High-efficiency retron-mediated single-stranded DNA production in plants

Abstract

Retrons are a class of retroelements that produce multicopy single-stranded DNA (ssDNA) and participate in anti-phage defenses in bacteria. Retrons have been harnessed for the overproduction of ssDNA, genome engineering and directed evolution in bacteria, yeast and mammalian cells. Retron-mediated ssDNA production in plants could unlock their potential applications in plant biotechnology. For example, ssDNA can be used as a template for homology-directed repair (HDR) in several organisms. However, current gene editing technologies rely on the physical delivery of synthetic ssDNA, which limits their applications. Here, we demonstrated retron-mediated overproduction of ssDNA in Nicotiana benthamiana. Additionally, we tested different retron architectures for improved ssDNA production and identified a new retron architecture that resulted in greater ssDNA abundance. Furthermore, co-expression of the gene encoding the ssDNA-protecting protein VirE2 from Agrobacterium tumefaciens with the retron systems resulted in a 10.7-fold increase in ssDNA production in vivo. We also demonstrated clustered regularly interspaced short palindromic repeats-retron-coupled ssDNA overproduction and targeted HDR in N. benthamiana. Overall, we present an efficient approach for in vivo ssDNA production in plants, which can be harnessed for biotechnological applications. Graphical Abstract.

Keywords: CRISPR-Cas9; HDR; VirE2; retron; ssDNA.

Figure 1.

Schematic representation of retron-mediated production of exogenous ssDNA in vivo in N. benthamiana and design of vectors and donor templates for ssDNA production and genome engineering in N. benthamiana. (a) Retron Ec86-RT and Retron Ec86-ncRNA are expressed independently. Later, the Ec86 RT recognizes the hairpin structure in msr and initiate reverse transcription from the priming guanosine to synthesize msDNA. The mature msDNA, a DNA-RNA hybrid, consists of ssDNA and msr ncRNA. (b) Different vector backbones (pK2-Cas9-XTEN-RT, pK2-Cas9-T2A-RT, pK2-RT-XTEN-Cas9, pK2-RT and pK2-Cas9) used for the transient expression studies in N. benthamiana. (c) The N. benthamiana PDS gene sequence and the DRT sequences employed for repair by the Retron-Editor system.

Figure 2.

Southern blot analysis for the detection of ssDNA. Total RNA was isolated from N. benthamiana to detect PDS-1 and PDS-4 targets and run without any nuclease treatment, with RNase treatment, and with DNase treatment on an agarose gel side by side, together with the 140-bp amplified PDS-1 or PDS-4 msd-DRT-msd as a positive control. We used a 100-bp DIG-labeled DRT as a probe for the corresponding target. The membrane was imaged with the ChemiDoc imaging system (Bio-Rad).

Figure 3.

Illustration of different retron structure modifications and Southern blot analysis for the detection of ssDNA in N. benthamiana containing different retron modifications. (a) Representation of the different retron structural modifications in msr and msd sequence lengths. (b)Total RNA was isolated from N. benthamiana to detect PDS-1 and PDS-4 targets and the RNase-treated samples were run on an agarose gel together with the 140-bp amplified PDS-1 or PDS-4 msd-DRT-msd as a positive control. We used a 100-bp DIG-labeled DRT as a probe for the corresponding target. The membrane was imaged with the ChemiDoc imaging system (Bio-Rad).

Figure 4.

Southern blot analysis for the detection of ssDNA in N. benthamiana co-expressing VirE2 and retron systems. Total RNA was isolated from N. benthamiana to detect the PDS-4 target, where RNase treated and the samples were run on an agarose gel together with the 140-bp amplified PDS-4 msd-DRT-msd as a positive control. We used a 100-bp DIG-labeled DRT as a probe. The membrane was imaged with the ChemiDoc imaging system (Bio-Rad).

Figure 5.

Examination of Retron-Editor-mediated precision HDR and Cas9 indel frequencies in N. benthamiana. Deep amplicon sequencing of PDS-1 and PDS-4 targets with different Retron-Editors showed variable (a) HDR and (b) indel frequencies. Data are expressed as mean ± standard deviation (n = 3). P values were obtained using two-tailed Student’s t-tests in e and f. *P < 0.05, **P < 0.01, ***P < 0.001.