Optimized protoplast isolation and transfection with a breakpoint: accelerating Cas9/sgRNA cleavage efficiency validation in monocot and dicot.

The CRISPR-Cas genome editing tools are revolutionizing agriculture and basic biology with their simplicity and precision ability to modify target genomic loci. Software-predicted guide RNAs (gRNAs) often fail to induce efficient cleavage at target loci. Many target loci are inaccessible due to complex chromatin structure. Currently, there is no suitable tool available to predict the architecture of genomic target sites and their accessibility. Hence, significant time and resources are spent on performing editing experiments with inefficient guides. Although in vitro-cleavage assay could provide a rough assessment of gRNA efficiency, it largely excludes the interference of native genomic context. Transient in-vivo testing gives a proper assessment of the cleavage ability of editing reagents in a native genomic context. Here, we developed a modified protocol that offers highly efficient protoplast isolation from rice, Arabidopsis, and chickpea, using a sucrose gradient, transfection using PEG (polyethylene glycol), and validation of single guide RNAs (sgRNAs) cleavage efficiency of CRISPR-Cas9. We have optimized various parameters for PEG-mediated protoplast transfection and achieved high transfection efficiency using our protocol in both monocots and dicots. We introduced plasmid vectors containing Cas9 and sgRNAs targeting genes in rice, Arabidopsis, and chickpea protoplasts. Using dual sgRNAs, our CRISPR-deletion strategy offers straightforward detection of genome editing success by simple agarose gel electrophoresis. Sanger sequencing of PCR products confirmed the editing efficiency of specific sgRNAs. Notably, we demonstrated that isolated protoplasts can be stored for up to 24/48 h with little loss of viability, allowing a pause between isolation and transfection. This high-efficiency protocol for protoplast isolation and transfection enables rapid (less than 7 days) validation of sgRNA cleavage efficiency before proceeding with stable transformation. The isolation and transfection method can also be utilized for rapid validation of editing strategies, evaluating diverse editing reagents, regenerating plants from transfected protoplasts, gene expression studies, protein localization and functional analysis, and other applications. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-024-00139-7.

Potential gene editing targets for developing haploid inducer stocks in rice and wheat with high haploid induction frequency.

Doubled haploid (DH) breeding is a powerful technique to ensure global food security via accelerated crop improvement. DH can be produced in planta by employing haploid inducer stock (HIS). Widely used HIS in maize is known to be governed by ZmPLA, ZmDMP, ZmPLD3, and ZmPOD65 genes. To develop such HIS in rice and wheat, we have identified putative orthologs of these genes using in silico approaches. The OsPLD1; TaPLD1, and OsPOD6; TaPOD8 were identified as putative orthologs of ZmPLD3 and ZmPOD65 in rice and wheat, respectively. Despite being closely related to ZmPLD3, OsPLD1 and TaPLD1 have shown higher anther-specific expression. Similarly, OsPOD6 and TaPOD8 were found closely related to the ZmPOD65 based on both phylogenetic and expression analysis. However, unlike ZmPLD3 and ZmPOD65, two ZmDMP orthologs have been found for each crop. OsDMP1 and OsDMP2 in rice and TaDMP3 and TaDMP13 in wheat have shown similarity to ZmDMP in terms of both sequence and expression pattern. Furthermore, analogs to maize DMP proteins, these genes possess four transmembrane helices making them best suited to be regarded as ZmDMP orthologs. Modifying these predicted orthologous genes by CRISPR/Cas9-based genome editing can produce a highly efficient HIS in both rice and wheat. Besides revealing the genetic mechanism of haploid induction, the development of HIS would advance the genetic improvement of these crops. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03857-9.

A detailed landscape of CRISPR-Cas-mediated plant disease and pest management.

Genome editing technology has rapidly evolved to knock-out genes, create targeted genetic variation, install precise insertion/deletion and single nucleotide changes, and perform large-scale alteration. The flexible and multipurpose editing technologies have started playing a substantial role in the field of plant disease management. CRISPR-Cas has reduced many limitations of earlier technologies and emerged as a versatile toolbox for genome manipulation. This review summarizes the phenomenal progress of the use of the CRISPR toolkit in the field of plant pathology. CRISPR-Cas toolbox aids in the basic studies on host-pathogen interaction, in identifying virulence genes in pathogens, deciphering resistance and susceptibility factors in host plants, and engineering host genome for developing resistance. We extensively reviewed the successful genome editing applications for host plant resistance against a wide range of biotic factors, including viruses, fungi, oomycetes, bacteria, nematodes, insect pests, and parasitic plants. Recent use of CRISPR-Cas gene drive to suppress the population of pathogens and pests has also been discussed. Furthermore, we highlight exciting new uses of the CRISPR-Cas system as diagnostic tools, which rapidly detect pathogenic microorganism. This comprehensive yet concise review discusses innumerable strategies to reduce the burden of crop protection.