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.

Keywords: Agrobacterium strains; CRISPR/Cas; OsMac3; genome editing; hairy root transformation; pKSEe401R; vector construction.

Figure 1

Relationships between Agrobacterium rhizogenes and Agrobacterium tumefaciens strains. A. rhizogenes strains are given in red, A. tumefaciens strains in black, transconjugants between A. rhizogenes and A. tumefaciens are given in both colors. The abbreviations of the four main ancestral wild type Agrobacterium strains are highlighted by bold print and underlining: A4, ATCC15834, NCPPB1855, C58. Arrows show the directions of isolation of derivaties. Parts of Agrobacterium strain abbreviations: AR—Agrobacterium rhizogenes; ARqua1—Agrobacterium rhizogenes strain obtained by H.J. Quandt; ATCC—American Type Culture Collection; A4RS—A4 derivative resistant to rifampicin and spectinomycin; C58—A. tumefaciens strain isolated from cherry gall in 1958; C58C1 or C58C9—C58 strain cured of Ti plasmid (pTi), the numeric designation 1 or 9 refers to a colony number; GV—Ghent University and Vrije Universiteit Brussel; LBA—Lugdunum Batavorum Agrobacterium; MSU—Michigan State University; NCPPB—National Collection of Plant Pathogenic Bacteria. Plus or minus indicates a transfer or loss of any component in the resulting Agrobacterium strain. The chromosomal background or the Ri plasmid transferred to a new A. rhizogenes strain are indicated next to the arrows in black or red, respectively. Antibiotic resistances or other modifications of the chromosomal background or of the Ri plasmids are designated as follows. C58 derivatives cured of the Ti plasmid are indicated with an asterisk. WT stands for wild type Agrobacterium strain or for its native plasmids (pTi or pRi). Antibiotic resistances appearing either in the chromosome (g) or as inserts in pRi are given in green; Carb—carbenicillin, Cm (CAT)—chloramphenicol, Ery—erythromycin, Km—kanamycin, Nal—nalidixic acid, Sp—spectinomycin, Str—streptomycin, Tet—tetracycline; a question mark denotes suggested resistance. Other modifications inserted into pRi (given in blue): pAR5—vector harboring genes encoding either β-glucuronidase (GUS) or chloramphenicol acetyltransferase (CAT) or luciferase (LUC), was integrated into wild type pRi15834 of the strain AR10 his⁻ via homologous recombination; pBR322—part of the pBR322 sequence inserted into wild type pRi15834 of the strain C58C1 (pRi15834) via homologous recombination; tmr—cytokinin synthesis locus encoding an isopentenyl transferase; vir reg.—part of the vir region from pTiBO342 (conferring the supervirulent phenotype to A. tumefaciens A348). Other modifications (given in brown): His^-—histidine auxotrophy acquired by random mutagenesis of C58C1 (pRi15834). The sequencer icon (created with BioRender) indicates Agrobacterium strains with sequenced genome.

Figure 2

Map of a CRISPR/Cas9 vector. Vector components (clockwise): RB—right T-DNA border; CRISPR cassette containing P—promoter of the small nucleolar RNA (snoRNA) gene; gRNA—guide RNA; crR—crisprRNA (target specific); tracrR—trans-activating crisprRNA (conserved, used as a binding scaffold for Cas9); T—terminator of the snoRNA gene; NLS—nuclear localization signal; Cas9—CRISPR associated nuclease 9; LB—left T-DNA border.

Figure 3

Localization of CsRALF34 expression in a Cucumis sativus root tip. Confocal laser scanning microscopy of vibratome longitudinal (A,B) and cross (C) sections of transgenic pCsRALF34::mNeonGreen-H2B roots. Green channel—fluorescence of mNeonGreen-H2B; magenta channel—cell walls are counter stained with SCRI Renaissance 2200. (A) An overview and (B) close-up of the parental root meristem shows the acropetal sequence of CsRALF34 promoter activity in protoxylem, pericycle and endodermis. CsRALF34 expression arises first in the protoxylem at a distance of 150 µm from the initial cells. (C) The establishment of activity in xylem, pericycle layers and endodermis on a cross section at a distance of 300 µm from the initial cells. Arrows indicate developing lateral root primordia. Scale bars: 100 µm in (A), and 40 μm in (B,C).

Figure 4

pKSEe401R plasmid. (A) Map of pKSEe401R; parts (clockwise): RB—right T-DNA border; pAtU6-26—promoter of the Arabidopsis small nucleolar RNA (snoRNA) U6-26 gene; SpR—gene encoding spectinomycin resistance; tracrRNA scaffold—trans-activating crisprRNA (conservative, used as a binding scaffold for Cas9); AtU6-26t—terminator of the Arabidopsis snoRNA U6-26 gene; p35S—35S promoter from the Cauliflower Mosaic Virus (CaMV); OsMac3—158 bp fragment of the OsMac3 5′-UTR; 3xFLAG—sequence encoding protein tag with the amino acid sequence DYKDHDGDYKDHDIDYKDDDDK; NLS—nuclear localization signal; SV40 NLS—NLS derived from Simian Virus 40 T antigen; zCas9—maize codon-optimized CRISPR associated nuclease 9; rbcS-E9 t—terminator from pea ribulose-1,5-bisphosphate carboxylase small subunit (rbcS) E9 gene; pAtUbi10—promoter from the Arabidopsis polyubiquitin 10 gene; DsRed1—gene encoding orange fluorescent protein DsRed1 from Discosoma sp.; NOS T—nopaline synthase terminator; KmR—gene encoding kanamycin resistance; CaMV poly(A) signal—polyadenylation signal from CaMV (often used as terminator sequence); LB—left T-DNA border. pKSEe401R nomenclature: p—plasmid; K—KmR; S—p35S; e4—zCas9 enhanced with OsMac3 5′ UTR; 01—pAtU6-26p; R—DsRed1. (B) pKSEe401R was constructed in two steps: (I) cloning of the 158 bp OsMac3 5′-UTR fragment into pKSE401 using Gibson Assembly resulted in pKSEe401; (II) insertion of the DsRed1 cassette into pKSEe401 using T4 ligation resulted in pKSEe401R with the DsRed1 cassette in either in tandem (Var. 1) or in reverse (Var. 2) orientation relative to the Cas9 cassette.

Figure 5

Types of the CRISPR/Cas9 systems. (A) System based on the Cas9 form producing a double-strand break, NHEJ—non-homologous end joining. (B) Approaches using nickase Cas9 (nCas9) activity resulting in a single-strand break, pegRNA—prime editing guide RNA comprised of a target specific crRNA, a conservative tracrRNA, a PBS—primer binding site, an RT template—RNA template for reverse transcription (direction of reverse transcription indicated by a dark blue arrow). The linker joining crRNA and tracrRNA with PBS and the RT template is indicated in orange. RT—reverse transcriptase; UGI—uracil-DNA glycosylase inhibitor domain. (C) Techniques using the catalytically inactive (dead) form of Cas9 (dCas9); types of effectors used in dCas9-based systems: (de)methyl.—methyltransferase or demethylase for epigenetic modifications of DNA; (de)acetyl.—acetyltransferase or deacetylase for epigenetic modifications of histones; F—fluorescent protein; VP64—tetrameric repeat of the minimal activation domain of herpes simplex viral protein 16; UGI—uracil-DNA glycosylase inhibitor domain; deaminase—cytidine or adenosine deaminase; SRDX—SUPERMAN Repression Domain X. For systems based on Cas9 and nCas9 activities, scissors indicated in red show the place where DNA strand breaks occur. Figures were created with BioRender.

Figure 6

Cucumber (Cucumis sativus) hairy roots co-transformed with pKSEe401R with a DsRed1 cassette in tandem (A–C) or reverse (D–F) orientation relative to the Cas9 cassette. (B) DsRed1 fluorescence was absent when the fluorescent protein (FP) cassette was in tandem orientation relative to the Cas9 cassette; (E) DsRed1 fluorescence was visible when the FP cassette was in reverse orientation relative to the Cas9 cassette. (A,D) bright field illumination; (C,F) root autofluorescence. Scale bars denote 5 mm.

Figure 7

Number of studies on CRISPR/Cas9 genome editing in hairy roots (analyzed on the 13 December 2021). Histogram is based on the Google-search of studies presented in Table S1.