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High Homology-Directed Repair Using Mitosis Phase and Nucleus Localizing Signal.

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.

journal_name

Int J Mol Sci

journal_title

International journal of molecular sciences

authors

Han JP,Chang YJ,Song DW,Choi BS,Koo OJ,Yi SY,Park TS,Yeom SC

doi

10.3390/ijms21113747

subject

Has Abstract

pub_date

2020-05-26 00:00:00

issue

11

issn

1422-0067

pii

ijms21113747

journal_volume

21

pub_type

杂志文章

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