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Gaillochet, Christophe; Peña Fernández, Alexandra; Goossens, Vera; D’Halluin, Katelijn; Drozdzecki, Andrzej; Shafie, Myriam; Van Duyse, Julie; Van Isterdael, Gert; Gonzalez, Camila; Vermeersch, Mattias; De Saeger, Jonas; Develtere, Ward; Audenaert, Dominique; De Vleesschauwer, David; Meulewaeter, Frank; Jacobs, Thomas B.

Systematic optimization of Cas12a base editors in wheat and maize using the ITER platform

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  • Medientyp: E-Artikel
  • Titel: Systematic optimization of Cas12a base editors in wheat and maize using the ITER platform
  • Beteiligte: Gaillochet, Christophe; Peña Fernández, Alexandra; Goossens, Vera; D’Halluin, Katelijn; Drozdzecki, Andrzej; Shafie, Myriam; Van Duyse, Julie; Van Isterdael, Gert; Gonzalez, Camila; Vermeersch, Mattias; De Saeger, Jonas; Develtere, Ward; Audenaert, Dominique; De Vleesschauwer, David; Meulewaeter, Frank; Jacobs, Thomas B.
  • Erschienen: Springer Science and Business Media LLC, 2023
  • Erschienen in: Genome Biology, 24 (2023) 1
  • Sprache: Englisch
  • DOI: 10.1186/s13059-022-02836-2
  • ISSN: 1474-760X
  • Entstehung:
  • Anmerkungen:
  • Beschreibung: Abstract Background Testing an ever-increasing number of CRISPR components is challenging when developing new genome engineering tools. Plant biotechnology has few high-throughput options to perform iterative design-build-test-learn cycles of gene-editing reagents. To bridge this gap, we develop ITER (Iterative Testing of Editing Reagents) based on 96-well arrayed protoplast transfections and high-content imaging. Results We validate ITER in wheat and maize protoplasts using Cas9 cytosine and adenine base editors (ABEs), allowing one optimization cycle — from design to results — within 3 weeks. Given that previous LbCas12a-ABEs have low or no activity in plants, we use ITER to develop an optimized LbCas12a-ABE. We show that sequential improvement of five components — NLS, crRNA, LbCas12a, adenine deaminase, and linker — leads to a remarkable increase in activity from almost undetectable levels to 40% on an extrachromosomal GFP reporter. We confirm the activity of LbCas12a-ABE at endogenous targets in protoplasts and obtain base-edited plants in up to 55% of stable wheat transformants and the edits are transmitted to T1 progeny. We leverage these improvements to develop a highly mutagenic LbCas12a nuclease and a LbCas12a-CBE demonstrating that the optimizations can be broadly applied to the Cas12a toolbox. Conclusion Our data show that ITER is a sensitive, versatile, and high-throughput platform that can be harnessed to accelerate the development of genome editing technologies in plants. We use ITER to create an efficient Cas12a-ABE by iteratively testing a large panel of vector components. ITER will likely be useful to create and optimize genome editing reagents in a wide range of plant species.
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