MRE11 and RAD50, but not NBS1, are essential for gene targeting in the moss Physcomitrella patens

Abstract

The moss Physcomitrella patens is unique among plant models for the high frequency with which targeted transgene insertion occurs via homologous recombination. Transgene integration is believed to utilize existing machinery for the detection and repair of DNA double-strand breaks (DSBs). We undertook targeted knockout of the Physcomitrella genes encoding components of the principal sensor of DNA DSBs, the MRN complex. Loss of function of PpMRE11 or PpRAD50 strongly and specifically inhibited gene targeting, whilst rates of untargeted transgene integration were relatively unaffected. In contrast, disruption of the PpNBS1 gene retained the wild-type capacity to integrate transforming DNA efficiently at homologous loci. Analysis of the kinetics of DNA-DSB repair in wild-type and mutant plants by single-nucleus agarose gel electrophoresis revealed that bleomycin-induced fragmentation of genomic DNA was repaired at approximately equal rates in each genotype, although both the Ppmre11 and Pprad50 mutants exhibited severely restricted growth and development and enhanced sensitivity to UV-B and bleomycin-induced DNA damage, compared with wild-type and Ppnbs1 plants. This implies that while extensive DNA repair can occur in the absence of a functional MRN complex; this is unsupervised in nature and results in the accumulation of deleterious mutations incompatible with normal growth and development.

Genotoxin Induced Mutagenesis in the Model PlantPhyscomitrella patens

“…Half-lives of DSB survival τ 1/2 1.5 min for wild type and 2.5 min for pplig4 are similar to τ 1/2 2.9 min for pprad50 , τ 1/2 4.1 min for ppmre11 , and τ 1/2 1.9 min for ppnbs1 previously reported in [6]. …”

“…Mutational inactivation can be used as selectable marker for mutator genotyping as well as analysis of mutations in APT locus on nucleotide level [4–6]. …”

Genetic analysis of Physcomitrella patens identifies ABSCISIC ACID NON-RESPONSIVE (ANR), a regulator of ABA responses unique to basal land plants and required for desiccation tolerance

“…The growth index was calculated by image analysis of digital photographs using ImageJ as previously described (Kamisugi et al, 2012), with average plant area normalized either to the area of the Petri dish (Figure 4) or to a 5-cm line (Figure 8). …”

Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens

“…The ratio between TGR and TGI events was identical, but the proportion of plants with a single copy replacement was significantly higher with CRISPR (40.5%) than without (15%). Classical GT in P. patens was recently described as dependent on the classical RAD51-mediated HR repair pathway [112,113,118]. Interestingly, with CRISPR, HDR-mediated knock-in was reduced but not abolished (as it is without CRISPRs) in the Pprad51-1-2 double mutant, reaching about 30% of the wild type level.…”

“…Three separate plasmids carrying the Cas9, the sgRNA and a donor template bearing an antibiotic resistance gene framed by homologies to the reporter gene PpAPT were co-transformed by PEG fusion of protoplasts [41]. As previously described in P. patens [111][112][113][114] and as frequently observed in other plants [115], the donor DNA template can be inserted in two different ways: either by homologous recombination on both sides leading to targeted gene replacement (TGR) or by HDR on one side and NHEJ on the other, upstream or downstream of the targeted locus, leading to targeted gene insertions (TGI). For both these types of events, the insertions frequently contain multiple copies of the donor template [116,117].…”