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Homology-Directed Repair of a Defective Glabrous Gene in Arabidopsis With Cas9-Based Gene Targeting

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Homology-Directed Repair of a Defective Glabrous Gene in Arabidopsis With Cas9-Based Gene Targeting

Florian Hahn et al. Front Plant Sci.

Abstract

The CRISPR/Cas9 system has emerged as a powerful tool for targeted genome editing in plants and beyond. Double-strand breaks induced by the Cas9 enzyme are repaired by the cell's own repair machinery either by the non-homologous end joining pathway or by homologous recombination (HR). While the first repair mechanism results in random mutations at the double-strand break site, HR uses the genetic information from a highly homologous repair template as blueprint for repair of the break. By offering an artificial repair template, this pathway can be exploited to introduce specific changes at a site of choice in the genome. However, frequencies of double-strand break repair by HR are very low. In this study, we compared two methods that have been reported to enhance frequencies of HR in plants. The first method boosts the repair template availability through the formation of viral replicons, the second method makes use of an in planta gene targeting (IPGT) approach. Additionally, we comparatively applied a nickase instead of a nuclease for target strand priming. To allow easy, visual detection of HR events, we aimed at restoring trichome formation in a glabrous Arabidopsis mutant by repairing a defective glabrous1 gene. Using this efficient visual marker, we were able to regenerate plants repaired by HR at frequencies of 0.12% using the IPGT approach, while both approaches using viral replicons did not yield any trichome-bearing plants.

Keywords: CRISPR/Cas9; Glabrous1; gene editing; homologous recombination; in planta gene targeting; marker; trichomes; viral replicons.

Figures

FIGURE 1
FIGURE 1
Vector design for repair of the gl1 gene. Single T-DNA vectors were designed to repair the dysfunctional gl1 gene. (A) pVIR-Nuc contains the Cas9 Nuclease gene (red box) under a constitutive Ubiquitin4-2 promoter from parsley (PcUBp) and the sgRNA cassette (petrol box) controlled by the U6-26 promoter (U6-26p). A Basta resistance cassette (Bar, dark blue box) is included as selection marker. The repair template itself consists of 10 nucleotides (10 bp, CTGCCGTTTA, orange box), which should restore the reading frame of the gl1 gene, flanked by homology arms (HA, purple box). Rolling circle replication of the homology template is ensured by the flanking long intergenic region (LIR, pink hairpin), short intergenic region (pink box, SIR), and the gene encoding the replicase Rep/RepA (light blue box). (B) pVIR-Nick contains similar features but harbors the gene for the Cas9 nickase instead of the nuclease and a kanamycin resistance cassette (Kan, dark blue box) as selection marker. (C) pIPGT-Nuc contains the gene for the Cas9 nuclease, the sgRNA cassette, the Basta resistance cassette and the repair template with homology arms flanked by target sites for the Cas9 nuclease (red lines). RB, right T-DNA border; 35Sp, Cauliflower Mosaic Virus 35S promoter; LB, left T-DNA border. Size not to scale.
FIGURE 2
FIGURE 2
Two different mechanisms enhance the availability of the homology template in the plant cell nucleus. (A) If the T-DNA from pVIR-Nuc is integrated into the plant genome, Rep is expressed and initiates rolling circle endoreplication of the homology template leading to thousands of viral replicons. The presence of viral replicons can be detected by PCR using primers that only generate an amplicon on the circular replicons but not on the T-DNA (green arrows). The homology template can then attach to the DSB site in the gl1 gene induced by Cas9 due to homology between the homology arms (HA) and the genomic regions next to the DSB (purple). Repair of the DSB by HR will then lead to integration of the 10 bp, which restores the ORF. pVIR-Nick functions accordingly, except that Cas9 nickase only induces a DNA nick in the gl1 gene. (B) The homology template in the T-DNA of pIPGT-Nuc is flanked by the same sgRNA target site that is present in the gl1 gene. Expression of Cas9 will therefore simultaneously target the gl1 gene and release the homology template, which can then attach to the DSB and function as repair template.
FIGURE 3
FIGURE 3
T2 generation plants show spots of trichomes. (A) Representative images of T2 plants transformed with pIPGT-Nuc (red frame) or pVIR-Nuc (blue frame) showing spots of trichomes (yellow circles) ranging from one single trichome to fully covered leaves. (B) The gl1 gene was amplified from leaves with trichomes, subcloned, and sequenced. Sequence analysis revealed clones with various indel mutations next to clones containing the desired 10 bp insertion (red letters). PAM sequence is underlined, STOP codon in gl1 line is marked in green letters, Cas9 target site is marked in blue letters.
FIGURE 4
FIGURE 4
Wild-type-like trichome patterning was detected in three T3 generation plants. (A) Exemplary picture of one of the three T3 plants (#1) with full trichome covering. (B) We verified the repair of the gl1 gene by amplifying the gl1 gene and sequencing the PCR amplicon. Double peaks appeared at the site of Cas9 cleavage hinting at a chimeric or biallelic mutation, the peaks corresponded to an insertion of the ten bp (red) from the homology template and to an adenine insertion. Sequence of the dysfunctional gl1 gene is given on top of the sequencing histogram for comparison. Peak colors: Adenine = green, cytosine = blue, guanine = black, thymine = red; PAM sequence underlined, premature STOP codon marked in green letters. (C) Presence of the Cas9 gene in the three non-glabrous plants was detected by PCR using Cas9-specific primers. All three plants revealed PCR amplicons at the expected size of 749 bp. In contrast, DNA from the gl1 background line did not yield a PCR Cas9 signal. Image of agarose gel was color inverted for better visibility. M, marker; N, water control.
FIGURE 5
FIGURE 5
Sequencing histogram of T4 generation plant reveals homozygous integration of the 10 nucleotides thereby restoring the GL1 reading frame. Sequence of the dysfunctional gl1 gene is given on top of the sequencing histogram for comparison. Peak colors: Adenine = green, cytosine = blue, guanine = black, thymine = red; PAM sequence underlined, premature STOP codon marked in green letters.

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