Abstract
In homology-directed repair, mediated knock-in single-stranded oligodeoxynucleotides (ssODNs) can be used as a homologous template and present high efficiency, but there is still a need to improve efficiency. Previous studies have mainly focused on controlling double-stranded break size, ssODN stability, and the DNA repair cycle. Nevertheless, there is a lack of research on the correlation between the cell cycle and single-strand template repair (SSTR) efficiency. Here, we investigated the relationship between cell cycle and SSTR efficiency. We found higher SSTR efficiency during mitosis, especially in the metaphase and anaphase. A Cas9 protein with a nuclear localization signal (NLS) readily migrated to the nucleus; however, the nuclear envelope inhibited the nuclear import of many nucleotide templates. This seemed to result in non-homologous end joining (NHEJ) before the arrival of the homologous template. Thus, we assessed whether NLS-tagged ssODNs and free NLS peptides could circumvent problems posed by the nuclear envelope. NLS-tagging ssODNs enhanced SSTR and indel efficiency by 4-fold compared to the control. Our results suggest the following: (1) mitosis is the optimal phase for SSTR, (2) the donor template needs to be delivered to the nucleus before nuclease delivery, and (3) NLS-tagging ssODNs improve SSTR efficiency, especially high in mitosis.
Highlights
Eukaryotic cells have two DNA repair systems, namely error-prone non-homologous end joining (NHEJ) and error-free homologous recombination (HR)
The cell cycle of eukaryote consists of gap 1 (G1), synthesis (S), gap 2 (G2), and mitosis
Early-stage embryos progress in the following order: pronuclear formation (PN 0~5), mitosis, and G1 [12]
Summary
Eukaryotic cells have two DNA repair systems, namely error-prone non-homologous end joining (NHEJ) and error-free homologous recombination (HR). HR was used for gene targeting with embryonic stem cells, but random genetic damage caused by electroporation results in low specificity and efficiency. Developed nucleases can recognize specific sequences and create double-stranded breaks (DSBs). Zinc finger nucleases and transcriptional activator-like effector nucleases can bind to particular sequences but cannot create DSBs without conjugating to Fok. Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) nucleases can recognize complementary sequences and induce DSBs by themselves. These nucleases are widely utilized for gene manipulation using non-homologous end joining (NHEJ) or homology-directed repair (HDR) [1]
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