Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Jan 5;7(1):193-202.
doi: 10.1534/g3.116.035204.

CRISPR/Cas9-Induced Double-Strand Break Repair in Arabidopsis Nonhomologous End-Joining Mutants

Hexi Shen  1 , Gary D Strunks  1 , Bart J P M Klemann  1 , Paul J J Hooykaas  1 , Sylvia de Pater  2
Affiliations

CRISPR/Cas9-Induced Double-Strand Break Repair in Arabidopsis Nonhomologous End-Joining Mutants

Hexi Shen et al. G3 (Bethesda). .

Abstract

Double-strand breaks (DSBs) are one of the most harmful DNA lesions. Cells utilize two main pathways for DSB repair: homologous recombination (HR) and nonhomologous end-joining (NHEJ). NHEJ can be subdivided into the KU-dependent classical NHEJ (c-NHEJ) and the more error-prone KU-independent backup-NHEJ (b-NHEJ) pathways, involving the poly (ADP-ribose) polymerases (PARPs). However, in the absence of these factors, cells still seem able to adequately maintain genome integrity, suggesting the presence of other b-NHEJ repair factors or pathways independent from KU and PARPs. The outcome of DSB repair by NHEJ pathways can be investigated by using artificial sequence-specific nucleases such as CRISPR/Cas9 to induce DSBs at a target of interest. Here, we used CRISPR/Cas9 for DSB induction at the Arabidopsis cruciferin 3 (CRU3) and protoporphyrinogen oxidase (PPO) genes. DSB repair outcomes via NHEJ were analyzed using footprint analysis in wild-type plants and plants deficient in key factors of c-NHEJ (ku80), b-NHEJ (parp1 parp2), or both (ku80 parp1 parp2). We found that larger deletions of >20 bp predominated after DSB repair in ku80 and ku80 parp1 parp2 mutants, corroborating with a role of KU in preventing DSB end resection. Deletion lengths did not significantly differ between ku80 and ku80 parp1 parp2 mutants, suggesting that a KU- and PARP-independent b-NHEJ mechanism becomes active in these mutants. Furthermore, microhomologies and templated insertions were observed at the repair junctions in the wild type and all mutants. Since these characteristics are hallmarks of polymerase θ-mediated DSB repair, we suggest a possible role for this recently discovered polymerase in DSB repair in plants.

Keywords: Arabidopsis thaliana; CRISPR/Cas9; KU80; double-strand break; nonhomologous end-joining.

PubMed Disclaimer

Figures

Figure 1
Figure 1
CRISPR/Cas9 endonucleases for DSB induction in CRU3 and PPO. Cas9-CRU (A) with its protospacer in the CRU3 locus and Cas9-PPO (B) with its protospacer in the PPO locus are shown. sgRNA DNA binding sequences are highlighted in yellow, the PAM sequence is highlighted in black and the PstI and FauI restriction sites are shown in red lettering. The primers (▔) used to amplify the target regions and the sizes are indicated. Red arrows indicate the position of DSB induction.
Figure 2
Figure 2
CRISPR/Cas9 endonucleases-induced mutagenesis. (A) The CRU3 target site was amplified using undigested genomic DNA from untransformed wild-type seedlings and Cas9-CRU-transformed T2 seedlings and digested with PstI. (B) The PPO target site was amplified from untransformed wild-type seedlings and Cas9-PPO-transformed T2 seedlings of wild-type and ku80, parp1 parp2 (p1p2), and ku80 parp1 parp2 (k80p1p2) mutant plant lines and digested with FauI. M is the 1 kb DNA marker, with sizes of the bands at the left, and the % resistant bands is shown below the lanes. (C) Sequences of CRU3 and PPO targets from Cas9-CRU transformant #2 and Cas9-PPO transformant #7. The sgRNA protospacer is in red, PAM sequence is in gray, restriction sites are underlined, deletions are shown by dashes, insertions are in green, and microhomologies used for repair are in purple. Number of multiple clones with the same sequence are shown at the right. Numbers are length of deletions (−) and insertions (+).
Figure 3
Figure 3
HRM analysis of the PPO target. HRM analyses were performed on 48 PCR clones from undigested DNA of a pool of 10 T2 seedlings of wild-type Cas9-PPO transformant #7. (A) Melt curves of samples 1–48 measured in relative fluorescence units (RFU). Numbers indicated in the graph refer to the sequences below. (B) Sequences of representative PPO targets. PPO sgRNA protospacer (red), the PAM sequence (gray), and FauI restriction site (underlined) are indicated in the wild-type (WT) sequence. Footprints included deletions (dashed lines) and insertions (green). Microhomologies used for repair are shown in purple.
Figure 4
Figure 4
Analysis of deletion length. (A) Distribution of deletion lengths of mutated sequences obtained for the indicated genotypes with Cas9 nucleases. (B) Scatter plot of deletion lengths of the sequences used in (A). Median deletion lengths are indicated by horizontal lines. P-values are derived from a two-tailed Mann–Whitney U-test. Asterisks (*) indicate a statistically significant difference from wild type (P < 0.05).
Figure 5
Figure 5
Analysis of insertions. (A) Scatter plot of insertion lengths for the indicated genotypes. Data are combined for both targets. (B) Scatter plot of deletion lengths with (+) or without (−) insertions. Data are combined from all genotypes for both targets. Median insertion or deletion lengths are indicated by horizontal lines. P-values are derived from a two-tailed Mann–Whitney U-test. The asterisk (*) indicates a statistical significant difference (P < 0.05). (C) Footprints consisting of deletions (dashes) accompanied by insertions. Insertions are shown in green, template sources for the insertions are shown in yellow (direct strand) or underlined (reverse complement). Homologies between sequences flanking the template and the insertion and probably used as primer are shown in gray. Footprints from 1 to 8 are examples of perfectly matching the template, 9–15 are partially matching the template, and 16–21 are reversely matching the template. Numbers are length of deletions (−) and insertions (+).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Alonso J. M., Stepanova A. N., Leisse T. J., Kim C. J., Chen H., et al. , 2003. Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301: 653–657. - PubMed
    1. Belhaj K., Chaparro-Garcia A., Kamoun S., Patron N. J., Nekrasov V., 2015. Editing plant genomes with CRISPR/Cas9. Curr. Opin. Biotechnol. 32: 76–84. - PubMed
    1. Clough S. J., Bent A. F., 1998. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16: 735–743. - PubMed
    1. Deriano L., Roth D. B., 2013. Modernizing the nonhomologous end-joining repertoire: alternative and classical NHEJ share the stage. Annu. Rev. Genet. 47: 433–455. - PubMed
    1. de Pater S., Neuteboom L. W., Pinas J. E., Hooykaas P. J. J., van der Zaal B. J., 2009. ZFN-induced mutagenesis and gene-targeting in Arabidopsis through Agrobacterium-mediated floral dip transformation. Plant Biotechnol. J. 7: 821–835. - PubMed

MeSH terms