Allele-specific genome editing of imprinting genes by preferentially targeting non-methylated loci using Staphylococcus aureus Cas9 (SaCas9)

doi:10.1016/j.scib.2019.08.023

	Allele-specific genome editing of imprinting genes by preferentially targeting non-methylated loci using Staphylococcus aureus Cas9 (SaCas9)
	Liu, Yajing1,2,3 ; Li, Jianan1,2,3 ; Zhou, Changyang 3,4; Meng, Bin5 ; Wei, Yu 3,4; Yang, Guang1,2,3 ; Lu, Zongyang1,2,3 ; Shen, Qingmei 6; Zhang, Yu1 ; Yang, Hui 4; Qiao, Yunbo 6
	2019-11-15
发表期刊	SCIENCE BULLETIN
ISSN	2095-9273
EISSN	2095-9281
卷号	64 期号:21 页码:1592-1600
发表状态	已发表
DOI	10.1016/j.scib.2019.08.023
摘要	Allele-specific DNA methylation is the most important imprinting marker localized to differentially methylated regions (DMRs), and aberrant genomic imprinted DNA methylation is associated with some human diseases, including Prader-Willi syndrome and cancer. Thus, the development of an effective strategy for the precise editing of allele-specific methylated genes is essential for the functional clarification of imprinting elements and the correction of imprinting disorders in human diseases. To discover a feasible allele-specific genome editing tool based on the CRISPR/Cas system, which is an efficient genetargeting technique in various organisms, we examined the targeting efficiency of Staphylococcus aureus Cas9 (SaCas9) and Streptococcus pyogenes Cas9 (SpCas9) in response to DNA methylation interference. We found that the targeting efficiency of SaCas9, but not SpCas9, was enhanced by targeted DNA demethylation using the dCas9-Tet1 catalytic domain (CD) but suppressed by targeted DNA methylation using Dnmt3l-Dnmt3a-dCas9. An in vitro cleavage assay further demonstrated that SaCas9 nuclease activity was inhibited by 5-methylcytosine (5mC) in a synthesized CpG-containing context. Further analysis with ChIP-Q-PCR demonstrated that the non-methylated sequence targeting of SaCas9 depends on the binding preference of SaCas9 to non-methylated sequences. Taking advantage of this feature of SaCas9, we have successfully obtained non-methylated allele-biased targeted embryos/mice for two imprinting genes, H19 and Snrpn, with relatively high efficiencies of 28.6% and 47.4%, respectively. These results indicate that the targeting efficiency of SaCas9 was strongly reduced by DNA methylation. By using SaCas9, we successfully achieved allele-specific genome editing of imprinting genes by preferentially targeting non-methylated loci. (C) 2019 Science China Press. Published by Elsevier B.V. and Science China Press. All rights reserved.
关键词	Allele-specific DNA methylation SaCas9 Imprinting gene
收录类别	SCI ; SCIE ; EI
语种	英语
资助项目	National Natural Science Foundation of China[31471400]
WOS研究方向	Science & Technology - Other Topics
WOS类目	Multidisciplinary Sciences
WOS记录号	WOS:000493351200008
出版者	ELSEVIER
EI主题词	Alkylation ; Bacteria ; DNA ; Efficiency ; Methylation ; Polymerase chain reaction
WOS关键词	DNA METHYLATION ; CRISPR-CAS9 ; EFFICIENT ; POLYMORPHISMS ; EXPRESSION ; DYNAMICS
原始文献类型	Article
引用统计
文献类型	期刊论文
条目标识符	https://kms.shanghaitech.edu.cn/handle/2MSLDSTB/80558
专题	生命科学与技术学院_博士生生命科学与技术学院_PI研究组_黄行许组免疫化学研究所_特聘教授组_抗体结构学实验室生命科学与技术学院_硕士生
通讯作者	Yang, Hui; Qiao, Yunbo
作者单位	1.ShanghaiTech Univ, Sch Life Sci & Technol, Shanghai 201210, Peoples R China 2.Chinese Acad Sci, Inst Biochem & Cell Biol, Shanghai 200031, Peoples R China 3.Univ Chinese Acad Sci, Beijing 100049, Peoples R China 4.Chinese Acad Sci, Shanghai Inst Biol Sci, CAS Ctr Excellence Brain Sci & Intelligence Techn, Inst Neurosci,State Key Lab Neurosci,Key Lab Prim, Shanghai 200031, Peoples R China 5.ShanghaiTech Univ, Shanghai Inst Adv Immunochem Studies, Shanghai 200031, Peoples R China 6.Guangzhou Univ, Precise Genome Engn Ctr, Sch Life Sci, Guangzhou 510006, Guangdong, Peoples R China
第一作者单位	生命科学与技术学院
第一作者的第一单位	生命科学与技术学院
推荐引用方式 GB/T 7714	Liu, Yajing,Li, Jianan,Zhou, Changyang,et al. Allele-specific genome editing of imprinting genes by preferentially targeting non-methylated loci using Staphylococcus aureus Cas9 (SaCas9)[J]. SCIENCE BULLETIN,2019,64(21):1592-1600.
APA	Liu, Yajing.,Li, Jianan.,Zhou, Changyang.,Meng, Bin.,Wei, Yu.,...&Qiao, Yunbo.(2019).Allele-specific genome editing of imprinting genes by preferentially targeting non-methylated loci using Staphylococcus aureus Cas9 (SaCas9).SCIENCE BULLETIN,64(21),1592-1600.
MLA	Liu, Yajing,et al."Allele-specific genome editing of imprinting genes by preferentially targeting non-methylated loci using Staphylococcus aureus Cas9 (SaCas9)".SCIENCE BULLETIN 64.21(2019):1592-1600.