CRISPR directed evolution of the spliceosome for resistance to splicing inhibitors

Butt, Haroon; Eid, Ayman; Momin, Afaque Ahmad Imtiyaz; Bazin, Jérémie; Crespi, Martín; Arold, Stefan T.; Mahfouz, Magdy M.

doi:10.1186/s13059-019-1680-9

Cited by 110 publications

(75 citation statements)

References 30 publications

(27 reference statements)

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“…The herbicide GEX1A was used for CDE selection of OsSF3B1 variants. The variants generated in this proof-of-concept study displayed different levels of GEX1A resistance, with plants harboring K1049R, K1050E, and G1051H mutations being almost insensitive to GEX1A treatment [9]. All of the observed transmitted mutations were either deletions or substitutions but were in frame.…”

Section: Cde Via Imprecise Dsb Repairmentioning

confidence: 90%

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CRISPR-Based Directed Evolution for Crop Improvement

Butt

Zaidi

Hassan

et al. 2020

Trends in Biotechnology

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Section: Cde Via Imprecise Dsb Repairmentioning

confidence: 90%

“…A recent study used NHEJ for CDE in rice [9] with an sgRNA library targeting SPLICING FACTOR 3B SUBUNIT 1 (OsSF3B1). OsSF3B1 is an essential gene that is conserved among eukaryotes and targeted by the splicing inhibitors pladienolide B and herboxidiene (GEX1A) [9].…”

Section: Cde Via Imprecise Dsb Repairmentioning

confidence: 99%

CRISPR-Based Directed Evolution for Crop Improvement

Butt

Zaidi

Hassan

et al. 2020

Trends in Biotechnology

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“…RuBisCO was engineered using directed evolution in Rhodobacter capsulatus (Smith & Tabita, 2003) and Escherichia coli (Parikh et al , 2006). More recently, a ‘Cas9‐mediated directed evolution approach’ was demonstrated in rice by deploying 119 sgRNAs to identify protein variants that confer resistance to a splicing inhibitor (Butt et al , 2019).…”

Section: Engineering Proteinsmentioning

confidence: 99%

Beyond natural: synthetic expansions of botanical form and function

Patron

2020

New Phytologist

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Summary Powered by developments that enabled genome‐scale investigations, systems biology emerged as a field aiming to understand how phenotypes emerge from network functions. These advances fuelled a new engineering discipline focussed on synthetic reconstructions of complex biological systems with the goal of predictable rational design and control. Initially, progress in the nascent field of synthetic biology was slow due to the ad hoc nature of molecular biology methods such as cloning. The application of engineering principles such as standardisation, together with several key technical advances, enabled a revolution in the speed and accuracy of genetic manipulation. Combined with mathematical and statistical modelling, this has improved the predictability of engineering biological systems of which nonlinearity and stochasticity are intrinsic features leading to remarkable achievements in biotechnology as well as novel insights into biological function. In the past decade, there has been slow but steady progress in establishing foundations for synthetic biology in plant systems. Recently, this has enabled model‐informed rational design to be successfully applied to the engineering of plant gene regulation and metabolism. Synthetic biology is now poised to transform the potential of plant biotechnology. However, reaching full potential will require conscious adjustments to the skillsets and mind sets of plant scientists.

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“…This DSB can be repaired by the nonhomologous end joining (NHEJ) repair pathway, which produces imprecise repairs, or by homology-directed repair (HDR), which produces precise repairs based on a nucleic acid template 7,8 . The CRISPR/ Cas9 system is widely used for gene editing in plants because of its ability to produce site-specific gene knockout (loss of function) or new functional variants of an allele via short insertions and deletions (Indels) from NHEJ repair 9,10 . Base editors can also produce single-base conversions in a short window of a target sequence 11,12 .…”

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confidence: 99%

Fusion of the Cas9 endonuclease and the VirD2 relaxase facilitates homology-directed repair for precise genome engineering in rice

et al. 2020

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Precise genome editing by systems such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) requires high-efficiency homologydirected repair (HDR). Different technologies have been developed to improve HDR but with limited success. Here, we generated a fusion between the Cas9 endonuclease and the Agrobacterium VirD2 relaxase (Cas9-VirD2). This chimeric protein combines the functions of Cas9, which produces targeted and specific DNA double-strand breaks (DSBs), and the VirD2 relaxase, which brings the repair template in close proximity to the DSBs, to facilitate HDR. We successfully employed our Cas9-VirD2 system for precise ACETOLACTATE SYN-THASE (OsALS) allele modification to generate herbicide-resistant rice (Oryza sativa) plants, CAROTENOID CLEAVAGE DIOXYGENASE-7 (OsCCD7) to engineer plant architecture, and generate in-frame fusions with the HA epitope at HISTONE DEACETYLASE (OsHDT) locus. The Cas9-VirD2 system expands our ability to improve agriculturally important traits in crops and opens new possibilities for precision genome engineering across diverse eukaryotic species.

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CRISPR directed evolution of the spliceosome for resistance to splicing inhibitors

Cited by 110 publications

References 30 publications

CRISPR-Based Directed Evolution for Crop Improvement

CRISPR-Based Directed Evolution for Crop Improvement

Beyond natural: synthetic expansions of botanical form and function

Fusion of the Cas9 endonuclease and the VirD2 relaxase facilitates homology-directed repair for precise genome engineering in rice

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