Advances in the Development of Gene-Targeting Vectors to Increase the Efficiency of Genetic Modification

Abstract

Gene targeting via homologous recombination, albeit highly inefficient in human cells, is considered a powerful tool for analyzing gene functions. Despite recent progress in the application of artificial nucleases for genome editing, safety issues remain a concern, particularly when genetic modification is used for therapeutic purposes. Therefore, the development of gene-targeting vectors is necessary for safe and sophisticated genetic modification. In this paper, we describe the effect of vector structure on random integration, which is a major obstacle in efficient gene targeting. In addition, we focus on the features of exon-trapping-type genetargeting vectors, and discuss a novel strategy for negative selection to enhance gene targeting in human cells.

Investigation of the molecular biology underlying the pronounced high gene targeting frequency at the Myh9 gene locus in mouse embryonic stem cells

“…Gene targeting experiments with these constructs were performed and HR events were identified as described above (Fig 1B and 1C). In contrast to previous studies [16,47,48], our results demonstrated that longer arms are not always associated with higher GC efficiency and also revealed that there is obvious difference in the GT efficiency at various sites even in the same locus. Briefly, the highest GT efficiency occurred at the exon2 site (91.7%), followed by that at the intron2 GC content represents the percentage of nucleotides in the strand that possesses either cytosine or guanine bases; Simple sequence repeat (SSR) consists of short, tandemly repeated di, tri-, tetra-or penta-nucleotide motifs; CpG island is a short stretch of DNA in which the frequency of the CG sequences is higher than other regions; SINEs denotes short interspersed nuclear elements; LINEs denotes long interspersed nuclear elements; � CpG island located at the position of 48-810.…”

“…Even in mouse ES cells, obtaining the desired ES clone has therefore been a critical but time-consuming and labor-intensive step in previous studies [3,10,11]. Numerous factors including the length of the homologous arms, and the structure of the targeting vector, the targeted loci, the utilization of isogenic DNA and the status of ES cells, can affect the HR efficiency have been investigated and described [12][13][14][15][16]. However, the HR efficiency varies greatly even when the general principles are complied with.…”

Guidelines for Fluorescent Guided Biallelic HDR Targeting Selection With PiggyBac System Removal for Gene Editing

“…The previous reported workflow faces challenges when trying to edit genomic regions that present a high density of repetitive elements since it increases the chances of having homologous recombination in other genomic regions (Saito and Adachi, 2016). In our previous work we modeled the influence of the different types of repetitive elements and showed that the presence of repetitive elements of the family Short Interspersed Nuclear Elements (SINE) in the homology arms present higher frequency of random integration (Arias-Fuenzalida et al, 2017).…”

Inactivation of Pol θ and C-NHEJ eliminates off-target integration of exogenous DNA

“…The mechanism of RI in higher eukaryotes has been enigmatic for decades, because it has evaded rigorous genetic definition. Integration of exogenous DNA at unpredictable positions in the genome can heavily impact on the functioning of the integrated DNA, as well as its genomic environment, and thus presents a safety risk 2–4 . With the advent of CRISPR-Cas9 technology and its seemingly inevitable implementation in clinically relevant gene-correction strategies 5 , it becomes all the more important to devise strategies to counteract the potentially detrimental outcomes of RI.…”