Profiling the impact of the promoters on CRISPR-Cas12a system in human cells.

The plasmid vector platform is the most commonly used vector for the expression of the versatile CRISPR-Cas technique and the promoter is a crucial element for the expression vector, thus profiling the impact of the promoters on CRISPR editors provides the basic information for the gene-editing toolkits and can be a guideline for its design. Herein, we made a parallel comparison among four commonly used promoters (CAG, ~ 1700 bp; EF1a core, ~ 210 bp; CMV, ~ 500 bp; and PGK, ~ 500 bp) in CRISPR-Cas12a system in mammalian cells to explore the impact of promoters on this powerful tool. We found that without badly damaging targeting specificity, the CAG promoter-driving Cas12a editor exhibited the most active (efficiency takes as 100%, specificity index = ~ 75%) in genomic cleavage, multiplex editing, transcriptional activation, and base editing, followed by promoter CMV (efficiency = 70 ~ 90% (vs CAG), specificity index = ~ 78%), and then EF1a core and PGK (both efficiency = 40-60%, vs CAG) but with higher specificity (specificity index = ~ 84% and ~ 82%, respectively). Therefore, CAG is recommended in the CRISPR-Cas12a system for the applications that need a robust editing activity but without size limitation, CMV mostly can be an alternative for CAG when requiring a smaller space, EF1a is similar to PGK with relatively high specificity, but has a smaller size, thus is more suitable for in vivo therapeutic applications. The data outlined the properties of the widely used promoters in the CRISPR-Cas12a system, which can be a guide for its applications and can be a useful resource for the gene-editing field.

Expression and clinical significance of a new neuroendocrine marker secretagogin in cervical neuroendocrine carcinoma.

AIMS: To explore the expression of secretagogin (SCGN) in neuroendocrine carcinoma of the uterine cervix and analyse its relationship with clinicopathological characteristics and prognosis. METHODS: From January 2010 to December 2017, 44 patients with cervical neuroendocrine carcinoma undergoing surgery were included in the study group, and 55 patients with cervical non-neuroendocrine carcinoma (including 30 cases of cervical squamous cell carcinoma and 25 cases of cervical adenocarcinoma) undergoing surgery were included in the control group. Immunohistochemical staining of SCGN was performed in both groups and compared with three common neuroendocrine markers, chromogranin A, synaptophysin (Syn) and CD56 in the study group. Detailed clinicopathological data of the two groups were analysed, and the patient survival in the study group was followed up. RESULTS: The positive expression of SCGN in cervical neuroendocrine carcinoma, cervical adenocarcinoma and squamous cell carcinoma was 65.9% (29/44), 8% (2/25) and 0%, respectively. The positive expression of SCGN in cervical neuroendocrine carcinoma was significantly higher than that in cervical adenocarcinoma and squamous cell carcinoma (χ2=44.5, p<0.001). There were no statistical differences among the positive expression of SCGN and three common neuroendocrine markers (p>0.05 for all). The intensity of SCGN staining in patients with cervical neuroendocrine carcinoma with lymph node metastasis was significantly higher than that in patients without lymph node metastasis (p=0.020). However, there was no significant association between SCGN expression and survival among patients with cervical neuroendocrine carcinoma (p=0.633). CONCLUSIONS: SCGN is a new neuroendocrine marker for cervical neuroendocrine carcinoma, whose expression correlates with lymph node metastasis.