A family of ROP proteins that suppress actin dynamics and are essential for polarized growth and cell adhesion

Burkart, Graham M.; Baskin, Tobias I.; Bezanilla, Magdalena

doi:10.1242/jcs.172445

Cited by 40 publications

(68 citation statements)

References 57 publications

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“…As shown in Figure 6B and 6C, ΔROP1-3; ROP4(W100R) mutant showed severe growth defects; in contrast, the ΔROP1-3 mutant exhibited only a moderate growth phenotype. Protonemal cells of ΔROP1-3; ROP4(W100R) mutant were dramatically shortened and appeared in a round shape, which is consistent with the observation of strong RNAi knockdown of all ROP genes (Figure 6D) (Burkart et al, 2015). We obtained eight independent lines.…”

Section: Resultssupporting

confidence: 86%

“…The Rho of Plants (ROP) GTPases are conserved molecules that play diverse roles in cell growth and signaling (Feiguelman et al, 2018). In P. patens, four ROP family members have been identified; however, no strong hypomorphic mutants are available (Eklund et al, 2010; Burkart et al, 2015). We constructed the ROP4(W100R) mutant on the basis that the mutated residue is highly conserved and its corresponding mutant in yeast is temperature-sensitive (Figure 2B & Figure 6A) (Miller and Johnson, 1997).…”

Section: Resultsmentioning

confidence: 99%

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Fast, Efficient, and Precise Gene Editing in the MossPhyscomitrella patens

Goshima

2019

Preprint

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12Recent years, the bryophyte moss Physcomitrella patens has become an emerging 13 model organism for studying conserved signaling pathways and developmental 14 processes during plant evolution. Its short life cycle, ease of cultivation, and high rate of 15 homologous recombination have made it an ideal system for genetic analysis. However, 16 the presence of highly redundant genes and the difficulty of isolating hypomorphic 17 mutants have limited its broader use. Here we developed a simple, fast, and efficient 18 method to generate customized mutants in P. patens. We show that transient 19 cotransformation of CRISPR/Cas9 and oligonucleotide templates enables microindel 20 knock-in with high efficiency and accuracy. Using this method, we generated strains 21 carrying various types of mutations, including amino acid substitution, out-of-frame 22 deletion/insertion, splice site alteration, and small tag integration. We also demonstrate 23 that multiplex gene editing can be efficiently achieved to generate putative null and 24 hypomorphic mutants of redundant genes in one step. Thus our method will not only 25 simplify multiple-gene knockout, but also allows the generation of hypomorphic mutants 26 of genes of interest, especially those that are essential for viability. 27 28 Keywords 29 Bryophyte, Physcomitrella patens, CRISPR/Cas9, Oligonucleotide, Knock-in 30 4 2017a, 2017b). By forming a complex, its components, the endonuclease Cas9 and the 62 single guide RNA (sgRNA), are capable of introducing double-strand breaks (DSBs) at 63 predicted genomic loci once the sgRNA is customized with a targeting sequence (Jiang 64 and Doudna, 2017). These DSBs are repaired through the non-homologous end joining 65 or homology directed repair pathways, which leads to subsequent nucleotide changes 66 (Jiang and Doudna, 2017). In P. patens, CRISPR/Cas9 has been exploited to generate 67 heritable mutants (Nomura et al., 2016; Lopez-Obando et al., 2016; Collonnier et al., 68 2017a). However, it is unclear whether precise mutation knock-in can be achieved to 69 generate hypomorphic mutants. In addition, as microhomology-mediated end joining 70 (MMEJ) pathway is active in P. patens (Kamisugi et al., 2006; Collonnier et al., 2017a), 71 template-free editing can generate specific deletions at a high frequency, which may not 72 necessarily introduce out-of-frame mutations. For instance, a previous study reported 73 that 74% of the mutations generated by one CRISPR target are identical in-frame 74 deletions (Collonnier et al., 2017a).75 76Here we report a fast and efficient method to generate customized mutants in P. patens. 77 We show that transient cotransformation of CRISPR/Cas9 and oligonucleotide 78 templates induces microindel knock-in at a high efficiency and accuracy. This allows us 79 to introduce various types of mutations into the genome including amino acid 80 substitution, out-of-frame insertion/deletion, splice site alteration, or small tag integration. 81 We also demonstrate that multiplex gene editing can be effici...

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Section: Resultssupporting

confidence: 86%

Section: Resultsmentioning

confidence: 99%

Fast, Efficient, and Precise Gene Editing in the MossPhyscomitrella patens

Goshima

2019

Preprint

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“…In the apical cell, the polarity of cytoplasmic microtubules between the nucleus and the cell apex is tipward, with an apical focus of microtubule plus ends (Hiwatashi et al, 2014). In contrast, the cortical microtubule array consists of shorter microtubules that lack polarity (Burkart et al, 2015;Nakaoka et al, 2015).…”

Section: Microtubule Cytoskeletonmentioning

confidence: 99%

Interplay between Ions, the Cytoskeleton, and Cell Wall Properties during Tip Growth

2017

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“…However, establishing RNAi stable lines can be challenging. Moreover, in most cases, RNAi seems to favor gene knockdown rather than knockout in this moss (Burkart et al 2015; Nakaoka et al 2012). …”

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confidence: 99%

Simple and Efficient Targeting of Multiple Genes Through CRISPR-Cas9 in Physcomitrella patens

López-Obando

Hoffmann

Géry

et al. 2016

G3 Genes|Genomes|Genetics

112

138

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Powerful genome editing technologies are needed for efficient gene function analysis. The CRISPR-Cas9 system has been adapted as an efficient gene-knock-out technology in a variety of species. However, in a number of situations, knocking out or modifying a single gene is not sufficient; this is particularly true for genes belonging to a common family, or for genes showing redundant functions. Like many plants, the model organism Physcomitrella patens has experienced multiple events of polyploidization during evolution that has resulted in a number of families of duplicated genes. Here, we report a robust CRISPR-Cas9 system, based on the codelivery of a CAS9 expressing cassette, multiple sgRNA vectors, and a cassette for transient transformation selection, for gene knock-out in multiple gene families. We demonstrate that CRISPR-Cas9-mediated targeting of five different genes allows the selection of a quintuple mutant, and all possible subcombinations of mutants, in one experiment, with no mutations detected in potential off-target sequences. Furthermore, we confirmed the observation that the presence of repeats in the vicinity of the cutting region favors deletion due to the alternative end joining pathway, for which induced frameshift mutations can be potentially predicted. Because the number of multiple gene families in Physcomitrella is substantial, this tool opens new perspectives to study the role of expanded gene families in the colonization of land by plants.

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A family of ROP proteins that suppress actin dynamics and are essential for polarized growth and cell adhesion

Cited by 40 publications

References 57 publications

Fast, Efficient, and Precise Gene Editing in the MossPhyscomitrella patens

Fast, Efficient, and Precise Gene Editing in the MossPhyscomitrella patens

Interplay between Ions, the Cytoskeleton, and Cell Wall Properties during Tip Growth

Simple and Efficient Targeting of Multiple Genes Through CRISPR-Cas9 in Physcomitrella patens

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