Gene editing technology is a genetic operation technology that can modify the DNA sequence at the genomic level. The precision gene editing technology based on CRISPR/Cas9 system is a gene editing technology that is easy to operate and widely used. Unlike the traditional CRISPR/Cas9 system, the precision gene editing technology can perform site-directed mutation of genes without DNA template. This review summarizes the recent development of precision gene editing technology based on CRISPR/Cas9, and prospects the challenges and opportunities of this technology.
Gene Editing ; CRISPR-Cas Systems/genetics* ; Mutation ; Genome

Objective:Establishment of a new model of human primary colon cancer transplantation tumor in normal immune mice and to provide a reliable experimental animal model for studying the pathogenesis of colon cancer under normal immunity.Methods:Human colon cancer cells come from colon cancer patients who underwent surgery in the Affiliated Hospital of Jining Medical College in 2017. The mice in the cell control group were inoculated with phosphate buffered solution (PBS) containing colon cancer cells, the microcarrier control group was inoculated with PBS containing microcarrier 6, and the cell-microcarrier complex group was inoculated with the PBS containing colon cancer cell-microcarrier complex. The cells of each group were inoculated under the skin of the right axilla of mice by subcutaneous injection, and the time, size, tumor formation rate and pathological changes under microscope were recorded. The transplanted tumor tissue was immunohistochemically stained with the EnVisiion two-step method, and the tumor formation rate of the transplanted tumor was judged according to the proportion of positive cells in the visual field. The polymerase chain reaction (PCR) method was used to detect the expression of human-specific Alu sequence in mice tumor tissue.Results:After inoculation with tumor cells, the mice in the cell control group and the microcarrier control group did not die and did not form tumors; the mice in the cell-microcarrier complex group had palpable subcutaneous tumors in the right axillary subcutaneously on the 5th to 7th days after inoculation, and tumor formation rate is 67% (10/15), and the tumor volume can reach about 500 mm 3 2 to 3 weeks after vaccination. The immunohistochemistry results showed that CK20, CDX-2 and carcinoembryonic antigen were all positively expressed. The PCR results showed that the expression of human-specific Alu sequence can be detected in the transplanted tumor tissue of tumor-bearing mice. Conclusion:Human primary colon cancer cells used microcarrier 6 as a carrier to form tumors in normal immunized mice, and successfully established a new model of human colon cancer transplantation tumor in normal immune mice.

Objective:Establishment of a new model of human primary colon cancer transplantation tumor in normal immune mice and to provide a reliable experimental animal model for studying the pathogenesis of colon cancer under normal immunity.Methods:Human colon cancer cells come from colon cancer patients who underwent surgery in the Affiliated Hospital of Jining Medical College in 2017. The mice in the cell control group were inoculated with phosphate buffered solution (PBS) containing colon cancer cells, the microcarrier control group was inoculated with PBS containing microcarrier 6, and the cell-microcarrier complex group was inoculated with the PBS containing colon cancer cell-microcarrier complex. The cells of each group were inoculated under the skin of the right axilla of mice by subcutaneous injection, and the time, size, tumor formation rate and pathological changes under microscope were recorded. The transplanted tumor tissue was immunohistochemically stained with the EnVisiion two-step method, and the tumor formation rate of the transplanted tumor was judged according to the proportion of positive cells in the visual field. The polymerase chain reaction (PCR) method was used to detect the expression of human-specific Alu sequence in mice tumor tissue.Results:After inoculation with tumor cells, the mice in the cell control group and the microcarrier control group did not die and did not form tumors; the mice in the cell-microcarrier complex group had palpable subcutaneous tumors in the right axillary subcutaneously on the 5th to 7th days after inoculation, and tumor formation rate is 67% (10/15), and the tumor volume can reach about 500 mm 3 2 to 3 weeks after vaccination. The immunohistochemistry results showed that CK20, CDX-2 and carcinoembryonic antigen were all positively expressed. The PCR results showed that the expression of human-specific Alu sequence can be detected in the transplanted tumor tissue of tumor-bearing mice. Conclusion:Human primary colon cancer cells used microcarrier 6 as a carrier to form tumors in normal immunized mice, and successfully established a new model of human colon cancer transplantation tumor in normal immune mice.

Objective:To investigate the related factors that affect the timing and prognosis of early tracheostomy in patients with multiple rib fractures.Methods:A retrospective case series study was conducted on medical data of 222 patients with multiple rib fractures who underwent tracheostomy in Affiliated Hospital of Yangzhou University from February 2013 to October 2019，including 160 males and 66 females，with the age of 18 to 85 years ［（49.5 ± 16.3）years］. According to the practice management guidelines for tracheostomy timing and the use of propensity score matching technology，there were 118 patients with tracheostomy within 7 days of tracheal intubation （early group） and 104 patients with tracheostomy after 7 days of tracheal intubation （late group） before matching，and there were 87 patients in early group and 87 patients in late group after matching. Data were compared between groups including the gender，age，underlying disease，injury severity score （ISS），Glasgow coma score （GCS），number of fractured ribs，total number of rib fractures （NTRF），first rib fracture，flail chest，traumatic brain injury，combined injuries （spine，maxillofacial，sternum），acute respiratory distress syndrome （ARDS），volume fraction of pulmonary contusion（VPC），blood lactic acid （within 24 hours of admission），hemothorax，pneumothorax，mechanical ventilation time，duration of tracheostomy，time from tracheal intubation to incision，length of hospital stay，length of stay in ICU，closed thoracic drainage，number of fiberoptic bronchoscopy，multi-drug resistant bacteria infection，ventilator-associated pneumonia，antibiotic use time，duration of sedative and analgesic drugs used and 28-day mortality. The multivariate Logistic regression analysis was used to predict independent risk factors for early tracheostomy. The Pearson method was used to compare the relationship between multiple factors. The receiver operating characteristic （ROC） curve was used to predict indicators that affect the prognosis of patients with early tracheostomy，and calculate the best cut-off value. The Kaplan-Meier single factor and COX multivariate survival were used to analyze the relevant factors affecting the 28-day mortality of patients.Results:（1） In early group，the NTRF，ARDS and VPC were higher than those in late group，and the time from tracheal intubation to incision and 28-day mortality rate were lower than those in late group （ P < 0.05），while the two groups showed no significant differences in the gender，age，underlying diseases and ISS （ P > 0.05）. （2） The multivariate Logistic regression analysis showed that there was statistical significance in NTRF （ OR = 1.775，95% CI 1.439-2.188），ARDS（ OR = 3.740，95% CI 1.441-9.711），VPC （ OR = 1.087，95% CI 1.052-1.124） （ P < 0.05）； the Pearson method analysis showed a significant correlation between VPC and NTRF （ r = 0.369， P < 0.05） and a low degree of correlation between ARDS and VPC （ r = 0.179， P < 0.05），but there was no significant correlation between ARDS and NTRF （ r = 0.132， P > 0.05）. （3） The ROC curve analysis showed that the area under the curve （AUC） of the VPC and NTRF ［AUC = 0.832 （95% CI 0.770-0.893），AUC = 0.804 （95% CI 0.740-0.868）］ were significantly higher than those of the number of rib fractures ［AUC = 0.437（95% CI 0.352-0.523），GCS ［AUC = 0.519 （95% CI 0.432-0.605）］ and ISS ［AUC = 0.484 （95% CI 0.398-0.571）］ （ P < 0.05）. After calculating the Yorden index，the best cut-off value for VPC was 23.9，and the best cut-off value for NTRF was 8.5. （4） The Kaplan-Meier single factor and multivariate COX model survival analysis showed that the 28-day survival ratio of patients with early tracheostomy was significantly better than that of late tracheostomy （ P < 0.05）. Conclusions:The NTRF，ADRS and VPC are independent risk factors for the timing and prognosis of early tracheostomy. There is a significant correlation between VPC and NTRF. The VPC ≥ 23.9% and or NTRF ≥ 8.5 can be used to predict early tracheostomy in patients with multiple rib fractures. Early tracheostomy may benefit the 28-day survival of patients with multiple rib fractures.

Objective:To explore the relationship between anxiety, depression and various dimensions of cognitive function in patients with mild cognitive impairment (MCI) .Methods:The convenient sampling method was adopted to conduct memory screening for 727 elderly people who visited the multidisciplinary joint memory clinic of a Class Ⅲ Grade A hospital in Beijing from January to December 2019. With their consent, 247 MCI patients were investigated using Montreal Cognitive Assessment (MoCA) , Generalized Anxiety Disorder (GAD-7) and Patient Health Questionnaire (PHQ-9) .Results:A total of 247 questionnaires were distributed and 233 valid questionnaires were returned. There were 67 cases (28.8%) of MCI patients with anxiety and depression, 11 cases (4.7%) with anxiety, 53 cases (22.7%) with depression and 102 cases (43.8%) without anxiety and depression. In MCI patients, anxiety was positively correlated with delayed recall ( P<0.05) , while depression was negatively correlated with language function ( P<0.05) . MCI patients with different emotional states had statistically significant differences in language function ( P<0.05) . Conclusions:MCI patients have a higher incidence of anxiety and depression, and anxiety and depression will affect different dimensions of cognitive function in MCI patients. Medical staff need to provide targeted interventions for MCI patients with different emotional characteristics.

Objective To explore the effects of magnetic field on peripheral blood by comparing the peripheral hemogram of the soldiers and officers of radar troops and the personnel in physical examination.Methods One hundred and eighty-nine soldiers and officers of radar stations whose average age was (23.2±3.5) yr,radar working experience was more than 2 years and the habitation was in radiation area without any special protection measures,were selected as test group.Other 53 soldiers and officers who were received physical examination in Air Force General Hospital were selected as control group.Their average age was (21.3 ± 2.6) yr and they had no history of electromagnetic radiation exposure.The blood routine examination was done for 2 groups to compare and analyze the effects of magnetic field on peripheral blood.Results ① The abnormal rate of peripheral blood of test group was 60.90％.The hematocrit,mean corpuscular volume,mean corpuscular hemoglobin,mean platelet volume,red blood cell distribution width,lymphocyte,lymphocyte count was respectively higher than normal value,but the neutrophilic granulocyte was lower than normal value.The higher abnormal rate of lymphocyte,hematocrit,mean platelet volume,neutrophilic in test group was respectively 39.68％,21.69％,17.46％,17.46％.The abnormal rate of hematocrit and lymphocyte of control group was respectively 3.77％ and 5.66％.②The values of hematocrit,mean corpuscular volume,mean corpuscular hemoglobin,red blood cell distribution width,lymphocyte,lymphocyte count,eosinophil granulocyte,eosinophil granulocyte count,platelet hematocrit,mean platelet volume and platelet distribution width of test group were higher than those of control group (P＜0.05).The values of mean corpuscular hemoglobin concentration,neutrophilic and neutrophilic count were lower than those of control group (P ＜ 0.05).Collusions The magnetic field environment has resulted in a certain impact on the peripheral blood of the soldiers and officers of radar troops.Active protective measures should be taken to protect magnetic radiation.

Objective To study the change law of immunoglobulin (Ig) (IgG,IgA,IgM) and complement levels (C3,C4) in the peripheral blood of pilots by quantitative detection and to suggest the preventive measures.Methods IMMAGE800 instrument was used to detect the immunoglobulin and complement levels in 383 active pilots and 100 ground crews.Pilots were divided into bomber,transporter,helicopter and fighter groups by aircraft type.The ground crews were chosen as control group.Results Comparing to the control group,pilots showed lower peripheral blood IgG and IgM but higher C3 and C4 levels.Bomber group had lower IgG and higher C3 and C4 levels (P＜0.05).Transporter group had lower IgG and IgM but higher C3 and C4 levels (P＜0.05).Helicopter group showed lower IgG and IgM but higher C4 level (P＜0.05).Fighter group showed lower IgG but higher C3 and C4 levels (P＜0.05).Conclusions The changes on immunoglobulin and complement levels indicate the certain harmful effects on pilot's immune system.The environment factors may be the main reason led to pilot's lower IgG and IgM but higher C3 and C4 levels.The prevention of electromagnetic radiation in flight should be strengthened as the basic measures for maintaining pilot's immune functions.

Objective To explore the effects of magnetic field on peripheral blood by comparing the peripheral hemogram of the soldiers and officers of radar troops and the personnel in physical examination.Methods One hundred and eighty-nine soldiers and officers of radar stations whose average age was (23.2±3.5) yr,radar working experience was more than 2 years and the habitation was in radiation area without any special protection measures,were selected as test group.Other 53 soldiers and officers who were received physical examination in Air Force General Hospital were selected as control group.Their average age was (21.3 ± 2.6) yr and they had no history of electromagnetic radiation exposure.The blood routine examination was done for 2 groups to compare and analyze the effects of magnetic field on peripheral blood.Results ① The abnormal rate of peripheral blood of test group was 60.90％.The hematocrit,mean corpuscular volume,mean corpuscular hemoglobin,mean platelet volume,red blood cell distribution width,lymphocyte,lymphocyte count was respectively higher than normal value,but the neutrophilic granulocyte was lower than normal value.The higher abnormal rate of lymphocyte,hematocrit,mean platelet volume,neutrophilic in test group was respectively 39.68％,21.69％,17.46％,17.46％.The abnormal rate of hematocrit and lymphocyte of control group was respectively 3.77％ and 5.66％.②The values of hematocrit,mean corpuscular volume,mean corpuscular hemoglobin,red blood cell distribution width,lymphocyte,lymphocyte count,eosinophil granulocyte,eosinophil granulocyte count,platelet hematocrit,mean platelet volume and platelet distribution width of test group were higher than those of control group (P＜0.05).The values of mean corpuscular hemoglobin concentration,neutrophilic and neutrophilic count were lower than those of control group (P ＜ 0.05).Collusions The magnetic field environment has resulted in a certain impact on the peripheral blood of the soldiers and officers of radar troops.Active protective measures should be taken to protect magnetic radiation.

Objective To study the change law of immunoglobulin (Ig) (IgG,IgA,IgM) and complement levels (C3,C4) in the peripheral blood of pilots by quantitative detection and to suggest the preventive measures.Methods IMMAGE800 instrument was used to detect the immunoglobulin and complement levels in 383 active pilots and 100 ground crews.Pilots were divided into bomber,transporter,helicopter and fighter groups by aircraft type.The ground crews were chosen as control group.Results Comparing to the control group,pilots showed lower peripheral blood IgG and IgM but higher C3 and C4 levels.Bomber group had lower IgG and higher C3 and C4 levels (P＜0.05).Transporter group had lower IgG and IgM but higher C3 and C4 levels (P＜0.05).Helicopter group showed lower IgG and IgM but higher C4 level (P＜0.05).Fighter group showed lower IgG but higher C3 and C4 levels (P＜0.05).Conclusions The changes on immunoglobulin and complement levels indicate the certain harmful effects on pilot's immune system.The environment factors may be the main reason led to pilot's lower IgG and IgM but higher C3 and C4 levels.The prevention of electromagnetic radiation in flight should be strengthened as the basic measures for maintaining pilot's immune functions.

Objective To explore whether hypermethylation in the promoter of p16 gene and protein of p16 were associated with development and clinicopathological characteristics of colorectal cancer. Methods Methylation-specific PCR ( MSP) and immunohistochemistry SP were used to detected hypermethylation of p16 gene and p16 protein in tumor tissues from 32 patients with colorectal cancer. Results The hypermethylation of p16 gene was detected in 40. 6% of tumor tissues. The protein of p16 promoter was detected in 75% of tumor tissues. The hypermethylation of p16 gene was detected in 63% in Dukes stages of C and D tumors. The protein of p16 promoter was detected in 69% of tumor tissues. The hypermethylation of p16 gene was detected in 25% in the stages of A and B tumors. The protein of p16 promoter was detected in 81% of tumor tissues. The hpermethylation of p16 gene was detected in 100% in low differentiated carcinomas. The protein of p16 promoter was detected in 20% , the hypermethylation of p16 gene was detected in 30% , in the high and mediate differentiated carcinomas, the protein of p16 promoter was detected in 85%. Furthermore, the hypermethylation of p16 gene was detected in 63% in the lymph node metastasis and 25% in without lymph node metastasis. The protein of p16 promoter was detected in 65% in rectum and 100% in colon. Conclusions Our study demonstrated that p16 hypermethylation and protein were associated with the development of colorectal cancer and could be used as a putative prognostic indicator for this malignancy.