In situ bioimaging is a powerful tool for directly observing the localization, expression, and interactions of nucleic acids or protein targets within cells, providing essential insights into cell function and disease mechanisms. In recent years, the CRISPR/Cas9 system, a revolutionary gene-editing tool, has been applied to develop efficient in situ imaging techniques. This paper reviews recent CRISPR/Cas9-based imaging methods utilizing Cas9 protein, engineered single-guide RNA (sgRNA), and coupled fluorescent tags, and compares their application in living and fixed cells. It focuses on the specificity, signal amplification efficiency, and multi-modal imaging capabilities of these methods, with further discussion based on current research, aiming to offer a comprehensive overview of CRISPR/Cas9-based in situ bioimaging techniques, with some valuable reference and guidance for research in related fields.

Fluorescence-guided surgery (FGS) with tumor-targeted imaging agents, particularly those using the near-infrared wavelength, has emerged as a real-time technique to highlight the tumor location and margins during a surgical procedure. For accurate visualization of prostate cancer (PCa) boundary and lymphatic metastasis, we developed a new approach involving an efficient self-quenched near-infrared fluorescence probe, Cy-KUE-OA, with dual PCa-membrane affinity. Cy-KUE-OA specifically targeted the prostate-specific membrane antigen (PSMA), anchored into the phospholipids of the cell membrane of PCa cells and consequently showed a strong Cy7-de-quenching effect. This dual-membrane-targeting probe allowed us to detect PSMA-expressing PCa cells both in vitro and in vivo and enabled clear visualization of the tumor boundary during fluorescence-guided laparoscopic surgery in PCa mouse models. Furthermore, the high PCa preference of Cy-KUE-OA was confirmed on surgically resected patient specimens of healthy tissues, PCa, and lymph node metastases. Taken together, our results serve as a bridge between preclinical and clinical research in FGS of PCa and lay a solid foundation for further clinical research.

Objective:To evaluate the clinical value of Xing's ureteroileal anastomosis technique in radical cystectomy.Methods:The data of 38 patients who underwent radical cystectomy with Xing's ureteroileal anastomosis technique at Cancer Hospital, Chinese Academy of Medical Sciences and Beijing Chaoyang Hospital from July 2013 to June 2021 were retrospectively reviewed. There were 30 males and 8 females. The mean age was 61.6±15.1 years old. The mean body mass index (BMI) was 25.1±2.7 kg/m 2. The American Society of Anesthesiology (ASA) graded 25 cases as grade 1, 10 cases as grade 2 and 3 cases as grade 3. There were 35 cases with stage cT 2N 0M 0 and 3 cases with cT 3N 0M 0. All patients underwent radical cystectomy and ileal conduit, and the ureteroileal anastomosis was performed using the Xing's ureteroileal anastomosis technique. Afferent loop entry was divided equally into two lumens. After 1.5 cm-long lengthwise incisions, each ureter was directly and end-to-end anastomosed to the aforementioned lumens. Postoperative information was recorded, including ureteric stricture, ureteric reflux, hydronephrosis, anastomotic leakage, renal calculus, urinary tract infection, and pyelonephritis. Results:Ureteroileal anastomosis was performed successfully in 38 cases with 76 units. The median follow-up time was 35.6 (17.0, 46.3) months. Three patients developed unilateral anastomotic stenosis after operation. Five patients had unilateral ureteral reflux. Two patients had unilateral hydronephrosis. No anastomotic leakage, urinary tract infection, or pyelonephritis occurred after the operation. Renal calculus appeared in 3 cases, all on the left unit.Conclusions:Xing's ureteroileal anastomosis technique is a simple method with few postoperative and good functional outcomes.

Objective To explore the feasibility and clinical effect of laparoscopic radical cystectomy with intracorporeal Xing's orthotopic neobladder.Methods Forty-one patients who underwent laparoscopic radical cystectomy with intracorporeal Xing's orthotopic neobladder from July 2013 to August 2019.There were 31 cases performed in Beijing Chaoyang hospital and 10 cases in National Cancer Center.Mean age was 59 (range 44-78) years,mean BMI was 25.3 (range 20.1-34.7) kg/m2,and mean CCI was 3 (range 2-6).No urethral stricture or urinary incontinence was found by preoperative examination.No distant metastasis was identified by bone scans,chest X-ray and sonography.Cystoscopy or TURBT was performed on all patients and biopsy was taken to confirm the diagnosis.Preoperative pathology showed 30 cases (73.2％) of MIBC,9 cases of NMIBC (22.0％) and 2 cases (4.9％) of in-situ cancer.Laparoscopic radical cystectomy and lymphadenectomy were performed under general anesthesia.Urinary diversion was completed in the peritoneal cavity,by intercepting the terminal ileum about 60 cm,and taking the proximal ileum 10 cm as input loop on the right side with proximal to distal way,and the middle 40 cm ileum was detubated.After u-shaped suture,the ileum was folded back and stitched into a sphere building a novd orthotopic neobladder with bilateral isoperistaltic afferent limbs.The prognosis of perioperative data and postoperative satisfaction regarding continence were analyzed,continence was defined as 0-1 pad/day.The 41 patients were divided into two groups to compare the difference in term of operation time and blood loss between the first 21 patients and the last 20 patients.Results Mean total operative time was 324.9 mins (range 210-480) mins,and mean estimated blood loss was 177.6(range 50-700) ml.There were significant statistical differences in term of total operation time,construction time and blood loss between the first 21 patients and the next 20 patients (P ＜ 0.05).Postoperative pathological results were urothelial carcinoma in 40 cases (2 in situ carcinoma) and small cell carcinoma in 1 case.Mean number of dissected lymph nodes was 19 (range 11-58),with 7 cases(17.1％)of positive lymph nodes,and 3 cases(7.3％) had positive surgical margin.At a mean follow up of 17.6 (range 2-64) months,36 patients (87.8％) survived,including 2 patients (4.9％) with metastasis and 1 patient (2.4％) with recurrence,and 5 cases (12.2％)died.All patients were able to urinate without catheterization.Thirty-seven patients (90.2％) were satisfied with voiding control during the daytime (0-1 urinal pad),and 29 patients (70.7％) were satisfied with voiding control at nighttime (0-1 urinal pad) by the follow-up 12 months after the operation.Conclusions Total laparoscopic radical cystectomy combined with Xing's orthotopic ileum neobladder is a simple method with fewer postoperative complications and a satisfactory continence rate.