Identification of RPGR ORF15 mutation for X-linked retinitis pigmentosa in a large Chinese family and in vitro correction with prime editor.

X-linked retinitis pigmentosa (XLRP) is the most severe form of Retinitis Pigmentosa (RP) and one of the leading causes of blindness in the world. Currently, there is no effective treatment for RP. In the present study, we recruited a XLRP family and identified a 4 bp deletion mutation (c. 2234_2237del) in RPGR ORF15 with Sanger sequencing, which was located in the exact same region as the missing XES (X chromosome exome sequencing) coverage. Then, we generated cell lines harboring the identified mutation and corrected it via enhanced prime editing system (ePE). Collectively, Sanger sequencing identified a pathogenic mutation in RPGR ORF15 for XLRP which was corrected with ePE. This study provides a valuable insight for genetic counseling of the afflicted family members and prenatal diagnosis, also paves a way for applying prime editing based gene therapy in those patients.

A precise and efficient adenine base editor.

Adenine base editors (ABEs) are novel genome-editing tools, and their activity has been greatly enhanced by eight additional mutations, thus named ABE8e. However, elevated catalytic activity was concomitant with frequent generation of bystander mutations. This bystander effect precludes its safe applications required in human gene therapy. To develop next-generation ABEs that are both catalytically efficient and positionally precise, we performed combinatorial engineering of NG-ABE8e. We identify a novel variant (NG-ABE9e), which harbors nine mutations. NG-ABE9e exhibits robust and precise base-editing activity in human cells, with more than 7-fold bystander editing reduction at some sites, compared with NG-ABE8e. To demonstrate its practical utility, we used NG-ABE9e to correct the frequent T17M mutation in Rhodopsin for autosomal dominant retinitis pigmentosa. It reduces bystander editing by ∼4-fold while maintaining comparable efficiency. NG-ABE9e possesses substantially higher activity than NG-ABEmax and significantly lower bystander editing than NG-ABE8e in rice. Therefore, this study provides a versatile and improved adenine base editor for genome editing.

A Study of Familial Amyloid Polyneuropathy Induced by the TTR Val30Leu Mutation in China.

INTRODUCTION: Familial amyloid polyneuropathy is currently prevalent worldwide as the transthyretin (TTR) Val30Met mutation, and there are other types of mutations. The purpose of this study was to understand the clinical manifestations, electrophysiological characteristics, and outcomes of hormone-related therapy in patients with the TTR Val30Leu mutation in China. METHODS: Clinical data were collected from 9 members of a family with the TTR Val30Leu mutation in China, and blood samples of 7 members of the family were sequenced. The electrophysiological examinations of 4 of them were collected and analysed. RESULTS: A total of 7 people had the TTR gene c.148G>T missense mutation and the TTR protein Val30Leu mutation in this family, and the positive members all had similar symptoms, such as limb paraesthesia and gastrointestinal symptoms. In addition, electrophysiological examination showed abnormal nerve conduction velocity in all 4 patients. CONCLUSIONS: The clinical manifestations of this mutation involve mainly limb sensory or motor disorders or gastrointestinal symptoms or both, and the electrophysiological examination shows neurogenic damage.

Two high-fidelity variants: efSaCas9 and SaCas9-HF, which one is better?

CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated endonuclease Cas9) nucleases have been widely applied for genome engineering. Staphylococcus aureus Cas9 (SaCas9) is compact, which can be packaged in AAV (adeno-associated virus) vector for in vivo gene editing. While, wild-type SaCas9 can induce unwanted off-target mutations and substantially limits the applications. So far, there are two reported SaCas9 variants with high-fidelity, including efSaCas9 from our previous study and SaCas9-HF. However, it remains unknown which one possessing the better fidelity and higher activity. Here, we performed a parallel comparison of efSaCas9 and SaCas9-HF in human cells through fluorescent reporter system and target deep sequencing, respectively. The results demonstrated that efSaCas9 possesses higher cleavage activity and fidelity than SaCas9-HF at the most endogenous sites in human cells. Collectively, our study provides insights for the rational selection of suitable SaCas9 for human genome editing.

Human cell based directed evolution of adenine base editors with improved efficiency.

Adenine base editors (ABE) are genome-editing tools that have been harnessed to introduce precise A•T to G•C conversion. However, the low activity of ABE at certain sites remains a major bottleneck that precludes efficacious applications. Here, to address it, we develop a directional screening system in human cells to evolve the deaminase component of the ABE, and identify three high-activity NG-ABEmax variants: NG-ABEmax-SGK (R101S/D139G/E140K), NG-ABEmax-R (Q154R) and NG-ABEmax-K (N127K). With further engineering, we create a consolidated variant [NG-ABEmax-KR (N127K/Q154R)] which exhibit superior editing activity both in human cells and in mouse disease models, compared to the original NG-ABEmax. We also find that NG-ABEmax-KR efficiently introduce natural mutations in gamma globin gene promoters with more than four-fold increase in editing activity. This work provides a broadly applicable, rapidly deployable platform to directionally screen and evolve user-specified traits in base editors that extend beyond augmented editing activity.

Small-molecule compounds boost genome-editing efficiency of cytosine base editor.

Cytosine base editor (CBE) enables targeted C-to-T conversions at single base-pair resolution and thus has potential therapeutic applications in humans. However, the low efficiency of the system limits practical use of this approach. We reported a high-throughput human cells-based reporter system that can be harnessed for quickly measuring editing activity of CBE. Screening of 1813 small-molecule compounds resulted in the identification of Ricolinostat (an HDAC6 inhibitor) that can enhance the efficiency of BE3 in human cells (2.45- to 9.21-fold improvement). Nexturastat A, another HDAC6 inhibitor, could also increase BE3-mediated gene editing by 2.18- to 9.95-fold. Ricolinostat and Nexturastat A also boost base editing activity of the other CBE variants (BE4max, YE1-BE4max, evoAPOBEC1-BE4max and SpRY-CBE4max, up to 8.32-fold). Meanwhile, combined application of BE3 and Ricolinostat led to >3-fold higher efficiency of correcting a pathogenic mutation in ABCA4 gene related to Stargardt disease in human cells. Moreover, we demonstrated that our strategy could be applied for efficient generation of mouse models through direct zygote injection and base editing in primary human T cells. Our study provides a new strategy to improve the activity and specificity of CBE in human cells. Ricolinostat and Nexturastat A augment the effectiveness and applicability of CBE.

Lb2Cas12a and its engineered variants mediate genome editing in human cells.

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.

Oxygen content-related DNA damage of graphene oxide on human retinal pigment epithelium cells.

Arguments regarding the biocompatibility of graphene-based materials (GBMs) have never ceased. Particularly, the genotoxicity (e.g., DNA damage) of GBMs has been considered the greatest risk to healthy cells. Detailed genotoxicity studies of GBMs are necessary and essential. Herein, we present our recent studies on the genotoxicity of most widely used GBMs such as graphene oxide (GO) and the chemically reduced graphene oxide (RGO) toward human retinal pigment epithelium (RPE) cells. The genotoxicity of GO and RGOs against ARPE-19 (a typical RPE cell line) cells was investigated using the alkaline comet assay, the expression level of phosphorylated p53 determined via Western blots, and the release level of reactive oxygen species (ROS). Our results suggested that both GO and RGOs induced ROS-dependent DNA damage. However, the DNA damage was enhanced following the reduction of the saturated C-O bonds in GO, suggesting that surface oxygen-containing groups played essential roles in the reduced genotoxicity of graphene and had the potential possibility to reduce the toxicity of GBMs via chemical modification.

Engineered FnCas12a with enhanced activity through directional evolution in human cells.

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.

CRISPR/Cas9-mediated mutagenesis at microhomologous regions of human mitochondrial genome.

Genetic manipulation of mitochondrial DNA (mtDNA) could be harnessed for deciphering the gene function of mitochondria; it also acts as a promising approach for the therapeutic correction of pathogenic mutation in mtDNA. However, there is still a lack of direct evidence showing the edited mutagenesis within human mtDNA by clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9). Here, using engineered CRISPR/Cas9, we observed numerous insertion/deletion (InDel) events at several mtDNA microhomologous regions, which were triggered specifically by double-strand break (DSB) lesions within mtDNA. InDel mutagenesis was significantly improved by sgRNA multiplexing and a DSB repair inhibitor, iniparib, demonstrating the evidence of rewiring DSB repair status to manipulate mtDNA using CRISPR/Cas9. These findings would provide novel insights into mtDNA mutagenesis and mitochondrial gene therapy for diseases involving pathogenic mtDNA.

Rational Selection of CRISPR-Cas Triggering Homology-Directed Repair in Human Cells.

The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated) nucleases have been widely applied for genome engineering. Cas9 (Streptococcus pyogenes Cas9 [SpCas9] and Staphylococcus aureus Cas9 [SaCas9]) and Cpf1 (i.e., Francisella novicida U112 Cpf1 [FnCpf1], also named FnCas12a) were harnessed to perform gene editing in human cells. Precise genetic modification by homology-directed repair (HDR) is an attractive approach for in situ gene correction. However, so far, the comparative efficiencies of HDR mediated by different CRISPR orthologs remain unknown. To address this question, in this study, we developed a reporter system to investigate HDR efficiencies triggered by various CRISPR orthologs. We found that SpCas9 and SaCas9, the two most commonly used Cas9 enzymes, possessed a similar ability to induce HDR. Interestingly, with the increasing amount of coding plasmids or additional nuclear localization sequences, FnCpf1 could improve the HDR efficacy. Collectively, our study provides insights for the rational selection of appropriate tools for human genome manipulation.

Aberrant expression of semaphorin 6B affects cell phenotypes in thyroid carcinoma by activating the Notch signalling pathway.

INTRODUCTION: Numerous semaphorins have been widely clarified to be involved in the development of multiple cancers. However, semaphorin 6B (SEMA6B) has not yet been extensively reported in cancers, especially in thyroid carcinoma. MATERIAL AND METHODS: Thyroid carcinoma RNA-Seq dataset from the TCGA database was used to assess the expression of SEMA6B in tissues, as well as its clinical significance. We adopted qRT-PCR and western blot analyses to measure the mRNA and protein expression of SEMA6B in thyroid carcinoma cells. The biological roles of SEMA6B in thyroid carcinoma cells were examined through cell counting kit 8, clone formation, and Transwell assays. Also, GSEA was used to identify the gene sets modulated by SEMA6B, which is further verified by western blot. RESULTS: According to the public dataset from the TCGA database, we found that the expression of SEMA6B was upregulated in thyroid carcinoma tissues compared to adjacent non-tumour tissues, and a high level of SEMA6B resulted in a poorer prognosis compared to the low-level SEMA6B group. Functional experiments showed that silencing SEMA6B suppressed the B-CPAP cells viability, invasiveness, and motility, whereas up-regulating SEMA6B in FTC-133 cells led to opposite outcomes. Furthermore, knockdown of SEMA6B in B-CPAP cells could significantly elevate the protein expression of NUMB and reduce the expression of NOTCH1, HES1, and Cyclin D1. Conversely, overexpression of SEMA6B in FTC-133 cells presented opposite results on the protein expression of these Notch signalling pathway-related markers. CONCLUSIONS: Our findings demonstrated that SEMA6B exerts a tumourigenic effect in thyroid carcinoma partly by activating Notch signalling pathway, which provides a possible biomarker for the therapeutic intervention in thyroid carcinoma.

High-fidelity SaCas9 identified by directional screening in human cells.

CRISPR-Staphylococcus aureus Cas9 (CRISPR-SaCas9) has been harnessed as an effective in vivo genome-editing tool to manipulate genomes. However, off-target effects remain a major bottleneck that precludes safe and reliable applications in genome editing. Here, we characterize the off-target effects of wild-type (WT) SaCas9 at single-nucleotide (single-nt) resolution and describe a directional screening system to identify novel SaCas9 variants with desired properties in human cells. Using this system, we identified enhanced-fidelity SaCas9 (efSaCas9) (variant Mut268 harboring the single mutation of N260D), which could effectively distinguish and reject single base-pair mismatches. We demonstrate dramatically reduced off-target effects (approximately 2- to 93-fold improvements) of Mut268 compared to WT using targeted deep-sequencing analyses. To understand the structural origin of the fidelity enhancement, we find that N260, located in the REC3 domain, orchestrates an extensive network of contacts between REC3 and the guide RNA-DNA heteroduplex. efSaCas9 can be broadly used in genome-editing applications that require high fidelity. Furthermore, this study provides a general strategy to rapidly evolve other desired CRISPR-Cas9 traits besides enhanced fidelity, to expand the utility of the CRISPR toolkit.

Development of a Simple and Quick Method to Assess Base Editing in Human Cells.

Base editing is a form of genome editing that can directly convert a single base (C or A) to another base (T or G), which is of great potential in biomedical applications. The broad application of base editing is limited by its low activity and specificity, which still needs to be resolved. To address this, a simple and quick method for the determination of its activity/specificity is highly desired. Here, we developed a novel system, which could be harnessed for quick detection of editing activity and specificity of base editors (BEs) in human cells. Specifically, multiple cloning sites (MCS) were inserted into the human genome via lentivirus, and base editing targeting the MCS was performed with BEs. The base editing activities were assessed by specific restriction enzymes. The whole process only includes nucleotide-based targeting the MCS, editing, PCR, and digestion, thus, we named it NOTEPAD. This straightforward approach could be easily accessed by molecular biology laboratories. With this method, we could easily determine the BEs editing efficiency and pattern. The results revealed that BEs triggered more off-target effects in the genome than on plasmids including genomic indels (insertions and deletions). We found that ABEs (adenine base editors) had better fidelity than CBEs (cytosine base editors). Our system could be harnessed as a base editing assessment platform, which would pave the way for the development of next-generation BEs.

Efficient Human Genome Editing Using SaCas9 Ribonucleoprotein Complexes.

Genome editing using RNA-guided nucleases in their ribonucleoprotein (RNP) form represents a promising strategy for gene modification and therapy because they are free of exogenous DNA integration and have reduced toxicity in vivo and ex vivo. However, genome editing by Cas9 nuclease from Staphylococcus aureus (SaCas9) has not been reported in its RNP form, which recognizes a longer protospacer adjacent motif (PAM), 5'-NNGRRT-3', compared with Streptococcus pyogenes Cas9 (SpCas9) of 5'-NGG-3' PAM. Here, SaCas9-RNP-mediated genome editing is reported in human cells. The SaCas9-RNP displayed efficient genome editing activities of enhanced green fluorescent protein (EGFP) coding gene as well as three endogenous genes (OPA1, RS1, and VEGFA). Further, SaCas9-RNP is successfully implemented to correct a pathogenic RS1 mutation for X-linked juvenile retinoschisis. It is also shown that off-target effects triggered by SaCas9-RNP are undetectable by targeted deep sequencing. Collectively, this study demonstrates the potential of SaCas9-RNP-mediated genome editing in human cells, which could facilitate genome-editing-based therapy.

A Single Multiplex crRNA Array for FnCpf1-Mediated Human Genome Editing.

Cpf1 has been harnessed as a tool for genome manipulation in various species because of its simplicity and high efficiency. Our recent study demonstrated that FnCpf1 could be utilized for human genome editing with notable advantages for target sequence selection due to the flexibility of the protospacer adjacent motif (PAM) sequence. Multiplex genome editing provides a powerful tool for targeting members of multigene families, dissecting gene networks, modeling multigenic disorders in vivo, and applying gene therapy. However, there are no reports at present that show FnCpf1-mediated multiplex genome editing via a single customized CRISPR RNA (crRNA) array. In the present study, we utilize a single customized crRNA array to simultaneously target multiple genes in human cells. In addition, we also demonstrate that a single customized crRNA array to target multiple sites in one gene could be achieved. Collectively, FnCpf1, a powerful genome-editing tool for multiple genomic targets, can be harnessed for effective manipulation of the human genome.

Cytotoxicity and genotoxicity of bacterial magnetosomes against human retinal pigment epithelium cells.

A variety of nanomaterials have been developed for ocular diseases. The ability of these nanomaterials to pass through the blood-ocular barrier and their biocompatibility are essential characteristics that must be considered. Bacterial magnetosomes (BMs) are a type of biogenic magnetic nanomaterials synthesized by magnetotactic bacteria. Due to their unique biomolecular membrane shell and narrow size distribution of approximately 30 nm, BMs can pass through the blood-brain barrier. The similarity of the blood-ocular barrier to the blood-brain barrier suggests that BMs have great potential as treatments for ocular diseases. In this work, BMs were isolated from magnetotactic bacteria and evaluated in various cytotoxicity and genotoxicity studies in human retinal pigment epithelium (ARPE-19) cells. The BMs entered ARPE-19 cells by endocytosis after a 6-h incubation and displayed much lower cytotoxicity than chemically synthesized magnetic nanoparticles (MNPs). MNPs exhibited significantly higher genotoxicity than BMs and promoted the expression of Bax (the programmed cell death acceleration protein) and the induction of greater cell necrosis. In BM-treated cells, apoptosis tended to be suppressed via increased expression of the Bcl-2 protein. In conclusion, BMs display excellent biocompatibility and potential for use in the treatment of ocular diseases.

The Relationship between Serum Osteocalcin Concentration and Glucose and Lipid Metabolism in Patients with Type 2 Diabetes Mellitus ­ The Role of Osteocalcin in Energy Metabolism.

BACKGROUND: Recent animal studies have found that the osteocalcin secreted by osteoblasts could participate in glucose and lipid metabolism. Our study aimed to investigate the relationship between serum osteocalcin concentration and glucose and lipid metabolism in patients with type 2 diabetes mellitus. METHODS: 985 patients with type 2 diabetes were divided into the male group (n = 495) and the postmenopausal female group (n = 490). The average ages were 54.42 ± 10.535 and 64.93 ± 9.277, respectively. We collected the parameters of age, duration, fasting plasma glucose, HbA1c, fasting insulin, fasting C peptide, blood lipid, 25 (OH) VD3, parathyroid hormone (PTH), Alkaline phosphatase (ALP), procollagen type 1 N-terminal propeptide (P1NP), ß-C-terminal telopeptide of type I collagen (ß-CTx), osteocalcin, HOMA-IR, HOMA-ß, body mass index (BMI), and waist-to-hip ratio (WHR). The relationship of osteocalcin and these parameters were analyzed by Pearson/Spearman correlation analysis and stepwise multiple regression analysis. RESULTS: Osteocalcin was negatively correlated with HbA1c (p < 0.05) and it was also an independent relevant factor affecting HbA1c in both groups. Osteocalcin was positively correlated with HOMA-ß and it was an independent relevant factor affecting HOMA-ß in male group (p < 0.01). CONCLUSIONS: These findings indicate the association between serum osteocalcin and glucose metabolism and beta cell function. No relationship was found between osteocalcin and insulin resistance and lipid metabolism in type 2 diabetes.