CRISPR Advancements for Human Health.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) has emerged as a powerful gene editing technology that is revolutionizing biomedical research and clinical medicine. The CRISPR system allows scientists to rewrite the genetic code in virtually any organism. This review provides a comprehensive overview of CRISPR and its clinical applications. We first introduce the CRISPR system and explain how it works as a gene editing tool. We then highlight current and potential clinical uses of CRISPR in areas such as genetic disorders, infectious diseases, cancer, and regenerative medicine. Challenges that need to be addressed for the successful translation of CRISPR to the clinic are also discussed. Overall, CRISPR holds great promise to advance precision medicine, but ongoing research is still required to optimize delivery, efficacy, and safety.

Comparison of Thermal and Mechanical Pain Testing Modalities in Sprague Dawley and Fischer 344 Rats (Rattus norvegicus).

While rodents are used extensively for studying pain, there is a lack of reported direct comparisons of thermal and mechanical pain testing methods in rats of different genetic backgrounds. Understanding the range of interindividual variability of withdrawal thresholds and thermal latencies based on these testing methods and/or genetic background is important for appropriate experimental design. Testing was performed in two common rat genetic backgrounds: outbred Sprague-Dawley (SD) and inbred Fischer 344 (F344). Male and female, 10- to 14-wk-old F344 and SD rats were used to assess withdrawal thresholds in 3 different modalities: the Randall-Selitto test (RST), Hargreaves test (HT), and tail flick test (TFT). The RST was performed by using an operator-controlled handheld instrument to generate a noxious pressure stimulus to the left hind paw. The HT and the TFT used an electronically controlled light source to deliver a noxious thermal stimulus to the left hind paw or tail tip, respectively. Rats of each sex and genetic background underwent one type of test on day 0 and day 7. Withdrawal thresholds and thermal latencies were compared among tests. No significant differences were observed. Our findings can serve as a guide for researchers considering these nociceptive tests for their experiments.

Increasing midtarsal joint stiffness reduces triceps surae metabolic costs in walking simulations but has little effect on total stance limb metabolic cost.

The human foot's arch is thought to be beneficial for efficient gait. This study addresses the extent to which arch stiffness changes alter the metabolic energy requirements of human gait. Computational musculoskeletal simulations of steady state walking using direct collocation were performed. Across a range of foot arch stiffnesses, the metabolic cost of transport decreased by less than 1% with increasing foot arch stiffness. Increasing arch stiffness increased the metabolic efficiency of the triceps surae during push-off, but these changes were almost entirely offset by other muscle groups consuming more energy with increasing foot arch stiffness.

Behavioral characterization of G-protein-coupled receptor 160 knockout mice.

ABSTRACT: Neuropathic pain is a devastating condition where current therapeutics offer little to no pain relief. Novel nonnarcotic therapeutic targets are needed to address this growing medical problem. Our work identified the G-protein-coupled receptor 160 (GPR160) as a potential target for therapeutic intervention. However, the lack of small-molecule ligands for GPR160 hampers our understanding of its role in health and disease. To address this void, we generated a global Gpr160 knockout (KO) mouse using CRISPR-Cas9 genome editing technology to validate the contributions of GPR160 in nociceptive behaviors in mice. Gpr160 KO mice are healthy and fertile, with no observable physical abnormalities. Gpr160 KO mice fail to develop behavioral hypersensitivities in a model of neuropathic pain caused by constriction of the sciatic nerve. On the other hand, responses of Gpr160 KO mice in the hot-plate and tail-flick assays are not affected. We recently deorphanized GPR160 and identified cocaine- and amphetamine-regulated transcript peptide (CARTp) as a potential ligand. Using Gpr160 KO mice, we now report that the development of behavioral hypersensitivities after intrathecal or intraplantar injections of CARTp are dependent on GPR160. Cocaine- and amphetamine-regulated transcript peptide plays a role in various affective behaviors, such as anxiety, depression, and cognition. There are no differences in learning, memory, and anxiety between Gpr160 KO mice and their age-matched and sex-matched control floxed mice. Results from these studies support the pronociceptive roles of CARTp/GPR160 and GPR160 as a potential therapeutic target for treatment of neuropathic pain.

Ablation of placental REST deregulates fetal brain metabolism and impacts gene expression of the offspring brain at the postnatal and adult stages.

In this study, the transcriptional repressor REST (Repressor Element 1 Silencing Transcription factor) was ablated in the mouse placenta to investigate molecular and cellular impacts on the offspring brain at different life stages. Ablation of placental REST deregulated several brain metabolites, including glucose and lactate that fuel brain energy, vitamin C (ascorbic acid) that functions in the epigenetic programming of the brain during postnatal development, and glutamate and creatine that help the brain to respond to stress conditions during adult life. Bulk RNA-seq analysis showed that a lack of placental REST persistently altered multiple transport genes, including those related to oxygen transportation in the offspring brain. While metabolic genes were impacted in the postnatal brain, different stress response genes were activated in the adult brain. DNA methylation was also impacted in the adult brain due to the loss of placental REST, but in a sex-biased manner. Single-nuclei RNA-seq analysis showed that specific cell types of the brain, particularly those of the choroid plexus and ependyma, which play critical roles in producing cerebrospinal fluid and maintaining metabolic homeostasis, were significantly impacted due to the loss of placental REST. These cells showed significant differential expression of genes associated with the metabotropic (G coupled protein) and ionotropic (ligand-gated ion channel) glutamate receptors, suggesting an impact of ablation of placental REST on the glutamatergic signaling of the offspring brain. The study expands our understanding of placental influences on the offspring brain.

Efficient DNA knock-in using AAV-mediated delivery with 2-cell embryo CRISPR-Cas9 electroporation.

Recent advances in CRISPR-Cas genome editing technology have been instrumental in improving the efficiency to produce genetically modified animal models. In this study we have combined four very promising approaches to come up with a highly effective pipeline to produce knock-in mouse and rat models. The four combined methods include: AAV-mediated DNA delivery, single-stranded DNA donor templates, 2-cell embryo modification, and CRISPR-Cas ribonucleoprotein (RNP) electroporation. Using this new combined approach, we were able to produce successfully targeted knock-in rat models containing either Cre or Flp recombinase sequences with knock-in efficiencies over 90%. Furthermore, we were able to produce a knock-in mouse model containing a Cre recombinase targeted insertion with over 50% knock-in efficiency directly comparing efficiencies to other commonly used approaches. Our modified AAV-mediated DNA delivery with 2-cell embryo CRISPR-Cas9 RNP electroporation technique has proven to be highly effective for generating both knock-in mouse and knock-in rat models.

Electroporation-Mediated CRISPR/Cas9 Genome Editing in Rat Zygotes.

Genetic engineering in the rat has been revolutionized by the development of CRISPR-based genome editing tools. Conventional methods for inserting genome editing elements such as CRISPR/Cas9 reagents into rat zygotes include cytoplasmic or pronuclear microinjections. These techniques are labor-intensive, require specialized micromanipulator equipment, and are technically challenging. Here, we describe a simple and effective method for zygote electroporation in which CRISPR/Cas9 reagents are introduced into rat zygotes via pores produced by precise electrical pulses applied to the cells. Zygote electroporation allows for high-throughput efficient genome editing in rat embryos.

Gene Targeting in Rat Embryonic Stem Cells.

Rat germline-competent embryonic stem (ES) cell lines have been available since 2008, and rat models with targeted mutations have been successfully generated using ES cell-based genome targeting technology. This chapter will focus on the procedures of gene targeting in rat ES cells.

Characterizing the mechanical function of the foot's arch across steady-state gait modes.

The arch of the human foot has historically been likened to either a truss, a rigid lever, or a spring. Growing evidence indicates that energy is stored, generated, and dissipated actively by structures crossing the arch, suggesting that the arch can further function in a motor- or spring-like manner. In the present study, participants walked, ran with a rearfoot strike pattern, and ran with a non-rearfoot strike pattern overground while foot segment motions and ground reaction forces were recorded. To quantify the midtarsal joint's (i.e., arch's) mechanical behavior, a brake-spring-motor index was defined as the ratio between midtarsal joint net work and the total magnitude of joint work. This index was statistically significantly different between each gait condition. Index values decreased from walking to rearfoot strike running to non-rearfoot strike running, indicating that the midtarsal joint was most motor-like when walking and most spring-like in non-rearfoot running. The mean magnitude of elastic strain energy stored in the plantar aponeurosis mirrored the increase in spring-like arch function from walking to non-rearfoot strike running. However, the behavior of the plantar aponeurosis could not account for a more motor-like arch in walking and rearfoot strike running, given the lack of main effect of gait condition on the ratio between net work and total work performed by force in the plantar aponeurosis about the midtarsal joint. Instead, the muscles of the foot are likely altering the motor-like mechanical function of the foot's arch, the operation of these muscles between gait conditions warrants further investigation.

Foot arch rigidity in walking: In vivo evidence for the contribution of metatarsophalangeal joint dorsiflexion.

Human foot rigidity is thought to provide a more effective lever with which to push against the ground. Tension of the plantar aponeurosis (PA) with increased metatarsophalangeal (MTP) joint dorsiflexion (i.e., the windlass mechanism) has been credited with providing some of this rigidity. However, there is growing debate on whether MTP joint dorsiflexion indeed increases arch rigidity. Further, the arch can be made more rigid independent of additional MTP joint dorsiflexion (e.g., when walking with added mass). The purpose of the present study was therefore to compare the influence of increased MTP joint dorsiflexion with the influence of added mass on the quasi-stiffness of the midtarsal joint in walking. Participants walked with a rounded wedge under their toes to increase MTP joint dorsiflexion in the toe-wedge condition, and wore a weighted vest with 15% of their body mass in the added mass condition. Plantar aponeurosis behavior, foot joint energetics, and midtarsal joint quasi-stiffness were compared between conditions to analyze the mechanisms and effects of arch rigidity differences. Midtarsal joint quasi-stiffness was increased in the toe-wedge and added mass conditions compared with the control condition (both p < 0.001). In the toe-wedge condition, the time-series profiles of MTP joint dorsiflexion and PA strain and force were increased throughout mid-stance (p < 0.001). When walking with added mass, the time-series profile of force in the PA did not increase compared with the control condition although quasi-stiffness did, supporting previous evidence that the rigidity of the foot can be actively modulated. Finally, more mechanical power was absorbed (p = 0.006) and negative work was performed (p < 0.001) by structures distal to the rearfoot in the toe-wedge condition, a condition which displayed increased midtarsal joint quasi-stiffness. This indicates that a more rigid foot may not necessarily transfer power to the ground more efficiently.

Prss29 Cre recombinase mice are useful to study adult uterine gland function.

All mammalian uteri contain glands in their endometrium that develop only or primarily after birth. In mice, those endometrial glands govern post implantation pregnancy establishment via regulation of blastocyst implantation, stromal cell decidualization, and placental development. Here, we describe a new uterine glandular epithelium (GE) specific Cre recombinase mouse line that is useful for the study of uterine gland function during pregnancy. Utilizing CRISPR-Cas9 genome editing, Cre recombinase was inserted into the endogenous serine protease 29 precursor (Prss29) gene. Both Prss29 mRNA and Cre recombinase activity was specific to the GE of the mouse uterus following implantation, but was absent from other areas of the female reproductive tract. Next, Prss29-Cre mice were crossed with floxed forkhead box A2 (Foxa2) mice to conditionally delete Foxa2 specifically in the endometrial glands. Foxa2 was absent in the glands of the post-implantation uterus, and Foxa2 deleted mice exhibited complete infertility after their first pregnancy. These results establish that Prss29-Cre mice are a valuable resource to elucidate and explore the functions of glands in the adult uterus.

Optimal Load Magnitude and Placement for Peak Power Production in a Vertical Jump: A Segmental Contribution Analysis.

ABSTRACT: Bordelon, NM, Jones, DH, Sweeney, KM, Davis, DJ, Critchley, ML, Rochelle, LE, George, AC, and Dai, B. Optimal load magnitude and placement for peak power production in a vertical jump: A segmental contribution analysis. J Strength Cond Res 36(4): 911-919, 2022-Weighted jumps are widely used in power training, however, there are discrepancies regarding which loading optimizes peak jump power. The purpose was to quantify the effects of load magnitudes and placements on the force, velocity, and power production in a countermovement vertical jump. Sixteen male and 15 female subjects performed vertical jumps in 7 conditions: no external load, 10 and 20% dumbbell loads, 10 and 20% vest loads, and 10 and 20% barbell loads with load percentages relative to body weight. Arm swing was encouraged for all, but the barbell load conditions. Kinematics were collected to quantify the whole-body (the person and external loads) forces, velocities, and power as well as segments' contributions to the whole-body forces and velocities. Repeated-measure analyses of variance were performed followed by paired comparisons. Jump heights were the greatest for the no external load and 10% dumbbell conditions. The 10 and 20% dumbbell conditions demonstrated the greatest peak whole-body power, while the 2 barbell conditions showed the lowest peak whole-body power. At the time of peak whole-body power, the 2 dumbbell and 2 vest conditions resulted in greater whole-body forces. Whole-body velocities were the greatest for the no external load and 10% dumbbell conditions. Holding the dumbbells in the hands magnified the effects of external loads in producing forces and velocities. The constraint of arm movements in the barbell conditions limited power production. These findings highlight the importance of load placement and arm swing in identifying the optimal configuration for power production in weighted jumps.

The Ighmbp2D564N mouse model is the first SMARD1 model to demonstrate respiratory defects.

Spinal muscular atrophy with respiratory distress type I (SMARD1) is a neurodegenerative disease defined by respiratory distress, muscle atrophy and sensory and autonomic nervous system defects. SMARD1 is a result of mutations within the IGHMBP2 gene. We have generated six Ighmbp2 mouse models based on patient-derived mutations that result in SMARD1 and/or Charcot-Marie Tooth Type 2 (CMT2S). Here we describe the characterization of one of these models, Ighmbp2D564N (human D565N). The Ighmbp2D564N/D564N mouse model mimics important aspects of the SMARD1 disease phenotype, including motor neuron degeneration and muscle atrophy. Ighmbp2D564N/D564N is the first SMARD1 mouse model to demonstrate respiratory defects based on quantified plethysmography analyses. SMARD1 disease phenotypes, including the respiratory defects, are significantly diminished by intracerebroventricular (ICV) injection of ssAAV9-IGHMBP2 and the extent of phenotypic restoration is dose-dependent. Collectively, this model provides important biological insight into SMARD1 disease development.

Molecular and culture-based assessment of the microbiome in a zebrafish (Danio rerio) housing system during set-up and equilibration.

BACKGROUND: Zebrafish used in research settings are often housed in recirculating aquaculture systems (RAS) which rely on the system microbiome, typically enriched in a biofiltration substrate, to remove the harmful ammonia generated by fish via oxidation. Commercial RAS must be allowed to equilibrate following installation, before fish can be introduced. There is little information available regarding the bacterial community structure in commercial zebrafish housing systems, or the time-point at which the system or biofilter reaches a microbiological equilibrium in RAS in general. METHODS: A zebrafish housing system was monitored at multiple different system sites including tank water in six different tanks, pre- and post-particulate filter water, the fluidized bed biofilter substrate, post-carbon filter water, and water leaving the ultra-violet (UV) disinfection unit and entering the tanks. All of these samples were collected in quadruplicate, from prior to population of the system with zebrafish through 18 weeks post-population, and analyzed using both 16S rRNA amplicon sequencing and culture using multiple agars and annotation of isolates via matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry. Sequencing data were analyzed using traditional methods, network analyses of longitudinal data, and integration of culture and sequence data. RESULTS: The water microbiome, dominated by Cutibacterium and Staphylococcus spp., reached a relatively stable richness and composition by approximately three to four weeks post-population, but continued to evolve in composition throughout the study duration. The microbiomes of the fluidized bed biofilter and water leaving the UV disinfection unit were distinct from water at all other sites. Core taxa detected using molecular methods comprised 36 amplicon sequence variants, 15 of which represented Proteobacteria including multiple members of the families Burkholderiaceae and Sphingomonadaceae. Culture-based screening yielded 36 distinct isolates, and showed moderate agreement with sequencing data. CONCLUSIONS: The microbiome of commercial RAS used for research zebrafish reaches a relatively stable state by four weeks post-population and would be expected to be suitable for experimental use following that time-point.

Mutational analyses of novel rat models with targeted modifications in inflammatory bowel disease susceptibility genes.

Mutations and single base pair polymorphisms in various genes have been associated with increased susceptibility to inflammatory bowel disease (IBD). We have created a series of rat strains carrying targeted genetic alterations within three IBD susceptibility genes: Nod2, Atg16l1, and Il23r, using CRISPR/Cas9 genome editing technology. Knock-out alleles and alleles with known human susceptibility polymorphisms were generated on three different genetic backgrounds: Fischer, Lewis and Sprague Dawley. The availability of these rat models will contribute to our understanding of the basic biological roles of these three genes as well as provide new potential IBD animal models.

Vertical Ground Reaction Force Estimation From Benchmark Nonstationary Kinematic Data.

Time-differentiating kinematic signals from optical motion capture amplifies the inherent noise content of those signals. Commonly, biomechanists address this problem by applying a Butterworth filter with the same cutoff frequency to all noisy displacement signals prior to differentiation. Nonstationary signals, those with time-varying frequency content, are widespread in biomechanics (eg, those containing an impact) and may necessitate a different filtering approach. A recently introduced signal filtering approach wherein signals are divided into sections based on their energy content and then Butterworth filtered with section-specific cutoff frequencies improved second derivative estimates in a nonstationary kinematic signal. Utilizing this signal-section filtering approach for estimating running vertical ground reaction forces saw more of the signal's high-frequency content surrounding heel strike maintained without allowing inappropriate amounts of noise contamination in the remainder of the signal. Thus, this signal-section filtering approach resulted in superior estimates of vertical ground reaction forces compared with approaches that either used the same filter cutoff frequency across the entirety of each signal or across the entirety of all signals. Filtering kinematic signals using this signal-section filtering approach is useful in processing data from tasks containing an impact when accurate signal second derivative estimation is of interest.

Automatic segment filtering procedure for processing non-stationary signals.

Computing time derivatives is a frequent stage in the processing of biomechanical data. Unfortunately, differentiation amplifies the high frequency noise inherent within the signal hampering the accuracy of signal derivatives. A low-pass Butterworth filter is commonly used to reduce the sampled signal noise prior to differentiation. One hurdle lies in selecting an appropriate filter cut-off frequency which retains the signal of interest while reducing deleterious noise. Most biomechanics data processing approaches utilize the same cut-off frequency for the whole sampled signal, but the frequency components of a signal can vary with time. To accommodate such signals, the Automatic Segment Filtering Procedure (ASFP) is proposed which uses different automatically determined Butterworth filter cut-off frequencies for separate segments of a sampled signal. The Teager-Kaiser Energy Operator of the signal is computed and used to determine segments of the signal with different energy content. The Autocorrelation-Based Procedure (ABP) is used on each of these segments to determine filter cut-off frequencies. This new procedure was evaluated by estimating acceleration values from the test data set of Dowling (1985). The ASFP produced a root mean square error (RMSE) of 16.4 rad s-2 (26.6%) whereas a single ABP determined filter cut-off frequency applied to the whole Dowling (1985) signal, representing the common approach, produced a RMSE of 25.5 rad s-2 (41.4%). As a point of comparison, a Generalized Cross-Validated Quintic Spline, a common non-Butterworth filter, produced a RMSE of 23.6 rad s-2 (38.4%). This new automatic approach is advantageous in biomechanics for preserving high frequency content of non-stationary signals.

The effects of mid-flight whole-body and trunk rotation on landing mechanics: implications for anterior cruciate ligament injuries.

The purpose was to quantify the effects of mid-flight whole-body and trunk rotation on knee mechanics in a double-leg landing. Eighteen male and 20 female participants completed a jump-landing-jump task in five conditions: no rotation, testing leg ipsilateral or contralateral (WBRC) to the whole-body rotation direction, and testing leg ipsilateral (TRI) or contralateral to the trunk rotation direction. The WBRC and TRI conditions demonstrated decreased knee flexion and increased knee abduction angles at initial contact (2.6 > Cohen's dz > 0.3) and increased peak vertical ground reaction forces and knee adduction moments during the 100 ms after landing (1.7 > Cohen's dz > 0.3). The TRI condition also showed the greatest knee internal rotation angles at initial contact and peak knee abduction and internal rotation angles and peak knee extension moments during the 100 ms after landing (2.0 > Cohen's dz > 0.5). Whole-body rotation increased contralateral knee loading because of its primary role in decelerating medial-lateral velocities. Trunk rotation resulted in the greatest knee loading for the ipsilateral knee due to weight shifting and mechanical coupling between the trunk and lower extremities. These findings may help understand altered trunk motion in anterior cruciate ligament injuries.

A novel conditional ZsGreen-expressing transgenic reporter rat strain for validating Cre recombinase expression.

The Cre/loxP recombination system has revolutionized the ability to genetically manipulate animal genomes in order to conditionally control gene expression. With recent advances in genome editing, barriers to manipulating the rat genome have been overcome and it is now possible to generate new rat strains (Cre drivers) in which Cre recombinase expression is carefully controlled temporally and/or spatially. However, the ability to evaluate and characterize these Cre driver strains is limited by the availability of reliable reporter rat strains. Here, we describe the generation and characterization of a new transgenic rat strain in which conditional expression of the ZsGreen fluorescent protein gene requires the presence of exogenous Cre recombinase. Breeding Cre-expressing rat strains to this stable ZsGreen reporter strain provides an ideal method for validating new rat Cre driver lines and will greatly accelerate the characterization pipeline.

Mid-flight trunk flexion and extension altered segment and lower extremity joint movements and subsequent landing mechanics.

OBJECTIVES: To assess the effect of mid-flight trunk flexion and extension on the movements of body segments and lower extremity joints and subsequent landing mechanics during a jump-landing task. DESIGN: Participants performed three jump-landing conditions in a randomized order. METHODS: Forty-one participants completed jump-landing trials when performing three different mid-flight trunk motion: reaching forward, reaching up, and reaching backward. Whole-body kinematic and ground reaction force data were collected. RESULTS: The reaching backward condition resulted in a more posteriorly positioned upper body center of mass (COM) and more anteriorly positioned pelvis COM, legs COM, hip, and knee joint positions relative to the whole-body COM in flight and at initial contact of landing. The reaching backward condition showed the least hip flexion and ankle plantarflexion angles at initial contact as well as the least hip and knee flexion angles and the greatest ankle dorsiflexion angles at 100ms after landing. The reaching backward condition also demonstrated the greatest peak posterior ground reaction forces, peak and average knee extension moments, peak and average hip flexion moments, and peak knee varus moments within the first 100ms after landing. Opposite changes were observed for the reaching forward condition. CONCLUSIONS: Mid-flight trunk extension resulted in body postures that predisposed individuals to land with increased knee extension and varus moments and decreased knee flexion angles, which are indirectly associated with increased ACL loading. These findings may help to understand altered trunk motion during certain ACL injury events and provide information for developing jump-landing training strategies.