Sophisticated CRISPR/Cas tools for fine-tuning plant performance

Capdeville, Niklas; Merker, Laura; Schindele, Patrick; Puchta, Holger

doi:10.1016/j.jplph.2020.153332

Cited by 10 publications

(8 citation statements)

References 171 publications

(190 reference statements)

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“…In contrast, precise gene editing still occurs at relatively low frequencies or it is limited to specific (cell wall-less) strains or experimental approaches ( Ferenczi et al, 2017 ; Greiner et al, 2017 ; Jiang and Weeks, 2017 ). In Chlamydomonas , as observed in land plants and other eukaryotes ( Gallagher and Haber, 2018 ; Kan et al, 2017 ; Paix et al, 2017 ; Boel et al, 2018 ; Richardson et al, 2018 ; Sansbury et al, 2019 ; Capdeville et al, 2020 ; Gallagher et al, 2020 ), the repair of DSBs induced by CRISPR/Cas9 RNPs likely occurs by several distinct pathways, partly determined by the repair machinery expressed in each cell, the complexity of the DSB, and the nature of the DNA molecules involved in the repair. As described for some mammalian cell lines ( Shy et al, 2016 ; Mitzelfelt et al, 2017 ), only a small subset of the population, in asynchronously grown Chlamydomonas , may be capable of HDR, possibly associated with being in a certain phase of the cell cycle ( Angstenberger et al, 2020 ).…”

Section: Discussionmentioning

confidence: 99%

“…precise nucleotide changes in a target genomic sequence) triggered by RNA-programmable SSNs remains fairly inefficient or limited to specific strains/experimental approaches ( Ferenczi et al, 2017 ; Greiner et al, 2017 ; Jiang and Weeks, 2017 ). As reported for somatic cells of land plants ( Capdeville et al, 2020 ), a major constraint is that, in Chlamydomonas , DSB repair by error-prone NHEJ pathways appears to be much more efficient than HR in the presence of donor DNA ( Sizova et al, 2013 ; Greiner et al, 2017 ). As described for some mammalian cell lines ( Shy et al, 2016 ; Mitzelfelt et al, 2017 ), only a small subset of the population, in asynchronously grown Chlamydomonas , may be capable of HDR, possibly associated with being in a certain phase of the cell cycle ( Angstenberger et al, 2020 ).…”

Section: Introductionmentioning

confidence: 90%

“…DSB repair by error-prone nonhomologous end-joining (NHEJ), including canonical and alternative pathways, may result in insertions/deletions (i.e. indels) and/or missense/nonsense mutations at the target sites ( Rodgers and McVey, 2016 ; Gallagher and Haber, 2018 ; Scully et al, 2019 ; Capdeville et al, 2020 ; Gallagher et al, 2020 ). Alternatively, homology directed repair (HDR) in the presence of template donor DNA, which can also occur by several pathways, may result in precise sequence changes ( Rodgers and McVey, 2016 ; Gallagher and Haber, 2018 ; Paix et al, 2017 ; Scully et al, 2019 ; Capdeville et al, 2020 ; Gallagher et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

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Co-targeting strategy for precise, scarless gene editing with CRISPR/Cas9 and donor ssODNs in Chlamydomonas

Akella

Bačová

et al. 2021

Plant Physiology

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Programmable site-specific nucleases, such as the CRISPR/Cas9 ribonucleoproteins (RNPs), have allowed creation of valuable knockout mutations and targeted gene modifications in Chlamydomonas (Chlamydomonas reinhardtii). However, in walled strains, present methods for editing genes lacking a selectable phenotype involve co-transfection of RNPs and exogenous double-stranded DNA (dsDNA) encoding a selectable marker gene. Repair of the double-stranded DNA breaks induced by the ribonucleoproteins is usually accompanied by genomic insertion of exogenous dsDNA fragments, hindering the recovery of precise, scarless mutations in target genes of interest. Here, we tested whether co-targeting two genes by electroporation of pairs of CRISPR/Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) would facilitate the recovery of precise edits in a gene of interest (lacking a selectable phenotype) by selection for precise editing of another gene (creating a selectable marker) - in a process completely lacking exogenous dsDNA. We used PPX1 (encoding protoporphyrinogen IX oxidase) as the generated selectable marker, conferring resistance to oxyfluorfen, and identified precise edits in the homolog of bacterial ftsY or the WD and TetratriCopeptide repeats protein 1 (WDTC1) genes in ∼1% of the oxyfluorfen resistant colonies. Analysis of the target site sequences in edited mutants suggested that ssODNs were used as templates for DNA synthesis during homology directed repair, a process prone to replicative errors. The Chlamydomonas acetolactate synthase gene could also be efficiently edited to serve as an alternative selectable marker. This transgene-free strategy may allow creation of individual strains containing precise mutations in multiple target genes, to study complex cellular processes, pathways or structures.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 90%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Co-targeting strategy for precise, scarless gene editing with CRISPR/Cas9 and donor ssODNs in Chlamydomonas

Akella

Bačová

et al. 2021

Plant Physiology

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“…The use of CRISPR/Cas in homologous recombination-based gene targeting (HR-GT) has demonstrated the potential to improve gene-targeting efficiency through precise DSB induction if a donor template is provided to promote the homology-directed repair (HDR) pathway ( Capdeville et al, 2021 ). The HDR-based GT mostly occurs in dividing cells and desired HR-GT products are often mixed with additional indels (insertions/deletions) due to preferred non-homologous end joining (NHEJ).…”

Section: Genome Editing Toolsmentioning

confidence: 99%

Genome Editing for Plasmodesmal Biology

Iswanto

Shelake

et al. 2021

Front. Plant Sci.

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Plasmodesmata (PD) are cytoplasmic canals that facilitate intercellular communication and molecular exchange between adjacent plant cells. PD-associated proteins are considered as one of the foremost factors in regulating PD function that is critical for plant development and stress responses. Although its potential to be used for crop engineering is enormous, our understanding of PD biology was relatively limited to model plants, demanding further studies in crop systems. Recently developed genome editing techniques such as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associate protein (CRISPR/Cas) might confer powerful approaches to dissect the molecular function of PD components and to engineer elite crops. Here, we assess several aspects of PD functioning to underline and highlight the potential applications of CRISPR/Cas that provide new insight into PD biology and crop improvement.

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“…The sgRNA and Cas protein form a Cas/sgRNA complex which is guided to a specific genomic locus site using Watson–Crick base pairing. This results in the cleavage of target DNA sequences adjacent to the PAM (protospacer-adjacent motif), a short, few-nucleotide-long sequence that is crucial for Cas binding [ 18 , 19 ]. Recently, Grützner et al [ 20 ] reported that the addition of 13 introns into the Cas9 coding sequence in combination with two nuclear localization signals (NLS) led to higher accumulation of the Cas9 in the nucleus and significant improvement of editing efficiency.…”

Section: Introductionmentioning

confidence: 99%

Combined fluorescent seed selection and multiplex CRISPR/Cas9 assembly for fast generation of multiple Arabidopsis mutants

et al. 2021

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Background Multiplex CRISPR-Cas9-based genome editing is an efficient method for targeted disruption of gene function in plants. Use of CRISPR-Cas9 has increased rapidly in recent years and is becoming a routine method for generating single and higher order Arabidopsis thaliana mutants. Low entry, reliable assembly of CRISPR/Cas9 vectors and efficient mutagenesis is necessary to enable a maximum of researchers to break through the genetic redundancy within plant multi-gene families and allow for a plethora of gene function studies that have been previously unachievable. It will also allow routine de novo generation of mutations in ever more complex genetic backgrounds that make introgression of pre-existing alleles highly cumbersome. Results To facilitate rapid and efficient use of CRISPR/Cas9 for Arabidopsis research, we developed a CRISPR/Cas9-based toolbox for generating mutations at multiple genomic loci, using two-color fluorescent seed selection. In our system, up-to eight gRNAs can be routinely introduced into a binary vector carrying either a FastRed, FastGreen or FastCyan fluorescent seed selection cassette. FastRed and FastGreen binary vectors can be co-transformed as a cocktail via floral dip to introduce sixteen gRNAs at the same time. The seeds can be screened either for red or green fluorescence, or for the presence of both colors. Importantly, in the second generation after transformation, Cas9 free plants are identified simply by screening the non-fluorescent seeds. Our collection of binary vectors allows to choose between two widely-used promoters to drive Cas enzymes, either the egg cell-specific (pEC1.2) from A. thaliana or the constitutive promoter from Petroselinum crispum (PcUBi4-2). Available enzymes are “classical” Cas9 codon-optimized for A. thaliana and a recently reported, intron-containing version of Cas9 codon-optimized for Zea mays, zCas9i. We observed the highest efficiency in producing knockout phenotypes by using intron-containing zCas9i driven under egg-cell specific pEC1.2 promoter. Finally, we introduced convenient restriction sites flanking promoter, Cas9 and fluorescent selection cassette in some of the T-DNA vectors, thus allowing straightforward swapping of all three elements for further adaptation and improvement of the system. Conclusion A rapid, simple and flexible CISPR/Cas9 cloning system was established that allows assembly of multi-guide RNA constructs in a robust and reproducible fashion, by avoiding generation of very big constructs. The system enables a flexible, fast and efficient screening of single or higher order A. thaliana mutants.

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Sophisticated CRISPR/Cas tools for fine-tuning plant performance

Cited by 10 publications

References 171 publications

Co-targeting strategy for precise, scarless gene editing with CRISPR/Cas9 and donor ssODNs in Chlamydomonas

Co-targeting strategy for precise, scarless gene editing with CRISPR/Cas9 and donor ssODNs in Chlamydomonas

Genome Editing for Plasmodesmal Biology

Combined fluorescent seed selection and multiplex CRISPR/Cas9 assembly for fast generation of multiple Arabidopsis mutants

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