CRISPR/Gal4BD-Cas donor adapting systems based on miniaturized Cas proteins for improved gene editing.

Targeted precise point editing and knock-in can be achieved by homology-directed repair(HDR) based gene editing strategies in mammalian cells. However, the inefficiency of HDR strategies seriously restricts their application in precision medicine and molecular design breeding. In view of the problem that exogenous donor DNA cannot be efficiently recruited autonomously at double-stranded breaks(DSBs) when using HDR strategies for gene editing, the concept of donor adapting system(DAS) was proposed and the CRISPR/Cas9-Gal4BD DAS was developed previously. Due to the large size of SpCas9 protein, its fusion with the Gal4BD adaptor is inconvenient for protein expression, virus vector packaging and in vivo delivery. In this study, two novel CRISPR/Gal4BD-SlugCas9 and CRISPR/Gal4BD-AsCas12a DASs were further developed, using two miniaturized Cas proteins, namely SlugCas9-HF derived from Staphylococcus lugdunensis and AsCas12a derived from Acidaminococcus sp. Firstly, the SSA reporter assay was used to assess the targeting activity of different Cas-Gal4BD fusions, and the results showed that the fusion of Gal4BD with SlugCas9 and AsCas12a N-terminals had minimal distraction on their activities. Secondly, the HDR efficiency reporter assay was conducted for the functional verification of the two DASs and the corresponding donor patterns were optimized simultaneously. The results demonstrated that the fusion of the Gal4BD adaptor binding sequence at the 5'-end of intent dsDNA template (BS-dsDNA) was better for the CRISPR/Gal4BD-AsCas12a DAS, while for the CRISPR/Gal4BD-SlugCas9 DAS, the dsDNA-BS donor pattern was recommended. Finally, CRISPR/Gal4BD-SlugCas9 DAS was used to achieve gene editing efficiency of 24%, 37% and 31% respectively for EMX1, NUDT5 and AAVS1 gene loci in HEK293T cells, which was significantly increased compared with the controls. In conclusion, this study provides a reference for the subsequent optimization of the donor adapting systems, and expands the gene editing technical toolbox for the researches on animal molecular design breeding.

A case report: Splenic abscess caused by Burkholderia pseudomallei.

RATIONALE: Melioidosis is an emerging infectious disease caused by Burkholderia pseudomallei. To our knowledge, there have been very few cases of splenic abscesses due to melioidosis in Hainan, China. PATIENT CONCERNS: The patient was a 55-year-old male farmer, who was admitted in our hospital with persistent left epigastric dull pain accompanied by chills and febrile. One month before, the patient presented with persistent abdominal pain. After received anti-infection therapy, the subjective symptoms eased slightly, but recently he suffered from intermittent abdominal pain again. DIAGNOSES: Bacteria isolated from splenic pus were identified as B. pseudomallei by the Phoenix-100 system and indirect immunofluorescence. INTERVENTIONS: The patient was treated by surgical excision and anti-infection therapy. OUTCOMES: The patient was then treated with intravenous ceftazidime and oral trimethoprim-sulfamethoxazole for 2 weeks and his clinical symptoms improved. LESSONS: In endemic areas, B. pseudomallei should be considered as a causative organism of splenic abscess in patients with established risk factors. The isolation of B. pseudomallei from abscess sites is crucial to improve clinical outcomes by appropriate antimicrobial therapy coupled with surgical drainage.

[Hemolytic disease of neonates due to anti-M: report of one case and review of reports of 21 cases].

OBJECTIVE: To analyze and summarize clinical manifestation of hemolytic disease of the newborn (HDN) due to anti-M. METHODS: Data of one case of HDN due to anti-M and the reports of 21 cases seen in the past 20 years at the home country were reviewed and analyzed. RESULTS: There was an increasing number of reports of cases with HDN due to anti-M. Among the 22 cases, four were the first fetus. Of 18 infants, ten were male, and eight were female. The blood group was MN in 19/21 infants, and was M in 2/21 infants. The blood group was N in 10/21 mothers, and was NN in 11/21 mothers. Among the 18 infants, the direct antiglobulin test of 7 infants were positive, of 4 infants were dubiously positive, and of 7 infants was negative. Among the 16 infants, the antibody release test of 13 infants was positive, and of 3 infants were negative. Among 17 infants, the free antibody test of all was positive. Among the 21 mothers, the anti-M of IgG were positive in all mothers, and along with IgM in 11 mothers. The anti-M of IgG was positive in all infants. Mild or severe anemia and icterus were found in all cases. Among the 15 cases, jaundice was evident on the 1st day of life in 11 cases. Among 13 cases, marked elevation of both indirect- and direct-reacting bilirubin levels was reported in 4 cases. Phototherapy was applied when jaundice became evident. High-dose intravenous immunoglobulin was given to 4/15 cases. Exchange transfusion were performed in 8 of 22 cases. Three cases died, and 19 cases were cured. CONCLUSION: HDN of varying degrees of severity has been reported in association with anti-M and can even lead to intrauterine deaths or requiring treatment with exchange transfusion. If the mother has a history of prior intrauterine deaths, abortion, hydrops fetalis, severe fetal anemia or infertile, MN blood group and anti-M antibodies should be tested after excluding the possibility of other causes and HDN due to ABO or Rh blood group incompatibility. As the efficacy of phototherapy increases, the role of exchange transfusion in acute management is rapidly decreasing. High-dose intravenous immunoglobulin and/or intramuscular metalloporphyrins may further reduce the need for exchange transfusion. The exchange transfusion may be performed through peripheral arterial (drawn out) and venous (infused in) lines.