 65 nm CMOS process for a fully reconfigurable architecture. It can operate at a frequency of 166 MHz, providing a maximum throughput up to 664 Mbps for a multistream system and 166 Mbps for single stream communication systems, respectively. One of the vital requirements for multimode operation is the fast switching between different standards, which is supported by this hardware with minimal cycle cost overheads. Maximum flexibility and fast switchability among multiple standards during run time makes the proposed architecture a right choice for the radio baseband processing platform.