The efficacy of CRISPR-mediated cytosine base editing with the RPS5a promoter in Arabidopsis thaliana

Choi, Myeon-Song; Yun, Jae-Young; Kim, Jun‐Hyuk

doi:10.1038/s41598-021-87669-y

Cited by 25 publications

(16 citation statements)

References 37 publications

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“…Some advantages to our strategy, i.e., using a single gRNA to target two genes, include faster preparation of mutant lines compared with obtaining different allelic mutant lines by crossing between single mutant lines and reduced the complexity of gRNA design compared with using multiple gRNAs. The efficiencies of various promoters used for Cas9 expression during CRISPR-mediated genome editing have been assessed [ 27 , 28 , 37 , 38 , 39 , 40 ]. Using more efficient promoters, such as RPS5a, may enhance mutation rates in the T 1 generation, thus increasing the chance of obtaining more inherited mutations in the T 2 generation.…”

Section: Discussionmentioning

confidence: 99%

The Functional Association of ACQOS/VICTR with Salt Stress Resistance in Arabidopsis thaliana Was Confirmed by CRISPR-Mediated Mutagenesis

Choi

Bae

2021

IJMS

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Clustered regularly interspaced palindromic repeat (CRISPR)-mediated mutagenesis has become an important tool in plant research, enabling the characterization of genes via gene knock-out. CRISPR genome editing tools can be applied to generate multi-gene knockout lines. Typically, multiple single-stranded, single guide RNAs (gRNAs) must be expressed in an organism to target multiple genes simultaneously; however, a single gRNA can target multiple genes if the target genes share similar sequences. A gene cluster comprising ACQUIRED OSMOTOLERANCE (ACQOS; AT5G46520) and neighboring nucleotide-binding leucine-rich repeats (NLRs; AT5G46510) is associated with osmotic tolerance. To investigate the role of ACQOS and the tandemly arranged NLR in osmotic tolerance, we introduced small insertion/deletion mutations into two target genes using a single gRNA and obtained transformant plant lines with three different combinations of mutant alleles. We then tested our mutant lines for osmotic tolerance after a salt-stress acclimation period by determining the chlorophyll contents of the mutant seedlings. Our results strongly suggest that ACQOS is directly associated with salt resistance, while the neighboring NLR is not. Here, we confirmed previous findings suggesting the involvement of ACQOS in salt tolerance and demonstrated the usefulness of CRISPR-mediated mutagenesis in validating the functions of genes in a single genetic background.

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Section: Discussionmentioning

confidence: 99%

The Functional Association of ACQOS/VICTR with Salt Stress Resistance in Arabidopsis thaliana Was Confirmed by CRISPR-Mediated Mutagenesis

Choi

Bae

2021

IJMS

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“…Besides, the prime editor can edit near or far from PAM sites making it less constrained by PAM availability in the target genome in the same way as other CRISPR-based methods. Overall, these new precise nucleotide-editing strategies could further expand the CRISPR-based applications for the development of novel quantitative traits with a gain-of-function mutation [184,185]. In addition to its numerous advantages over conventional CRISPR-based systems, there are still certain aspects (e.g., on-target editing efficiency, unwanted mutations, optimal experiment design [186]) that need to be improved further for a more efficient and robust genome editing application.…”

Section: Development Of Highly Efficient and Precision Genome Editing...mentioning

confidence: 99%

From Genome Sequencing to CRISPR-Based Genome Editing for Climate-Resilient Forest Trees

Cao

Gailing

2022

IJMS

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Due to the economic and ecological importance of forest trees, modern breeding and genetic manipulation of forest trees have become increasingly prevalent. The CRISPR-based technology provides a versatile, powerful, and widely accepted tool for analyzing gene function and precise genetic modification in virtually any species but remains largely unexplored in forest species. Rapidly accumulating genetic and genomic resources for forest trees enabled the identification of numerous genes and biological processes that are associated with important traits such as wood quality, drought, or pest resistance, facilitating the selection of suitable gene editing targets. Here, we introduce and discuss the latest progress, opportunities, and challenges of genome sequencing and editing for improving forest sustainability.

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“…NGS output files were further processed using a modified python script (Choi et al 2021). The files of the NGS data generated after processing can be found in Supplemental Dataset 1.…”

Section: Genotyping By Sequencingmentioning

confidence: 99%

Generation of inheritable A-to-G transitions using adenine base editing and NG-PAM Cas9 in Arabidopsis thaliana

Chae

Tan

Liew

et al. 2022

Preprint

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CRISPR/Cas9 technology is an important tool for functional genomics and crop improvement. It can be used to generate mutations at precise positions in the genome. Base editors consist of deaminase components and Cas9 to specify the type of mutation, such as C-to-T (cytosine base editors) or A-to-G (adenine base editors) transition. Available adenine base editor vectors usually make use of canonical Cas9, which limits their use to 5'-NGG-3' containing targets. We combined a relaxed variant of SpCas9 that uses 5'-NG-3' containing targets with the adenine base editor containing TadA7.10 or TadA8e to make a set of vectors. By using a phenotype-based screen, we found that our vectors efficiently induce A-to-G somatic mutations in a 5'-NGG-3' PAM context in Arabidopsis thaliana up to 81% efficiency. Such mutations are inheritable at the homozygous stage in T2. Among tested vectors, pECNUS4 (Addgene #184887), which carries TadA8e, showed highest efficiency at generating a stable A-to-G transition in a 5'-NGH-3' PAM context in the gene DM3. Using this vector, we were able to recreate a naturally occurring allele of DM3 in two generations without the transgene. pECNUS4 is a new component of the CRISPR toolbox to be used for introducing desired adenine base transitions with an expanded target window for functional genomic research and trait improvement.

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The efficacy of CRISPR-mediated cytosine base editing with the RPS5a promoter in Arabidopsis thaliana

Cited by 25 publications

References 37 publications

The Functional Association of ACQOS/VICTR with Salt Stress Resistance in Arabidopsis thaliana Was Confirmed by CRISPR-Mediated Mutagenesis

The Functional Association of ACQOS/VICTR with Salt Stress Resistance in Arabidopsis thaliana Was Confirmed by CRISPR-Mediated Mutagenesis

From Genome Sequencing to CRISPR-Based Genome Editing for Climate-Resilient Forest Trees

Generation of inheritable A-to-G transitions using adenine base editing and NG-PAM Cas9 in Arabidopsis thaliana

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