A DNA-Free Editing Platform for Genetic Screens in Soybean via CRISPR/Cas9 Ribonucleoprotein Delivery

Abstract

CRISPR/Cas9-based ribonucleoprotein (RNP)-mediated system has the property of minimizing the effects related to the unwanted introduction of vector DNA and random integration of recombinant DNA. Here, we describe a platform based on the direct delivery of Cas9 RNPs to soybean protoplasts for genetic screens in knockout gene-edited soybean lines without the transfection of DNA vectors. The platform is based on the isolation of soybean protoplasts and delivery of Cas RNP complex. To empirically test our platform, we have chosen a model gene from the soybean genetic toolbox. We have used five different guide RNA (gRNA) sequences that targeted the constitutive pathogen response 5 (CPR5) gene associated with the growth of trichomes in soybean. In addition, efficient protoplast transformation, concentration, and ratio of Cas9 and gRNAs were optimized for soybean for the first time. Targeted mutagenesis insertion and deletion frequency and sequences were analyzed using both Sanger and targeted deep sequencing strategies. We were able to identify different mutation patterns within insertions and deletions (InDels) between + 5 nt and -30 bp and mutation frequency ranging from 4.2 to 18.1% in the GmCPR5 locus. Our results showed that DNA-free delivery of Cas9 complexes to protoplasts is a useful approach to perform early-stage genetic screens and anticipated analysis of Cas9 activity in soybeans.

Keywords: breeding; gene editing; genetic screening; genetically modified organism; genetically modified plants; mutagenesis; target deep sequencing; transgenesis.

FIGURE 1

Schematic protocol for CRISPR/Cas9-mediated DNA-free/transient genome editing in the soybean. The protoplasts were isolated from young seedlings of unifoliate leaf strips with enzyme solution. Isolated protoplasts were transformed with preassembled CRISPR-RNP complexes via the PEG-mediated method. The transformed protoplasts were subjected to either genomic DNA extraction or fluorescence-activated cell sorting (FACS) of Cas9-GFP expressing protoplasts for an enrichment followed by DNA extraction or to cultivation. Target sites are amplified by PCR followed by T7E1 validation of mutation and high-throughput sequencing for estimation of mutation efficiency.

FIGURE 2

Schematic representation of the GmCPR5 locus and design of gRNAs. The location of target sites was shown by engineered gRNAs 1, 2, 3, 4, and 5 along with their sequences. The PAM motifs are indicated in red.

FIGURE 3

Isolation, purification, and cultivation of protoplasts from Glycine max cv. OAC Bayfield. (A) Unifoliate leaves of 10-day-old soybean seedlings. (B) Freshly isolated protoplasts. (C) Washed and purified protoplasts. (D) FDA-stained protoplasts subjected to confocal fluorescence microscopy to visualize viable cells (GFP-positive). (E) Merged image of green channel (GFP) and ESID channel (transmitted light detection) showing all protoplasts. (F) Protoplasts undergoing cell division (indicated by white arrows) in culture medium after 3 days of isolation.

FIGURE 4

Cellular localization of GFP-Cas9 RNP complexes in transfected protoplasts from Glycine max cv Bayfield. (A,D) Confocal fluorescence microscopy showing GFP-Cas9 located inside transfected protoplasts using the green channel. White arrows indicate internalized localization of GFP-Cas9. (B,E) The same protoplasts as depicted in (A,D) showing bright-field images using the ESID channel. (C,F) Are overlay images of green and ESID channels. White arrows indicate internalized localization of GFP-Cas9.

FIGURE 5

In vitro cleavage assay. The in vitro transcribed or purchased sgRNAs at GmCPR5 loci were mixed with SpCas9 and PCR templates of target sites (gRNA1–5) for in vitro digestion and resolved on 2% agarose gel. Lanes M, DNA ladders; C, PCR wild type (control untreated); T, treated with sgRNAs and SpCas9. The parental and cleaved fragments are indicated with blue and red arrows, respectively.

FIGURE 6

Detection of site-directed mutagenesis at target sites on GmCPR5 loci using direct delivery of RGEN RNPs. (A–E) T7E1 digestion resulting gel images for the transformants of T1–T5. Lanes M, DNA ladder; WT, untransformed wild type (control) to each target site; Cas9, transformed with SpCas9 only; T1–T5, transformed with RNPs; T3T7E1(–), negative control (undigested). Cleaved fragments are indicated with red arrows. Images 6E and 6F have been processed for better visualization of bands. This process is in accordance with Frontiers Policies and Publication Ethics Guidelines.

FIGURE 7

Mutation patterns observed by targeted deep and Sanger sequencing for the corresponding target sites at GmCPR5 loci are shown. (A) Distribution of the five most frequent alleles along with their mutation pattern, contribution percentage, and read count observed with the Cas-Analyzer around targeted sites in GmCPR5 for T1, T3, and T5. Wild-type (WT) nuclease target sequences were in bold and underlined. PAM sites are denoted by the red font. Insertions and deletions are shown in bold font (blue) and horizontal dashed lines, respectively. (B) The result of editing efficiency and mutation patterns analyzed with DECODR software for T2 and T4. The top panels display the graphs for the InDel distribution rate. The bottom panel shows the list of deconvoluted InDel-containing sequences as alignments along with InDel types and percentages (%). Insertion (highlighted with purple rectangles) and deletion (marked with horizontal dashed lines) of mutations are shown in alignments. A 20 bp target and 3 bp PAM site are depicted with green and red lines, respectively.