Identification and Efficiency Testing of Cas9 Variants Targeting NG PAM Sequences for Editing the ZmSCD Gene in Maize

doi:10.7668/hbnxb.20194671

Abstract

Abstract:

To evaluate the potential of SpCas9-NG in maize genome editing,the key gene for chlorophyll synthesis,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 sequences 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 transformation,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 demonstrates that SpCas9-NG had comparable editing efficiency to traditional SpCas9 in maize,showing similar gene editing 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.

Key words: Maize, SpCas9-NG, Editing efficiency, ZmSCD

QIAN Buxuan, PAN Hong, WANG Qi, CHEN Ziqi, YANG Yawen, XU Jieting, XIA Hanchao, ZHAO Rengui, LIU Xiangguo. Identification and Efficiency Testing of Cas9 Variants Targeting NG PAM Sequences for Editing the ZmSCD Gene in Maize[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(5): 9-14. doi: 10.7668/hbnxb.20194671.

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Figures/Tables 5

Fig.1 Structure and carrier map of the ZmSCD gene in maize A.ZmSCD gene structure and target design sites in maize;B.pXG51.1 and pXG51.4 carrier map.

Fig.2 Maize callus with albino seedling phenomenon in differentiation medium The box represents the callus tissue of transgenic albino seedlings.

Tab.1 Statistics of maize genetic transformation callus

不同编辑器 Different editors	愈伤组织数量/个 Number of callus tissue	白化叶片愈伤组织数量/个 Number of callus tissue from albino leaves	白化率 /% Whitening rate
SpCas9	76	11	14.47
	125	17	13.60
	28	3	10.71
SpCas9-NG	100	12	12.00
	69	7	10.14
	30	4	13.33

Fig.3 Sequencing of albino seedlings and statistics of albino rate A.Sequencing maps of albino seedlings edited by SpCas9 and SpCas9-NG;B.Albino seedling rate of SpCas9 and SpCas9-NG editing systems.

Fig.4 The identifiable count of NGG and NG PAM sequences on the first to fourth exons of the ZmSCD gene in maize

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