Identification and Efficiency Testing of Cas9 Variants Targeting NG PAM Sequences for Editing the ZmSCD Gene in Maize
To evaluate the potential of SpCas9-NG in maize genome editing,the key gene for chlorophyll syn-thesis,ZmSCD,was selected as the target.The absence of this gene causes seedlings to exhibit albinism,providing a visual assessment of editing efficiency.Based on the PAM sequence recognition rules of SpCas9(5'-NGG-3')and SpCas9-NG(5'-NG-3'),target sites were designed on the second and third exons of ZmSCD.These target se-quences were successfully constructed into SpCas9 and SpCas9-NG knockout vectors,which were then introduced into the maize inbred line KN5585 using Agrobacterium-mediated genetic transformation.The callus tissues were cultured until leaf tissues differentiated,and the albinism rate was recorded to determine the editing efficiency of the different editors.The genomes of albino seedlings were then sequenced.Through three rounds of genetic transforma-tion,SpCas9 produced 76,125 and 28 callus tissues,while SpCas9-NG produced 100,69 and 30 callus tissues.The results showed that the gene editing efficiency of SpCas9 across the three transformations was 14.47％,13.60％,and 10.71％,respectively,while SpCas9-NG editing efficiency was 12.00％,10.14％and 13.33％.Sequencing results of the albino seedlings revealed overlapping peaks near the target sites for both editors.The study demon-strates that SpCas9-NG had comparable editing efficiency to traditional SpCas9 in maize,showing similar gene edi-ting capabilities.In contrast,SpCas9-NG has a broader PAM sequence adaptability,allowing for more flexible target design.This flexibility enables more precise and diverse editing within the maize genome.
万方数据
维普
