Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice
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TLDR
Adaptations of the type II CRISPR/Cas system leading to successful expression of the Cas9/sgRNA system in model plant and crop species bodes well for its near-term use as a facile and powerful means of plant genetic engineering for scientific and agricultural applications.Abstract:
The type II CRISPR/Cas system from Streptococcus pyogenes and its simplified derivative, the Cas9/single guide RNA (sgRNA) system, have emerged as potent new tools for targeted gene knockout in bacteria, yeast, fruit fly, zebrafish and human cells. Here, we describe adaptations of these systems leading to successful expression of the Cas9/sgRNA system in two dicot plant species, Arabidopsis and tobacco, and two monocot crop species, rice and sorghum. Agrobacterium tumefaciens was used for delivery of genes encoding Cas9, sgRNA and a non-fuctional, mutant green fluorescence protein (GFP) to Arabidopsis and tobacco. The mutant GFP gene contained target sites in its 5' coding regions that were successfully cleaved by a CAS9/sgRNA complex that, along with error-prone DNA repair, resulted in creation of functional GFP genes. DNA sequencing confirmed Cas9/sgRNA-mediated mutagenesis at the target site. Rice protoplast cells transformed with Cas9/sgRNA constructs targeting the promoter region of the bacterial blight susceptibility genes, OsSWEET14 and OsSWEET11, were confirmed by DNA sequencing to contain mutated DNA sequences at the target sites. Successful demonstration of the Cas9/sgRNA system in model plant and crop species bodes well for its near-term use as a facile and powerful means of plant genetic engineering for scientific and agricultural applications.read more
Citations
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Delivery of CRISPR/Cas Components into Higher Plant Cells for Genome Editing
Bulat Kuluev,G. R. Gumerova,E. V. Mikhaylova,G. A. Gerashchenkov,N. A. Rozhnova,Z. R. Vershinina,A. V. Khyazev,R.T. Matniyazov,An. Kh. Baymiev,Al. Kh. Baymiev,A. V. Chemeris +10 more
TL;DR: There are a number of cases in which new organisms created by a CRISPR/Cas genome editing without any introduction of alien DNA were not considered as transgenic ones; it is quite possible that such plants will not fall under Russian legislation prohibiting GMO cultivation.
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Intron-Based Single Transcript Unit CRISPR Systems for Plant Genome Editing
Zhaohui Zhong,Shishi Liu,Xiaopei Liu,Binglin Liu,Xu Tang,Qiurong Ren,Jianping Zhou,Xuelian Zheng,Yiping Qi,Yong Zhang,Yong Zhang +10 more
TL;DR: Multiple iSTU-CRISPR/Cas9 and Cas12a systems for plant genome editing are developed and it is demonstrated that singular genome editing and multiplexed genome editing with these iSTUs are demonstrated.
Journal ArticleDOI
Agrobacterium-mediated transient transformation of sorghum leaves for accelerating functional genomics and genome editing studies.
Rita Sharma,Rita Sharma,Rita Sharma,Yan Liang,Yan Liang,Mi Yeon Lee,Mi Yeon Lee,Venkataramana R. Pidatala,Venkataramana R. Pidatala,Jenny C. Mortimer,Jenny C. Mortimer,Henrik Vibe Scheller,Henrik Vibe Scheller,Henrik Vibe Scheller +13 more
TL;DR: An Agrobacterium-mediated transient transformation assay with intact sorghum leaves using green fluorescent protein as marker is reported, providing a good monocot alternative to tobacco and protoplast assays with a direct, native and more reliable system for testing single guide RNA (sgRNA) expression construct efficiency.
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