Phenotyping first-generation genome editing mutants: a new standard?

Genome editing tools have rapidly been adopted by plant scientists for gene function discovery and crop improvement. The current technical challenge is to efficiently induce precise and predictable targeted point mutations valuable for crop breeding purposes. Cytidine base editors (CBEs) are CRISPR/Cas9 derived tools recently developed to direct a C-to-T base conversion. Stable genomic integration of CRISPR/Cas9 components through Agrobacterium-mediated transformation is the most widely used approach in dicotyledonous plants. However, elimination of foreign DNA may be difficult to achieve, especially in vegetatively propagated plants. In this study, we targeted the acetolactate synthase (ALS) gene in tomato and potato by a CBE using Agrobacterium-mediated transformation. We successfully and efficiently edited the targeted cytidine bases, leading to chlorsulfuron-resistant plants with precise base edition efficiency up to 71% in tomato. More importantly, we produced 12.9% and 10% edited but transgene-free plants in the first generation in tomato and potato, respectively. Such an approach is expected to decrease deleterious effects due to the random integration of transgene(s) into the host genome. Our successful approach opens up new perspectives for genome engineering by the co-edition of the ALS with other gene(s), leading to transgene-free plants harboring new traits of interest.

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Genome editing in plants using CRISPR type I-D nuclease

Communications Biology ◽

10.1038/s42003-020-01366-6 ◽

2020 ◽

Vol 3 (1) ◽

Author(s):

Keishi Osakabe ◽

Naoki Wada ◽

Tomoko Miyaji ◽

Emi Murakami ◽

Kazuya Marui ◽

...

Keyword(s):

Genome Editing ◽

First Generation ◽

Genome Engineering ◽

Targeted Mutagenesis ◽

Plant Genome ◽

Type I ◽

Crispr System ◽

Target Dna ◽

Short Indels

Abstract Genome editing in plants has advanced greatly by applying the clustered regularly interspaced short palindromic repeats (CRISPRs)-Cas system, especially CRISPR-Cas9. However, CRISPR type I—the most abundant CRISPR system in bacteria—has not been exploited for plant genome modification. In type I CRISPR-Cas systems, e.g., type I-E, Cas3 nucleases degrade the target DNA in mammals. Here, we present a type I-D (TiD) CRISPR-Cas genome editing system in plants. TiD lacks the Cas3 nuclease domain; instead, Cas10d is the functional nuclease in vivo. TiD was active in targeted mutagenesis of tomato genomic DNA. The mutations generated by TiD differed from those of CRISPR/Cas9; both bi-directional long-range deletions and short indels mutations were detected in tomato cells. Furthermore, TiD can be used to efficiently generate bi-allelic mutant plants in the first generation. These findings indicate that TiD is a unique CRISPR system that can be used for genome engineering in plants.

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Robust CRISPR/Cas9 mediated genome editing and its application in manipulating plant height in the first generation of hexaploid Ma bamboo ( Dendrocalamus latiflorus Munro )

Plant Biotechnology Journal ◽

10.1111/pbi.13320 ◽

2020 ◽

Vol 18 (7) ◽

pp. 1501-1503 ◽

Cited By ~ 5

Author(s):

Shanwen Ye ◽

Gang Chen ◽

Markus V. Kohnen ◽

Wenjia Wang ◽

Changyang Cai ◽

...

Keyword(s):

Genome Editing ◽

Plant Height ◽

First Generation ◽

Dendrocalamus Latiflorus ◽

Ma Bamboo

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Non-GM Genome Editing Approaches in Crops

Frontiers in Genome Editing ◽

10.3389/fgeed.2021.817279 ◽

2021 ◽

Vol 3 ◽

Author(s):

Zheng Gong ◽

Ming Cheng ◽

Jose R. Botella

Keyword(s):

Genome Editing ◽

Viral Vectors ◽

Large Scale ◽

First Generation ◽

Transformation Process ◽

Plant Genome ◽

Crop Species ◽

Breeding Programs ◽

Public Controversy ◽

Rigid Cell Wall

CRISPR/Cas-based genome editing technologies have the potential to fast-track large-scale crop breeding programs. However, the rigid cell wall limits the delivery of CRISPR/Cas components into plant cells, decreasing genome editing efficiency. Established methods, such as Agrobacterium tumefaciens-mediated or biolistic transformation have been used to integrate genetic cassettes containing CRISPR components into the plant genome. Although efficient, these methods pose several problems, including 1) The transformation process requires laborious and time-consuming tissue culture and regeneration steps; 2) many crop species and elite varieties are recalcitrant to transformation; 3) The segregation of transgenes in vegetatively propagated or highly heterozygous crops, such as pineapple, is either difficult or impossible; and 4) The production of a genetically modified first generation can lead to public controversy and onerous government regulations. The development of transgene-free genome editing technologies can address many problems associated with transgenic-based approaches. Transgene-free genome editing have been achieved through the delivery of preassembled CRISPR/Cas ribonucleoproteins, although its application is limited. The use of viral vectors for delivery of CRISPR/Cas components has recently emerged as a powerful alternative but it requires further exploration. In this review, we discuss the different strategies, principles, applications, and future directions of transgene-free genome editing methods.

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