Establishment of targeted mutagenesis in soybean protoplasts using CRISPR/Cas9 RNP delivery via electro-transfection

Abstract

The soybean (Glycine max L.) is an important crop with high agronomic value. The improvement of agronomic traits through gene editing techniques has broad application prospects in soybean. The polyethylene glycol (PEG)-mediated cell transfection has been successfully used to deliver the CRISPR/Cas9-based ribonucleoprotein (RNP) into soybean protoplasts. However, several downstream analyses or further cell regeneration protocols might be hampered by PEG contamination within the samples. Here in this study, we attempted to transfect CRISPR/Cas9 RNPs into trifoliate leaf-derived soybean protoplasts using Neon electroporation to overcome the need for PEG transfection for the first time. We investigated different electroporation parameters including pulsing voltage (V), strength and duration of pulses regarding protoplast morphology, viability, and delivery of CRISPR/Cas9. Electroporation at various pulsing voltages with 3 pulses and 10 ms per pulse was found optimal for protoplast electro-transfection. Following electro-transfection at various pulsing voltages (500 V, 700 V, 1,000 V, and 1,300 V), intact protoplasts were observed at all treatments. However, the relative frequency of cell viability and initial cell divisions decreased with increasing voltages. Confocal laser scanning microscopy (CLSM) confirmed that the green fluorescent protein (GFP)-tagged Cas9 was successfully internalized into the protoplasts. Targeted deep sequencing results revealed that on-target insertion/deletion (InDel) frequencies were increased with increasing voltages in protoplasts electro-transfected with CRISPR/Cas9 RNPs targeting constitutive pathogen response 5 (CPR5). InDel patterns ranged from +1 bp to -6 bp at three different target sites in CPR5 locus with frequencies ranging from 3.8% to 8.1% following electro-transfection at 1,300 V and 2.1% to 3.8% for 700 V and 1,000 V, respectively. Taken together, our results demonstrate that the CRISPR/Cas9 RNP system can be delivered into soybean protoplasts by the Neon electroporation system for efficient and effective gene editing. The electro-transfection system developed in this study would also further facilitate and serve as an alternative delivery method for DNA-free genome editing of soybean and other related species for genetic screens and potential trait improvement.

Keywords: CRISPR/Cas9 RNPs; gene-editing; neon electroporation system; protoplasts; soybean; target-deep sequencing; trait improvement.

Figure 1

Isolation of protoplasts from trifoliate leaves of soybean plantlets. (A) Fifteen-day-old plants showing trifoliate leaves of suitable size. (B, C) Protoplasts of freshly extracted (B) and purified cells (C) under the Motic AE2000 inverted microscope with × 20 and × 40 objectives, respectively. Black scale bar, 30 µm. (D, E) The protoplast viability was assessed by FDA staining and observed under both bright field (D) and fluorescence channel, and simultaneously merged images are depicted (E) using Axio Vert.A1 inverted microscope with a × 20 objective. (F) Division of protoplasts (shown by white arrows) at 4 days after isolation in culture medium. FDA, fluorescein diacetate.

Figure 2

Effect of various pulsing voltage on the protoplasts morphology, viability, and cell division efficiency following electro-transfection. Left panel: representative images of electroporated protoplasts after 24 h are shown. Middle panel: the merged fluorescence images (under bright and fluorescence field using Axio Vert.A1 inverted microscope with a × 20 objective) showing the FDA-stained viable cells after 24 h of electroporation. Right panel: images showing primary divisions (shown by black arrows) of electroporated protoplasts at 4 days after culture initiation. FDA, fluorescein diacetate.

Figure 3

Delivery and cellular localization of Cas9-GFP to soybean protoplasts through electro-transfection. GFP-Cas9 in electro-transfected (0 V to 1,300 V) protoplasts at 24 h after electroporation was seen using a laser scanning confocal microscope under GFP (left panel) and bright field of ESID channel (middle panel). Right panel: representative overlay images of GFP and bright field are shown. White arrows show the location of internalized GFP-Cas9. GFP, green fluorescent protein; ESID, electronically switchable illumination and detection module.

Figure 4

CRISPR/Cas9-mediated editing of the exogenous GmCPR5 gene in soybean protoplasts using electro-transfection. (A) GmCPR5 locus, location of target sites (T1, T3, and T5), and their gRNA sequences. (B–D) Results of T7E1 endonuclease assay for target sites T1, T3 and T5. Lane M: a DNA ladder. Lane WT: non- transfected wild type (control). Lane Cas9: transfected with SpCas9 only. Lanes T1–T5: electro-transfected with RNPs at 700 V, 1,000 V, and 1,300 V. Red arrows indicate the T7E1-mediated cleaved bands. The mutation patterns observed by targeted deep sequencing for the corresponding target sites of T1–T5 at GmCPR5 loci by electro-transfection at 1,300 V are shown on the right panel. Wild-type (WT) nuclease target sequences are in bold and underlined. PAM sites are denoted in red. RNPs, ribonucleoproteins; gRNA, guide RNA.