First‐generation genome editing in potato using hairy root transformation

CRISPR/Cas-mediated genome editing is a powerful tool of plant functional genomics. Hairy root transformation is a rapid and convenient approach for obtaining transgenic roots. When combined, these techniques represent a fast and effective means of studying gene function. In this review, we outline the current state of the art reached by the combination of these approaches over seven years. Additionally, we discuss the origins of different Agrobacterium rhizogenes strains that are widely used for hairy root transformation; the components of CRISPR/Cas vectors, such as the promoters that drive Cas or gRNA expression, the types of Cas nuclease, and selectable and screenable markers; and the application of CRISPR/Cas genome editing in hairy roots. The modification of the already known vector pKSE401 with the addition of the rice translational enhancer OsMac3 and the gene encoding the fluorescent protein DsRed1 is also described.

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Efficient Genome Editing in Potato Using a Hairy Root Transformation System

Springer Protocols Handbooks - CRISPR-Cas Methods ◽

10.1007/978-1-0716-1657-4_11 ◽

2021 ◽

pp. 149-158

Author(s):

Nathaniel M. Butler ◽

Jiming Jiang

Keyword(s):

Genome Editing ◽

Hairy Root ◽

Transformation System ◽

Root Transformation

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HAIRY ROOT TRANSFORMATION SYSTEM IN LARGE-SEEDED GRAIN LEGUMES

Israel Journal of Plant Sciences ◽

10.1080/07929978.1995.10676585 ◽

1995 ◽

Vol 43 (1) ◽

pp. 1-5 ◽

Cited By ~ 9

Author(s):

H.J. Siefkes-Boer ◽

M.J. Noonan ◽

D.W. Bullock ◽

A.J. Conner

Keyword(s):

Vicia Faba ◽

Hairy Root ◽

Hairy Roots ◽

Faba Bean ◽

Grain Legumes ◽

Transformation System ◽

Root Transformation ◽

Specific Strain ◽

Size And Morphology ◽

Root Size

Hairy roots were produced on faba bean (Vicia faba L.) and chickpea (Cicer arietinum L.) plants by inoculation with Agrobacterium root-inducing strains. Examination of 14 plant genotypes and eight Agrobacterium strains in all possible combinations revealed specific strain/genotype interactions. Hairy root size and morphology differed substantially between faba bean and chickpea hairy roots. Sixty percent of chickpea hairy roots were 10–15 mm in length and forty percent, 15–25 mm. All were <1.0 mm in thickness. Sixty-three percent of faba bean hairy roots were 15–25 mm long and thirty-seven percent, 25–40 mm. All faba bean hairy roots were between 1.0 and 1.5 mm in thickness.

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

Mammalian Genome ◽

10.1007/s00335-017-9711-x ◽

2017 ◽

Vol 28 (7-8) ◽

pp. 377-382 ◽

Cited By ~ 14

Author(s):

Lydia Teboul ◽

Stephen A. Murray ◽

Patrick M. Nolan

Keyword(s):

Genome Editing ◽

First Generation

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Root isoflavonoids and hairy root transformation influence key bacterial taxa in the soybean rhizosphere

Environmental Microbiology ◽

10.1111/1462-2920.13602 ◽

2017 ◽

Vol 19 (4) ◽

pp. 1391-1406 ◽

Cited By ~ 13

Author(s):

Laura J. White ◽

Xijin Ge ◽

Volker S. Brözel ◽

Senthil Subramanian

Keyword(s):

Hairy Root ◽

Root Transformation

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Hairy Root Transformation in Lotus japonicus

BIO-PROTOCOL ◽

10.21769/bioprotoc.795 ◽

2013 ◽

Vol 3 (12) ◽

Cited By ~ 7

Author(s):

Satoru Okamoto ◽

Emiko Yoro ◽

Takuya Suzaki ◽

Masayoshi Kawaguchi

Keyword(s):

Hairy Root ◽

Lotus Japonicus ◽

Root Transformation

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Detection of Target Sites Edited in Upland Cotton By The CRISPR/Cas9 System Mediated By Agrobacterium Rhizogenes

10.21203/rs.3.rs-1105254/v1 ◽

2021 ◽

Author(s):

Lili Zhou ◽

Yali Wang ◽

Peilin Wang ◽

Jiamin Wang ◽

Hongmei Cheng

Keyword(s):

Agrobacterium Rhizogenes ◽

Hairy Root ◽

Hairy Roots ◽

Upland Cotton ◽

Gene Editing ◽

Target Site ◽

Shoot Meristem ◽

Multiple Target ◽

Root Transformation ◽

Target Sites

Abstract Background CRIPSR/Cas9 gene editing has the ability to effectively modify plant genomes. Multiple target sites usually were designed and the effective target sites were selected for editing. However, upland cotton is allotetraploid and is commonly considered as difficult and inefficient to transform. Therefore, it’s important to quickly identify feasibility of the target site. In this study, we use Agrobacterium rhizogenes K599 strain to infect cotton shoot meristem and induce them to grow hairy roots to detect the feasibility of a selected target designed in GhMYB25-like gene. Results We designed a sgRNA within the second exons of GhMYB25-likeA and GhMYB25-likeD and constructed the CRISPR vector. Transient hairy root transformation using A. rhizogenes K599 with four OD600s (0.4, 0.6,0.8, 1.0) was performed in Coker 312 (R15). The results show that A. rhizogenes at OD600 = 0.6–0.8 is the best concentration range for inducing cotton hairy roots. The other three cultivars (TM-1, Lumian 21, Zhongmian 49) were injected using A. rhizogenes K599 with OD600 = 0.6-0.8 and all produced hairy roots. We characterized ten R15 plants with hairy roots and detected different degrees of base deletions and insert at the target site in five R15 plants. Conclusion Overall, our data show A. rhizogenes-mediated transient hairy root transformation offers a rapid and efficient method to detect sgRNA feasibility in cotton.

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Transgene-Free Genome Editing in Tomato and Potato Plants Using Agrobacterium-Mediated Delivery of a CRISPR/Cas9 Cytidine Base Editor

International Journal of Molecular Sciences ◽

10.3390/ijms20020402 ◽

2019 ◽

Vol 20 (2) ◽

pp. 402 ◽

Cited By ~ 57

Author(s):

Florian Veillet ◽

Laura Perrot ◽

Laura Chauvin ◽

Marie-Paule Kermarrec ◽

Anouchka Guyon-Debast ◽

...

Keyword(s):

Genome Editing ◽

First Generation ◽

Genome Engineering ◽

Crop Improvement ◽

Point Mutations ◽

Acetolactate Synthase ◽

Agrobacterium Mediated Transformation ◽

Dicotyledonous Plants ◽

Function Discovery ◽

Vegetatively Propagated Plants

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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