Strategies for site-specific recombination with high efficiency and precise spatiotemporal resolution

Abstract

Site-specific recombinases (SSRs) are invaluable genome engineering tools that have enormously boosted our understanding of gene functions and cell lineage relationships in developmental biology, stem cell biology, regenerative medicine, and multiple diseases. However, the ever-increasing complexity of biomedical research requires the development of novel site-specific genetic recombination technologies that can manipulate genomic DNA with high efficiency and fine spatiotemporal control. Here, we review the latest innovative strategies of the commonly used Cre-loxP recombination system and its combinatorial strategies with other site-specific recombinase systems. We also highlight recent progress with a focus on the new generation of chemical- and light-inducible genetic systems and discuss the merits and limitations of each new and established system. Finally, we provide the future perspectives of combining various recombination systems or improving well-established site-specific genetic tools to achieve more efficient and precise spatiotemporal genetic manipulation.

Site-Specific Recombination and the Development of Cisgenic Plants

“…Site-specific recombination systems have been studied since the 1980s. These include Cre-lox P ('causes/cyclization recombination/locus of crossing over, x, in P1'), FLP/FRP (flippase/flippase recognition target) and 𝛌 integrase [9]. Zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats-CRISPR-associated gene 9 (CRISPR-Cas9) is another group of nucleases that has been adapted for manipulation of DNA at specific sites [10].…”

“…The loxP site is composed of 34 bp consensus sequence consisting of an 8 bp nonsymmetrical central region flanked by two 13 bp palindromic sequences. Cre recombinase is a 38 kDa protein that catalyses the recombination of two loxP recognition sites on the same or different DNA strands using tyrosine 324 for the nucleophilic attack [9]. The recombination takes place via a Holliday junction intermediate formed by two antiparallel DNA molecules/segments to which a dimer of Cre recombinase subunits is bound to each loxP site.…”

Recent Advances in Implantation-Based Genetic Modeling of Biliary Carcinogenesis in Mice

“…The Cre-LoxP system has been widely used to identify tumor-initiating cells and develop tumors in a tissue- or organ-specific manner [ 25 , 26 , 27 , 28 ]. In mice with conditional alleles that contain two LoxP sequences, flanked lesions were spliced upon the induced expression of Cre recombinase.…”

Expression of mammalian proteins for diagnostics and therapeutics: a review

“…Thus, there are strategies to eliminate clonal variance while generating stable high expressing cell lines expressing protein of interest in a controllable, quantifiable, and efficient fashion. Site-specific targeting based on recombinases manipulate genomic DNA with high efficiency and fine spatiotemporal control by inserting the GOI into the chromatin in a transcriptionally active region, or genomic hot spot [ 99 , 100 ]. These systems do not possess preference for specific cell types but require genetically engineered parental cell lines with “landing pads” which are suitably arranged recombinase recognition sites as chromosomal targets [ 101 ].…”

“…Site-specific recombination is a precise genome editing tool to produce more controllable and predictable stable cell clones with high productivity and is likely to dramatically reduce the time needed for cell line screening, thus making them indispensable for biomedical research [ 79 ]. Due to their exceptional ability to excise, insert, inverse, and translocate genomic DNA in living organisms, site-specific recombinases are vital genome engineering techniques [ 100 ]. Anyhow, there are some technical issues that comes with the use of site-specific recombinase.…”