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

Overview

Journal Int J Mol Sci

Publisher MDPI

Specialties Biochemistry
Chemistry
Molecular Biology

Date 2020 May 30

PMID 32466470

Citations 8

Authors

Jeong Pil Han

Yoo Jin Chang

Dong Woo Song

Beom Seok Choi

Ok Jae Koo

Seung Youn Yi

Tae Sub Park

Su Cheong Yeom

Affiliations

Soon will be listed here.

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.

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References

Giraud G, Stadhouders R, Conidi A, Dekkers D, Huylebroeck D, Demmers J . NLS-tagging: an alternative strategy to tag nuclear proteins. Nucleic Acids Res. 2014; 42(21). PMC: 4245968. DOI: 10.1093/nar/gku869. View

Gu B, Posfai E, Rossant J . Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos. Nat Biotechnol. 2018; 36(7):632-637. DOI: 10.1038/nbt.4166. View

Rodriguez A, DAndrea A . Fanconi anemia pathway. Curr Biol. 2017; 27(18):R986-R988. DOI: 10.1016/j.cub.2017.07.043. View

Chazaud C, Yamanaka Y . Lineage specification in the mouse preimplantation embryo. Development. 2016; 143(7):1063-74. DOI: 10.1242/dev.128314. View

Bertoni C, Rustagi A, Rando T . Enhanced gene repair mediated by methyl-CpG-modified single-stranded oligonucleotides. Nucleic Acids Res. 2009; 37(22):7468-82. PMC: 2794159. DOI: 10.1093/nar/gkp757. View

Park J, Lim K, Kim J, Bae S . Cas-analyzer: an online tool for assessing genome editing results using NGS data. Bioinformatics. 2016; 33(2):286-288. PMC: 5254075. DOI: 10.1093/bioinformatics/btw561. View

Jang D, Lee J, Lee J, Koo O, Bae H, Jung M . Multiple sgRNAs with overlapping sequences enhance CRISPR/Cas9-mediated knock-in efficiency. Exp Mol Med. 2018; 50(4):1-9. PMC: 5938013. DOI: 10.1038/s12276-018-0037-x. View

Aird E, Lovendahl K, Martin A, Harris R, Gordon W . Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template. Commun Biol. 2018; 1:54. PMC: 6123678. DOI: 10.1038/s42003-018-0054-2. View

Nalepa G, Clapp D . Fanconi anemia and the cell cycle: new perspectives on aneuploidy. F1000Prime Rep. 2014; 6:23. PMC: 3974572. DOI: 10.12703/P6-23. View

10.

Riesenberg S, Maricic T . Targeting repair pathways with small molecules increases precise genome editing in pluripotent stem cells. Nat Commun. 2018; 9(1):2164. PMC: 5986859. DOI: 10.1038/s41467-018-04609-7. View

11.

Schwinn M, Machleidt T, Zimmerman K, Eggers C, Dixon A, Hurst R . CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide. ACS Chem Biol. 2017; 13(2):467-474. DOI: 10.1021/acschembio.7b00549. View

12.

Wienert B, Nguyen D, Guenther A, Feng S, Locke M, Wyman S . Timed inhibition of CDC7 increases CRISPR-Cas9 mediated templated repair. Nat Commun. 2020; 11(1):2109. PMC: 7193628. DOI: 10.1038/s41467-020-15845-1. View

13.

Yang D, Scavuzzo M, Chmielowiec J, Sharp R, Bajic A, Borowiak M . Enrichment of G2/M cell cycle phase in human pluripotent stem cells enhances HDR-mediated gene repair with customizable endonucleases. Sci Rep. 2016; 6:21264. PMC: 4757933. DOI: 10.1038/srep21264. View

14.

Gavet O, Pines J . Activation of cyclin B1-Cdk1 synchronizes events in the nucleus and the cytoplasm at mitosis. J Cell Biol. 2010; 189(2):247-59. PMC: 2856909. DOI: 10.1083/jcb.200909144. View

15.

Rothkamm K, Kruger I, Thompson L, Lobrich M . Pathways of DNA double-strand break repair during the mammalian cell cycle. Mol Cell Biol. 2003; 23(16):5706-15. PMC: 166351. DOI: 10.1128/MCB.23.16.5706-5715.2003. View

16.

Renaud J, Boix C, Charpentier M, De Cian A, Cochennec J, Duvernois-Berthet E . Improved Genome Editing Efficiency and Flexibility Using Modified Oligonucleotides with TALEN and CRISPR-Cas9 Nucleases. Cell Rep. 2016; 14(9):2263-2272. DOI: 10.1016/j.celrep.2016.02.018. View

17.

Zhu J, Wan S, Zheng D, Lei Q, Zhuo R, Feng J . Propelled Transnuclear Gene Transport Achieved through Intracellularly Redox-Responsive and Acidity-Accelerative Decomposition of Supramolecular Florescence-Quenchable Vectors. ACS Appl Mater Interfaces. 2016; 9(1):255-265. DOI: 10.1021/acsami.6b14730. View

18.

Wang D, Tai P, Gao G . Adeno-associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discov. 2019; 18(5):358-378. PMC: 6927556. DOI: 10.1038/s41573-019-0012-9. View

19.

Savic N, Ringnalda F, Lindsay H, Berk C, Bargsten K, Li Y . Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair. Elife. 2018; 7. PMC: 6023611. DOI: 10.7554/eLife.33761. View

20.

Wossidlo M, Nakamura T, Lepikhov K, Marques C, Zakhartchenko V, Boiani M . 5-Hydroxymethylcytosine in the mammalian zygote is linked with epigenetic reprogramming. Nat Commun. 2011; 2:241. DOI: 10.1038/ncomms1240. View