Abstract
Precise genome editing of plants has the potential to reshape global agriculture through the targeted engineering of endogenous pathways or the introduction of new traits. To develop a CRISPR nuclease-based platform that would enable higher efficiencies of precise gene insertion or replacement, we screened the Cpf1 nucleases from Francisella novicida and Lachnospiraceae bacterium ND2006 for their capability to induce targeted gene insertion via homology directed repair. Both nucleases, in the presence of a guide RNA and repairing DNA template flanked by homology DNA fragments to the target site, were demonstrated to generate precise gene insertions as well as indel mutations at the target site in the rice genome. The frequency of targeted insertion for these Cpf1 nucleases, up to 8%, is higher than most other genome editing nucleases, indicative of its effective enzymatic chemistry. Further refinements and broad adoption of the Cpf1 genome editing technology have the potential to make a dramatic impact on plant biotechnology.
Highlights
Plant biotechnology using traditional molecular biology and transformation technologies has resulted in crops that have reduced energy intensive inputs, improved yields, and increased food, fuel, and fiber security across the globe
The Cpf[1] nucleases from Fransicella novicida (FnCpf1) and Lachnospiraceae bacterium ND2006 (LbCpf1) were selected for investigation in plants with the goal of identifying CRISPR nuclease activities, when combined with the plant endogenous repairing events, that lead to gene knockouts and targeted insertion, both of which are inherently important to a robust plant genome editing platform
To test the capability of each Cpf[1] enzyme to generate targeted gene insertion via homology directed repair (HDR), a screen was developed that would result in a visual phenotype upon genome editing
Summary
To develop a CRISPR nucleasebased platform that would enable higher efficiencies of precise gene insertion or replacement, we screened the Cpf[1] nucleases from Francisella novicida and Lachnospiraceae bacterium ND2006 for their capability to induce targeted gene insertion via homology directed repair. The adoption of genome editing in industrial plant biotechnology has resulted in new products with improved disease tolerance, yield, and food quality[2,3,4] These advanced products were developed at a fraction of the time and cost of traditional ag-biotech traits. The Cpf[1] nucleases from Fransicella novicida (FnCpf1) and Lachnospiraceae bacterium ND2006 (LbCpf1) were selected for investigation in plants with the goal of identifying CRISPR nuclease activities, when combined with the plant endogenous repairing events, that lead to gene knockouts and targeted insertion, both of which are inherently important to a robust plant genome editing platform. Multiple Cpf[1] nucleases were tested in our plant transformation system and both HDR and NHEJ repair genome edits were observed
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