Noninvasive Light Flicker Stimulation Promotes Optic Nerve Regeneration by Activating Microglia and Enhancing Neural Plasticity in Zebrafish.

Purpose: Forty-hertz light flicker stimulation has been proven to reduce neurodegeneration, but its effect on optic nerve regeneration is unclear. This study explores the effect of 40-Hz light flicker in promoting optic nerve regeneration in zebrafish and investigates the underlying mechanisms. Methods: Wild-type and mpeg1:EGFP zebrafish were used to establish a model of optic nerve crush. Biocytin tracing and hematoxylin and eosin staining were employed to observe whether 40-Hz light flicker promotes regeneration of retinal ganglion cell axons and dendrites. Optomotor and optokinetic responses were evaluated to assess recovery of visual function. Immunofluorescence staining of mpeg1:EGFP zebrafish was performed to observe changes in microglia. Differentially expressed genes that promote optic nerve regeneration following 40-Hz light flicker stimulation were identified and validated through RNA-sequencing analysis and quantitative real-time PCR (qRT-PCR). Results: Zebrafish exhibited spontaneous optic nerve regeneration after optic nerve injury and restored visual function. We observed that 40-Hz light flicker significantly activated microglia following optic nerve injury and promoted regeneration of retinal ganglion cell axons and dendrites, as well as recovery of visual function. Transcriptomics and qRT-PCR analyses revealed that 40-Hz light flicker increased the expression of genes associated with neuronal plasticity, including bdnf, npas4a, fosab, fosb, egr4, and ier2a. Conclusions: To our knowledge, this study is the first to demonstrate that 40-Hz light flicker stimulation promotes regeneration of retinal ganglion cell axons and dendrites and recovery of visual function in zebrafish, which is associated with microglial activation and enhancement of neural plasticity.

EGR1 and EGR2 positively regulate plant ABA signaling by modulating the phosphorylation of SnRK2.2.

During abscisic acid (ABA) signaling, reversible phosphorylation controls the activity and accumulation of class III SNF1-RELATED PROTEIN KINASE 2s (SnRK2s). While protein phosphatases that negatively regulate SnRK2s have been identified, those that positively regulate ABA signaling through SnRK2s are less understood. In this study, Arabidopsis thaliana mutants of Clade E Growth-Regulating 1 and 2 (EGR1/2), which belong to the protein phosphatase 2C family, exhibited reduced ABA sensitivity in terms of seed germination, cotyledon greening, and ABI5 accumulation. Conversely, overexpression increased these ABA-induced responses. Transcriptomic data revealed that most ABA-regulated genes in egr1 egr2 plants were expressed at reduced levels compared with those in Col-0 after ABA treatment. Abscisic acid up-regulated EGR1/2, which interact directly with SnRK2.2 through its C-terminal domain I. Genetic analysis demonstrated that EGR1/2 function through SnRK2.2 during ABA response. Furthermore, SnRK2.2 de-phosphorylation by EGR1/2 was identified at serine 31 within the ATP-binding pocket. A phospho-mimic mutation confirmed that phosphorylation at serine 31 inhibited SnRK2.2 activity and reduced ABA responsiveness in plants. Our findings highlight the positive role of EGR1/2 in regulating ABA signaling, they reveal a new mechanism for modulating SnRK2.2 activity, and provide novel insight into how plants fine-tune their responses to ABA.

Protective effect of the natural flavonoid naringenin in mouse models of retinal injury.

Glaucoma is an eye disease with a high rate of blindness and a complex pathogenesis. Ocular hypertension (OHT) is a critical risk factor, and retinal ischemia/reperfusion (I/R) is an important pathophysiological basis. This study was designed to investigate the retinal neuroprotective effect of oral naringenin in an acute retinal I/R model and a chronic OHT model and the possible mechanism involved. After the I/R and OHT models were established, mice were given vehicle or naringenin (100 mg/kg or 300 mg/kg). Hematoxylin-eosin (HE) staining and immunostaining of RBPMS and glial fibrillary acidic protein (GFAP) were used to evaluate retinal injury. GFAP, CD38, Sirtuin1 (SIRT1), and NOD-like receptor protein 3 (NLRP3) expression levels were measured by Western blotting. In the OHT model, intraocular pressure (IOP) was dynamically maintained at approximately 20-25 mmHg after injury. The retinal structure was damaged, and retinal ganglion cells (RGCs) were lost in both models. Naringenin ameliorated the abovementioned indications but also demonstrated that high concentrations of naringenin significantly inhibited retinal astrocyte activation and inhibited damage-induced increases in the expression of GFAP, NLRP3, and CD38 proteins, while SIRT1 protein expression was upregulated. This study showed for the first time that naringenin can reduce microbead-induced IOP elevation in the OHT model, providing new evidence for the application of naringenin in glaucoma. Naringenin may mediate the CD38/SIRT1 signaling pathway, inhibit astrocyte activation, and ultimately exert an anti-inflammatory effect to achieve retinal neuroprotection.

Resveratrol Alleviates Retinal Ischemia-Reperfusion Injury by Inhibiting the NLRP3/Gasdermin D/Caspase-1/Interleukin-1ß Pyroptosis Pathway.

Purpose: The purpose of this study is to investigate the anti-pyroptotic effect of resveratrol in the context of ischemia-reperfusion (I/R)-induced retinal injury, with a particular focus on Müller glial cells (MGCs) and to elucidate the underlying molecular mechanisms. Methods: The retinal I/R model was constructed in mice and pyroptotic markers were measured at six, 12, 24, 48, and 72 hours after I/R injury to determine the peak of pyroptotic activity. The effects of resveratrol on pyroptosis, inflammasomes, and the activation of MGCs after I/R injury were observed on the retina of mice. Moreover, induction of pyroptosis in rat Müller glial cells (r-MC) via lipopolysaccharide was used to explore the effects of resveratrol on pyroptosis of r-MC in vitro. Results: After the induction of retinal I/R injury in mice, the intricate involvement of pyroptosis in the progressive degeneration of the retina was observed, reaching its zenith at the onset of 24 hours after I/R injury. Resveratrol treatment alleviated I/R injury on the retina, relieved retinal ganglion cells death. In addition, resveratrol inhibited Caspase-1 activation, gasdermin D (GSDMD-N) cleavage, the inflammasome assembly, and the release of inflammatory cytokines, simultaneously relieving the MGCs activation. Furthermore, resveratrol inhibited the pyroptosis-related NLRP3/GSDMD-N/TMS1/ASC/Caspase-1/IL-1ß pathway in r-MC cells, and mitigated cells death in vitro. Conclusions: Pyroptosis plays an important role in the pathogenesis of retinal I/R injury. Resveratrol can attenuate pyroptotic-driven damage in the retina and MGC by inhibiting the NLRP3/GSDMD-N/TMS1/ASC/Caspase-1/IL-1ß pyroptosis pathway.

Multifunctional agents against Alzheimer's disease based on oxidative stress: Polysubstituted pyrazine derivatives synthesized by multicomponent reactions.

As Alzheimer's disease (AD) is a neurodegenerative disease with a complex pathogenesis, the exploration of multi-target drugs may be an effective strategy for AD treatment. Multifunctional small molecular agents can be obtained by connecting two or more active drugs or privileged pharmacophores by multicomponent reactions (MCRs). In this paper, two series of polysubstituted pyrazine derivatives with multifunctional moieties were designed as anti-AD agents and synthesized by Passerini-3CR and Ugi-4CR. Since the oxidative stress plays an important role in the pathological process of AD, the antioxidant activities of the newly synthesized compounds were first evaluated. Subsequently, selected active compounds were further screened in a series of AD-related bioassays, including Aß1-42 self-aggregation and deaggregation, BACE-1 inhibition, metal chelation, and protection of SH-SY5Y cells from H2O2-induced oxidative damage. Compound A3B3C1 represented the best one with multifunctional potencies. Mechanism study showed that A3B3C1 acted on Nrf2/ARE signaling pathway, thus increasing the expression of related antioxidant proteins NQO1 and HO-1 to normal cell level. Furthermore, A3B3C1 showed good in vitro human plasma and liver microsome stability, indicating a potential for further development as multifunctional anti-AD agent.

Recent advances in novel mutation genes of Parkinson's disease.

With increasing life expectancy, a growing number of individuals are being affected by Parkinson's Disease (PD), a Neurodegenerative Disease (ND). Approximately, 5-10% of PD is explained by genetic causes linked to known PD genes. With improvements in genetic testing and high-throughput technologies, more PD-associated susceptibility genes have been reported in recent years. However, a comprehensive review of the pathogenic mechanisms and physiological roles of these genes is still lacking. This article reviews novel genes with putative or confirmed pathogenic mutations in PD reported since 2019, summarizes the physiological functions and potential associations with PD. Newly reported PD-related genes include ANK2, DNAH1, STAB1, NOTCH2NLC, UQCRC1, ATP10B, TFG, CHMP1A, GIPC1, KIF21B, KIF24, SLC25A39, SPTBN1 and TOMM22. However, the evidence for pathogenic effects of many of these genes is inconclusive. A variety of novel PD-associated genes have been identified through clinical cases of PD patients and analysis of Genome-Wide Association Studies (GWAS). However, more evidence is needed in confirm the strong association of novel genes with disease.

YIPF5 (p.W218R) mutation induced primary microcephaly in rabbits.

Primary microcephaly (PMCPH) is a rare autosomal recessive neurodevelopmental disorder with a global prevalence of PMCPH ranging from 0.0013% to 0.15%. Recently, a homozygous missense mutation in YIPF5 (p.W218R) was identified as a causative mutation of severe microcephaly. In this study, we constructed a rabbit PMCPH model harboring YIPF5 (p.W218R) mutation using SpRY-ABEmax mediated base substitution, which precisely recapitulated the typical symptoms of human PMCPH. Compared with wild-type controls, the mutant rabbits exhibited stunted growth, reduced head circumference, altered motor ability, and decreased survival rates. Further investigation based on model rabbit elucidated that altered YIPF5 function in cortical neurons could lead to endoplasmic reticulum stress and neurodevelopmental disorders, interference of the generation of apical progenitors (APs), the first generation of progenitors in the developing cortex. Furthermore, these YIPF5-mutant rabbits support a correlation between unfolded protein responses (UPR) induced by endoplasmic reticulum stress (ERS), and the development of PMCPH, thus providing a new perspective on the role of YIPF5 in human brain development and a theoretical basis for the differential diagnosis and clinical treatment of PMCPH. To our knowledge, this is the first gene-edited rabbit model of PMCPH. The model better mimics the clinical features of human microcephaly than the traditional mouse models. Hence, it provides great potential for understanding the pathogenesis and developing novel diagnostic and therapeutic approaches for PMCPH.

Selective extended dissection for pancreaticoduodenectomy is associated with better survival in pancreatic cancer patients: retrospective cohort study.

BACKGROUND: Pancreatic ductal adenocarcinoma is a highly malignant tumor with relatively poor survival. Surgery is the first choice for treating patients with early pancreatic cancer. However, the surgical approach and the extent of resection for patients with pancreatic cancer are currently controversial. METHODS: The authors optimized the procedure of standard pancreaticoduodenectomy to selective extended dissection (SED), which is based on the extrapancreatic nerve plexus potentially invaded by the tumor. The authors retrospectively analyzed the clinicopathological data of patients with pancreatic adenocarcinoma who underwent radical surgery in our center from 2011 to 2020. Patients who underwent standard dissection (SD) were matched 2:1 to those who underwent SED using propensity score matching. The log-rank test and Cox regression model were used to analyze survival data. In addition, statistical analyses were performed for the perioperative complications, postoperative pathology, and recurrence pattern. RESULTS: A total of 520 patients were included in the analysis. Among patients with extrapancreatic perineural invasion (EPNI), disease-free survival was significantly longer in those who received SED than in those who received SD (14.5 months vs. 10 months, P <0.05). The incidence of metastasis in No. 9 and No. 14 lymph nodes was significantly higher in patients with EPNI. In addition, there was no significant difference in the incidence rate of perioperative complications between the two surgical procedures. CONCLUSION: Compared with SD, SED exhibits a significant prognostic benefit for patients with EPNI. The SED procedure aiming at specific nerve plexus dissection displayed particular efficacy and safety in resectable pancreatic ductal adenocarcinoma patients.

Rapamycin suppresses neuroinflammation and protects retinal ganglion cell loss after optic nerve crush.

Pyroptosis, an inflammasome-mediated mode of death, plays an important role in glaucoma. It has been shown that regulating the mTOR pathway can inhibit pyroptosis. Unfortunately, whether rapamycin (RAPA), a specific inhibitor of the mTOR pathway, can inhibit optic nerve crush (ONC)-induced pyroptosis to protect retinal ganglion cells (RGCs) has not been investigated. Our research aimed to confirm the effect of intravitreal injection of RAPA on RGCs. Furthermore, we used the ONC model to explore the underlying mechanisms. First, we observed that intravitreal injection of RAPA alleviated RGC damage induced by various types of injury. We then used the ONC model to further explore the potential mechanism of RAPA. Mechanistically, RAPA not only reduced the activation of glial cells in the retina but also inhibited retinal pyroptosis-induced expression of inflammatory factors such as nucleotide-binding oligomeric domain-like receptor 3 (NLRP3), apoptosis-associated speckle-like protein containing a CARD (ASC), N-terminal of gasdermin-D (GSDMD-N), IL-18 and IL-1ß. Moreover, RAPA exerted protective effects on RGC axons, possibly by inhibiting glial activation and regulating the mTOR/ROCK pathway. Therefore, this study demonstrates a novel mechanism by which RAPA protects against glaucoma and provides further evidence for its application in preclinical studies.

Rapid discovery and crystallography study of highly potent and selective butylcholinesterase inhibitors based on oxime-containing libraries and conformational restriction strategies.

Butyrylcholinesterase is regarded as a promising drug target in advanced Alzheimer's disease. In order to identify highly selective and potent BuChE inhibitors, a 53-membered compound library was constructed via the oxime-based tethering approach based on microscale synthesis. Although A2Q17 and A3Q12 exhibited higher BuChE selectivity versus acetylcholinesterase, the inhibitory activities were unsatisfactory and A3Q12 did not inhibit Aß1-42 peptide self-induced aggregation. With A2Q17 and A3Q12 as leads, a novel series of tacrine derivatives with nitrogen-containing heterocycles were designed based on conformation restriction strategy. The results demonstrated that 39 (IC50 = 3.49 nM) and 43 (IC50 = 7.44 nM) yielded much improved hBuChE inhibitory activity compared to the lead A3Q12 (IC50 = 63 nM). Besides, the selectivity indexes (SI = AChE IC50 / BChE IC50) of 39 (SI = 33) and 43 (SI = 20) were also higher than A3Q12 (SI = 14). The results of the kinetic study showed that 39 and 43 exhibited a mixed-type inhibition against eqBuChE with respective Ki values of 1.715 nM and 0.781 nM. And 39 and 43 could inhibit Aß1-42 peptide self-induced aggregation into fibril. X-ray crystallography structures of 39 or 43 complexes with BuChE revealed the molecular basis for their high potency. Thus, 39 and 43 are deserve for further study to develop potential drug candidates for the treatment of Alzheimer's disease.

Contributions of NR1H3 genetic polymorphisms to susceptibility and effects of narrowband UVB phototherapy to nonsegmental vitiligo.

Vitiligo is the most common depigmenting disorder to which both genetic and environmental factors contribute. The aim of the current work was to evaluate the relationship between polymorphisms of the gene nuclear receptor subfamily 1 Group H member 3 (NR1H3) and the risk of vitiligo and phototherapy effects in the Chinese Han population. Two independent samples were enrolled to form the discovery set (comprised of 1668 nonsegmental vitiligo [NSV] patients and 2542 controls) and the validation set (comprised of 745 NSV patients and 1492 controls). A total of 13 tag single nucleotide polymorphisms (SNPs) were genotyped in the samples from the discovery stage. SNPs that achieved nominal significance were validated in another independent sample set. The serum level of NR1H3 protein was assayed using enzyme-linked immunosorbent assay kits in the validation set. Genetic association analysis was carried out at allelic and genotypic levels. The therapeutic effects of significant SNPs were examined in the validation set. The SNP rs3758672 was significantly associated with NSV. The A allele was correlated with NSV risk and poorer therapeutic effects. The A allele was strongly correlated with the increased level of serum NR1H3 in both controls and patients. In summary, SNP rs3758672 in NR1H3 was significantly associated with both disease susceptibility and individualized therapeutic effects of NSV in study participants with Han Chinese ancestry.

Clinical and genetic findings in a Chinese cohort with choroideremia.

OBJECTIVE: Choroideremia (CHM) is an X-linked chorioretinal dystrophy caused by variants in the CHM gene. The aim of this study was to report the clinical and genetic features of a cohort of affected males with CHM and establish the relationship between best correct visual acuity (BCVA) and age. METHOD: Twenty-seven patients from 24 unrelated families underwent detailed ophthalmic examinations and comprehensive molecular genetic analysis. We combined the 27 patients in our own cohort with 68 Chinese patients from six previously reported studies to determine a transition age for BCVA rapid decline in 95 patients. RESULTS: Twenty-three causal (9 novel) CHM variants were identified in the 27 patients, who had a mean age of 30.5 ± 17.4 years and a mean BCVA (LogMAR) of 0.61 ± 0.79. Patients at different disease stages showed different extents of retinal pigment epithelium (RPE) and choroid abnormalities. Central retinal optical coherence tomography (OCT) scanning revealed defects in the ellipsoid zone and RPE in all patients and outer retinal tubulations in 75%. The 95 patients had a mean age of 33.27 ± 16.27 years and an average (LogMAR) of 0.72 ± 0.82. The BCVA did not decline rapidly before age 25, but decreased at a mean rate of 0.037logMAR/year after that age. CONCLUSIONS: Our results indicated Chinese patients with CHM variants have a younger transition age for rapid BCVA decline than previously reported for other ethnic groups. Central retinal OCT scanning can identify different abnormalities in the retinal structures, and these might be used as other parameters for monitoring disease progression in patients with CHM.

Current situation and progress of drugs for reducing intraocular pressure.

Glaucoma, the most common cause of irreversible blindness worldwide, usually causes characteristic optic nerve damage. Pathological intraocular pressure (IOP) elevation is a major risk factor. Drug reduction of IOP is the preferred treatment for clinicians because it can delay the progression of disease. However, the traditional IOP-lowering drugs currently used by patients may be poorly tolerated. Therefore, in recent years, some new drugs have been put into clinical application or in clinical phase I-III studies. They have a better IOP-lowering effect and fewer adverse reactions. Because glaucoma is a chronic disease, drugs need to be administered continuously for a long time. For patients, good compliance and high drug bioavailability have a positive effect on the prognosis of the disease. Therefore, clinicians and scientists have developed drug delivery systems to solve this complex problem. In addition, natural compounds and dietary supplements have a good effect of reducing IOP, and they can also protect the optic nerve through antioxidant action. We summarize the current traditional drugs, new drugs, sustained-release drug delivery systems, and complementary drugs and outline the mechanism of action and clinical effects of these drugs on glaucoma and their recent advances.

CRISPR/Cas9-mediated deletion of Fam83h induces defective tooth mineralization and hair development in rabbits.

Family with sequence similarity 83 members H (Fam83h) is essential for dental enamel formation. Fam83h mutations cause human amelogenesis imperfecta (AI), an inherited disorder characterized by severe hardness defects in dental enamel. Nevertheless, previous studies showed no enamel defects in Fam83h-knockout/lacZ-knockin mice. In this study, a large deletion of the Fam83h gene (900 bp) was generated via a dual sgRNA-directed CRISPR/Cas9 system in rabbits. Abnormal tooth mineralization and loose dentine were found in homozygous Fam83h knockout (Fam83h-/- ) rabbits compared with WT rabbits. In addition, reduced hair follicle counts in dorsal skin, hair cycling dysfunction and hair shaft differentiation deficiency were observed in Fam83h-/- rabbits. Moreover, X-rays and staining of bone sections showed abnormal bending of the ulna and radius and an ulnar articular surface with insufficient trabecular bone in Fam83h-/- rabbits. Taken together, these data are the first report of defective hair cycling, hair shaft differentiation and abnormal bending of the ulna and radius in Fam83h-/- rabbits. This novel Fam83h-/- rabbit model may facilitate understanding the function of Fam83h and the pathogenic mechanism of the Fam83h mutation.

Loss of BAF (mSWI/SNF) chromatin-remodeling ATPase Brg1 causes multiple malformations of cortical development in mice.

Mutations in genes encoding subunits of the BAF (BRG1/BRM-associated factor) complex cause various neurodevelopmental diseases. However, the underlying pathophysiology remains largely unknown. Here, we analyzed the function of Brahma-related gene 1 (Brg1), a core ATPase of BAF complexes, in the developing cerebral cortex. Loss of Brg1 causes several morphological defects resembling human malformations of cortical developments (MCDs), including microcephaly, cortical dysplasia, cobblestone lissencephaly and periventricular heterotopia. We demonstrated that neural progenitor cell renewal, neuronal differentiation, neuronal migration, apoptotic cell death, pial basement membrane and apical junctional complexes, which are associated with MCD formation, were impaired after Brg1 deletion. Furthermore, transcriptome profiling indicated that a large number of genes were deregulated. The deregulated genes were closely related to MCD formation, and most of these genes were bound by Brg1. Cumulatively, our study indicates an essential role of Brg1 in cortical development and provides a new possible pathogenesis underlying Brg1-based BAF complex-related neurodevelopmental disorders.

Phenotype-Based Genetic Analysis Reveals Missing Heritability of ABCA4-Related Retinopathy: Deep Intronic Variants and Copy Number Variations.

Purpose: To identify the missing heritability of ABCA4-related retinopathy in a Chinese cohort. Methods: We recruited 33 unrelated patients with ABCA4-related retinopathy carrying a monoallelic variant in ABCA4. All patients underwent ophthalmic examinations. Next-generation sequencing of the whole ABCA4 sequence, including coding and noncoding regions, was performed to detect deep intronic variants (DIVs) and copy number variations (CNVs). Results: We identified eight missing pathogenic ABCA4 variants in 60.6% of the patients (20/33), which comprised five DIVs and three CNVs. The five DIVs, including four novel (c.1555-816T>G, c.2919-169T>G, c.2919-884G>T, and c.5461-1321A>G) and one reported (c.4539+1100A>G), accounted for the missing alleles in 51.5% of the patients. Minigene assays showed that four novel DIVs activated cryptic splice sites leading to the insertions of pseudoexons. The three novel CNVs consisted of one gross deletion of 1273 bp (exon 2) and two gross duplications covering 25.2 kb (exons 28-43) and 9.4 kb (exons 38-44). The microhomology domains were identified at the breakpoints and revealed the potential mechanisms of CNV formation. Conclusions: DIVs and CNVs explained approximately two-thirds of the unresolved Chinese cases with ABCA4-related retinopathy. Combining results from phenotypic-directed screening, targeting the whole ABCA4 sequencing and in silico tools can help to identify the missing heritability.

Activation of Rictor/mTORC2 signaling acts as a pivotal strategy to protect against sensorineural hearing loss.

The Food and Drug Administration­approved drug sirolimus, which inhibits mechanistic target of rapamycin (mTOR), is the leading candidate for targeting aging in rodents and humans. We previously demonstrated that sirolimus could treat ARHL in mice. In this study, we further demonstrate that sirolimus protects mice against cocaine-induced hearing loss. However, using efficacy and safety tests, we discovered that mice developed substantial hearing loss when administered high doses of sirolimus. Using pharmacological and genetic interventions in murine models, we demonstrate that the inactivation of mTORC2 is the major driver underlying hearing loss. Mechanistically, mTORC2 exerts its effects primarily through phosphorylating in the AKT/PKB signaling pathway, and ablation of P53 activity greatly attenuated the severity of the hearing phenotype in mTORC2-deficient mice. We also found that the selective activation of mTORC2 could protect mice from acoustic trauma and cisplatin-induced ototoxicity. Thus, in this study, we discover a function of mTORC2 and suggest that its therapeutic activation could represent a potentially effective and promising strategy to prevent sensorineural hearing loss. More importantly, we elucidate the side effects of sirolimus and provide an evaluation criterion for the rational use of this drug in a clinical setting.

Versatile and efficient in vivo genome editing with compact Streptococcus pasteurianus Cas9.

Compact CRISPR-Cas9 systems that can be packaged into an adeno-associated virus (AAV) show promise for gene therapy. However, the requirement of protospacer adjacent motifs (PAMs) restricts the target scope. To expand this repertoire, we revisited and optimized a small Cas9 ortholog derived from Streptococcus pasteurianus (SpaCas9) for efficient genome editing in vivo. We found that SpaCas9 enables potent targeting of 5'-NNGYRA-3' PAMs, which are distinct from those recognized by currently used small Cas9s; the Spa-cytosine base editor (CBE) and Spa-adenine base editor (ABE) systems efficiently generated robust C-to-T and A-to-G conversions both in vitro and in vivo. In addition, by exploiting natural variation in the PAM-interacting domain, we engineered three SpaCas9 variants to further expand the targeting scope of compact Cas9 systems. Moreover, mutant mice with efficient disruption of the Tyr gene were successfully generated by microinjection of SpaCas9 mRNA and the corresponding single guide RNA (sgRNA) into zygotes. Notably, all-in-one AAV delivery of SpaCas9 targeting the Pcsk9 gene in adult mouse liver produced efficient genome-editing events and reduced its serum cholesterol. Thus, with distinct PAMs and a small size, SpaCas9 will broaden the CRISPR-Cas9 toolsets for efficient gene modifications and therapeutic applications.

Reduced off-target effect of NG-BE4max by using NG-HiFi system.

Recently, a rationally engineered SpCas9 variant (SpCas9-NG) that can recognize a minimal NG protospacer adjacent motif (PAM) was reported to expand the targeting scope in genome editing. However, increased genome-wide off-target mutations with this variant compared with SpCas9 were reported in previous studies. In addition, lower base editing frequencies and higher unintended off-target mutations were also found in Hoxc13-ablated rabbits generated by NG-BE4max in our study. Here, a high-fidelity base editor, NG-HiFi, in comparison to NG-BE4max, showed retention of on-target activity while exhibiting significantly decreased off-target activity in Hoxc13-ablated rabbits. Collectively, the improved specificity and reduced off-target effect of SpCas9-NG assisted in cytidine base editing with the NG-HiFi system, providing a promising tool to precisely model human diseases in rabbits.