Practical Application Strategies of Cre-LoxP Conditional Gene Knockout / 中国生物化学与分子生物学报

ABSTRACT

Cre-loxP is an efficient recombination system originated from P1 phage. Its specific recombination patterns based on the locus of X-over P1 make it one of the most commonly used tools for gene editing in recent years. This paper focuses on the practical application of Cre-loxP system. Firstly, the functions and advantages of CRISPR/Cas9 system in Cre sequence insertion and loxP sequence insertion are analyzed. Then, a series of practical application problems of Cre-loxP system are described. For example, in this paper, the selection of Cre recombinase sequence in situ and safe site, the strategy of loxP sequence insertion, the identification of Cre recombinase tag protein, fluorescence identification of "ectopic" expression, primer design of PCR identification and reproductive strategy of mouse were described. At the same time, the optimization of Cre-loxP system in conditional gene knockout is introduced, such as ligand-induced Cre, promoter activated Cre, photo-induced Cre and activity modification of Cre. Through these optimized applications, we can obtain time-controlled conditional gene knockout, regulate the activity of Cre recombinase, and even avoid the toxicity of Cre recombinase itself. Finally, this paper discusses the defects and challenges of Cre-loxP system, and looks into the future development direction of Cre-loxP system. In summary, this paper reviews the practical application of gene knockout based on Cre-loxP system, summarizes the latest research progress and optimization strategies of Cre-loxP system, and prospects the future gene editing based on Cre-loxP system. This paper aims to provide theoretical guidance for solving practical operation problems based on Cre-loxP system, and to provide new research ideas for more accurate, more controllable and more adaptive gene editing in the future.