DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

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

Journal Article · · Nature Communications

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.

Research Organization:
Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Sponsoring Organization:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
Grant/Contract Number:
AC02-05CH11231
OSTI ID:
1615316
Journal Information:
Nature Communications, Vol. 11, Issue 1; ISSN 2041-1723
Publisher:
Nature Publishing GroupCopyright Statement
Country of Publication:
United States
Language:
English
Citation Metrics:
Cited by: 119 works
Citation information provided by
Web of Science

References (101)

Nonenzymatic β-Carotene Degradation in Provitamin A-Biofortified Crop Plants journal February 2017
Genome Engineering with Targetable Nucleases journal June 2014
Cloning and comparative analysis of carotenoid β-hydroxylase genes provides new insights into carotenoid metabolism in tetraploid (Triticum turgidum ssp. durum) and hexaploid (Triticum aestivum) wheat grains journal September 2012
Genetically modified (GM) crops: milestones and new advances in crop improvement journal July 2016
Application of the CRISPR–Cas System for Efficient Genome Engineering in Plants journal November 2013
Gene replacements and insertions in rice by intron targeting using CRISPR–Cas9 journal September 2016
Fast and accurate short read alignment with Burrows-Wheeler transform journal May 2009
Making ends meet: targeted integration of DNA fragments by genome editing journal July 2018
Optimizing the DNA Donor Template for Homology-Directed Repair of Double-Strand Breaks journal June 2017
Progress and prospects in plant genome editing journal July 2017
High-frequency, precise modification of the tomato genome journal November 2015
Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens journal May 2017
Genome editing in maize directed by CRISPR–Cas9 ribonucleoprotein complexes journal November 2016
Back to the future: revisiting MAS as a tool for modern plant breeding journal December 2018
CRISPR/Cas brings plant biology and breeding into the fast lane journal February 2020
Precision Genome Engineering and Agriculture: Opportunities and Regulatory Challenges journal June 2014
Applications of CRISPR technologies in research and beyond journal September 2016
Provitamin A biofortification of crop plants: a gold rush with many miners journal April 2017
Expression and inheritance of multiple transgenes in rice plants journal November 1998
Genome editing in rice and wheat using the CRISPR/Cas system journal September 2014
ARGOS8 variants generated by CRISPR-Cas9 improve maize grain yield under field drought stress conditions journal August 2016
Homology-mediated end joining-based targeted integration using CRISPR/Cas9 journal May 2017
Targeted mutagenesis in rice using CRISPR-Cas system journal September 2013
Targeted Mutagenesis, Precise Gene Editing, and Site-Specific Gene Insertion in Maize Using Cas9 and Guide RNA journal August 2015
Trait stacking in transgenic crops: Challenges and opportunities journal July 2010
The Sequences of 1504 Mutants in the Model Rice Variety Kitaake Facilitate Rapid Functional Genomic Studies journal June 2017
Genome editing in rice and wheat using the CRISPR/Cas system journal September 2014
Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis journal June 2007
Precision Genome Engineering and Agriculture: Opportunities and Regulatory Challenges journal June 2014
Precision genetic modifications: a new era in molecular biology and crop improvement journal February 2014
Efficient in planta gene targeting in tomato using geminiviral replicons and the CRISPR/Cas9 system journal May 2018
Precise Genome Modification via Sequence-Specific Nucleases-Mediated Gene Targeting for Crop Improvement journal December 2016
Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation journal February 1992
Genome sequence of the model rice variety KitaakeX journal November 2019
Large dataset enables prediction of repair after CRISPR–Cas9 editing in primary T cells journal July 2019
CRISPR/Cas9-mediated gene targeting in Arabidopsis using sequential transformation journal May 2018
Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases journal January 2014
Compositional Analysis of Genetically Engineered GR2E “Golden Rice” in Comparison to That of Conventional Rice journal June 2019
A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants journal August 2015
Genome-Scale Sequence Disruption Following Biolistic Transformation in Rice and Maize journal January 2019
Expression and inheritance of multiple transgenes in rice plants journal November 1998
Molecular and Functional Characterization of GR2-R1 Event Based Backcross Derived Lines of Golden Rice in the Genetic Background of a Mega Rice Variety Swarna journal January 2017
High-frequency, precise modification of the tomato genome journal November 2015
Towards a better bowl of rice: assigning function to tens of thousands of rice genes journal February 2008
Making ends meet: targeted integration of DNA fragments by genome editing journal July 2018
Targeted genome modification of crop plants using a CRISPR-Cas system journal August 2013
Multiplex precise base editing in cynomolgus monkeys journal May 2020
Pindel: a pattern growth approach to detect break points of large deletions and medium sized insertions from paired-end short reads journal June 2009
Engineering Herbicide-Resistant Rice Plants through CRISPR/Cas9-Mediated Homologous Recombination of Acetolactate Synthase journal April 2016
BreakDancer: an algorithm for high-resolution mapping of genomic structural variation journal August 2009
Nonenzymatic β-Carotene Degradation in Provitamin A-Biofortified Crop Plants journal February 2017
Application of the CRISPR–Cas System for Efficient Genome Engineering in Plants journal November 2013
A large-scale whole-genome sequencing analysis reveals highly specific genome editing by both Cas9 and Cpf1 (Cas12a) nucleases in rice journal July 2018
Genetically modified (GM) crops: milestones and new advances in crop improvement journal July 2016
Genome Engineering with Targetable Nucleases journal June 2014
Gene Insertion Into Genomic Safe Harbors for Human Gene Therapy journal April 2016
Homology-mediated end joining-based targeted integration using CRISPR/Cas9 journal May 2017
Gene replacements and insertions in rice by intron targeting using CRISPR–Cas9 journal September 2016
A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants journal August 2015
Compositional Analysis of Genetically Engineered GR2E “Golden Rice” in Comparison to That of Conventional Rice journal June 2019
Genome-Scale Sequence Disruption Following Biolistic Transformation in Rice and Maize journal January 2019
Gene Targeting by Homology-Directed Repair in Rice Using a Geminivirus-Based CRISPR/Cas9 System journal July 2017
Applications of CRISPR technologies in research and beyond journal September 2016
Large dataset enables prediction of repair after CRISPR–Cas9 editing in primary T cells journal July 2019
Cas9-Guide RNA Directed Genome Editing in Soybean journal August 2015
CRISPR/Cas brings plant biology and breeding into the fast lane journal February 2020
Genome-Wide Sequencing of 41 Rice (Oryza sativa L.) Mutated Lines Reveals Diverse Mutations Induced by Fast-Neutron Irradiation journal July 2016
Genome editing in maize directed by CRISPR–Cas9 ribonucleoprotein complexes journal November 2016
Genome-Wide Prediction of Highly Specific Guide RNA Spacers for CRISPR–Cas9-Mediated Genome Editing in Model Plants and Major Crops journal May 2014
CRISPR/Cas9-mediated targeted T-DNA integration in rice journal January 2019
Pindel: a pattern growth approach to detect break points of large deletions and medium sized insertions from paired-end short reads journal June 2009
Targeted genome modification of crop plants using a CRISPR-Cas system journal August 2013
Targeted A-to-G base editing of chloroplast DNA in plants journal December 2022
A One Pot, One Step, Precision Cloning Method with High Throughput Capability journal November 2008
Targeted Mutagenesis, Precise Gene Editing, and Site-Specific Gene Insertion in Maize Using Cas9 and Guide RNA journal August 2015
Bottlenecks in carotenoid biosynthesis and accumulation in rice endosperm are influenced by the precursor-product balance journal April 2015
Trait stacking in transgenic crops: Challenges and opportunities journal July 2010
Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis journal June 2007
Towards a better bowl of rice: assigning function to tens of thousands of rice genes journal February 2008
BreakDancer: an algorithm for high-resolution mapping of genomic structural variation journal August 2009
Engineering Herbicide-Resistant Rice Plants through CRISPR/Cas9-Mediated Homologous Recombination of Acetolactate Synthase journal April 2016
The Sequences of 1504 Mutants in the Model Rice Variety Kitaake Facilitate Rapid Functional Genomic Studies journal June 2017
CRISPR/Cas9-mediated gene targeting in Arabidopsis using sequential transformation journal May 2018
Molecular and Functional Characterization of GR2-R1 Event Based Backcross Derived Lines of Golden Rice in the Genetic Background of a Mega Rice Variety Swarna journal January 2017
Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens journal May 2017
Cas9-Guide RNA Directed Genome Editing in Soybean journal August 2015
Improving the nutritional value of Golden Rice through increased pro-vitamin A content journal March 2005
Gene Targeting by Homology-Directed Repair in Rice Using a Geminivirus-Based CRISPR/Cas9 System journal July 2017
ARGOS8 variants generated by CRISPR-Cas9 improve maize grain yield under field drought stress conditions journal August 2016
Engineering the Provitamin A (-Carotene) Biosynthetic Pathway into (Carotenoid-Free) Rice Endosperm journal January 2000
Precision genetic modifications: a new era in molecular biology and crop improvement journal February 2014
Gene Insertion Into Genomic Safe Harbors for Human Gene Therapy journal April 2016
A One Pot, One Step, Precision Cloning Method with High Throughput Capability journal November 2008
The Sequence Alignment/Map format and SAMtools journal June 2009
CRISPR/Cas9-mediated targeted T-DNA integration in rice journal January 2019
Cloning and comparative analysis of carotenoid β-hydroxylase genes provides new insights into carotenoid metabolism in tetraploid (Triticum turgidum ssp. durum) and hexaploid (Triticum aestivum) wheat grains journal September 2012
Optimizing the DNA Donor Template for Homology-Directed Repair of Double-Strand Breaks journal June 2017
Precise insertion and guided editing of higher plant genomes using Cpf1 CRISPR nucleases journal September 2017
Efficient in planta gene targeting in tomato using geminiviral replicons and the CRISPR/Cas9 system journal May 2018
Effect of drying and storage on the degradation of total carotenoids in orange-fleshed sweetpotato cultivars journal December 2009
A large-scale whole-genome sequencing analysis reveals highly specific genome editing by both Cas9 and Cpf1 (Cas12a) nucleases in rice collection January 2018

Figures / Tables (5)