Biology subjects

Tei, C.

Publications and source records attributed to Tei, C..

3 recordsLinked to original sources

bioRxiv2024

Comprehensive analysis of end-modified long dsDNA donors in CRISPR-mediated endogenous tagging

CRISPR-mediated endogenous tagging is a powerful gene editing technique for studying protein dynamics and function in their native cellular environment. While the use of 5 modified DNA donors has emerged as a promising strategy to improve the typically low efficiency of knock-in gene editing, the underlying mechanisms remain poorly understood. In this study, we conducted a comprehensive analysis of end-modified long linear dsDNA donors in CRISPR-mediated endogenous tagging in human non-transformed cells. In-depth analysis of repair patterns reveals that 5 biotinylation of dsDNA donors significantly reduces imprecise insertions, thereby enhancing homology-directed repair (HDR)-mediated precise insertion efficiency. Notably, the impact of biotinylation on repair patterns resembles that of non-homologous end joining (NHEJ) pathway inhibition, suggesting its role in preventing NHEJ-mediated mis-integration. Moreover, combining biotin modification with NHEJ inhibitor treatment further improves bi-allelic knock-in efficiency. Overall, this study provides novel insights into the mechanisms by which 5 modifications enhance precise knock-ins and demonstrates their potential for achieving high-efficient, prercise endogenous tagging in human cells.

cell biology↗

bioRxiv2023

Comparable analysis of multiple DNA double-strand break repair pathways in CRISPR-mediated endogenous tagging

CRISPR-mediated endogenous tagging, utilizing the homology-directed repair (HDR) of DNA double-strand breaks (DSBs) with exogenously incorporated donor DNA, is a powerful tool in biological research. Inhibition of the non-homologous end joining (NHEJ) pathway has been proposed as a promising strategy for improving the low efficiency of accurate knock-in via the HDR pathway. However, the influence of alternative DSB repair pathways on gene knock-in remains to be fully explored. In this study, our long-read amplicon sequencing analysis reveals various patterns of imprecise repair in CRISPR/Cas-mediated knock-in, even under conditions where NHEJ is inhibited. Suppression of the microhomology-mediated end joining (MMEJ) or the single strand annealing (SSA) repair mechanisms leads to a reduction in distinct patterns of imprecise repair, thereby elevating the efficiency of accurate knock-in. Furthermore, a novel reporter system shows that the SSA pathway contributes to a specific pattern of imprecise repair, known as asymmetric HDR. Collectively, our study uncovers the involvement of multiple DSB repair pathways in CRISPR/Cas-mediated gene knock-in and proposes alternative approaches to enhance the efficiency of precise gene knock-in.

genomics↗

bioRxiv2022

ssDNA is not superior to dsDNA as long HDR donors for CRISPR-mediated endogenous gene tagging in human diploid cells

Recent advances in CRISPR technology have enabled us to perform gene knock-in in various species and cell lines. CRISPR-mediated knock-in requires donor DNA which serves as a template for homology-directed repair (HDR). For knock-in of short sequences or base substitutions, ssDNA donors are frequently used among various other forms of HDR donors, such as linear dsDNA. However, for insertion of long transgenes such as fluorescent reporters in human cells, the optimal type of HDR donors remains unclear. In this study, we established a simple and efficient CRISPR-mediated knock-in method for long transgenes using linear dsDNA and ssDNA donors, and systematically compared the performance of these two donors for endogenous gene tagging in human non-transformed diploid cells. Quantification using flow cytometry revealed higher efficiency of fluorescent tagging with dsDNA donors than with ssDNA. By analyzing knock-in outcomes using long-read amplicon sequencing and a classification framework, a variety of mis-integration events were detected regardless of the donor type. Importantly, the ratio of precise insertion was higher with dsDNA donors than with ssDNA. Moreover, in off-target integration analyses, dsDNA and ssDNA were comparably prone to non-homologous integration. These results indicate that ssDNA is not superior to dsDNA as long HDR donors for gene knock-in in human cells.

cell biology↗