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Canonical CRISPR-Cas9 genome editing technique has profoundly impacted the fields of plant biology, biotechnology, and crop improvement. Since non-homologous end joining (NHEJ) is usually considered to generate random indels, its high efficiency mutation is generally not pertinent to precise editing. Homology-directed repair (HDR) can mediate precise editing with supplied donor DNA, but it suffers from extreme low efficiency in higher plants. Therefore, precision editing in plants will be facilitated by the ability to predict NHEJ repair outcome and to improve HDR efficiency. Here, we report that NHEJ-mediated single nucleotide insertion at different rice genes is predictable based on DNA sequences at the target loci. Three mutation prediction tools (inDelphi, FORECasT, and SPROUT) have been validated in the rice plant system. We also evaluated the chimeric guide RNA (cgRNA) and Cas9-Retron precISe Parallel Editing via homologY (CRISPEY) strategies to facilitate donor template supply for improving HDR efficiency in Nicotiana benthamiana and rice. However, neither cgRNA nor CRISPEY improved plant HDR editing efficiency in this study. Interestingly, our data indicate that tethering of 200–250 nucleotides long sequence to either 5′ or 3′ ends of guide RNA did not significantly affect Cas9 cleavage activity. 

The Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein system (CRISPR/Cas) has recently become the most powerful tool available for genome engineering in various organisms. With efficient and proper expression of multiple guide RNAs (gRNAs), the CRISPR/Cas system is particularly suitable for multiplex genome editing. During the past several years, different CRISPR/Cas expression strategies, such as two-component transcriptional unit, single transcriptional unit, and bidirectional promoter systems, have been developed to efficiently express gRNAs as well as Cas nucleases. Significant progress has been made to optimize gRNA production using different types of promoters and RNA processing strategies such as ribozymes, endogenous RNases, and exogenous endoribonuclease (Csy4). Besides being constitutively and ubiquitously expressed, inducible and spa- tiotemporal regulations of gRNA expression have been demonstrated using inducible, tissue-specific, and/or synthetic promoters for specific research purposes. Most recently, the emergence of CRISPR/Cas ribonucleoprotein delivery methods, such as engineered nanoparticles, further revolutionized trans- gene-free and multiplex genome editing. In this review, we discuss current strategies and future per- spectives for efficient expression and engineering of gRNAs with a goal to facilitate CRISPR/Cas-based multiplex genome editing. 

Human life intimately depends on plants for food, biomaterials, health, energy, and a sustainable environment. Various plants have been genetically improved mostly through breeding, along with limited modification via genetic engineering, yet they are still not able to meet the ever-increasing needs, in terms of both quantity and quality, resulting from the rapid increase in world population and expected standards of living. A step change that may address these challenges would be to expand the potential of plants using biosystems design approaches. This represents a shift in plant science research from relatively simple trial-and-error approaches to innovative strategies based on predictive models of biological systems. Plant biosystems design seeks to accelerate plant genetic improvement using genome editing and genetic circuit engineering or create novel plant systems through de novo synthesis of plant genomes. From this perspective, we present a comprehensive roadmap of plant biosystems design covering theories, principles, and technical methods, along with potential applications in basic and applied plant biology research. We highlight current challenges, future opportunities, and research priorities, along with a framework for international collaboration, towards rapid advancement of this emerging interdisciplinary area of research. Finally, we discuss the importance of social responsibility in utilizing plant biosystems design and suggest strategies for improving public perception, trust, and acceptance.