Inhibition of KU70 and KU80 by CRISPR interference, not NgAgo interference, increases the efficiency of homologous recombination in pig fetal fibroblasts

doi:10.1016/S2095-3119(18)62150-1

Journal of Integrative Agriculture ›› 2019, Vol. 18 ›› Issue (2): 438-448.DOI: 10.1016/S2095-3119(18)62150-1

收稿日期:2017-11-27 出版日期:2019-02-01 发布日期:2019-01-29

Inhibition of KU70 and KU80 by CRISPR interference, not NgAgo interference, increases the efficiency of homologous recombination in pig fetal fibroblasts

LI Guo-ling^1*, QUAN Rong^1*, WANG Hao-qiang¹, RUAN Xiao-fang¹, MO Jian-xin¹, ZHONG Cui-li¹, YANG Hua-qiang^{1, 2}, LI Zi-cong¹, GU Ting1, LIU De-wu¹, WU Zhen-fang^{1, 2}, CAI Geng-yuan^{1, 2}, ZHANG Xian-wei^{1, 2}

¹National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, P.R.China
² Wens Foodstuff Group Co., Ltd., Yunfu 527400, P.R.China

Received:2017-11-27 Online:2019-02-01 Published:2019-01-29
Contact: Correspondence ZHANG Xian-wei, E-mail: zxianw@163.com * These authors contributed equally to this study.
About author: LI Guo-ling, E-mail: liguoling191520@163.com;
Supported by:
This work was supported by the National Science and Technology Major Project for Breeding of New Transgenic Organisms, China (2016ZX08006002) and the Guangdong Province “Flying Sail Program” Postdoctoral Foundation, China (2016).

摘要/Abstract

Abstract:

Non-homologous end-joining (NHEJ) is a predominant pathway for the repair of DNA double-strand breaks (DSB). It inhibits the efficiency of homologous recombination (HR) by competing for DSB targets. To improve the efficiency of HR, multiple CRISPR interference (CRISPRi) and Natronobacterium gregoryi Argonaute (NgAgo) interference (NgAgoi) systems have been designed for the knockdown of NHEJ key molecules, KU70, KU80, polynucleotide kinase/phosphatase (PNKP), DNA ligase IV (LIG4), and NHEJ1. Suppression of KU70 and KU80 by CRISPRi dramatically promoted (P<0.05) the efficiency of HR to 1.85- and 1.58-fold, respectively, whereas knockdown of PNKP, LIG4, and NHEJ1 repair factors did not significantly increase (P>0.05) HR efficiency. Interestingly, although the NgAgoi system significantly suppressed (P<0.05) KU70, KU80, PNKP, LIG4, and NHEJ1 expression, it did not improve (P>0.05) HR efficiency in primary fetal fibroblasts. Our result showed that both NgAgo and catalytically inactive Cas9 (dCas9) could interfere with the expression of target genes, but the downstream factors appear to be more active following CRISPR-mediated interference than that of NgAgo.

Key words: homologous recombination , non-homologous end-joining , CRISPRi , NgAgoi , KU70 , KU80

. [J]. Journal of Integrative Agriculture, 2019, 18(2): 438-448.

LI Guo-ling, QUAN Rong, WANG Hao-qiang, RUAN Xiao-fang, MO Jian-xin, ZHONG Cui-li, YANG Huaqiang, LI Zi-cong, GU Ting, LIU De-wu, WU Zhen-fang, CAI Geng-yuan, ZHANG Xian-wei. Inhibition of KU70 and KU80 by CRISPR interference, not NgAgo interference, increases the efficiency of homologous recombination in pig fetal fibroblasts[J]. Journal of Integrative Agriculture, 2019, 18(2): 438-448.

参考文献

Alshareeda A T, Negm O H, Albarakati N, Green A R, Nolan C, Sultana R, Madhusudan S, BenHasouna A, Tighe P, Ellis I O, Rakha E A. 2013. Clinicopathological significance of KU70/KU80, a key DNA damage repair protein in breast cancer. Breast Cancer Research and Treatment, 139, 301–310.
Bertolini L R, Bertolini M, Maga E A, Madden K R, Murray J D. 2009. Increased gene targeting in KU70 and Xrcc4 transiently deficient human somatic cells. Molecular Biotechnology, 41, 106–114.
Bikard D, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini L A. 2013. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Research, 41, 7429–7437.
Britton S, Coates J, Jackson S P. 2013. A new method for high-resolution imaging of Ku foci to decipher mechanisms of DNA double-strand break repair. Journal of Cell Biology, 202, 579–595.
Chang H, Pannunzio N R, Adachi N, Lieber M R. 2017. Non-homologous DNA end joining and alternative pathways to double-strand break repair. Nature Reviews Molecular Cell Biology, 18, 495–506.
Chu V T, Weber T, Wefers B, Wurst W, Sander S, Rajewsky K, Kühn R. 2015. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nature Biotechnology, 33, 543–548.
Gottlieb T M, Jackson S P. 1993. The DNA-dependent protein kinase: Requirement for DNA ends and association with Ku antigen. Cell, 72, 131–142.
Hammel M, Rey M, Yu Y, Mani R S, Classen S, Liu M, Pique M E, Fang S, Mahaney B L, Weinfeld M, Schriemer D C, Lees-Miller S P, Tainer J A. 2011. XRCC4 protein interactions with XRCC4-like factor XLF create an extended grooved scaffold for DNA ligation and double strand break repair. Journal of Biological Chemistry, 286, 32638–32650.
Jilani A, Ramotar D, Slack C, Ong C, Yang X M, Scherer S W, Lasko D D. 1999. Molecular cloning of the human gene, PNKP, encoding a polynucleotide kinase 3´-phosphatase and evidence for its role in repair of DNA strand breaks caused by oxidative damage. Journal of Biological Chemistry, 274, 24176–24186.
Li G, Liu D, Zhang X, Quan R, Zhong C, Mo J, Huang Y, Wang H, Ruan X, Xu Z, Zheng E, Gu T, Hong L, Li Z, Wu Z, Yang H. 2018. Suppressing Ku70/Ku80 expression elevates homology-directed repair efficiency in primary fibroblasts. International Journal of Biochemistry and Cell Biology, 99, 154–160.
Lin S, Staahl B T, Alla R K, Doudna J A. 2014. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. Elife, 3, e04766.
Ma Y, Pannicke U, Schwarz K, Lieber M R. 2002. Hairpin opening and overhang processing by an Artemis/DNA-dependent protein kinase complex in nonhomologous end joining and VDJ recombination. Cell, 108, 781–792.
Maruyama T, Dougan S K, Truttmann M C, Bilate A M, Ingram J R, Ploegh H L. 2015. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joing. Nature Biotechnology, 33, 538–542.
Mullins E A, Shi R, Parsons Z D, Yuen P K, David S S, Igarashi Y, Eichman B F. 2015. The DNA glycosylase AlkD uses a non-base-flipping mechanism to excise bulky lesions. Nature, 527, 254–258.
Qi J, Dong Z, Shi Y, Wang X, Qin Y, Wang Y, Liu D. 2016. NgAgo-based fabp11a gene knockdown causes eye developmental defects in zebrafish. Cell Research, 26, 1349–1352.
Qi L S, Larson M H, Gilbert L A, Doudna J A, Weissman J S, Arkin A P, Lim W A. 2013. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell, 152, 1173–1183.
Rosenbluh J, Xu H, Harrington W, Gill S, Wang X, Vazquez F, Root D E, Tsherniak A, Hahn W C. 2017. Complementary information derived from CRISPR Cas9 mediated gene deletion and suppression. Nature Communications, 8, 15403.
Sheng G, Zhao H, Wang J, Rao Y, Tian W, Swarts D C, Van der Oost J, Patel D J, Wang Y. 2014. Structure-based cleavage mechanism of Thermus thermophilus Argonaute DNA guide strand-mediated DNA target cleavage. Proceedings of the National Academy of Sciences of the United States of America, 111, 652–657.
Swarts D C, Hegge J W, Hinojo I, Shiimori M, Ellis M A, Dumrongkulraksa J, Terns R M, Terns M P, Van der Oost J. 2015. Argonaute of the archaeon Pyrococcus furiosus is a DNA-guided nuclease that targets cognate DNA. Nucleic Acids Research, 43, 5120–5129.
Swarts D C, Jore M M, Westra E R, Zhu Y, Janssen J H, Snijders A P, Wang Y, Patel D J, Berenguer J, Brouns S J J, Van der Oost J. 2014a. DNA-guided DNA interference by a prokaryotic Argonaute. Nature, 507, 258–261.
Swarts D C, Makarova K, Wang Y, Nakanishi K, Ketting R F, Koonin E V, Patel D J, Van der Oost J. 2014b. The evolutionary journey of Argonaute proteins. Nature Structural and Molecular Biology, 21, 743–753.
Wu P Y, Frit P, Meesala S, Dauvillier S, Modesti M, Andres S N, Huang Y, Sekiguchi J, Calsou P, Salles B, Junop M S. 2009. Structural and functional interaction between the human DNA repair proteins DNA ligase IV and XRCC4. Molecular and Cellular Biology, 29, 3163–3172.
Ye S, Bae T, Kim K, Omer H, Lee S H, Kim Y Y, Lee K-I, Kim S, Kim J-S. 2017. DNA-dependent RNA cleavage by the Natronobacterium gregoryi Argonaute. BioRxiv, doi: 10.1101/101923

Inhibition of KU70 and KU80 by CRISPR interference, not NgAgo interference, increases the efficiency of homologous recombination in pig fetal fibroblasts

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