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. 2021 Jul 1;12:689937. doi: 10.3389/fpls.2021.689937

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Copyright © 2021 Vazquez-Vilar, Garcia-Carpintero, Selma, Bernabé-Orts, Sanchez-Vicente, Salazar-Sarasua, Ressa, de Paola, Ajenjo, Quintela, Fernández-del-Carmen, Granell and Orzáez.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Flowchart and functional validation of the software-assisted procedure for gRNAs cloning with GoldenBraid. (A) Schematic representation of the software tools for Cas9 gRNAs cloning. Single Cas9 gRNAs cloning is assisted via the Single Cas9_gRNA Domesticator (tS9D) and the Single Cas9_gRNA Assembler (tS9A) (top). Cas9 multiplexing gRNAs are assembled with the use of three consecutive software tools: the Multi Cas9_gRNA Domesticator 1 (tM9D1), the Multi Cas9 gRNA Domesticator 2 (tM9D2), and the Multiple Cas9_gRNA Assembler (tM9A) that creates Level 1 gRNA arrays (middle). The CRISPR for Dummies tool takes 1–6 protospacers as input and generates Level 1 gRNA arrays in a single step (bottom). (B) Correlation of Cas9 gRNAs predicted on-target scores and editing efficiencies tested in N. benthamiana transient expression. Schematic representation of the plasmids co-infiltrated in this experiment (top) and Cas9 guide RNAs editing efficiencies (left axis, bars, determined with ICE) and their corresponding on-target score (right axis, line) determined with the “Rule Set2 scoring” (bottom). (C) Correlation of Cas12a crRNAs predicted on-target scores and editing efficiencies tested in N. benthamiana transient expression. Schematic representation of the plasmids co-infiltrated in this experiment (top) and Cas12a guide RNAs editing efficiencies (left axis, bars, determined with TIDE) and their corresponding on-target score (right axis, line) determined with CINDEL (bottom).