Developing a highly efficient and wildly adaptive <scp>CRISPR</scp>‐<i>Sa</i>Cas9 toolset for plant genome editing

Qin, Ruiying; Li, Juan; Li, Hao; Zhang, Yuandi; Liu, Xiaoshuang; Miao, Yuxin; Zhang, Xiuqing; Wei, Pengcheng

doi:10.1111/pbi.13047

Cited by 54 publications

(40 citation statements)

References 11 publications

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“…To improve the base editing efficiency, more effective cytidine deaminases such as the human APOBEC3A have been tested (Zong et al, 2018). On the other hand, for expanding the base editing scope in plants, several SpCas9 and SaCas9 variants such as VQR-Cas9, VRER-Cas9 and SaKKH-Cas9 that recognize PAMs other than the canonical NGG motif were introduced into the CBE and ABE toolbox (Hua et al, 2018a;Qin et al, 2018). However, relative to the widely used CRISPR/Cas gene editing technologies for inducing DSBs and subsequent repair-caused mutations, the efficiency of base editing is still low.…”

mentioning

confidence: 99%

Optimizing base editors for improved efficiency and expanded editing scope in rice

Wang

Mao

et al. 2019

Plant Biotechnology Journal

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mentioning

confidence: 99%

Optimizing base editors for improved efficiency and expanded editing scope in rice

Wang

Mao

et al. 2019

Plant Biotechnology Journal

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“…SaCas9 showed a comparable editing efficiency to SpCas9 in Arabidopsis, rice and tobacco (Steinert et al 2015;Kaya et al 2016). Both SaCas9 and its KKH variant have been shown to induce high mutagenesis frequencies in rice (Qin et al 2019). In another study, a 1092 aa Cas9 ortholog was identified in Brevibacillus laterosporus (BlatCas9), with the PAM preference of 5 0 -NNNNCNDD-3 0 (D = A, G or T) (Karvelis et al 2015).…”

Section: Targeting Altered Pams With Cas9 Orthologs and Engineered Vamentioning

confidence: 99%

“…Recently, a series of adenine and cytosine base editors were developed using both SpCas9 and SaCas9 variants to expand the targeting scope in rice (Hua et al 2019b;Qin et al 2019). Similarly, adenine and cytosine base editors based on Cas9-NG were also reported to edit relaxed PAM sites in plants (Negishi et al 2019;Ren et al 2019;Hua et al 2019a).…”

Section: Base Editorsmentioning

confidence: 99%

CRISPR-Cas nucleases and base editors for plant genome editing

et al. 2019

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Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) and base editors are fundamental tools in plant genome editing. Cas9 from Streptococcus pyogenes (SpCas9), recognizing an NGG protospacer adjacent motif (PAM), is a widely used nuclease for genome editing in living cells. Cas12a nucleases, targeting T-rich PAMs, have also been recently demonstrated in several plant species. Furthermore, multiple Cas9 and Cas12a engineered variants and orthologs, with different PAM recognition sites, editing efficiencies and fidelity, have been explored in plants. These RNA-guided sequence-specific nucleases (SSN) generate double-stranded breaks (DSBs) in DNA, which trigger non-homologous end-joining (NHEJ) repair or homology-directed repair (HDR), resulting in insertion and deletion (indel) mutations or precise gene replacement, respectively. Alternatively, genome editing can be achieved by base editors without introducing DSBs. So far, several base editors have been applied in plants to introduce C-to-T or A-to-G transitions, but they are still undergoing improvement in editing window size, targeting scope, off-target effects in DNA and RNA, product purity and overall activity. Here, we summarize recent progress on the application of Cas nucleases, engineered Cas variants and base editors in plants.

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“…SaCas9 and its engineered variant SaKKH (E782K/N968K/R1015H) (Kleinstiver et al 2015), which relaxes the canonical NNGRRT PAM of SaCas9 to NNNRRT, have been used to achieve the efficient targeted mutagenesis of Arabidopsis thaliana, tobacco (Nicotiana tabacum), rice, and citrus (Citrus sp.) (Kaya et al 2016;Steinert et al 2015;Jia et al 2017;Qin et al 2019). SaCas9 and SaKKH have also been used to develop plant base editors (BEs), including the cytosine BE (CBE) responsible for a C•G to T•A conversion and the adenine BE (ABE) responsible for the reverse substitution of A•T to G•C.…”

Section: Type II Cas9 Systemsmentioning

confidence: 99%

“…SaCas9 and SaKKH have also been used to develop plant base editors (BEs), including the cytosine BE (CBE) responsible for a C•G to T•A conversion and the adenine BE (ABE) responsible for the reverse substitution of A•T to G•C. Similar to the BEs derived from SpCas9, the SaCas9 BEs have been successfully used to induce specific base conversions in the rice genome (Hua et al 2018;Hua et al 2019;Qin et al 2019). Notably, the editing windows of the SaCas9 BEs are much broader than those of the SpCas9 BEs, possibly due to differences in the formation of the R-loop complex (Kim et al 2017d).…”

Section: Type II Cas9 Systemsmentioning

confidence: 99%

How to start your monocot CRISPR/Cas project: plasmid design, efficiency detection, and offspring analysis

Yue

Hong

Wei

et al. 2020

Rice

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The breakthrough CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-mediated genome-editing technology has led to great progress in monocot research; however, several factors need to be considered for the efficient implementation of this technology. To generate genome-edited crops, single guide (sg)RNA and Cas9 DNA are delivered into plant cells and expressed, and the predicted position is targeted. Analyses of successful targeted mutations have revealed that the expression levels, expression timing, and variants of both sgRNA and Cas9 need to be sophisticatedly regulated; therefore, the promoters of these genes and the target site positions are the key factors for genome-editing efficiency. Currently, various vectors and online tools are available to aid sgRNA design. Furthermore, to reduce the sequence limitation of the protospacer adjacent motif (PAM) and for other purposes, many Cas protein variants and base editors can be used in plants. Before the stable transformation of a plant, the evaluation of vectors and target sites is therefore very important. Moreover, the delivery of Cas9-sgRNA ribonucleoproteins (RNPs) is one strategy that can be used to prevent transgene issues with the expression of sgRNA and Cas proteins. RNPs can be used to efficiently generate transgene-free genome-edited crops that can reduce transgene issues related to the generation of genetically modified organisms. In this review, we introduce new techniques for genome editing and identifying marker-free genome-edited mutants in monocot crops. Four topics are covered: the design and construction of plasmids for genome editing in monocots; alternatives to SpCas9; protoplasts and CRISPR; and screening for marker-free CRISPR/Cas9-induced mutants. We have aimed to encompass a full spectrum of information for genome editing in monocot crops.

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Developing a highly efficient and wildly adaptive CRISPR‐SaCas9 toolset for plant genome editing

Cited by 54 publications

References 11 publications

Optimizing base editors for improved efficiency and expanded editing scope in rice

Optimizing base editors for improved efficiency and expanded editing scope in rice

CRISPR-Cas nucleases and base editors for plant genome editing

How to start your monocot CRISPR/Cas project: plasmid design, efficiency detection, and offspring analysis

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