CRISPR-Cas nucleases and base editors for plant genome editing

Gürel, Filiz; Zhang, Yingxiao; Sretenovic, Simon; Qi, Yiping

doi:10.1007/s42994-019-00010-0

Cited by 23 publications

(13 citation statements)

References 109 publications

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“…Engineered CBEs with minimized off‐target effects have been developed and demonstrated in human cells, such as eA3A‐BE3 (Gehrke et al ., 2018 ), A3G‐BEs (Lee et al ., 2020 ), and YE1‐BE3‐FNLS (Zuo et al ., 2020 ). Despite the fact that a variety of CBEs have been demonstrated in plants (Gurel et al ., 2020 ; Molla and Yang, 2019 ; Zhang et al ., 2019b ), the genome‐wide off‐target effects in plants remain untested for most of them. It is hence imperative to further develop CBE systems of high editing activity and purity, coupled with a genome‐wide assessment of off‐target effects of these improved CBEs and other widely used CBEs in plants.…”

Section: Introductionmentioning

confidence: 99%

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

Ren

Sretenovic

Liu

et al. 2021

Plant Biotechnology Journal

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Cytosine base editors (CBEs) are great additions to the expanding genome editing toolbox. To improve C-to-T base editing in plants, we first compared seven cytidine deaminases in the BE3like configuration in rice. We found A3A/Y130F-CBE_V01 resulted in the highest C-to-T base editing efficiency in both rice and Arabidopsis. Furthermore, we demonstrated this A3A/Y130F cytidine deaminase could be used to improve iSpyMacCas9-mediated C-to-T base editing at Arich PAMs. To showcase its applications, we first applied A3A/Y130F-CBE_V01 for multiplexed editing to generate microRNA-resistant mRNA transcripts as well as pre-mature stop codons in multiple seed trait genes. In addition, we harnessed A3A/Y130F-CBE_V01 for efficient artificial evolution of novel ALS and EPSPS alleles which conferred herbicide resistance in rice. To further improve C-to-T base editing, multiple CBE_V02, CBE_V03 and CBE_V04 systems were developed and tested in rice protoplasts. The CBE_V04 systems were found to have improved editing activity and purity with focal recruitment of more uracil DNA glycosylase inhibitors (UGIs) by the engineered single guide RNA 2.0 scaffold. Finally, we used whole-genome sequencing (WGS) to compare six CBE_V01 systems and four CBE_V04 systems for genome-wide off-target effects in rice. Different levels of cytidine deaminase-dependent and sgRNA-independent off-target effects were indeed revealed by WGS among edited lines by these CBE systems. We also investigated genome-wide sgRNA-dependent off-target effects by different CBEs in rice. This comprehensive study compared 21 different CBE systems, and benchmarked PmCDA1-CBE_V04 and A3A/ Y130F-CBE_V04 as next-generation plant CBEs with high editing efficiency, purity, and specificity.

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Section: Introductionmentioning

confidence: 99%

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

Ren

Sretenovic

Liu

et al. 2021

Plant Biotechnology Journal

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“…Since 2016, numerous CRISPR-Cas9-derived base editors have been reported and were first used to edit mammalian genomes, and more recently for editing plant genomes ( Molla and Yang, 2019 ; Zhang et al, 2019 ; Gurel et al, 2020 ). Currently, there are two major types of base editors used to edit plant genomes.…”

Section: Introductionmentioning

confidence: 99%

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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“…Different versions of CBE include base editor 1 (BE1), base editor 2 (BE2), base editor 3 (BE3), and base editor 4 (BE4) (Box 1). 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].…”

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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CRISPR-Cas nucleases and base editors for plant genome editing

Cited by 23 publications

References 109 publications

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

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

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

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

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