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| 1 | Improving Plant Genome Editing with High-Fidelity xCas9 and Non-canonical PAM-Targeting Cas9-NG显示文摘Two recently engineered SpCas9 variants, namely xCas9 and Cas9-NG, show promising potential in improving targeting specificity and broadening the targeting range. In this study, we evaluated these Cas9 variants in the model and crop plant, rice. We first tested xCas9-3.7, the most effective xCas9 variant in mammalian cells, for targeted mutagenesis at 16 possible NGN PAM (protospacer adjacent motif) combinations in duplicates. xCas9 exhibited nearly equivalent editing efficiency to wild-type Cas9 (Cas9-WT) at most canonical NGG PAM sites tested, whereas it showed limited activity at non-canonical NGH (H = A, C, T) PAM sites. High editing efficiency of xCas9 at NGG PAMs was further demonstrated with C to T base editing by both rAPOBECI and PmCDAI cytidine deaminases. With mismatched sgRNAs, we found that xCas9 had improved targeting specificity over the Cas9-WT. Furthermore, we tested two Cas9-NG variants, Cas9-NGv1 and Cas9-NG, for targeting NGN PAMs. Both Cas9-NG variants showed higher editing efficiency at most non-canonical NG PAM sites tested, and enabled much more efficient editing than xCas9 at AT-rich PAM sites such as GAT, GAA, and CAA. Nevertheless, we found that Cas9-NG variants showed significant reduced activity at the canonical NGG PAM sites. In stable transgenic rice lines, we demonstrated that Cas9-NG had much higher editing efficiency than Cas9-NGv1 and xCas9 at NG PAM sites. To expand the base-editing scope, we developed an efficient C to T base-editing system by making fusion of Cas9-NG nickase (D10A version), PmCDAI, and UGI. Taken together, our work benchmarked xCas9 as a high-fidelity nuclease for targeting canonical NGG PAMs and Cas9-NG as a preferred variant for targeting relaxed PAMs for plant genome editing. | Zhaohui Zhong Simon Sretenovic Qiurong Ren Lijia Yang Yu Bao Caiyan Qi Mingzhu Yuan Yao He Shishi Liu Xiaopei Liu Jiaheng Wang Lan Huang Yan Wang Dibin Baby David Wang Tao Zhang Yiping Qi Yong Zhang | 2019 | Molecular Plant2019，12，7： | 13 |
| 2 | Active ammonia-oxidizing bacteria and archaea in wastewater treatment systems显示文摘Ammonia-oxidizing bacteria(AOB)and archaea(AOA)are two microbial groups mediating nitrification,yet little is presently known about their abundances and community structures at the transcriptional level in wastewater treatment systems(WWTSs).This is a significant issue,as the numerical abundance of AOA or AOB at the gene level may not necessarily represent their functional role in ammonia oxidation.Using amo A genes as molecular markers,this study investigated the transcriptional abundance and community structure of active AOA and AOB in 14 WWTSs.Quantitative PCR results indicated that the transcriptional abundances of AOB amo A(averaged:1.6×10^(8)copies g^(-1)dry sludge)were higher than those of AOA(averaged:3.4×10^(7)copies g^(-1)dry sludge)in all WWTSs despite several higher abundances of AOA amo A at the gene level.Moreover,phylogenetic analysis demonstrated that Nitrosomonas europaea and unknown clusters accounted for 37.66%and 49.96%of the total AOB amo A transcripts,respectively,suggesting their dominant role in driving ammonia oxidation.Meanwhile,AOA amo A transcripts were only successfully retrieved from 3 samples,and the Nitrosospaera sister cluster dominated,accounting for 83.46%.Finally,the substrate utilization kinetics of different AOA and AOB species might play a fundamental role in shaping their niche differentiation,community composition,and functional activity.This study provides a basis for evaluating the relative contributions of ammonia-oxidizing microorganisms(AOMs)to nitrogen conversions in WWTSs. | Maosheng Zheng Shishi He Yueqi Feng Mingyuan Wang Yong-Xin Liu Chenyuan Dang Jiawen Wang | 2021 | Journal of Environmental Sciences2021，33，4： | 2 |
| 3 | Hypercompact CRISPR–Cas12j2 (CasF) enables genome editing, gene activation, and epigenome editing in plants显示文摘CRISPR-Cas9,-Cas12a,-Cas12b,and-Cas13 have been harnessed for genome engineering in human and plant cells(Liu et al.,2022).However,the large size of these Cas proteins(e.g.190 kDa for SpCas9)makes them difficult to deliver into cells via a viral vector.The development of smaller Cas proteins will lead to reduced viral vector sizes that can be more widely adopted in versatile genome engineering systems.Recently,a CRISPR-Cas12j2(CasF)system was discovered in huge phages and developed into a hypercompact genome editor due to the small size of Cas12j2(80 kDa)(Pausch et al.,2020).Unfortunately,the gene editing efficiency of Cas12j2 in Arabidopsis protoplasts using ribonucleoprotein delivery was less than one percent(Pausch et al.,2020).Further optimization of this system is clearly required if CRISPR-Cas12j2-mediated editing in plant genomes is to be adopted by the plant sciences community. | Shishi Liu Simon Sretenovic Tingting Fan Yanhao Cheng Gen Li Aileen Qi Xu Tang Yang Xu Weijun Guo Zhaohui Zhong Yao He Yanling Liang Qinqin Han Xuelian Zheng Xiaofeng Gu Yiping Qi Yong Zhang | 2022 | Plant Communications2022，3，6： | 0 |
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