CRISPR/Cas9技术介导猪基因组单碱基编辑效率的研究

doi:10.14088/j.cnki.issn0439-8114.2020.18.029

湖北农业科学 ›› 2020, Vol. 59 ›› Issue (18): 143-149.doi: 10.14088/j.cnki.issn0439-8114.2020.18.029

CRISPR/Cas9技术介导猪基因组单碱基编辑效率的研究

张婷婷^1,2, 陈涛^2,3, 李燕莉^1,2, 杨漫漫^2,3, 魏强^2,3, 王然^2,3, 李林^2,3, 李勇^1,2,3

1.深圳华大三生园科技有限公司,广东深圳 518000;
2.深圳市华大农业应用研究院,广东深圳 518000;
3.深圳动物基因组辅助育种工程实验室,广东深圳 518000

收稿日期:2019-11-06 发布日期:2020-11-05
通讯作者: 李勇（1981-）,男,副研究员,（电子信箱）liyong3@genomics.cn。
作者简介:张婷婷（1991-）,女,广东湛江人,主要从事生物技术研究,（电话）13570488346（电子信箱）2276087932@qq.com;
基金资助:
大鹏新区产业发展专项资金项目（KY20160305）; 深圳市卫计委医疗卫生“三名工程”（SZSM201512003）; 深圳市基因组辅助育种工程实验室提升项目

Programmable base editing efficiency study of CRISPR/Cas9-guided DNA base editors in pig genome

ZHANG Ting-ting^1,2, CHEN Tao^2,3, LI Yan-li^1,2, YANG Man-man^2,3, WEI Qiang^2,3, WANG Ran^2,3, LI Lin^2,3, LI Yong^1,2,3

1. BGI-Shenzhen Sanshengyuan Technology Co., Ltd., Shenzhen 518000, Guangdong, China;
2. BGI-Agricultural Application Research Institute, Shenzhen 518000, Guangdong, China;
3. Shenzhen Engineering Laboratory for Genomics-Assisted Animal Breeding, Shenzhen 518000, Guangdong, China

Received:2019-11-06 Published:2020-11-05

摘要/Abstract

摘要： 利用国际上最新的两种基于CRISPR/Cas9的BE3（Cytosine base editor,CBE）和ABE7.10（Adenine base editor,ABE）单碱基修饰技术对猪基因组靶基因位点进行编辑效率的分析研究。设计、合成并构建4个猪基因组靶基因位点gRNA表达载体,分别与CBE或ABE共转染PK15细胞,继续培养48 h,结合二代测序技术测定单碱基替换效率和indels发生率。结果表明,单碱基编辑系统BE3和ABE7.10对猪基因组靶位点碱基修饰的活性编辑窗口主要分别为5个核苷酸和4个核苷酸;两套单碱基编辑系统主要对编辑窗口内的目标碱基进行单碱基转换而非indel;两套单碱基编辑系统对猪基因组碱基置换有一定偏好性,其中CMAH、MC1R（1-2）、MC1R（2-1）基因编辑窗口内C→T的效率分别为2.2%、0.4%和1.3%;猪GGTA、MSTN-2窗口内A→G的效率分别为1.4%、1.4%。表明单碱基编辑系统BE3和ABE7.10均能够对猪细胞的基因组靶位点序列进行有效的单碱基置换。

关键词: 猪, 单碱基编辑, CRISPR/Cas9, BE3, ABE7.10

Abstract: The CRISPR/Cas9-based BE3（Cytosine base editor,CBE） and ABE7.10（Adenine base editor,ABE） were used to analyze and study the editing efficiency of target gene loci in pig genome. Four porcine genomic target gene loci gRNA expression vectors were designed, synthesized and constructed, which were co-transfected into PK15 cells with CBE or ABE, respectively. The PK15 cells were further cultured for 48 h, and single-base replacement efficiency and indels incidence were determined by second-generation sequencing technology. The results showed that the active editing Windows of the single base editing system BE3 and ABE7.10 were 5 nucleotides and 4 nucleotides, respectively; Both BE3 and ABE7.10 were mainly contributed to single base conversion but not indel; The two sets of single-base editing systems showed certain preference for base replacement in pig genome. In the CMAH, MC1R（1-2） and MC1R（2-1） gene editing Windows, the efficiency of C→T was 2.2%, 0.4% and 1.3%, respectively; The efficiency of A→G in pig GGTA and MSTN-2 was 1.4% and 1.4%, respectively. It is shown that both BE3 and ABE7.10 can perform effective single base substitutions for genomic target sequences in pig cells.

Key words: pig, single base editing, CRISPR/Cas9, BE3, ABE7.10

中图分类号:

Q812

张婷婷, 陈涛, 李燕莉, 杨漫漫, 魏强, 王然, 李林, 李勇. CRISPR/Cas9技术介导猪基因组单碱基编辑效率的研究[J]. 湖北农业科学, 2020, 59(18): 143-149.

ZHANG Ting-ting, CHEN Tao, LI Yan-li, YANG Man-man, WEI Qiang, WANG Ran, LI Lin, LI Yong. Programmable base editing efficiency study of CRISPR/Cas9-guided DNA base editors in pig genome[J]. HUBEI AGRICULTURAL SCIENCES, 2020, 59(18): 143-149.

参考文献

[1] BEIL-WAGNER J,DÖSSINGER G,SCHOBER K,et al. T cell-specific inactivation of mouse CD2 by CRISPR/Cas9[J]. Sci Rep,2016,6:21377.
[2] YIN Y J,HAO H Y,XU X B,et al.Generation of an MC3R knock-out pig by CRSPR/Cas9 combined with somatic cell nuclear transfer (SCNT) technology[J]. Lipids Health Dis,2019,18:122.
[3] IKEDA M,MATSUYAMA S,AKAGI S,et al.Correction of a disease mutation using CRISPR/Cas9-assisted genome editing in Japanese black cattle[J]. Sci Rep,2017,7:17827.
[4] VILARINO M,RASHID S T,SUCHY F P,et al.CRISPR/Cas9 microinjection in oocytes disables pancreas development in sheep[J]. Sci Rep,2017,7:17472.
[5] MA K,HAN J L,HAO Y,et al.An effective strategy to establish a male sterility mutant mini-library by CRISPR/Cas9-mediated knockout of anther-specific genes in rice[J]. J Genet Genomics,2019,46(5):273-275.
[6] RYDER P,MCHALE M,FORT A,et al.Generation of stable nulliplex autopolyploid lines of Arabidopsis thaliana using CRISPR/Cas9 genome editing[J]. Plant Cell Rep,2017,36(6):1005-1008.
[7] HIROHATA A,SATO I,KAINO V,et al.CRISPR/Cas9-mediated homologous recombination in tobacco[J]. Plant Cell Rep,2019,38(4):463-473.
[8] FENG C,SU H D,BAI H,et al.High-efficiency genome editing using a dmc1 promoter-controlled CRISPR/Cas9 system in maize[J]. Plant Biotechnol J,2018,16(11):1848-1857.
[9] LIANG Z,CHEN K L,LI T D,et al.Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes[J]. Nat Commun,2017,8:14261.
[10] OTA S,HISANO Y,IKAWA Y,et al.Multiple genome modifications by the CRISPR/Cas9 system in zebrafish[J]. Genes to cells,2014,19(7):555-564.
[11] LIU M J,REHMAN S,TANG X D,et al.Methodologies for improving HDR efficiency[J]. Front Genet,2019,9:691.
[12] KOMOR A C,KIM Y B,PACKER M S,et al.Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage[J]. Nature,2016,533:420-424.
[13] GAUDELLI N M,KOMOR A C,REES H A,et al.Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage[J]. Nature,2017,551:464-471.
[14] JIANG W,FENG S J,HUANG S S,et al.BE-PLUS: A new base editing tool with broadened editing window and enhanced fidelity[J]. Cell Res,2018,28(8):855-861.
[15] FAGAGNA DI F D,WELLER G R,DOHERTY A J,et al. The Gam protein of bacteriophage Mu is an orthologue of eukaryotic Ku[J]. EMBO Rep,2003,4(1):47-52.
[16] LIU Z,LU Z Y,YANG G,et al.Efficient generation of mouse models of human diseases via ABE- and BE-mediated base editing[J]. Nat Commun,2018,9(1):2338.
[17] ZHANG Y H,QIN W,LU X C,et al.Programmable base editing of zebrafish genome using a modified CRISPR-Cas9 system[J]. Nat Commun,2017,8(1):118.
[18] YANG J P,LI J Y,SUZUKI K,et al.Genetic enhancement in cultured human adult stem cells conferred by a single nucleotide recoding[J]. Cell Res,2017,27(9):1178-1181.
[19] YAN F,KUANG Y J,REN B,et al.Highly efficient A·T to G·C base editing by Cas9n-Guided tRNA adenosine deaminase in rice[J]. Mol Plant,2018,11(4):631-634.
[20] ZONG Y,WANG Y P,LI C,et al.Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion[J]. Nat Biotechnol,2017,35(5):438-440.
[21] NIU D,WEI H J,LIN L,et al.Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9[J]. Science,2017,357(6357):1303-1307.
[22] YAN S,TU Z C,LIU Z M,et al. A huntingtin knockin pig model recapitulates features of selective neurodegeneration in Huntington's disease[J]. Cell,2018,173(4):989-1002.e13.
[23] HUANG L,HUA Z D,XIAO H W,et al.CRISPR/Cas9-mediated ApoE-/- and LDLR-/- double gene knockout in pigs elevates serum LDL-C and TC levels[J]. Oncotarget,2017,8(23):37751-37760.
[24] YU H H,ZHAO H,QING Y B,et al.Porcine zygote injection with Cas9/sgRNA results in DMD-Modified pig with muscle dystrophy[J]. Int J Mol Sci,2016,17(10):1668.
[25] LI Z F,DUAN X Y,AN X M,et al.Efficient RNA-guided base editing for disease modeling in pigs[J]. Cell Discov,2018,4:64.
[26] XIE J K,GE W K,LI N,et al.Efficient base editing for multiple genes and loci in pigs using base editors[J]. Nat Commun,2019,10(1):2852.
[27] NISHIDA K,ARAZOE T,YACHIE N,et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems[J]. Science,2016,353(6305):aaf8729.
[28] LU Y M,ZHU J K.Precise editing of a target base in the rice genome using a modified CRISPR/Cas9 system[J]. Mol Plant,2017,10(3):523-525.
[29] KIM K,RYU S M,KIM S T,et al.Highly efficient RNA-guided base editing in mouse embryos[J]. Nat Biotechnol,2017,35(5):435-437.
[30] CAO F,XIE X Y,GOLLAN T,et al.Comparison of gene-transfer efficiency in human embryonic stem cells[J]. Mol Imaging Biol,2010,12(1):15-24.
[31] ROSS J W,WHYTE J J,ZHAO J G,et al.Optimization of square-wave electroporation for transfection of porcine fetal fibroblasts[J]. Transgenic Res,2010,19(4):611-620.
[32] MAURISSE R,DE SEMIR D,EMAMEKHOO H,et al.Comparative transfection of DNA into primary and transformed mammalian cells from different lineages[J]. BMC Biotechnol,2010,10:9.
[33] LIANG P P,SUN H W,ZHANG X Y,et al.Effective and precise adenine base editing in mouse zygotes[J]. Protein & cell,2018,9(9):808-813.

CRISPR/Cas9技术介导猪基因组单碱基编辑效率的研究

Programmable base editing efficiency study of CRISPR/Cas9-guided DNA base editors in pig genome

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