GFP Loss-of-Function Mutations in<i>Arabidopsis thaliana</i>

Fu, Jason L; Kanno, Tohru; Liang, Shuli; Matzke, A. J. M.; Matzke, Marjori

doi:10.1534/g3.115.019604

Cited by 26 publications

(40 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The alleles generated were largely consistent across events, with 1-bp insertions being the predominant outcome (58 to 94%) followed by 1-bp deletions (3 to 38%; Supplemental Data Set 2) for the GFP-1 target locus. A small but significant proportion of alleles were in-frame (3-bp deletions), but, as the GFP-1 gRNA targets the essential residue Gly67 (Fu et al, 2015), these alleles likely result in no GFP fluorescence. For the two SMB-targeting gRNAs, 1-bp insertions were the predominant repair outcome (78 to 88%), with a minority (;10%) of alleles being 1-bp deletions for SMB-1 and 3-bp deletions for SMB-2 (Supplemental Data Set 2).…”

Section: Root-cap-specific Gene Knockoutmentioning

confidence: 99%

“…Users should also consider targeting functional domains, as is generally recommended with any standard knockout strategy. For example, the gRNA GFP-1 used here targets the essential Gly67 residue for GFP fluorescence, so that even in-frame mutations result in a loss of fluorescence (Fu et al, 2015). Hence, the use of gRNAs targeting genes of interest in particularly sensitive sites, such as crucial interaction domains or active sites, could further increase the likelihood of CRISPR-TSKO being effective.…”

Section: Considerations For the Use Of Crispr-tskomentioning

confidence: 99%

See 1 more Smart Citation

CRISPR-TSKO: A Technique for Efficient Mutagenesis in Specific Cell Types, Tissues, or Organs in Arabidopsis

et al. 2019

View full text Add to dashboard Cite

Detailed functional analyses of many fundamentally important plant genes via conventional loss-of-function approaches are impeded by the severe pleiotropic phenotypes resulting from these losses. In particular, mutations in genes that are required for basic cellular functions and/or reproduction often interfere with the generation of homozygous mutant plants, precluding further functional studies. To overcome this limitation, we devised a clustered regularly interspaced short palindromic repeats (CRISPR)-based tissue-specific knockout system, CRISPR-TSKO, enabling the generation of somatic mutations in particular plant cell types, tissues, and organs. In Arabidopsis (Arabidopsis thaliana), CRISPR-TSKO mutations in essential genes caused well-defined, localized phenotypes in the root cap, stomatal lineage, or entire lateral roots. The modular cloning system developed in this study allows for the efficient selection, identification, and functional analysis of mutant lines directly in the first transgenic generation. The efficacy of CRISPR-TSKO opens avenues for discovering and analyzing gene functions in the spatial and temporal contexts of plant life while avoiding the pleiotropic effects of systemwide losses of gene function.

show abstract

Section: Root-cap-specific Gene Knockoutmentioning

confidence: 99%

Section: Considerations For the Use Of Crispr-tskomentioning

confidence: 99%

CRISPR-TSKO: A Technique for Efficient Mutagenesis in Specific Cell Types, Tissues, or Organs in Arabidopsis

et al. 2019

View full text Add to dashboard Cite

show abstract

“…Total proteins were extracted from ∼2-wk-old seedlings, separated by SDS-PAGE, and blotted onto a PVDF membrane. The blot was probed sequentially with antibodies to GFP protein and tubulin as a loading control (Fu et al 2015). Sample names are indicated at the top.…”

Section: Genome-wide Analysis Of Alternative Splicingmentioning

confidence: 99%

“…GFP protein was detected by Western blotting using protein extracts isolated from 2-wk-old mutant and wild-type seedlings according to a previously published protocol (Fu et al 2015).…”

Section: Western Blotsmentioning

confidence: 99%

A genetic screen implicates a CWC16/Yju2/CCDC130 protein and SMU1 in alternative splicing in Arabidopsis thaliana

Kanno

Lin

et al. 2017

RNA

Self Cite

View full text Add to dashboard Cite

To identify regulators of pre-mRNA splicing in plants, we developed a forward genetic screen based on an alternatively spliced GFP reporter gene in Arabidopsis thaliana. In wild-type plants, three major splice variants issue from the GFP gene but only one represents a translatable GFP mRNA. Compared to wild-type seedlings, which exhibit an intermediate level of GFP expression, mutants identified in the screen feature either a "GFP-weak" or "Hyper-GFP" phenotype depending on the ratio of the three splice variants. GFP-weak mutants, including previously identified prp8 and rtf2, contain a higher proportion of unspliced transcript or canonically spliced transcript, neither of which is translatable into GFP protein. In contrast, the coilindeficient hyper-gfp1 (hgf1) mutant displays a higher proportion of translatable GFP mRNA, which arises from enhanced splicing of a U2-type intron with noncanonical AT-AC splice sites. Here we report three new hgf mutants that are defective, respectively, in spliceosome-associated proteins SMU1, SmF, and CWC16, an Yju2/CCDC130-related protein that has not yet been described in plants. The smu1 and cwc16 mutants have substantially increased levels of translatable GFP transcript owing to preferential splicing of the U2-type AT-AC intron, suggesting that SMU1 and CWC16 influence splice site selection in GFP pre-mRNA. Genome-wide analyses of splicing in smu1 and cwc16 mutants revealed a number of introns that were variably spliced from endogenous pre-mRNAs. These results indicate that SMU1 and CWC16, which are predicted to act directly prior to and during the first catalytic step of splicing, respectively, function more generally to modulate splicing patterns in plants.

show abstract

“…the essential Gly67 residue for GFP fluorescence, so that even in-frame mutations result646 in a loss of fluorescence(Fu et al, 2015). Hence, gRNAs targeting genes of interest in 647 particularly sensitive sites, such as crucial interaction domains or active sites, can further 648 increase the likelihood of CRISPR-TSKO being effective.649 We observed a strong correlation between gene knockout and Cas9-mCherry expression,650 which can be used to facilitate event selection.…”

mentioning

confidence: 88%

CRISPR-TSKO facilitates efficient cell type-, tissue-, or organ-specific mutagenesis in Arabidopsis

Decaestecker

Buono

Pfeiffer

et al. 2018

Preprint

View full text Add to dashboard Cite

17Detailed functional analyses of many fundamentally-important plant genes via 18 conventional loss-of-function approaches are impeded by severe pleiotropic phenotypes. 19 In particular, mutations in genes that are required for basic cellular functions and/or 20 reproduction often interfere with the generation of homozygous mutant plants, precluding 21 further functional studies. To overcome this limitation, we devised a CRISPR-based 22 tissue-specific knockout system, CRISPR-TSKO, enabling the generation of somatic 23 mutations in particular plant cell types, tissues, and organs. In Arabidopsis, CRISPR-24 TSKO mutations in essential genes caused well-defined, localized phenotypes in the root 25 cap, stomatal lineage, or entire lateral roots. The underlying modular cloning system 26 allows for efficient selection, identification, and functional analysis of mutant lines directly 27 in the first transgenic generation. The efficacy of CRISPR-TSKO opens new avenues to 28 discover and analyze gene functions in spatial and temporal contexts of plant life while 29 avoiding pleiotropic effects of system-wide loss of gene function.30 31 32 88 and organs. We also detail important considerations and limitations on the use of 89 CRISPR-TSKO and provide best practices for researchers. Our approach opens new 90 opportunities to study the function of fundamentally-important genes in specific contexts 91 of plant development and creates new possibilities to investigate post-embryonic 92 developmental processes.93 4 Results 94Proof-of-concept: tissue-specific GFP knockout in the lateral root cap 95 We reasoned that by using tissue-specific, somatic promoters to drive Cas9 expression, 96CRISPR could be used to generate cell type-, tissue-, and organ-specific DNA mutations 97 in plants. To test this hypothesis, T-DNA vectors were constructed with Cas9 expression 98 controlled by the promoter region of SOMBRERO/ANAC033 (SMB; AT1G79580). The 99 SMB promoter (pSMB) is highly root cap-specific and activated directly after the formative 100 division of root cap stem cells (Willemsen et al., 2008; Fendrych et al., 2014). A 101 pSMB:Cas9 expression cassette was combined with one of two gRNAs targeting the GFP 102 coding sequence, GFP-1 and GFP-2, and transformed into a homozygous Arabidopsis 103 line with ubiquitous expression of a nuclear-localized GFP and β-glucuronidase (GUS) 104 fusion protein (pHTR5:NLS-GFP-GUS (Ingouff et al., 2017), henceforth, NLS-GFP).105Primary transgenic plants (T1 seedlings) were selected via resistance to the herbicide 106 glufosinate and investigated for loss of GFP signal in the root tips of five-day-old seedlings.

show abstract

GFP Loss-of-Function Mutations inArabidopsis thaliana

Cited by 26 publications

References 25 publications

CRISPR-TSKO: A Technique for Efficient Mutagenesis in Specific Cell Types, Tissues, or Organs in Arabidopsis

CRISPR-TSKO: A Technique for Efficient Mutagenesis in Specific Cell Types, Tissues, or Organs in Arabidopsis

A genetic screen implicates a CWC16/Yju2/CCDC130 protein and SMU1 in alternative splicing in Arabidopsis thaliana

CRISPR-TSKO facilitates efficient cell type-, tissue-, or organ-specific mutagenesis in Arabidopsis

Contact Info

Product

Resources

About