CRISPR/Cas9 mediated homology directed repair enables precise modification in sugarcane ALS gene

Gisela Giampaoli¹, Maximiliano Martín Sosa², Amalia Beatriz Saavedra Pons¹ and Germán Serino¹

¹Chacra Experimental Agrícola Santa Rosa, Camino Vecinal no. 8 km 6, Colonia Santa Rosa, Salta 4531, Argentina; ggiampaoli@chacraexperimental.org

²Centro de Estudios Fotosintéticos y Bioquímicos – CONICET, Suipacha 570, S2000RLJ, Rosario, Argentina We investigated CRISPR/Cas9 homology-directed repair (HDR)-mediated introduction of the W574L mutation aimed at obtaining tolerance to acetolactate synthase (ALS) inhibiting herbicides. Specific sgRNAs were designed and inserted into Cas9-expressing gene editing vectors. Single-stranded oligonucleotides, 90 to 120 nt long, were designed as repair templates to introduce the W574L mutation and to eliminate PAM-sites and a BtsCI restriction site to facilitate molecular diagnosis of HDR. Combinations of editing vectors and repair templates were co-delivered into sugarcane calli by biolistic gene transfer with an nptII expression vector for in vitro selection, resulting in the integration of the transgenes. To assess editing of the target sequences, a PCR fragment containing the editing target was digested with the diagnostic restriction enzyme BtsCI. BtsCI restriction-resistant PCR fragments (404 bp, Hi-Fi-polymerase-amplified) were cloned and sequenced. Modification of the target W574L, PAM and BtsCI sites, indicating the occurrence of HDR editing, was observed in various independent transgenic events. A series of 1 to 61 nucleotide deletions and a single, one-nucleotide insertion were also observed, indicating non-homologous end joining (NHEJ) editing. Simultaneous HDR and NHEJ editing was observed. Co-editing of at least two alleles of the W574L codon substitution was observed. Edited clones were sprayed with the imidazolinone at label rate, and no clones survived the applications. Sixteen ALS alleles were identified based on the sequence of a 404-base-pair gene fragment from WT NA 05-860, suggesting that edited gene dosage may be insufficient for imidazolinone resistance in the field. Further work is required to achieve editing of additional copies of ALS and assess possibly higher resistance levels