Towards DNA-free CRISPR/Cas9 genome editing for sustainable oil palm improvement.

The CRISPR/Cas9 genome editing system has been in the spotlight compared to programmable nucleases such as ZFNs and TALENs due to its simplicity, versatility, and high efficiency. CRISPR/Cas9 has revolutionized plant genetic engineering and is broadly used to edit various plants' genomes, including those transformation-recalcitrant species such as oil palm. This review will comprehensively present the CRISPR-Cas9 system's brief history and underlying mechanisms. We then highlighted the establishment of the CRISPR/Cas9 system in plants with an emphasis on the strategies of highly efficient guide RNA design, the establishment of various CRISPR/Cas9 vector systems, approaches of multiplex editing, methods of transformation for stable and transient techniques, available methods for detecting and analyzing mutations, which have been applied and could be adopted for CRISPR/Cas9 genome editing in oil palm. In addition, we also provide insight into the strategy of DNA-free genome editing and its potential application in oil palm.

A streamlined guide RNA screening system for genome editing in Sorghum bicolor.

BACKGROUND: Genome editing tools derived from clustered regularly interspaced short palindromic repeats (CRISPR) systems have been developed for generating targeted mutations in plants. Although these tools hold promise for rapid crop improvement, target-specific guide RNAs exhibit variable activity. To improve genome editing, a rapid and precise method for evaluating their efficiency is necessary. RESULTS: Here we report an efficient system for screening single guide RNAs (sgRNAs) for genome editing in sorghum using a transient protoplast transfection assay. Protoplasts were isolated from leaves from sorghum plants cultivated under three different conditions. Cultivation for three days of continuous darkness following seven days with a 16-h light and 8-h dark photoperiod resulted in the highest yield of viable protoplasts and the highest protoplast transfection efficiency. We tested both plasmid-mediated and ribonucleoprotein-based delivery to protoplasts, via polyethylene glycol-mediated transfection, of CRISPR components targeting the sorghum genome. The frequencies of small insertions and deletions induced by a set of sgRNAs targeting four endogenous sorghum genes were analyzed via targeted deep sequencing. Our screening system induced indels in sorghum protoplasts at frequencies of up to 77.8% (plasmid) and 18.5% (RNP). The entire screening system was completed within 16 days. CONCLUSIONS: The screening system optimized in this study for predicting sgRNA activity for genome editing in sorghum is efficient and straightforward. This system will reduce the time and effort needed for sorghum genome editing.

CRISPR-Based Genome Editing Tools: Insights into Technological Breakthroughs and Future Challenges.

Genome-editing (GE) is having a tremendous influence around the globe in the life science community. Among its versatile uses, the desired modifications of genes, and more importantly the transgene (DNA)-free approach to develop genetically modified organism (GMO), are of special interest. The recent and rapid developments in genome-editing technology have given rise to hopes to achieve global food security in a sustainable manner. We here discuss recent developments in CRISPR-based genome-editing tools for crop improvement concerning adaptation, opportunities, and challenges. Some of the notable advances highlighted here include the development of transgene (DNA)-free genome plants, the availability of compatible nucleases, and the development of safe and effective CRISPR delivery vehicles for plant genome editing, multi-gene targeting and complex genome editing, base editing and prime editing to achieve more complex genetic engineering. Additionally, new avenues that facilitate fine-tuning plant gene regulation have also been addressed. In spite of the tremendous potential of CRISPR and other gene editing tools, major challenges remain. Some of the challenges are related to the practical advances required for the efficient delivery of CRISPR reagents and for precision genome editing, while others come from government policies and public acceptance. This review will therefore be helpful to gain insights into technological advances, its applications, and future challenges for crop improvement.