OBJECTIVETo detect the distribution of clustered regularly interspaced short palindromic repeat (CRISPR) associated protein genes cas1 and cas2 in Shigella and to understand the characteristics of CRISPR with relationship between CRISPR and related characteristics on drug resistance.

METHODSCRISPR associated protein genes cas1 and cas2 in Shigella were detected by PCR, with its products sequenced and compared.

RESULTSThe CRISPR-associated protein genes cas1 and cas2 were found in all the 196 Shigella isolates which were isolated at different times and locations in China. Consistencies showed through related sequencing appeared as follows: cas2, cas1 (a) and cas1 (b) were 96.44%, 97.61% and 96.97%, respectively. There were two mutations including 3177129 site(C→G)and 3177126 site (G→C) of cas1 (b) gene in 2003135 strain which were not found in the corresponding sites of Z23 and 2008113.

RESULTSshowed that in terms of both susceptibility and antibiotic-resistance, strain 2003135 was stronger than Z23 and 2008113.

CONCLUSIONCRISPR system widely existed in Shigella, with the level of drug resistance in cas1 (b) gene mutant strains higher than in wild strains. Cas1 (b) gene mutation might be one of the reasons causing the different levels of resistance.

Bacterial Proteins ; genetics ; CRISPR-Associated Proteins ; genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Drug Resistance, Bacterial ; genetics ; Mutation ; Shigella ; genetics

In the recent years, the shortage of allo-skin sources has resulted in great challenges for salvage of patients with large area severe burns. Although being similar to human skin in construction and function, the clinical application of xenogenic porcine skin in burn wound management is limited due to factors including immuno-rejection, porcine endogenous retroviruses infection, etc. With the development of gene editing technology, especially the emerge of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein-9 system, multiple target genes could be possibly edited at the same time, which will bring broad prospect for the application of xenogenic porcine skin in the treatment of burn wounds. The paper mainly discusses the development, the existed barrier, the strategies of gene modification/editing, and the applications and research of xenogenic porcine skin xenografts in the clinical treatment of burn wound.
Animals ; Burns/surgery* ; CRISPR-Associated Proteins ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Humans ; Skin Transplantation/methods* ; Swine

Targeted point mutagenesis through homologous recombination has been widely used in genetic studies and holds considerable promise for repairing disease-causing mutations in patients. However, problems such as mosaicism and low mutagenesis efficiency continue to pose challenges to clinical application of such approaches. Recently, a base editor (BE) system built on cytidine (C) deaminase and CRISPR/Cas9 technology was developed as an alternative method for targeted point mutagenesis in plant, yeast, and human cells. Base editors convert C in the deamination window to thymidine (T) efficiently, however, it remains unclear whether targeted base editing in mouse embryos is feasible. In this report, we generated a modified high-fidelity version of base editor 2 (HF2-BE2), and investigated its base editing efficacy in mouse embryos. We found that HF2-BE2 could convert C to T efficiently, with up to 100% biallelic mutation efficiency in mouse embryos. Unlike BE3, HF2-BE2 could convert C to T on both the target and non-target strand, expanding the editing scope of base editors. Surprisingly, we found HF2-BE2 could also deaminate C that was proximal to the gRNA-binding region. Taken together, our work demonstrates the feasibility of generating point mutations in mouse by base editing, and underscores the need to carefully optimize base editing systems in order to eliminate proximal-site deamination.
APOBEC-1 Deaminase ; genetics ; metabolism ; Animals ; Bacterial Proteins ; genetics ; metabolism ; Base Sequence ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cytidine ; genetics ; metabolism ; Embryo Transfer ; Embryo, Mammalian ; Endonucleases ; genetics ; metabolism ; Gene Editing ; methods ; HEK293 Cells ; High-Throughput Nucleotide Sequencing ; Humans ; Mice ; Mice, Inbred C57BL ; Microinjections ; Plasmids ; chemistry ; metabolism ; Point Mutation ; RNA, Guide ; genetics ; metabolism ; Thymidine ; genetics ; metabolism ; Zygote ; growth & development ; metabolism ; transplantation