Hairy CRISPR: Genome Editing in Plants Using Hairy Root Transformation

Abstract

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.

Hairy root transformation system as a tool for CRISPR/Cas9-directed genome editing in oilseed rape (Brassica napus)

“…The hairy root transformation system as a tool for CRISPR/Cas9-based genome editing has been established recently for various species (reviewed in Kiryushkin et al, 2022 ). Among Brassica crops, B. carinata hairy roots were successfully edited using a CRISPR/Cas9 system ( Kirchner et al, 2017 ).…”

“…Hairy root transformation has been recently discovered as an efficient tool for studying gene function using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs)/CRISPR-associated protein 9 (Cas9)-mediated gene editing in a wide range of plant taxa (reviewed in Kiryushkin et al, 2022 ). This bacterial system has been exploited to develop a powerful RNA-guided genome editing tool in various species.…”

Gene-Editing Technologies and Applications in Legumes: Progress, Evolution, and Future Prospects

“…For this reason, it was suggested that RNA interference studies be replaced with this method. According to the literature, Cas9 protein is the most preferred and used for the development of genome-edited plants ( Kiryushkin et al, 2022 ). The expression of Cas9 under the cauliflower mosaic virus (CaMV) 35S promoter (p35S) was observed in a total of 78 plant-related genome-editing studies ( Amack and Antunes, 2020 ).…”

“…It is mainly chosen when there are no protocols existing for stable transformation and regeneration, or the desired traits were only seen in roots ( Christey, 2001 ). Recently, a study described the possibility of editing the genomes of transgenic hairy roots using CRISPR/Cas9 ( Kiryushkin et al, 2022 ). This study concluded that by combining these techniques, it is possible to study gene function quickly and efficiently.…”

Hairy root transformation system as a tool for CRISPR/Cas9-directed genome editing in oilseed rape (Brassica napus)

“…Hairy root transformation has been recently discovered as an efficient tool for studying gene function using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing in a wide range of plant taxa (reviewed in Kiryushkin et al, 2021). This bacterial system has been exploited to develop a powerful RNA-guided genome editing tool in various species.…”