The T-shaped radial spoke (RS) is a protein complex attached to the A microtubule of the outer doublet microtubules, and it extends toward the central pair (CP). It modulates the beat frequency, amplitude, and waveform of the flagella and cilia by serving as a mechanochemical signal transducer between the CP and dyneins. In humans, RS defects cause primary ciliary dyskinesia, but the structural components of triple RSs (RS1, RS2, and RS3) in mammals remain to be elucidated in detail. Here, we introduce a mouse model that lacked the entire RS1 in sperm flagella, due to the deletion of Iqub, while the tracheal cilia possessed intact triple RSs. Furthermore, the absence of IQUB only resulted in male infertility, owing to the sperm motility defect. Based on the mouse model, the RS1 compositions are identified in sperm flagella. In summary, this study elucidates the RS1 components and function in mammalian flagella.

Purpose: We aim to analyze the clinical and genetic features in a Chinese family with congenital retinoschisis by whole-exome sequencing and comprehensive clinical examination. Methods: Six members were recruited from a Chinese family. Three of them were diagnosed as congenital retinoschisis, including two twin siblings. All subjects received a full eye examination. Whole-exome sequencing (WES) and Sanger sequencing were performed on two twin probands and all participants, respectively. Results: A novel splice site mutation RS1.c.53-1G>A was identified in a Chinese congenital retinoschisis family. The mean onset age was 16.7 ± 2.4 years old. The average BCVA in patients was 0.37 ± 0.05. A typical spoke-wheel pattern was observed in all affected eyes. OCT examination results showed fovea schisis and schisis cavities were located in the inner nuclear layer in 100% eyes (6/6). ERG b/a ratio was decreased markedly, but was still more than 1 in the four eyes that were available. Conclusion: The present study discovered a new pathogenic splice cite variant of RS1 in congenital retinoschisis, which expands the mutational spectrum. In contrast to previous research, the phenotype of patients with the same mutation within one family was highly similar. Early molecular testing is crucial for early diagnosis, clinical management, and genetic counseling of patients with congenital retinoschisis.

X-linked retinoschisis (XLRS) is among the most commonly inherited degenerative retinopathies. XLRS is caused by functional impairment of RS1. However, the molecular mechanisms underlying RS1 malfunction remain largely uncharacterized. Here, we performed a data-independent acquisition-mass spectrometry-based proteomic analysis in RS1-null mouse retina with different postal days (Ps), including the onset (P15) and early progression stage (P56). Gene set enrichment analysis showed that type I interferon-mediated signaling was upregulated and photoreceptor proteins responsible for detection of light stimuli were downregulated at P15. Positive regulation of Tor signaling was downregulated and nuclear transcribed mRNA catabolic process nonsense-mediated decay was upregulated at P56. Moreover, the differentially expressed proteins at P15 were enriched in metabolism of RNA and RNA destabilization. A broader subcellular localization distribution and enriched proteins in visual perception and phototransduction were evident at P56. Combined transcriptomic-proteomic analysis revealed that functional impairments, including detection of visible light, visual perception, and visual phototransduction, occurred at P21 and continued until P56. Our work provides insights into the molecular mechanisms underlying the onset and progression of an XLRS mouse model during the early stages, thus enhancing the understanding of the mechanism of XLRS.

UNASSIGNED: To study the long-term photoreceptor changes and to evaluate the effects of topical application of a carbonic anhydrase inhibitor (CAI) in a mouse model of X-linked retinoschisis (XLRS).
UNASSIGNED: Conventional electroretinograms (ERGs) and dark-adapted 10-Hz flicker ERGs were recorded in control and Rs1 -/Y mice generated with CRISPR/Cas9. ON-pathway blocker 2-amino-4-phosphobutyric acid (APB) was injected intravitreally. Morphology was evaluated with histology and optical coherence tomography (OCT). Mice were treated with a CAI inhibitor brinzolamide eye drops (10 mg/ml) three times a day for 3 months. OCT and ERG findings at 1, 4, and 10 months were analyzed.
UNASSIGNED: Negative ERGs and retinal cavities were evident in Rs1 -/Y mice. Both a-wave and b-wave amplitudes decreased with age when compared with age-matched controls. The APB-isolated a-wave (a\') amplitudes of Rs1 -/Y mice were reduced in all age groups. In dark-adapted 10-Hz flicker ERG, the amplitude-intensity curve of Rs1 -/Y mice shifted down. The thickness of ONL and IS/OS decreased in Rs1 -/Y mice. CAI reduced the splitting retinal cavities but didn\'t affect the ERG.
UNASSIGNED: In addition to post receptoral impairments, photoreceptor cells underwent progressive dysfunction since early age in Rs1 -/Y mice. Long-term CAI treatment improved the shrinkage of the splitting retinal cavity, while no functional improvement was observed.

Although it has a poor prognosis, patients with lung adenocarcinoma (LUAD) have a relatively higher 5-year survival period. Thus, it is necessary to identify effective prognostic markers to evaluate the effect of early treatment. RS1 gene encodes retinoschisin, a key protein in congenital retinoschisis, while few studies have been reported on the association between RS1 and cancer prognosis.
We performed bioinformatic analyses based on the data obtained from The Cancer Genome Atlas and Gene Expression Omnibus databases to demonstrate the expression level of RS1 was related to the LUAD prognosis and our findings were verified in-vitro and clinical samples. Then, we explored the potential mechanism of how RS1 expression influenced the prognosis of LUAD.
Compared with normal tissues, the RS1 expression was significantly lower in tumor tissues. The Multivariate Cox regression model showed that RS1 could be used as an independent prognostic indicator. Furthermore, we found significant differences in immune cell infiltration between RS1 high and low expression groups, and the proteasome pathway was found enriched in RS1 low expression samples.
In conclusion, our study suggests that RS1 is a novel prognostic biomarker for LUAD. Differences in immune cell infiltration and signaling pathways may contribute to the poor prognosis of LUAD caused by low RS1 expression.

A notable behavioural feature of X-linked retinoschisis (XLRS) is extensive structural schisis (splitting) of the outer plexiform and inner nuclear layers of the neurosensory retina, which is partly combined with complications related to vitreous hemorrhage, macular holes and retinal detachment. The present study aimed to identify the pathogenic gene mutation in a three-generation Chinese family with XLRS by whole-exome sequencing (WES). The clinical information of a three-generation Chinese family with cases of XLRS was collected. WES was performed for the proband. A comparison with the human reference genome sequence (hg38) and bioinformatic analysis were performed to reveal putative variants and Sanger sequencing was applied to verify mutations in this family and healthy control participants. Intraretinal cystic spaces were detected by optical coherence tomography imaging. Structures of the wild-type and mutant retinoschisin 1 (RS1) protein were modelled by PyMol. Almost all patients had a history of vision loss and abnormal blue-purple colour vision; however, the phenotypes of the 4 patients were distinctly different. There was no linear correlation between phenotypic severity and age. A recurrent RS1 (Xp22.2) mutation (NM_000330: c.559C>T) was detected, resulting in the p.Q187X variant. According to the protein model, this variant is likely pathogenic. The present study was the first to report that RS1:c.559C>T induces XLRS in a three-generation Chinese pedigree, with the mutation leading to premature termination of translation of the RS1 protein. WES was able to diagnose XLRS, which has the characteristics of clinical and genetic heterogeneity.

Cas12a-mediated targeted genome engineering strategies have enabled a broad range of research and clinical applications. However, the limited target-selection spectrum and low activity/fidelity remain a bottleneck for its widespread application in precision site-specific human genome editing. Therefore, there exists an acute need to identify novel Cas12a nucleases with improved features for genome editing. By screening a range of candidate Cas12a nucleases, here we demonstrate that Lb2Cas12a possesses genome editing activity in human cells and it has greater flexibility in PAM (5\'-BYYV-3\') selection. Furthermore, we engineered Lb2Cas12a to generate variants (Lb2Cas12a-RVR and Lb2Cas12a-RR), which greatly expands the target-selection spectrum. Our study illustrated that Lb2Cas12a could be harnessed as additional genome editing tool for the manipulation of human genome.

Clustered regularly interspaced short palindromic repeat-Cas12a has been harnessed to manipulate the human genome; however, low cleavage efficiency and stringent protospacer adjacent motif hinder the use of Cas12a-based therapy and applications. Here, we have described a directional evolving and screening system in human cells to identify novel FnCas12a variants with high activity. By using this system, we identified IV-79 (enhanced activity FnCas12a, eaFnCas12a), which possessed higher DNA cleavage activity than WT FnCas12a. Furthermore, to widen the target selection spectrum, eaFnCas12a was engineered through site-directed mutagenesis. eaFnCas12a and one engineered variant (eaFnCas12a-RR), used for correcting human RS1 mutation responsible for X-linked retinoschisis, had a 3.28- to 4.04-fold improved activity compared with WT. Collectively, eaFnCas12a and its engineered variants can be used for genome-editing applications that requires high activity.