Transforming plant biology and breeding with <scp>CRISPR</scp>/Cas9, Cas12 and Cas13

Schindele, Patrick; Wolter, Felix; Puchta, Holger

doi:10.1002/1873-3468.13073

Cited by 78 publications

(49 citation statements)

References 107 publications

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“…Post‐transcriptional gene knockdown, instead of transcriptional regulation, can be interesting when only certain splicing variants are to be regulated, as all isoforms are repressed during transcriptional regulation (Mahas et al ). Additionally, gene activity suppression by post‐transcriptional regulation provides a fast, effective and highly specific means of regulation: existing cytoplasmic mRNAs get cleaved, whereas transcriptional regulation suppresses the production of further mRNA generations, while already produced mRNAs remain active (Schindele et al ; Wolter and Puchta ). An applied example of such post‐transcriptional repression was recently shown to successfully combat an RNA virus in plants, using the Cas13a ribonuclease from Leptotrichia shahii (LshCas13a).…”

Section: Modifications and Extensions Of Cas Endonucleasesmentioning

confidence: 99%

“…While initial efforts for targeted genome rearrangements, applying rare cutters like I-SceI, were limited by the need of artificially inserting the target site as part of a transgene, the development and availability of engineered zinc-finger nucleases (ZFN) (Kim et al 1996;Smith et al 2000), transcription activator-like effector nucleases (TALENs) (Boch et al 2009;Moscou and Bogdanove 2009;Cermak et al 2011) and, most recently, the CRISPR (clustered regularly interspaced palindromic repeat)-associated (Cas) system, enabled the generation of new genome engineering tools for addressing nearly all genomic loci with unprecedented accuracy and efficiency (Voytas 2013;Mahfouz et al 2014;Belhaj et al 2015;Schaeffer and Nakata 2015;Lee et al 2016;Weeks et al 2016;Pacher and Puchta 2017;Puchta 2017;Schindele et al 2018).…”

Section: Sequence-specific Induction Of Dna Double-strand Breaksmentioning

confidence: 99%

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The CRISPR/Cas revolution continues: From efficient gene editing for crop breeding to plant synthetic biology

Kumlehn

Pietralla

Hensel

et al. 2018

JIPB

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Since the discovery that nucleases of the bacterial CRISPR (clustered regularly interspaced palindromic repeat)‐associated (Cas) system can be used as easily programmable tools for genome engineering, their application massively transformed different areas of plant biology. In this review, we assess the current state of their use for crop breeding to incorporate attractive new agronomical traits into specific cultivars of various crop plants. This can be achieved by the use of Cas9/12 nucleases for double‐strand break induction, resulting in mutations by non‐homologous recombination. Strategies for performing such experiments − from the design of guide RNA to the use of different transformation technologies − are evaluated. Furthermore, we sum up recent developments regarding the use of nuclease‐deficient Cas9/12 proteins, as DNA‐binding moieties for targeting different kinds of enzyme activities to specific sites within the genome. Progress in base deamination, transcriptional induction and transcriptional repression, as well as in imaging in plants, is also discussed. As different Cas9/12 enzymes are at hand, the simultaneous application of various enzyme activities, to multiple genomic sites, is now in reach to redirect plant metabolism in a multifunctional manner and pave the way for a new level of plant synthetic biology.

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Section: Modifications and Extensions Of Cas Endonucleasesmentioning

confidence: 99%

Section: Sequence-specific Induction Of Dna Double-strand Breaksmentioning

confidence: 99%

The CRISPR/Cas revolution continues: From efficient gene editing for crop breeding to plant synthetic biology

Kumlehn

Pietralla

Hensel

et al. 2018

JIPB

Self Cite

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“…Similarly, CRISPR/Cas12a, an alternative gene editing tool, can be deployed on sequences ending with TTTN motifs (Endo et al, 2016;Schindele et al, 2018;Wang et al, 2017;Zetsche et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

Heat-shock inducible CRISPR/Cas9 system generates heritable mutations in rice

Nandy

Pathak

Zhao

et al. 2018

Preprint

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Transient expression of CRISPR/Cas9 is an effective approach for limiting its activities and improving its precision in genome editing. Here, we describe the heat-shock inducible CRISPR/Cas9 system for controlled genome editing, and demonstrate its efficiency in the model crop, rice. Using a soybean heatshock protein gene promoter and the rice U3 promoter to express Cas9 and sgRNA, respectively, we developed the heat-shock (HS) inducible CRISPR/Cas9 system, and tested its efficacy in targeted mutagenesis. Two loci were targeted by transforming rice with HS-CRISPR/Cas9 vectors, and the presence of targeted mutations was determined before and after the HS treatment. We found only a low rate of targeted mutagenesis before HS, but an increased rate of mutagenesis after HS treatment among the transgenic lines. Specifically, only ~11% of transformants showed characteristic insertions-deletions at the ambient room temperature, but a higher percentage (~45%) of callus lines developed mutations after a few days of HS treatment. Analysis of regenerated plants harboring HS-CRISPR/Cas9 revealed that targeted mutagenesis was suppressed in the plants but induced by HS, which was detectable by Sanger sequencing after several weeks of HS treatments. Most importantly, the HS-induced mutations were transmitted to the progeny at a high rate, generating monoallelic and biallelic mutant lines that independently segregated from Cas9. Taken together, this work shows that HS-CRISPR/Cas9 is a controlled and reasonably efficient platform for genome editing, and therefore, a promising tool for limiting genome-wide off-target effects and improving the precision of genome editing. Significance Statement:A method for the temporal control on gene editing based on the use of heat-shock induced expression of CRISPR/Cas9 is described, which was efficient in producing heritable mutations in the rice genome. We assume this method will be useful for targeting essential genes and improving the precision of CRISPR/Cas9.

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“…However, precise and efficient base editing via HR in 92 plants suffers from low efficiency and the delivery of template DNA is still challenging 93 (Schindele et al 2018). A new CRISPR-Cas9-based genome editing system has recently been 94 developed based on fusing either a cytidine or an adenine deaminase to a Cas9 nickase (nCas9 95 D10A), leading to a C-to-T or an A-to-G conversion on the edited strand, respectively 96 (Schindele et al 2018). The sgRNA directs the nCas9/deaminase fusion to the target locus, 97 enabling edition on the non-complementary strand.…”

Section: Tubers 32mentioning

confidence: 99%

The Solanum tuberosum GBSSI gene: a target for assessing gene and base editing in tetraploid potato

Veillet

Chauvin

Kermarrec

et al. 2019

Preprint

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The StGBSSI gene was successfully and precisely edited in the tetraploid potato using gene and base editing strategies, leading to plants with impaired amylose biosynthesis. AbstractGenome editing has recently become a method of choice for basic research and functional 4 genomics, and holds great potential for molecular plant breeding applications. The powerful 5 CRISPR-Cas9 system that typically produces double-strand DNA breaks is mainly used to 6 generate knockout mutants. Recently, the development of base editors has broadened the 7 scope of genome editing, allowing precise and efficient nucleotide substitutions. In this study, 8we produced mutants in two cultivated elite cultivars of the tetraploid potato (Solanum 9 tuberosum) using stable or transient expression of the CRISPR-Cas9 components to knockout 10 the amylose-producing StGBSSI gene. We set up a rapid, highly sensitive and cost-effective 11 screening strategy based on high-resolution melting analysis followed by direct Sanger 12 sequencing and trace chromatogram analysis. Most mutations consisted of small indels, but 13 unwanted insertions of plasmid DNA were also observed. We successfully created tetra-14 allelic mutants with impaired amylose biosynthesis, confirming the loss-of-function of the 15 StGBSSI protein. The second main objective of this work was to demonstrate the proof of 16 concept of CRISPR-Cas9 base editing in the tetraploid potato by targeting two loci encoding 17 catalytic motifs of the StGBSSI enzyme. Using a cytidine base editor (CBE), we efficiently 18 and precisely induced DNA substitutions in the KTGGL-encoding locus, leading to discrete 19 variation in the amino acid sequence and generating a loss-of-function allele. The successful 20 application of base editing in the tetraploid potato opens up new avenues for genome 21 engineering in this species. 22 23 24 tuberosum. We identified single-and multi-allelic edited plants, and mutations in all four 113 alleles were observed, resulting in amylose biosynthesis impairment. To induce this 114 modification, we used protoplast transformation, which can produce non-transgenic plants. 115 Finally, we assessed the efficiency of the cytidine base editor (CBE) in two loci encoding 116 catalytic motifs of the StGBSSI enzyme, resulting in precise base conversion and amino-acid 117 substitution. Our results highlight that CRISPR-Cas9 gene and base editing can be efficiently 118 developed in a genetically complex and vegetatively propagated crop such as potato to 119 modify agronomic traits. 120 121 6 Materials and methods 122 Plant material 123 Potato cultivars Desiree (ZPC, the Netherlands) and Furia (Germicopa, France) were in vitro 124 propagated in 1X Murashige and Skoog (MS) medium (pH 5.8) including vitamins (Duchefa, 125 the Netherlands), 0.4 mg/L thiamine hydrochloride (Sigma-Aldrich, USA), 2.5% sucrose and 126 0.8% agar powder (VWR, USA). Plants were cultivated in a growth chamber at 19°C with a 127 16:8 h light:dark photoperiod. For the production of in vitro microtubers, plants w...

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Transforming plant biology and breeding with CRISPR/Cas9, Cas12 and Cas13

Cited by 78 publications

References 107 publications

The CRISPR/Cas revolution continues: From efficient gene editing for crop breeding to plant synthetic biology

The CRISPR/Cas revolution continues: From efficient gene editing for crop breeding to plant synthetic biology

Heat-shock inducible CRISPR/Cas9 system generates heritable mutations in rice

The Solanum tuberosum GBSSI gene: a target for assessing gene and base editing in tetraploid potato

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