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.

A novel miR160a–GmARF16–GmMYC2 module determines soybean salt tolerance and adaptation

“…Editing GmARF16 and GmMYC2 has potential for enhancing salt tolerance Gene-editing technology has provided scientists with an opportunity to change or introduce single-nucleotide changes in plant genomes with high precision and without the need for double-strand breaks (Molla et al, 2021;Wang & Doudna, 2023). Base editing is a promising tool for plant breeding because it can be used to introduce bespoke edits that can, or already has, significantly enhanced yields, improve nutritional value or confer stress resistance (Hua et al, 2018;Eraslan et al, 2019;Dutta et al, 2023;Subburaj & Agapito-Tenfen, 2023). For instance, a point mutation in the miR156 binding site of OsSPL14 leads to rice plants with improved architecture and higher seed yields (Jiao et al, 2010).…”

Plant biomacromolecule delivery methods in the 21st century

“…Protoplast isolation and transformation with PEG were performed in many crop plants (Bilang et al, 1994) including but not limited to wheat (Brandt et al, 2020), rice (Lin et al, 2020;Bes et al, 2021), maize (Svitashev et al, 2016), potato (Carlsen et al, 2022), and soybean (Patil et al, 2022). Additionally, in recent years, the PEG-mediated protoplast transfection of Cas9 protein and Cas9 complexed with in vitro synthesized guide RNA (ribonucleoprotein, RNP) was successfully used in major row crop and horticultural crops in a quest to establish a DNA-free genome editing platform (Sant'Ana et al, 2020;Subburaj et al, 2022;Liu et al, 2022).…”