Improved plant cytosine base editors with high editing activity, purity, and specificity

Ren, Qiao; Sretenovic, Simon; Liu, Guanqing; Zhong, Zhiyong; Wang, Jiaheng; Huang, Lan; Tang, Xu; Guo, Yachong; Liu, Li; Wu, Yuechao; Zhou, Jié; Zhao, Yuxin; Han, Yang; He, Yao; Liu, Shishi; Yin, Desuo; Mayorga, Rocio; Zheng, Xuelian; Zhang, Tao; Qi, Yiping; Zhang, Yong

doi:10.1111/pbi.13635

Cited by 76 publications

(73 citation statements)

References 77 publications

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“…To develop plant CGBEs, we decided to compare the best performing CGBEs from the three recent studies used to edit in human cells ( Chen et al, 2021 ; Kurt et al, 2021 ; Zhao et al, 2021 ). Since these CGBEs were all based on rAPOBEC1, the rAPOBEC1-based CBE-BE3 (pYPQ265, BE3) ( Ren et al, 2021a ) was included as a control ( Figure 1A ). We used a maize codon optimized Cas9 (zCas9) which was previously shown to be very efficient for genome editing in Arabidopsis ( Wang et al, 2015 ), maize ( Lee et al, 2019 ), and wheat ( Li et al, 2021c ), and recently used for efficient base editing in rice ( Ren et al, 2021a ; Ren et al, 2021b ), tomato ( Randall et al, 2021 ), and poplar ( Li et al, 2021a ).…”

Section: Resultsmentioning

confidence: 99%

“…All the primers used in this study are listed in Supplementary Table S1 . The pYPQ265 vector (Addgene # 164712) was reported in our recent publication ( Ren et al, 2021a ). To prepare Gateway compatible attL1-attR5 entry clone pYPQ265K (Addgene #173997), the backbone obtained from pYPQ166-D10A plasmid after restriction digestion with BsrGI-HF (NEB, catalog # R3575*) and NcoI-HF (NEB, catalog # R3193*) and CGBE1-gBk synthetic DNA (IDT gBlock) digested with BsrGI-HF and NcoI-HF were ligated together.…”

Section: Methodsmentioning

confidence: 99%

“…The first type is cytosine base editors (CBEs) which direct C-to-T transition base changes ( Komor et al, 2016 ; Nishida et al, 2016 ). Many CBEs based on different cytidine deaminases were reported for use in plants including rABOBEC1 ( Li et al, 2017 ; Lu and Zhu, 2017 ; Zong et al, 2017 ), PmCDA1 ( Shimatani et al, 2017 ; Tang et al, 2019 ; Zhong et al, 2019 ), hAID ( Ren et al, 2018 ), human APOBEC3A (A3A) ( Zong et al, 2018 ; Cheng et al, 2021 ), APOBEC3B (A3B) ( Jin et al, 2020 ), and A3A/Y130F ( Li et al, 2021a ; Ren et al, 2021a ; Randall et al, 2021 ). The second type is adenine base editors (ABEs) which confer A-to-G transition base changes ( Gaudelli et al, 2017 ).…”

Section: Introductionmentioning

confidence: 99%

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Exploring C-To-G Base Editing in Rice, Tomato, and Poplar

Sretenovic

Liu

et al. 2021

Front. Genome Ed.

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As a precise genome editing technology, base editing is broadly used in both basic and applied plant research. Cytosine base editors (CBEs) and adenine base editors (ABEs) represent the two commonly used base editor types that mediate C-to-T and A-to-G base transition changes at the target sites, respectively. To date, no transversion base editors have been described in plants. Here, we assessed three C-to-G base editors (CGBEs) for targeting sequences with SpCas9’s canonical NGG protospacer adjacent motifs (PAMs) as well as three PAM-less SpRY-based CGBEs for targeting sequences with relaxed PAM requirements. The analyses in rice and tomato protoplasts showed that these CGBEs could make C-to-G conversions at the target sites, and they preferentially edited the C6 position in the 20-nucleotide target sequence. C-to-T edits, insertions and deletions (indels) were major byproducts induced by these CGBEs in the protoplast systems. Further assessment of these CGBEs in stably transformed rice and poplar plants revealed the preference for editing of non-GC sites, and C-to-T edits are major byproducts. Successful C-to-G editing in stably transgenic rice plants was achieved by rXRCC1-based CGBEs with monoallelic editing efficiencies up to 38% in T0 lines. The UNG-rAPOBEC1 (R33A)-based CGBE resulted in successful C-to-G editing in polar, with monoallelic editing efficiencies up to 6.25% in T0 lines. Overall, this study revealed that different CGBEs have different preference on preferred editing sequence context, which could be influenced by cell cycles, DNA repair pathways, and plant species.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Exploring C-To-G Base Editing in Rice, Tomato, and Poplar

Sretenovic

Liu

et al. 2021

Front. Genome Ed.

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“…Efficient C/G to T/A base editing has been widely achieved in many plant species and the most used system is BE3 [54][55][56]. Further improved CBEs, such as PmCDA1-CBE_V04 and A3A/Y130F-CBE_V04, were recently developed with high editing activity and specificity as well as reduced indel byproducts [57]. TadA8e and TadA9 are the most active ABEs, with the widest sequence compatibility among the ABE series developed and recommended for converting A/T to G/C in a variety of targets with improved performance and product purity [38,58].…”

Section: Base Editorsmentioning

confidence: 99%

“…Notably, CBEs can induce Cas-independent genome-wide off-target mutations in plants and mammalian systems, while ABEs have minimal off-target effects [59][60][61]. However, CBEs can be engineered to reduce off-target editing while maintaining comparable on-target editing [57,59,62] (Box 1). Therefore, we recommend using the improved version of CBEs.…”

Section: Base Editorsmentioning

confidence: 99%

Construct design for CRISPR/Cas-based genome editing in plants

Hassan

Zhang

Yuan

et al. 2021

Trends in Plant Science

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CRISPR construct design is a key step in the practice of genome editing, which includes identification of appropriate Cas proteins, design and selection of guide RNAs (gRNAs), and selection of regulatory elements to express gRNAs and Cas proteins. Here, we review the choices of CRISPR-based genome editors suited for different needs in plant genome editing applications. We consider the technical aspects of gRNA design and the associated computational tools. We also discuss strategies for the design of multiplex CRISPR constructs for highthroughput manipulation of complex biological processes or polygenic traits. We provide recommendations for different elements of CRISPR constructs and discuss the remaining challenges of CRISPR construct optimization in plant genome editing. Genome editing and associated technologiesGenome editing can be defined as a targeted intervention of genetic materials (i.e., DNA or RNA) in living organisms to deliberately alter their sequences. Although genome editing can target both DNA and RNA, here we only review DNA editing. DNA editing mainly relies on the introduction of in vivo DNA double-stranded breaks (DSBs) induced by the engineered sequence-specific nucleases (SSNs) programmed to recognize predefined sites in a genome. The induced DSBs are then repaired by cellular DNA repair mechanisms, namely non-homologous end-joining (NHEJ) and homologydirected repair (HDR) (Figure 1). The repair of DSBs by NHEJ results in mutation at the break site, largely via imprecise sequence insertions or deletions (indels), disrupting the native structure and function of the targeted sequences (e.g., genes, promoters). In addition, NHEJ can mediate targeted sequence insertion or replacement when a suitable DNA fragment is provided [1]. By contrast, repair by HDR can precisely introduce predefined sequences carried by a donor DNA template (Figure 1).The SSNs, with the capacity to introduce DSB in DNA, are referred to as the key elements in genome editing technologies and include meganucleases [2], zinc finger nucleases (ZFNs) [3], transcription activator-like effector nucleases (TALENs) [4], and clustered regularly interspaced short palindromic repeat (CRISPR) systems [5][6][7][8]. Unlike ZFNs and TALENs, which rely on protein-DNA interaction to define target specificity, CRISPR systems use RNA-DNA interaction to guide the DNA targeting and cleavage, making it a simple, efficient, and inexpensive technology for genetic manipulation. CRISPR systems have now become the leading genome editing technology and have been applied in a wide variety of plant species. Efficient genome editing has been achieved in many dicot and monocot species using diverse CRISPR-Cas systems for fundamental research and crop improvement and the application of CRISPR-Cas technology in plants has been increased dramatically over the past few years [9][10][11][12].Three classes of CRISPR technology are currently available for editing plant genomes [10,13]. These are CRISPR-Cas nucleases, base editors, and prime editors. CRISPR-Cas...

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