One-year outcomes and safety assessment of faricimab in treatment-naïve patients with neovascular age-related macular degeneration in Japan.

This multicentre retrospective study evaluated the 1-year outcomes and safety profile of faricimab in treatment-naïve patients with neovascular age-related macular degeneration (nAMD). Fifty-five patients (57 eyes) underwent loading therapy comprising three monthly faricimab injections. If dryness was achieved by the third month, subsequent treat-and-extend (TAE) follow-up continued at a minimum 8-week interval thereafter. If wet macula persisted at the third month, a fourth dose was administered, followed by the TAE regimen. After 1 year, improvements in visual acuity (0.44 ± 0.46 [baseline] to 0.34 ± 0.48; p < 0.01) and central foveal thickness (326 ± 149 [baseline] to 195 ± 82 µm; p < 0.0001) were significant. Dry macula, characterised by the absence of intraretinal or subretinal fluid, was achieved in 65% of cases. Treatment intervals varied, ranging from 8 to 16 weeks, with 44% of eyes extending to a 16-week interval, followed by 33% at 8 weeks, 16% at 12 weeks, 5% at 14 weeks, and 2% at 10 weeks. Notably, 50% of the polypoidal choroidal vasculopathy patients exhibited complete regression of polypoidal lesions between 12 and 15 months. Faricimab treatment in nAMD patients induced significant improvements in central vision and retinal morphology. Two cases of retinal pigment epithelial tears and one case of iritis were reported as ocular complications.

Text-Guided Image Editing Based on Post Score for Gaining Attention on Social Media.

Text-guided image editing has been highlighted in the fields of computer vision and natural language processing in recent years. The approach takes an image and text prompt as input and aims to edit the image in accordance with the text prompt while preserving text-unrelated regions. The results of text-guided image editing differ depending on the way the text prompt is represented, even if it has the same meaning. It is up to the user to decide which result best matches the intended use of the edited image. This paper assumes a situation in which edited images are posted to social media and proposes a novel text-guided image editing method to help the edited images gain attention from a greater audience. In the proposed method, we apply the pre-trained text-guided image editing method and obtain multiple edited images from the multiple text prompts generated from a large language model. The proposed method leverages the novel model that predicts post scores representing engagement rates and selects one image that will gain the most attention from the audience on social media among these edited images. Subject experiments on a dataset of real Instagram posts demonstrate that the edited images of the proposed method accurately reflect the content of the text prompts and provide a positive impression to the audience on social media compared to those of previous text-guided image editing methods.

Development of T cell receptor-engineered T cells targeting the sarcoma-associated antigen papillomavirus binding factor.

We previously identified papillomavirus binding factor (PBF) as an osteosarcoma antigen recognized by an autologous cytotoxic T lymphocyte clone. Vaccination with PBF-derived peptide presented by HLA-A24 (PBF peptide) elicited strong immune responses. In the present study, we generated T cell receptor-engineered T cells (TCR-T cells) directed against the PBF peptide (PBF TCR-T cells). PBF TCR was successfully transduced into T cells and detected using HLA-A*24:02/PBF peptide tetramer. PBF TCR-T cells generated from a healthy donor were highly expanded and recognized T2-A24 cells pulsed with PBF peptide, HLA-A24+ 293T cells transfected with PBF cDNA, and sarcoma cell lines. To establish an adoptive cell therapy model, we modified the PBF TCR by replacing both α and ß constant regions with those of mice (hybrid PBF TCR). Hybrid PBF TCR-T cells also showed reactivity against T2-A24 cells pulsed with PBF peptide and to HLA-A24+ 293T cells transfected with various lengths of PBF cDNA including the PBF peptide sequence. Subsequently, we generated target cell lines highly expressing PBF (MFH03-PBF [short] epitope [+]) containing PBF peptide with in vivo tumorigenicity. Hybrid PBF TCR-T cells exhibited antitumor effects compared with mock T cells in NSG mice xenografted with MFH03-PBF (short) epitope (+) cells. CD45+ T cells significantly infiltrated xenografted tumors only in the hybrid PBF TCR T cell group and most of these cells were CD8-positive. CD8+ T cells also showed Ki-67 expression and surrounded the CD8-negative tumor cells expressing Ki-67. These findings suggest that PBF TCR-T cell therapy might be a candidate immunotherapy for sarcoma highly expressing PBF.

Low-Temperature Chiral Crystal Structure and Superconductivity in (Pt0.2Ir0.8)3Zr5.

We report the observation of superconductivity in (Pt0.2Ir0.8)3Zr5 with a chiral space group (P6122) at low temperatures. The bulk nature of the superconductivity at a transition temperature of 2.2 K was confirmed using specific heat measurements. We revealed that (Pt0.2Ir0.8)3Zr5 obeys the weak-coupling Bardeen-Cooper-Schrieffer model, and the dominant mechanism in the upper critical field is the orbital pair-breaking limit rather than the Pauli-Clogston limit. This indicates that the antisymmetric spin-orbit coupling caused by the chiral crystal structure does not significantly affect the superconductivity of (Pt0.2Ir0.8)3Zr5.

Observation of superconductivity and enhanced upper critical field of η-carbide-type oxide Zr4Pd2O.

We report the first observation of bulk superconductivity of a η-carbide-type oxide Zr4Pd2O. The crystal structure and the superconducting properties were studied through synchrotron X-ray diffraction, magnetization, electrical resistivity, and specific heat measurement. The superconducting transition was observed at Tc = 2.73 K. Our measurement revealed that the η-carbide-type oxide superconductor Zr4Pd2O shows an enhanced upper critical field µ0Hc2(0) = 6.72 T, which violates the Pauli-Clogston limit µ0HP = 5.29 T. On the other hand, we found that the enhanced upper critical field is absent in a Rh analogue Zr4Rh2O. The large µ0Hc2(0) of Zr4Pd2O would be raised from strong spin-orbit coupling with Pd-4d electrons. The discovery of new superconducting properties for Zr4Pd2O would shed light on the further development of η-carbide-type oxide superconductors.

Manipulation Direction: Evaluating Text-Guided Image Manipulation Based on Similarity between Changes in Image and Text Modalities.

At present, text-guided image manipulation is a notable subject of study in the vision and language field. Given an image and text as inputs, these methods aim to manipulate the image according to the text, while preserving text-irrelevant regions. Although there has been extensive research to improve the versatility and performance of text-guided image manipulation, research on its performance evaluation is inadequate. This study proposes Manipulation Direction (MD), a logical and robust metric, which evaluates the performance of text-guided image manipulation by focusing on changes between image and text modalities. Specifically, we define MD as the consistency of changes between images and texts occurring before and after manipulation. By using MD to evaluate the performance of text-guided image manipulation, we can comprehensively evaluate how an image has changed before and after the image manipulation and whether this change agrees with the text. Extensive experiments on Multi-Modal-CelebA-HQ and Caltech-UCSD Birds confirmed that there was an impressive correlation between our calculated MD scores and subjective scores for the manipulated images compared to the existing metrics.

Three-month outcomes of faricimab loading therapy for wet age-related macular degeneration in Japan.

This multicenter study aimed to assess the short-term effectiveness and safety of faricimab in treatment-naïve patients with wet age-related macular degeneration (wAMD) in Japan. We retrospectively reviewed 63 eyes of 61 patients with wAMD, including types 1, 2, and 3 macular neovascularization as well as polypoidal choroidal vasculopathy (PCV). Patients received three consecutive monthly intravitreal injections of faricimab as loading therapy. Over these 3 months, visual acuity improved gradually compared to baseline. Moreover, the central foveal thickness decreased significantly at 1, 2, and 3 months compared to baseline (p < 0.0001). At 3 months after initiation of faricimab therapy, a dry macula (defined as absence of intraretinal or subretinal fluid) was achieved in 82% of the eyes. Complete regression of polypoidal lesions was observed in 52% of eyes with PCV. Subfoveal choroidal thickness also decreased significantly at 1, 2, and 3 months compared to baseline (p < 0.0001). Although retinal pigment epithelium tears developed in two eyes, there were no other ocular or systemic complications observed during the 3 months of loading therapy. In conclusion, loading therapy using faricimab resulted in improved visual acuity and retinal morphology in Japanese patients with wAMD without particular safety issues.

Sign change in c-axis thermal expansion constant and lattice collapse by Ni substitution in transition-metal zirconide superconductor Co1-xNixZr2.

Recently, c-axis negative thermal expansion (NTE) was observed in a CoZr2 superconductor and related transition-metal zirconides. Here, we investigated the structural, electronic, and superconducting properties of Co1-xNixZr2 to achieve systematic control of c-axis NTE and switching from NTE to positive thermal expansion (PTE) by Ni substitution. At x ≤ 0.3, c-axis NTE was observed, and the thermal expansion constant αc approached zero with increasing x. At x = 0.4-0.6, c-axis thermal expansion close to zero thermal expansion (ZTE) was observed, and PTE appeared for x ≥ 0.7. On the superconducting properties, we observed bulk superconductivity for x ≤ 0.6, and bulk nature of superconductivity is suppressed by Ni heavy doping (x ≥ 0.7). For x ≤ 0.6, the evolution of the electronic density of states well explains the change in the superconducting transition temperature (Tc), which suggests conventional phonon-mediated superconductivity in the system. By analyzing the c/a ratio, we observed a possible collapsed transition in the tetragonal lattice at around x = 0.6-0.8. The lattice collapse would be the cause of the suppression of superconductivity in Ni-rich Co1-xNixZr2 and the switching from NTE to PTE.

Development of CAR-T cells specifically targeting cancer stem cell antigen DNAJB8 against solid tumours.

BACKGROUND: As therapy for solid tumours, various tumour antigens have been selected as targets, but CAR-T cells targeting these antigens have shown limited efficacy, in contrast to the effectiveness of CAR-T cells targeting haematological malignancies. In a previous report, we identified a cancer-testis antigen, DNAJB8. DNAJB8 plays a major role in tumorigenicity in cancer stem-like cells/cancer-initiating cells (CSCs/CICs). Here, we report a DNAJB8-reactive CAR yielding anti-tumour effects against renal cell carcinoma (RCC) and osteosarcoma. METHODS: We constructed a second-generation chimeric antigen receptor (CAR) against HLA-A*24:02/DNAJB8-derived peptide (DNAJB_143) complex (B10 CAR). The reactivity of B10-CAR T cells against T2-A24 cells pulsed with the cognate peptide and an RCC and osteosarcoma cell lines were quantified. The effects of adoptive cell transfer (ACT) therapy were assessed using in vivo xenografted mice models. RESULTS: B10 CAR-T cells recognised DNAJB8_143-pulsed T2-A24 cells and HLA-A*24:02(+)/DNAJB8(+) renal cell carcinoma and osteosarcoma cell lines. Moreover, ACT using B10 CAR-T cells showed anti-tumour effects against RCC and osteosarcoma cells. CONCLUSION: B10 CAR-T cells could show specific cytotoxicity against RCC and osteosarcoma cells in vitro and in vivo. B10 CAR-T cells targeting the CSC/CIC antigen DNAJB8 might be a candidate immunotherapy for carcinoma and sarcoma.

Reliability and accuracy of the critical shoulder angle measured by anteroposterior radiographs: using digitally reconstructed radiograph from 3-dimensional computed tomography images.

BACKGROUND: Accuracy of current standard radiographic measurement of the critical shoulder angle (CSA) is not well established. This study analyzed the reliability and accuracy of the CSA measurements obtained via anteroposterior (AP) radiographs, using a digitally reconstructed radiograph (true AP view) generated from a computed tomography image as the gold standard. METHODS: The CSA was measured on the radiographs and true AP views of 88 consecutive patients who had undergone shoulder arthroscopy for rotator cuff tears. Intraobserver and interobserver reliabilities of the CSA, measured by 2 orthopedic surgeons, were evaluated, and the average deviation of the CSA between radiographs and true AP views was calculated. Moreover, we compared the deviation of CSA between standard AP films (types A1 and C1) and nonstandard AP films (other types) against the Suter-Henninger criteria. RESULTS: Intraobserver and interobserver reliabilities were almost perfect on radiographs (0.96, 0.86) and true AP views (0.93, 0.85). The average deviation of CSA was 2.1° ± 1.6° for observer 1 and 2.2° ± 1.9° for observer 2. The percentage of cases with deviations of 2° or more when compared with the true AP view was 42% (37 of 88) for observer 1 and 53% (47 of 88) for observer 2. Only 22% (19 of 88) of films were standard AP films. The average deviation of CSA was not significantly different between standard and nonstandard AP films for observer 1 (standard 1.9° ± 1.3°; nonstandard 2.1° ± 1.7°; P = .76) and observer 2 (standard 1.6° ± 1.5°; nonstandard 2.4° ± 1.9°; P = .09). CONCLUSION: The CSA measurements using radiography were highly congruent, but a large measurement deviation occurred between radiographs and true AP views. The clinical usefulness and role of CSA in diagnosis require careful consideration.

Diagnostic accuracy of magnetic resonance imaging for partial tears of the long head of the biceps tendon in patients with rotator cuff tears.

Background: Magnetic resonance imaging (MRI) is useful for diagnosing shoulder diseases preoperatively. However, detection of partial tears of the long head of the biceps tendon (LHBT) using current clinical tests and imaging modalities is difficult. We aimed to evaluate the accuracy of radial-slice MRI for diagnosing partial tears of the LHBT. We hypothesized that radial-slice MRI may be a valuable diagnostic tool for assessing diagnosing tears of the LHBT. Methods: We retrospectively investigated 118 patients who underwent shoulder arthroscopy for rotator cuff tears. Intraoperative LHBT findings were compared with the identification of partial tears of the LHBT on conventional-slice MRI and radial-slice MRI, using a 3.0-T system. We calculated sensitivity, specificity, accuracy, and positive and negative predictive values for the detection of LHBT tears. Inter- and intraobserver reliability for radial-slice MRI was calculated using kappa statistics. Results: We diagnosed 69 patients (58%) without any LHBT tears and 49 with partial tears (42%), arthroscopically. Sensitivity, specificity, accuracy, and positive and negative predictive values of conventional-slice MRI for detection of partial tears of the LHBT were 52%, 94%, 78%, 92%, and 58%, respectively. Radial-slice MRI had 84% sensitivity, 90% specificity, 86% accuracy, and 92% positive and 80% negative predictive values for partial tears of the LHBT. Inter- and intraobserver reliability for radial-slice MRI was 0.69 and 0.74, respectively, corresponding to high reproducibility and defined as good. Conclusion: Radial-slice MRI demonstrated significantly higher sensitivity than conventional-slice MRI. These results indicate that radial-slice MRI is useful for diagnosing LHBT partial tears.

Relationship between preoperative size of rotator cuff tears measured using radial-slice magnetic resonance images and postoperative rotator cuff integrity: a prospective case-control study.

BACKGROUND: Magnetic resonance imaging (MRI) is useful for diagnosing shoulder diseases preoperatively. However, preoperative risk factors for retears have not been previously reported using a radial-slice MRI. Here, we investigated the relationship between the preoperative tear area of the rotator cuff evaluated using radial-slice MRI and the postoperative rotator cuff integrity. Our hypothesis is that larger tear area of the rotator cuff measured using radial-slice MRI would be associated with increased retear rates. METHODS: From June 2010 to October 2015, we treated 102 consecutive patients who underwent shoulder arthroscopy for reparable rotator cuff tears. The patient demographics, medical comorbidities, radiologic factors, tear size, fatty infiltration, muscle atrophy measured using oblique coronal and oblique sagittal MRI, and the tear area calculated using radial-slice MRI were assessed to compare the intact and retear groups in univariate and multivariate logistic regression analyses. The cutoff values of the independent factors were obtained using the receiver operating characteristic curve. RESULTS: Retears occurred in 15 of 102 (14.7%) patients. In the univariate analysis, significant differences were found between the two groups for tear size, fatty infiltration of the supraspinatus and infraspinatus, muscle atrophy, and tear area. In the multivariate analysis, the tear area was the independent factor that significantly affected the rate of retear. A tear area of 6.3 cm2 was the strongest predictor of retear with an area under the curve of 0.965, sensitivity of 86.7%, and specificity of 96.6%. CONCLUSION: The tear area was the independent factor that most significantly affected the rate of retear and showed excellent accuracy with a cutoff value of 6.3 cm2. Radial-slice MRI may be a valuable diagnostic tool for assessing the postoperative rotator cuff integrity.

Real-World Virological Efficacy and Safety of Ledipasvir and Sofosbuvir in Patients with Chronic Hepatitis C Virus Genotype 2 Infection: A Multicenter Study.

INTRODUCTION: The real-world virological efficacy and safety of interferon-free direct-acting antiviral (DAA) therapy with ledipasvir (LDV) plus sofosbuvir (SOF) were assessed in patients who were chronically infected with hepatitis C virus (HCV) genotype 2. METHODS: A total of 126 patients with chronic hepatitis C due to HCV genotype 2 infection who were treated with the LDV/SOF regimen were enrolled. The sustained virological response (SVR) rate and safety were analyzed. SVR was assessed in the intention-to-treat (ITT) population as well as in the modified intention-to-treat (mITT) population, which excluded patients with non-virological failure, including those who dropped out before the SVR assessment. RESULTS: The overall SVR rates of the ITT and mITT populations were 87.3% (95% confidence interval [CI] 80.2-92.6) (110/126) and 97.3% (95% CI 92.4-99.4) (110/113), respectively. In the mITT population, the percentages of patients with undetectable HCV RNA at 4, 8, and 12 weeks after the start of therapy were 92.9% (95% CI 86.5-96.9) (105/113), 99.1% (95% CI 95.2-100.0) (112/113), and 100.0% (95% CI 97.4-100.0) (113/113), respectively. Subgroup analyses of the mITT population showed no significant differences in SVR rates according to age, sex, HCV genotype (subtype), history of interferon-based therapy, baseline FIB-4 index, or baseline estimated glomerular filtration rate. In all subpopulations, the SVR rates were > 90%. There were no severe adverse events associated with the treatment. CONCLUSION: The LDV/SOF regimen showed high virological efficacy and acceptable safety in patients with HCV genotype 2 infection. TRIAL REGISTRATION: UMIN registration no. 000038604.

Osteosarcoma-initiating cells show high aerobic glycolysis and attenuation of oxidative phosphorylation mediated by LIN28B.

Osteosarcoma (OS) is a highly malignant bone tumor and the prognosis for non-responders to chemotherapy remains poor. Previous studies have shown that human sarcomas contain sarcoma-initiating cells (SIC), which have the characteristics of high tumorigenesis and resistance to chemotherapy. In the present study, we characterized SIC of a novel OS cell line, screened for SIC-related genes, and tried to regulate the proliferation of OS by metabolic interference. Initially, we established a new human OS cell line (OS13) and isolated clones showing higher tumorigenesis as SIC (OSHIGH ) and counterpart clones. OSHIGH cells showed chemoresistance and their metabolism highly depended on aerobic glycolysis and suppressed oxidative phosphorylation. Using RNA-sequencing, we identified LIN28B as a SIC-related gene highly expressed in OSHIGH cells. mRNA of LIN28B was expressed in sarcoma cell lines including OS13, but its expression was not detectable in normal organs other than the testis and placenta. LIN28B protein was also detected in various sarcoma tissues. Knockdown of LIN28B in OS13 cells reduced tumorigenesis, decreased chemoresistance, and reversed oxidative phosphorylation function. Combination therapy consisting of a glycolysis inhibitor and low-dose chemotherapy had antitumor effects. In conclusion, manipulation of glycolysis combined with chemotherapy might be a good adjuvant treatment for OS. Development of immunotherapy targeting LIN28B, a so-called cancer/testis antigen, might be a good approach.

Dorsal-to-Ventral Cortical Expansion Is Physically Primed by Ventral Streaming of Early Embryonic Preplate Neurons.

Despite recent studies elucidating the molecular mechanisms underlying cortical patterning and map formation, very little is known about how the embryonic pallium expands ventrally to form the future cortex and the nature of the underlying force-generating events. We find that neurons born at embryonic day 10 (E10) in the mouse dorsal pallium ventrally stream until E13, thereby superficially spreading the preplate, and then constitute the subplate from E14. From E11 to E12, the preplate neurons migrate, exerting pulling and pushing forces at the process and the soma, respectively. At E13, they are morphologically heterogeneous, with ∼40% possessing corticofugal axons, which are found to be in tension. Ablation of these E10-born neurons attenuates both deflection of radial glial fibers (by E13) and extension of the cortical plate (by E14), which should occur ventrally, and subsequently shrinks the postnatal neocortical map dorsally. Thus, the preplate stream physically primes neocortical expansion and arealization.

Differentiating cells mechanically limit the interkinetic nuclear migration of progenitor cells to secure apical cytogenesis.

Many proliferative epithelia are pseudostratified because of cell cycle-dependent interkinetic nuclear migration (IKNM, basal during G1 and apical during G2). Although most epithelia, including early embryonic neuroepithelia (≤100 µm thick), undergo IKNM over the entire apicobasal extent, more apicobasally elongated (300 µm) neural progenitor cells (radial glial cells) in the mid-embryonic mouse cerebral wall move their nuclei only within its apical (100 µm) compartment, leaving the remaining basal region nucleus-free (fiber-like). How this IKNM range [i.e. the thickness of a pseudostratified ventricular zone (VZ)] is determined remains unknown. Here, we report external fencing of IKNM and the VZ by differentiating cells. When a tight stack of multipolar cells immediately basal to the VZ was 'drilled' via acute neuron-directed expression of diphtheria toxin, IKNM of apicobasally connected progenitor cells continued further towards the basal region of the cell (200 µm). The unfencing-induced basally overshot nuclei stay in S phase for too long and do not move apically, suggesting that external limitation of IKNM is necessary for progenitors to undergo normal cytogenetic behaviors. Thus, physical collaboration between progenitors and differentiating cells, including neurons, underlies brain development.

Transpancreatic precut papillotomy versus double-guidewire technique in difficult biliary cannulation: prospective randomized study.

BACKGROUND AND STUDY AIMS: Difficult biliary cannulation and unintentional pancreatic duct cannulation are thought to be important contributors to pancreatitis occurring after endoscopic retrograde cholangiopancreatography. Our aim was to compare and evaluate the rates of success and complications of transpancreatic precut papillotomy (TPPP) and the double-guidewire technique (DGT), both with prophylactic pancreatic stenting. PATIENTS AND METHODS: From April 2011 to March 2014, patients with difficult biliary cannulation, in whom we planned to first position a guidewire in the pancreatic duct, were enrolled, and 68 patients were prospectively randomly allocated to two groups (TPPP 34, DGT 34). We evaluated the rates of success and complications for each group. RESULTS: TPPP had a significantly higher success rate (94.1 %) than DGT (58.8 %). The rate of post-ERCP pancreatitis was 2.9 % in both groups. There was no significant difference between the two groups in the overall rate of complications related to cannulation. CONCLUSION : If biliary cannulation cannot be achieved, TPPP should be selected first after unintentional pancreatic duct cannulation.

Short- and long-term results of endoscopic ultrasound-guided transmural drainage for pancreatic pseudocysts and walled-off necrosis.

AIM: To evaluate the short- and long-term results of endoscopic ultrasound-guided transmural drainage (EUS-GTD) for pancreatic fluid collection (PFC) and identify the predictive factors of treatment outcome for walled-off necrosis (WON) managed by EUS-GTD alone. METHODS: We investigated 103 consecutive patients with PFC who underwent EUS-GTD between September 1999 and August 2015. Patients were divided into four groups as follows: WON (n = 40), pancreatic pseudocyst (PPC; n = 11), chronic pseudocyst (n = 33), and others (n = 19). We evaluated the short- and long-term outcomes of the treatment. In cases of WON, multiple logistic regression analyses were performed to identify the predictor variables associated with the treatment success. In addition, PFC recurrence was examined in patients followed up for more than 6 mo and internal stent removal after successful EUS-GTD was confirmed. RESULTS: In this study, the total technical success rate was 96.1%. The treatment success rate of WON, PPC, chronic pseudocyst, and others was 57.5%, 90.9%, 91.0%, and 89.5%, respectively. Contrast-enhanced computed tomography using the multivariate logistic regression analysis revealed that the treatment success rate of WON was significantly lower in patients with more than 50% pancreatic parenchymal necrosis (OR = 17.0; 95%CI: 1.9-150.7; P = 0.011) and in patients with more than 150 mm of PFC (OR = 27.9; 95%CI: 3.4-227.7; P = 0.002).The recurrence of PFC in the long term was 13.3% (median observation time, 38.8 mo). Mean amylase level in the cavity was significantly higher in the recurrence group than in the no recurrence group (P = 0.02). CONCLUSION: The reduction of WON by EUS-GTD alone was associated with the proportion of necrotic tissue and extent of the cavity. The amylase level in the cavity may be a predictive factor for recurrence of PFC.

Crystal Structure of the Minimal Cas9 from Campylobacter jejuni Reveals the Molecular Diversity in the CRISPR-Cas9 Systems.

The RNA-guided endonuclease Cas9 generates a double-strand break at DNA target sites complementary to the guide RNA and has been harnessed for the development of a variety of new technologies, such as genome editing. Here, we report the crystal structures of Campylobacter jejuni Cas9 (CjCas9), one of the smallest Cas9 orthologs, in complex with an sgRNA and its target DNA. The structures provided insights into a minimal Cas9 scaffold and revealed the remarkable mechanistic diversity of the CRISPR-Cas9 systems. The CjCas9 guide RNA contains a triple-helix structure, which is distinct from known RNA triple helices, thereby expanding the natural repertoire of RNA triple helices. Furthermore, unlike the other Cas9 orthologs, CjCas9 contacts the nucleotide sequences in both the target and non-target DNA strands and recognizes the 5'-NNNVRYM-3' as the protospacer-adjacent motif. Collectively, these findings improve our mechanistic understanding of the CRISPR-Cas9 systems and may facilitate Cas9 engineering.