Efficient Targeted Mutagenesis in Apple and First Time Edition of Pear Using the CRISPR-Cas9 System

Frontiers in Plant Science
Aurélie CharrierElisabeth Chevreau

Abstract

Targeted genome engineering has emerged as an alternative to classical plant breeding and transgenic methods to improve crop plants. Among other methods (zinc finger nucleases or TAL effector nucleases) the CRISPR-Cas system proved to be the most effective, convenient and least expensive method. In this study, we optimized the conditions of application of this system on apple and explored its feasibility on pear. As a proof of concept, we chose to knock-out the Phytoene Desaturase (PDS) and Terminal Flower 1 (TFL1) genes. To improve the edition efficiency, two different single guide RNAs (gRNAs) were associated to the Cas9 nuclease for each target gene. These gRNAs were placed under the control of the U3 and U6 apple promoters. Characteristic albino phenotype was obtained for 85% of the apple transgenic lines targeted in MdPDS gene. Early flowering was observed in 93% of the apple transgenic lines targeted in MdTFL1.1 gene and 9% of the pear transgenic lines targeted in PcTFL1.1. Sequencing of the target zones in apple and pear CRISPR-PDS and CRISPR-TFL1.1 transgenic lines showed that the two gRNAs induced mutations but at variable frequencies. In most cases, Cas9 nuclease cut the DNA in the twenty targeted base pairs near the ...Continue Reading

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Citations

Oct 28, 2019·Horticulture Research·Jiemeng XuZhaobo Lang
Jul 30, 2019·Frontiers in Plant Science·Tanushri KaulPawan Kumar Agrawal
Nov 21, 2019·Scientific Reports·Isidre HooghvorstSalvador Nogués
Aug 14, 2020·International Journal of Molecular Sciences·Sunny AhmarKi-Hong Jung
Apr 10, 2019·Horticulture Research·Cameron P PeaceStijn Vanderzande
Nov 5, 2019·Transgenic Research·Rishikesh GhogareAmit Dhingra
Sep 15, 2020·Frontiers in Plant Science·Fabrizio SaloniaConcetta Licciardello
Sep 21, 2019·Transgenic Research·Aurélie CharrierElisabeth Chevreau
Jun 13, 2020·Frontiers in Plant Science·Yuanyuan JiangShunquan Lin
May 14, 2020·BMC Biotechnology·Zsófia BánfalviMihály Kondrák
Aug 22, 2020·Plant Biotechnology·Masato WadaSadao Komori
Jan 7, 2020·Critical Reviews in Biotechnology·Nikolay E ZlobinVasiliy V Taranov
Oct 8, 2020·Plant Methods·Irene N GentzelGuo-Liang Wang
Nov 11, 2020·BMC Biotechnology·Manjul DuttJude W Grosser
Jan 6, 2021·International Journal of Molecular Sciences·Jaiana MalabarbaEmilie Vergne
Mar 2, 2021·Horticulture Research·Chong RenZhenchang Liang
Apr 8, 2021·Transgenic Research·Fabiola Ramirez-TorresAmit Dhingra
Apr 10, 2021·Journal of Zhejiang University. Science. B·Chao LiBaohong Zhang
May 2, 2021·Horticulture Research·Xianhang WangXiping Wang
Jun 5, 2021·Plant Cell Reports·Xinrong LiFengqing Wang
Aug 25, 2021·Molecular Biology Reports·Mahpara KashtwariSajad Majeed Zargar
Sep 14, 2021·Frontiers in Plant Science·Vera PaveseRoberto Botta
Nov 28, 2021·International Journal of Molecular Sciences·Susan Schröpfer, Henryk Flachowsky
Jan 16, 2022·Horticulture Research·Jiaming LiJun Wu

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Datasets Mentioned

BETA
AJ223969
CP014260.1

Methods Mentioned

BETA
gene knock-out
gene knock-outs
transgenic
PCR
targeted mutation

Software Mentioned

CRISPOR
MultAlin
BLAST

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