Marker-free carotenoid-enriched rice generated through targeted gene insertion using CRISPR-Cas9.

Nature Communications
Oliver Xiaoou DongPamela C Ronald

Abstract

Targeted insertion of transgenes at pre-determined plant genomic safe harbors provides a desirable alternative to insertions at random sites achieved through conventional methods. Most existing cases of targeted gene insertion in plants have either relied on the presence of a selectable marker gene in the insertion cassette or occurred at low frequency with relatively small DNA fragments (<1.8 kb). Here, we report the use of an optimized CRISPR-Cas9-based method to achieve the targeted insertion of a 5.2 kb carotenoid biosynthesis cassette at two genomic safe harbors in rice. We obtain marker-free rice plants with high carotenoid content in the seeds and no detectable penalty in morphology or yield. Whole-genome sequencing reveals the absence of off-target mutations by Cas9 in the engineered plants. These results demonstrate targeted gene insertion of marker-free DNA in rice using CRISPR-Cas9 genome editing, and offer a promising strategy for genetic improvement of rice and other crops.

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Citations

Jun 20, 2020·The Journal of Biological Chemistry·Teresa B Fitzpatrick, Lottie M Chapman
Sep 26, 2020·Nature Reviews. Molecular Cell Biology·Haocheng ZhuCaixia Gao
Aug 10, 2020·Angewandte Chemie·Benjamin D BergstromLucas W Souza
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Dec 9, 2020·Journal of Integrative Plant Biology·Yufei LiJie Luo
Nov 23, 2020·Molecular Biology Reports·Ibrahim Ilker Ozyigit, Kuaybe Yucebilgili Kurtoglu
May 1, 2020·Molecular Plant·Jingying LiLanqin Xia
May 1, 2021·Advanced Healthcare Materials·Kyun Kyu KimSeung Hwan Ko
May 1, 2021·International Journal of Molecular Sciences·Qier LiuMaoyun She
May 13, 2021·Plant Cell Reports·Sukumar BiswasJianxin Shi
May 27, 2021·Critical Reviews in Food Science and Nutrition·Deepak KasoteAhmed Regina
May 30, 2021·Proceedings of the National Academy of Sciences of the United States of America·Oliver Xiaoou Dong, Pamela C Ronald
Jun 11, 2021·3 Biotech·Athiappan SelviKasirajan Lakshmi
Jun 15, 2021·Frontiers in Plant Science·Sunny AhmarKi-Hung Jung
Jul 16, 2021·Transgenic Research·Jerlie Mhay MatresInez H Slamet-Loedin
Jul 22, 2021·Journal of Agricultural and Food Chemistry·Shakeel AhmadShaoqing Tang
Jan 2, 2021·Trends in Biotechnology·Xiongjie ZhengSalim Al-Babili
Aug 27, 2021·Annals of the New York Academy of Sciences·Jennifer CableRammyani Bagchi
Sep 28, 2021·The CRISPR Journal·Chen-Tran HsuChoun-Sea Lin

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Methods Mentioned

BETA
gene knockout
PCR
genotyping
transgenic
electrophoresis

Software Mentioned

Cas
Borrow Wheeler Aligner ( BWA
BLAST
SAMtools mpileup
Image J
Pindel
GeneRuler
OFFinder
SAMtools
bcftools

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