The structural basis for 2'-5'/3'-5'-cGAMP synthesis by cGAS.

cGAS activates innate immune responses against cytosolic double-stranded DNA. Here, by determining crystal structures of cGAS at various reaction stages, we report a unifying catalytic mechanism. apo-cGAS assumes an array of inactive conformations and binds NTPs nonproductively. Dimerization-coupled double-stranded DNA-binding then affixes the active site into a rigid lock for productive metal•substrate binding. A web-like network of protein•NTP, intra-NTP, and inter-NTP interactions ensures the stepwise synthesis of 2'-5'/3'-5'-linked cGAMP while discriminating against noncognate NTPs and off-pathway intermediates. One divalent metal is sufficient for productive substrate binding, and capturing the second divalent metal is tightly coupled to nucleotide and linkage specificities, a process which manganese is preferred over magnesium by 100-fold. Additionally, we elucidate how mouse cGAS achieves more stringent NTP and linkage specificities than human cGAS. Together, our results reveal that an adaptable, yet precise lock-and-key-like mechanism underpins cGAS catalysis.

Pharmacological potential of cyclic nucleotide signaling in immunity.

Cyclic nucleotides are important signaling molecules that play many critical physiological roles including controlling cell fate and development, regulation of metabolic processes, and responding to changes in the environment. Cyclic nucleotides are also pivotal regulators in immune signaling, orchestrating intricate processes that maintain homeostasis and defend against pathogenic threats. This review provides a comprehensive examination of the pharmacological potential of cyclic nucleotide signaling pathways within the realm of immunity. Beginning with an overview of the fundamental roles of cAMP and cGMP as ubiquitous second messengers, this review delves into the complexities of their involvement in immune responses. Special attention is given to the challenges associated with modulating these signaling pathways for therapeutic purposes, emphasizing the necessity for achieving cell-type specificity to avert unintended consequences. A major focus of the review is on the recent paradigm-shifting discoveries regarding specialized cyclic nucleotide signals in the innate immune system, notably the cGAS-STING pathway. The significance of cyclic dinucleotides, exemplified by 2'3'-cGAMP, in controlling immune responses against pathogens and cancer, is explored. The evolutionarily conserved nature of cyclic dinucleotides as antiviral agents, spanning across diverse organisms, underscores their potential as targets for innovative immunotherapies. Findings from the last several years have revealed a striking diversity of novel bacterial cyclic nucleotide second messengers which are involved in antiviral responses. Knowledge of the existence and precise identity of these molecules coupled with accurate descriptions of their associated immune defense pathways will be essential to the future development of novel antibacterial therapeutic strategies. The insights presented herein may help researchers navigate the evolving landscape of immunopharmacology as it pertains to cyclic nucleotides and point toward new avenues or lines of thinking about development of therapeutics against the pathways they regulate.

The role of cGAMP via the STING pathway in modulating germinal center responses and CD4 T cell differentiation.

Germinal center (GC) responses are essential for establishing protective, long-lasting immunity through the differentiation of GC B cells (BGC) and plasma cells (BPC), along with the generation of antigen-specific antibodies. Among the various pathways influencing immune responses, the STING (Stimulator of Interferon Genes) pathway has emerged as significant, especially in innate immunity, and extends its influence to adaptive responses. In this study, we examined how the STING ligand cGAMP can modulate these key elements of the adaptive immune response, particularly in enhancing GC reactions and the differentiation of BGC, BPC, and follicular helper T cells (TFH). Employing in vivo models, we evaluated various antigens and the administration of cGAMP in Alum adjuvant, investigating the differentiation of BGC, BPC, and TFH cells, along with the production of antigen-specific antibodies. cGAMP enhances the differentiation of BGC and BPC, leading to increased antigen-specific antibody production. This effect is shown to be type I Interferon-dependent, with a substantial reduction in BPC frequency upon interferon (IFN)-ß blockade. Additionally, cGAMP's influence on TFH differentiation varies over time, which may be critical for refining vaccine strategies. The findings elucidate a complex, antigen-specific influence of cGAMP on T and B cell responses, providing insights that could optimize vaccine efficacy.

Redundant role of OsCNGC4 and OsCNGC5 encoding cyclic nucleotide-gated channels in rice pollen germination and tube growth.

In staple crops, such as rice (Oryza sativa L.), pollen plays a crucial role in seed production. However, the molecular mechanisms underlying rice pollen germination and tube growth remain underexplored. Notably, we recently uncovered the redundant expression and mutual interaction of two rice genes encoding cyclic nucleotide-gated channels (CNGCs), OsCNGC4 and OsCNGC5, in mature pollen. Building on these findings, the current study focused on clarifying the functional roles of these two genes in pollen germination and tube growth. To overcome functional redundancy, we produced gene-edited rice plants with mutations in both genes using the CRISPR-Cas9 system. The resulting homozygous OsCNGC4 and OsCNGC5 gene-edited mutants (oscngc4/5) exhibited significantly lower pollen germination rates than the wild type (WT), along with severely reduced fertility. Transcriptome analysis of the double oscngc4/5 mutant revealed downregulation of genes related to receptor kinases, transporters, and cell wall metabolism. To identify the direct regulators of OsCNGC4, which form a heterodimer with OsCNGC5, we screened a yeast two-hybrid library containing rice cDNAs from mature anthers. Subsequently, we identified two calmodulin isoforms (CaM1-1 and CaM1-2), NETWORKED 2 A (NET2A), and proline-rich extension-like receptor kinase 13 (PERK13) proteins as interactors of OsCNGC4, suggesting its roles in regulating Ca2+ channel activity and F-actin organization. Overall, our results suggest that OsCNGC4 and OsCNGC5 may play critical roles in pollen germination and elongation by regulating the Ca2+ gradient in growing pollen tubes.

How enzyme-centered approaches are advancing research on cyclic oligo-nucleotides.

Cyclic nucleotides are the most diversified category of second messengers and are found in all organisms modulating diverse pathways. While cAMP and cGMP have been studied over 50 years, cyclic di-nucleotide signaling in eukaryotes emerged only recently with the anti-viral molecule 2´3´cGAMP. Recent breakthrough discoveries have revealed not only the astonishing chemical diversity of cyclic nucleotides but also surprisingly deep-rooted evolutionary origins of cyclic oligo-nucleotide signaling pathways and structural conservation of the proteins involved in their synthesis and signaling. Here we discuss how enzyme-centered approaches have paved the way for the identification of several cyclic nucleotide signals, focusing on the advantages and challenges associated with deciphering the activation mechanisms of such enzymes.

Ginkgetin effectively mitigates collagen and AA-induced platelet activation via PLCγ2 but not cyclic nucleotide-dependent pathway in human.

Platelets assume a pivotal role in the cardiovascular diseases (CVDs). Thus, targeting platelet activation is imperative for mitigating CVDs. Ginkgetin (GK), from Ginkgo biloba L, renowned for its anticancer and neuroprotective properties, remains unexplored concerning its impact on platelet activation, particularly in humans. In this investigation, we delved into the intricate mechanisms through which GK influences human platelets. At low concentrations (0.5-1 µM), GK exhibited robust inhibition of collagen and arachidonic acid (AA)-induced platelet aggregation. Intriguingly, thrombin and U46619 remained impervious to GK's influence. GK's modulatory effect extended to ATP release, P-selectin expression, intracellular calcium ([Ca2+ ]i) levels and thromboxane A2 formation. It significantly curtailed the activation of various signaling cascades, encompassing phospholipase Cγ2 (PLCγ2)/protein kinase C (PKC), phosphoinositide 3-kinase/Akt/glycogen synthase kinase-3ß and mitogen-activated protein kinases. GK's antiplatelet effect was not reversed by SQ22536 (an adenylate cyclase inhibitor) or ODQ (a guanylate cyclase inhibitor), and GK had no effect on the phosphorylation of vasodilator-stimulated phosphoproteinSer157 or Ser239 . Moreover, neither cyclic AMP nor cyclic GMP levels were significantly increased after GK treatment. In mouse studies, GK notably extended occlusion time in mesenteric vessels, while sparing bleeding time. In conclusion, GK's profound impact on platelet activation, achieved through inhibiting PLCγ2-PKC cascade, culminates in the suppression of downstream signaling and, ultimately, the inhibition of platelet aggregation. These findings underscore the promising therapeutic potential of GK in the CVDs.

Fluorinated cGAMP analogs, which act as STING agonists and are not cleavable by poxins: Structural basis of their function.

The cGAS-STING pathway is a crucial part of innate immunity; it serves to detect DNA in the cytoplasm and to defend against certain cancers, viruses, and bacteria. We designed and synthesized fluorinated carbocyclic cGAMP analogs, MD1203 and MD1202D (MDs), to enhance their stability and their affinity for STING. These compounds demonstrated exceptional activity against STING. Despite their distinct chemical modifications relative to the canonical cyclic dinucleotides (CDNs), crystallographic analysis revealed a binding mode with STING that was consistent with the canonical CDNs. Importantly, MDs were resistant to cleavage by viral poxin nucleases and MDs-bound poxin adopted an unliganded-like conformation. Moreover, MDs complexed with poxin showed a conformation distinct from cGAMP bound to poxin, closely resembling their conformation when bound to STING. In conclusion, the development of MD1203 and MD1202D showcases their potential as potent STING activators with remarkable stability against poxin-mediated degradation-a crucial characteristic for future development of antivirals.

Second messenger 2'3'-cyclic GMP-AMP (2'3'-cGAMP): the cell autonomous and non-autonomous roles in cancer progression.

Cytosolic double-stranded DNA (dsDNA) is frequently accumulated in cancer cells due to chromosomal instability or exogenous stimulation. Cyclic GMP-AMP synthase (cGAS) acts as a cytosolic DNA sensor, which is activated upon binding to dsDNA to synthesize the crucial second messenger 2'3'-cyclic GMP-AMP (2'3'-cGAMP) that in turn triggers stimulator of interferon genes (STING) signaling. The canonical role of cGAS-cGAMP-STING pathway is essential for innate immunity and viral defense. Recent emerging evidence indicates that 2'3'-cGAMP plays an important role in cancer progression via cell autonomous and non-autonomous mechanisms. Beyond its role as an intracellular messenger to activate STING signaling in tumor cells, 2'3'-cGAMP also serves as an immunotransmitter produced by cancer cells to modulate the functions of non-tumor cells especially immune cells in the tumor microenvironment by activating STING signaling. In this review, we summarize the synthesis, transmission, and degradation of 2'3'-cGAMP as well as the dual functions of 2'3'-cGAMP in a STING-dependent manner. Additionally, we discuss the potential therapeutic strategies that harness the cGAMP-mediated antitumor response for cancer therapy.

Natural variation in CYCLIC NUCLEOTIDE-GATED ION CHANNEL 4 reveals a novel role of calcium signaling in vegetative phase change in Arabidopsis.

The timing of vegetative phase change (VPC) in plants is regulated by a temporal decline in the expression of miR156. Both exogenous cues and endogenous factors, such as temperature, light, sugar, nutrients, and epigenetic regulators, have been shown to affect VPC by altering miR156 expression. However, the genetic basis of natural variation in VPC remains largely unexplored. Here, we conducted a genome-wide association study on the variation of the timing of VPC in Arabidopsis. We identified CYCLIC NUCLEOTIDE-GATED ION CHANNEL 4 (CNGC4) as a significant locus associated with the diversity of VPC. Mutations in CNGC4 delayed VPC, accompanied by an increased expression level of miR156 and a corresponding decrease in SQUAMOSA PROMOTER BINDING-LIKE (SPL) gene expression. Furthermore, mutations in CNGC2 and CATION EXCHANGER 1/3 (CAX1/3) also led to a delay in VPC. Polymorphisms in the CNGC4 promoter contribute to the natural variation in CNGC4 expression and the diversity of VPC. Specifically, the early CNGC4 variant promotes VPC and enhances plant adaptation to local environments. In summary, our findings offer genetic insights into the natural variation in VPC in Arabidopsis, and reveal a previously unidentified role of calcium signaling in the regulation of VPC.

The CRISPR effector Cam1 mediates membrane depolarization for phage defence.

Prokaryotic type III CRISPR-Cas systems provide immunity against viruses and plasmids using CRISPR-associated Rossman fold (CARF) protein effectors1-5. Recognition of transcripts of these invaders with sequences that are complementary to CRISPR RNA guides leads to the production of cyclic oligoadenylate second messengers, which bind CARF domains and trigger the activity of an effector domain6,7. Whereas most effectors degrade host and invader nucleic acids, some are predicted to contain transmembrane helices without an enzymatic function. Whether and how these CARF-transmembrane helix fusion proteins facilitate the type III CRISPR-Cas immune response remains unknown. Here we investigate the role of cyclic oligoadenylate-activated membrane protein 1 (Cam1) during type III CRISPR immunity. Structural and biochemical analyses reveal that the CARF domains of a Cam1 dimer bind cyclic tetra-adenylate second messengers. In vivo, Cam1 localizes to the membrane, is predicted to form a tetrameric transmembrane pore, and provides defence against viral infection through the induction of membrane depolarization and growth arrest. These results reveal that CRISPR immunity does not always operate through the degradation of nucleic acids, but is instead mediated via a wider range of cellular responses.

Synthesis of Original Cyclic Dinucleotide Analogues Using the Sulfo-click Reaction.

The stimulator of interferon genes (STING) protein plays a crucial role in the activation of the innate immune response. Activation of STING is initiated by cyclic dinucleotides (CDNs) which prompted the community to synthesize structural analogues to enhance their biological properties. We present here the synthesis and biological evaluation of four novel CDN analogues composed of an N-acylsulfonamide linkage. These CDNs were obtained in high overall yields via the sulfo-click reaction as a key step.

Calcium-sensing receptor activator cinacalcet for treatment of cyclic nucleotide-mediated secretory diarrheas.

Cyclic nucleotide elevation in intestinal epithelial cells is the key pathology causing intestinal fluid loss in secretory diarrheas such as cholera. Current secretory diarrhea treatment is primarily supportive, and oral rehydration solution is the mainstay of cholera treatment. There is an unmet need for safe, simple and effective diarrhea treatments. By promoting cAMP hydrolysis, extracellular calcium-sensing receptor (CaSR) is a regulator of intestinal fluid transport. We studied the antidiarrheal mechanisms of FDA-approved CaSR activator cinacalcet and tested its efficacy in clinically relevant human cell, mouse and intestinal organoid models of secretory diarrhea. By using selective inhibitors, we found that cAMP agonists-induced secretory short-circuit currents (Isc) in human intestinal T84 cells are mediated by collective actions of apical membrane cystic fibrosis transmembrane conductance regulator (CFTR) and Clc-2 Cl- channels, and basolateral membrane K+ channels. 30 µM cinacalcet pretreatment inhibited all 3 components of forskolin and cholera toxin-induced secretory Isc by ∼75%. In mouse jejunal mucosa, cinacalcet inhibited forskolin-induced secretory Isc by ∼60% in wild type mice, with no antisecretory effect in intestinal epithelia-specific Casr knockout mice (Casr-flox; Vil1-cre). In suckling mouse model of cholera induced by oral cholera toxin, single dose (30 mg/kg) oral cinacalcet treatment reduced intestinal fluid accumulation by ∼55% at 20 hours. Lastly, cinacalcet inhibited forskolin-induced secretory Isc by ∼75% in human colonic and ileal organoids. Our findings suggest that CaSR activator cinacalcet has antidiarrheal efficacy in distinct human cell, organoid and mouse models of secretory diarrhea. Considering its excellent clinical safety profile, cinacalcet can be repurposed as a treatment for cyclic nucleotide-mediated secretory diarrheas including cholera.

Oomycete effector AVRblb2 targets cyclic nucleotide-gated channels through calcium sensors to suppress pattern-triggered immunity.

Transient and rapid increase in cytosolic Ca2+ plays a crucial role in plant-pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). Cyclic nucleotide-gated channels (CNGCs) have been implicated in mediating this Ca2+ influx; however, their regulatory mechanisms remain poorly understood. Here, we have found that AVRblb2 requires the calmodulin (CaM) and calmodulin-like (CML) proteins as co-factors to interact with the NbCNGCs, resulting in the formation of AVRblb2-CaM/CML-NbCNGCs complex. Furthermore, CaM and CML are dissociated from NbCNGC18 during PTI response to increase Ca2+ influx; however, Avrblb2 inhibits calcium channel activation by disrupting the release of CaM and CML from NbCNGC18. Following recognition of PAMP, NbCNGC18 forms active heteromeric channels with other NbCNGCs, which may give selectivity of CNGC complex against diverse signals for fine-tuning of cytosolic Ca2+ level to mediate appropriate responses. Silencing of multiple NbCNGCs compromised the function of AVRblb2 on the pathogenicity of Phytophthora infestans, confirming that AVRblb2 contributes to pathogen virulence by targeting CNGCs. Our findings provide new insights into the regulation of CNGCs in PTI and the role of pathogen effectors in manipulating host cell physiology to promote infection.

Phage anti-CBASS protein simultaneously sequesters cyclic trinucleotides and dinucleotides.

Cyclic-oligonucleotide-based anti-phage signaling system (CBASS) is a common immune system that uses cyclic oligonucleotide signals to limit phage replication. In turn, phages encode anti-CBASS (Acb) proteins such as Acb2, which can sequester some cyclic dinucleotides (CDNs) and limit downstream effector activation. Here, we identified that Acb2 sequesters many CDNs produced by CBASS systems and inhibits stimulator of interferon genes (STING) activity in human cells. Surprisingly, the Acb2 hexamer also binds with high affinity to CBASS cyclic trinucleotides (CTNs) 3'3'3'-cyclic AMP-AMP-AMP and 3'3'3'-cAAG at a distinct site from CDNs. One Acb2 hexamer can simultaneously bind two CTNs and three CDNs. Phage-encoded Acb2 provides protection from type III-C CBASS that uses cA3 signaling molecules. Moreover, phylogenetic analysis of >2,000 Acb2 homologs encoded by diverse phages and prophages revealed that most are expected to bind both CTNs and CDNs. Altogether, Acb2 sequesters nearly all known CBASS signaling molecules through two distinct binding pockets and therefore serves as a broad-spectrum inhibitor of cGAS-based immunity.

Insights into the metabolism, signaling, and physiological effects of 2',3'-cyclic nucleotide monophosphates in bacteria.

2',3'-cyclic nucleotide monophosphates (2',3'-cNMPs) have been discovered within both prokaryotes and eukaryotes in the past decade and a half, raising questions about their conserved existence in cells. In plants and mammals, wounding has been found to cause increased levels of 2',3'-cNMPs. Roles for 2',3'-cNMPs in plant immunity suggest that their regulation may be valuable for both plant hosts and microbial pathogens. In support of this hypothesis, a plethora of microbial enzymes have been found with activities related to these molecules. Studies in bacteria suggest that 2',3'-cNMPs are also produced in response to cellular stress and modulate expression of numerous genes. 2',3'-cNMP levels affect bacterial phenotypes, including biofilm formation, motility, and growth. Within E. coli and Salmonella enterica, 2',3'-cNMPs are produced by RNA degradation by RNase I, highlighting potential roles for Type 2 RNases producing 2',3'-cNMPs in a range of organisms. Development of cellular tools to modulate 2',3'-cNMP levels in bacteria has allowed for interrogation of the effects of 2',3'-cNMP concentration on bacterial transcriptomes and physiology. Pull-downs of cellular 2',3'-cNMP binding proteins have identified the ribosome and in vitro studies demonstrated that 2',3'-cNMPs decrease translation, suggesting a direct mechanism for 2',3-cNMP-dependent control of bacterial phenotypes. Future studies dissecting the cellular roles of 2',3'-cNMPs will highlight novel signaling pathways within prokaryotes and which can potentially be engineered to control bacterial physiology.

SMPDL3A is a cGAMP-degrading enzyme induced by LXR-mediated lipid metabolism to restrict cGAS-STING DNA sensing.

Lipid metabolism has been associated with the cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS) stimulator of interferon genes (STING) DNA-sensing pathway, but our understanding of how these signals are integrated into a cohesive immunometabolic program is lacking. Here, we have identified liver X receptor (LXR) agonists as potent inhibitors of STING signaling. We show that stimulation of lipid metabolism by LXR agonists specifically suppressed cyclic GMP-AMP (cGAMP)-STING signaling. Moreover, we developed cyclic dinucleotide-conjugated beads to biochemically isolate host effectors for cGAMP inhibition, and we found that LXR ligands stimulated the expression of sphingomyelin phosphodiesterase acid-like 3A (SMPDL3A), which is a 2'3'-cGAMP-degrading enzyme. Results of crystal structures suggest that cGAMP analog induces dimerization of SMPDL3A, and the dimerization is critical for cGAMP degradation. Additionally, we have provided evidence that SMPDL3A cleaves cGAMP to restrict STING signaling in cell culture and mouse models. Our results reveal SMPDL3A as a cGAMP-specific nuclease and demonstrate a mechanism for how LXR-associated lipid metabolism modulates STING-mediated innate immunity.

African swine fever virus B175L inhibits the type I interferon pathway by targeting STING and 2'3'-cGAMP.

IMPORTANCE: African swine fever virus (ASFV), the only known DNA arbovirus, is the causative agent of African swine fever (ASF), an acutely contagious disease in pigs. ASF has recently become a crisis in the pig industry in recent years, but there are no commercially available vaccines. Studying the immune evasion mechanisms of ASFV proteins is important for the understanding the pathogenesis of ASFV and essential information for the development of an effective live-attenuated ASFV vaccines. Here, we identified ASFV B175L, previously uncharacterized proteins that inhibit type I interferon signaling by targeting STING and 2'3'-cGAMP. The conserved B175L-zf-FCS motif specifically interacted with both cGAMP and the R238 and Y240 amino acids of STING. Consequently, this interaction interferes with the interaction of cGAMP and STING, thereby inhibiting downstream signaling of IFN-mediated antiviral responses. This novel mechanism of B175L opens a new avenue as one of the ASFV virulent genes that can contribute to the advancement of ASFV live-attenuated vaccines.

Vinylphosphonate-based cyclic dinucleotides enhance STING-mediated cancer immunotherapy.

Cyclic dinucleotides (CDNs) trigger the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway, which plays a key role in cytosolic DNA sensing and thus in immunomodulation against infections, cell damage and cancer. However, cancer immunotherapy trials with CDNs have shown immune activation, but not complete tumor regression. Nevertheless, we designed a novel class of CDNs containing vinylphosphonate based on a STING-affinity screening assay. In vitro, acyloxymethyl phosphate/phosphonate prodrugs of these vinylphosphonate CDNs were up to 1000-fold more potent than the clinical candidate ADU-S100. In vivo, the lead prodrug induced tumor-specific T cell priming and facilitated tumor regression in the 4T1 syngeneic mouse model of breast cancer. Moreover, we solved the crystal structure of this ligand bound to the STING protein. Therefore, our findings not only validate the therapeutic potential of vinylphosphonate CDNs but also open up opportunities for drug development in cancer immunotherapy bridging innate and adaptive immunity.

Arabinose- and xylose-modified analogs of 2',3'-cGAMP act as STING agonists.

Stimulator of interferon genes (STING) agonists are promising candidates for vaccine adjuvants and antitumor immune stimulants. The most potent natural agonist of STING, 2',3'-cyclic GMP-AMP (2',3'-cGAMP), is subject to nuclease-mediated inherent metabolic instability, thereby placing limits on its clinical efficacy. Here, we report on a new class of chemically synthesized sugar-modified analogs of 2',3'-cGAMP containing arabinose and xylose sugar derivatives that bind mouse and human STING alleles with high affinity. The co-crystal structures demonstrate that such analogs act as 2',3'-cGAMP mimetics that induce the "closed" conformation of human STING. These analogs show significant resistance to hydrolysis mediated by ENPP1 and increased stability in human serum, while retaining similar potency as 2',3'-cGAMP at inducing IFN-ß secretion from human THP1 cells. The arabinose- and xylose-modified 2',3'-cGAMP analogs open a new strategy for overcoming the inherent nuclease-mediated vulnerability of natural ribose cyclic nucleotides, with the additional benefit of high translational potential as cancer therapeutics and vaccine adjuvants.

Inhibition of Ryanodine Receptor 1 Reduces Endoplasmic Reticulum (ER) Stress and Promotes ER Protein Degradation in Cyclic Nucleotide-Gated Channel Deficiency.

The cone photoreceptor cyclic nucleotide-gated (CNG) channel plays a pivotal role in cone phototransduction. Mutations in genes encoding the channel subunits CNGA3 and CNGB3 account for about 80% of all cases of achromatopsia and are associated with progressive cone dystrophies. CNG channel deficiency leads to cellular/endoplasmic reticulum (ER) calcium dysregulation and ER stress-associated cone apoptosis. This work investigated the role of the ER calcium channel ryanodine receptor 1 (Ryr1) in ER stress and cone degeneration in CNG channel deficiency. The AAV-mediated CRISPR/SaCas9 genome editing was used to knock down Ryr1 specifically in cones. CNG channel-deficient mice displayed improved cone survival after subretinal injection of AAV2-SaCas9/gRNA-Ryr1, manifested as increased expression levels of cone proteins M-opsin, S-opsin, and cone arrestin. Knockdown of Ryr1 also led to reduced ER stress and increased expression levels of the ER-associated degradation proteins. This work demonstrates a role of Ryr1 in ER stress and cone degeneration in CNG channel deficiency, and supports strategies targeting ER calcium regulation for cone preservation.