Efficient genome editing in plants using a CRISPR/Cas system

Feng, Zhengyan; Zhang, Botao; Ding, Wona; Liu, Xiaodong; Yang, Donglei; Wei, Pengliang; Cao, Fengming; Zhu, Shihua; Zhang, Feng; Mao, Yanfei; Zhu, Jian‐Kang

doi:10.1038/cr.2013.114

Cited by 928 publications

(702 citation statements)

References 10 publications

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“…However, it remained possible that a functional redundancy between DCL1 and DCL2, 3 and 4 may have masked the requirement for the DCLs. To further test the requirement for DCLs in RdDM, we constructed dcl1/2/3/4 quadruple mutant plants using CRISPR/Cas9 [38,39] to mutate DCL1 in the dcl2/3/4 background. CRISPR/Cas9 caused different 1-bp insertions at two target sites near the dcl1-100 T-DNA insertion site [40] in the DCL1 coding region, resulting in frameshifts (Supplementary information, Figure S4A).…”

Section: Dicer-independent Dna Methylation At Rddm Target Locimentioning

confidence: 99%

Dicer-independent RNA-directed DNA methylation in Arabidopsis

et al. 2015

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RNA-directed DNA methylation (RdDM) is an important de novo DNA methylation pathway in plants. Small interfering RNAs (siRNAs) generated by Dicers from RNA polymerase IV (Pol IV) transcripts are thought to guide sequence-specific DNA methylation. To gain insight into the mechanism of RdDM, we performed whole-genome bisulfite sequencing of a collection of Arabidopsis mutants, including plants deficient in Pol IV (nrpd1) or Dicer (dcl1/2/3/4) activity. Unexpectedly, of the RdDM target loci that required Pol IV and/or Pol V, only 16% were fully dependent on Dicer activity. DNA methylation was partly or completely independent of Dicer activity at the remaining Pol IV-and/ or Pol V-dependent loci, despite the loss of 24-nt siRNAs. Instead, DNA methylation levels correlated with the accumulation of Pol IV-dependent 25-50 nt RNAs at most loci in Dicer mutant plants. Our results suggest that RdDM in plants is largely guided by a previously unappreciated class of Dicer-independent non-coding RNAs, and that siRNAs are required to maintain DNA methylation at only a subset of loci.

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Section: Dicer-independent Dna Methylation At Rddm Target Locimentioning

confidence: 99%

Dicer-independent RNA-directed DNA methylation in Arabidopsis

et al. 2015

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“…It is also possible to create OsmiR396d-resistant OsGRF4 and OsGRF6 alleles through CRISPR-Cas9 nickase-cytidine deaminase fusion system (Zong et al, 2017) to convert C to T in OsmiR396 targeted DNA regions. An additional approach may also be achieved through combining different members of OsmiR396 deleted "nontransgenic" mutants by CRISPR-CAS9 technology (Feng et al, 2013) to improve rice yield.…”

Section: Improving Agronomic Traits Through Manipulating Mir396-grf-rmentioning

confidence: 99%

OsmiR396d Affects Gibberellin and Brassinosteroid Signaling to Regulate Plant Architecture in Rice

et al. 2017

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“…It was shown that engineered CRISPR/Cas caused mutations in target genes or corrections in transgenes in transient expression assays in plant protoplasts and tobacco leaves (10). Importantly, stable expression of the CRISPR/ Cas in transgenic Arabidopsis, tobacco, and rice plants led to mutations (mostly indels) in target genes and correction of a transgene (4)(5)(6)(7)(8)(9). However, it was not known whether the gene mutations and corrections occurred in somatic cells only or whether some of the mutations and corrections happened in germ-line cells and thus may be heritable.…”

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“…The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) system has been successfully used for efficient genome editing in human cell lines, zebrafish, and mouse (1)(2)(3) and recently applied to gene modification in plants (4)(5)(6)(7)(8)(9)(10). In this system a short RNA molecule guides the associated endonuclease Cas9 to generate double strand breaks (DSBs) in the target genomic DNA, which lead to sequence mutations as a result of error-prone nonhomologous end-joining (NHEJ) DNA damage repair or to gene correction or replacement as a result of homologydependent recombination (HR) (11).…”

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Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis

Feng

Mao

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) system has emerged as a powerful tool for targeted gene editing in many organisms, including plants. However, all of the reported studies in plants focused on either transient systems or the first generation after the CRISPR/ Cas system was stably transformed into plants. In this study we examined several plant generations with seven genes at 12 different target sites to determine the patterns, efficiency, specificity, and heritability of CRISPR/Cas-induced gene mutations or corrections in Arabidopsis. The proportion of plants bearing any mutations (chimeric, heterozygous, biallelic, or homozygous) was 71.2% at T1, 58.3% at T2, and 79.4% at T3 generations. CRISPR/Cas-induced mutations were predominantly 1 bp insertion and short deletions. Gene modifications detected in T1 plants occurred mostly in somatic cells, and consequently there were no T1 plants that were homozygous for a gene modification event. In contrast, ∼22% of T2 plants were found to be homozygous for a modified gene. All homozygotes were stable to the next generation, without any new modifications at the target sites. There was no indication of any off-target mutations by examining the target sites and sequences highly homologous to the target sites and by in-depth whole-genome sequencing. Together our results show that the CRISPR/Cas system is a useful tool for generating versatile and heritable modifications specifically at target genes in plants.

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Efficient genome editing in plants using a CRISPR/Cas system

Cited by 928 publications

References 10 publications

Dicer-independent RNA-directed DNA methylation in Arabidopsis

Dicer-independent RNA-directed DNA methylation in Arabidopsis

OsmiR396d Affects Gibberellin and Brassinosteroid Signaling to Regulate Plant Architecture in Rice

Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis

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