Rapid and cloning‑free screening of edited alfalfa via next‑generation sequencing

Abstract

The identification of full knockout plants is frequently the rate-limiting step of the research and commercial projects searching the complete loss of the function of target genes via the CRISPR/Cas9 system. This problem is particularly acute in polyploid crops such as alfalfa, where the common screening needs cloning and Sanger sequencing. Here, we produced and analyzed fifteen edited events containing different doses of edited alleles for three unrelated target genes. This analysis showed that next-generation sequencing (NGS) technology largely increases the efficiency of the determination of the exact genotype of an individual plant and that this increased performance is related to the high output of sequences produced by this alternative genotyping approach rather than to a higher balanced amplification of the alleles. Finally, we discuss the possible applications of NGS technology in other polyploid crops with complex genetics.