The current method to induce haploids in rice is anther culture, which is time-consuming and labor intensive and only works for some varieties. Here we describe a seed-based haploid induction system created by CRISPR/Cas9 technology. By editing OsMATL, we generate rice haploid inducer lines with a 2-5% haploid induction rate in different germplasms.CRISPR-Cas9 system is one sequence-specific nuclease (SSN) that has several advantages over zinc finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN), such as multiplex genome editing. With multiplex genome editing, CRISPR-Cas9 becomes a preferred SSN to edit many quantitative trait loci (QTL) simultaneously for trait improvement in major crops. A multiplexed CRISPR system is also important for deletion of a large fragment within a chromosome, analysis of the function of gene families, exon exchange, gene activation, and repression. Therefore, assembly of several single guide RNAs (sgRNAs) into one binary vector is the main step in multigene editing by CRISPR-Cas9. Different vector construction methods have been practiced including Golden Gate assembly. This chapter provides a detailed protocol for the construction of a T-DNA binary vector for expressing Cas9 and three sgRNAs for simultaneous targeting of three QTL genes for improving seed trait in rice.CRISPR-Cas9 and Cas12a (formerly Cpf1), RNA-guided DNA endonucleases found from adaptive immune system in prokaryotes, have been engineered and widely adopted as two of the most powerful genome editing systems in plants. Recently, we developed a single transcript unit (STU) CRISPR 2.0 toolbox for applications in plants, which contains two STU-Cas9 systems and one STU-Cas12a system. Here, we describe a detailed protocol about using the STU CRISPR 2.0 systems to achieve single and multiplex genome editing in rice.Genome editing technologies, mainly CRISPR/CAS9, are revolutionizing plant biology and breeding. Since the demonstration of its effectiveness in eukaryotic cells, a very large number of derived technologies has emerged. Demonstrating and comparing the effectiveness of all these new technologies in entire plants is a long, tedious, and labor-intensive process that generally involves the production of transgenic plants and their analysis. Protoplasts, plant cells free of their walls, offer a simple, high-throughput system to test the efficiency of these editing technologies in a few weeks' time span. We have developed a routine protocol using protoplasts to test editing technologies in rice. Our protocol allows to test more than 30 constructs in protoplasts prepared from leaf tissues of 100, 9-11-day-old seedlings. CRISPR/CAS9 construct effectiveness can be clearly established within less than a week. We provide here a full protocol, from designing sgRNA to mutation analysis.The CRISPR/Cas systems have become the most widely used tool for genome editing in plants and beyond. However, CRISPR/Cas systems may cause unexpected off-target mutations due to sgRNA recognizing highly homologous DNA sequence elsewhere in the genome. Whole-genome sequencing (WGS) can be used to identify on- and off-target mutation. Here, we describe a pipeline of analyzing WGS data using a series of open source software for analysis of off-target mutations in CRISPR-edited rice plants. In this pipeline, the adapter is trimmed using SKEWER. Then, the cleaned reads are mapped to reference genome by applying BWA. To avoid mapping bias, the GATK is used to realign reads near indels (insertions and deletions) and recalibrate base quality controls. Whole-genome single nucleotide variations (SNVs) and indels are detected by LoFreq*, Mutect2, VarScan2, and Pindel. Last, SNVs and indels are compared with in silico off-target sites using Cas-OFFinder.The fast-moving CRISPR technology has allowed plant scientists to manipulate plant genomes in a targeted manner. So far, most of the applications were focused on gene knocking out by creating indels. However, more precise genome editing tools are demanded to assist the introduction of functional single nucleotide polymorphisms (SNPs) in breeding programs. The CRISPR base editing tools were developed to meet this need. In this chapter, we present a cytidine deaminase base editing method for editing the point mutations that control the grain size and seed coat color in rice.CRISPR-Cas resonates a revolutionary genome editing technology applicable through a horizon spreading across microbial organism to higher plant and animal. This technology can be harnessed with ease to understand the basic genetics of a living system by altering sequence of individual genes and characterizing their functions. The precision of this technology is unparallel. It allows very precise and targeted base pair level edits in the genome. Here, in the current chapter, we have provided end-to-end process outline on how to generate genome edited plants in crops like rice to evaluate for agronomic traits associated with yield, disease resistance and abiotic stress tolerance, etc. Genome editing process includes designing of gene editing strategy, vector construction, plant transformation, molecular screening, and phenotyping under control environment conditions. Furthermore, its application for development of commercial crop product may require additional processes, including field trials in the target geography for evaluation of product efficacy. Evaluation of genome edited lines in controlled greenhouse/net house or open field condition requires few generations for outcrossing with wild-type parent to eliminate and/or reduce any potential pleiotropic effect in the edited genome which may arise during the process. The genome edited plant selected for advancement shall harbor the genome with only the intended changes, which can be analyzed by various molecular techniques, advanced sequencing methods, and genomic data analysis tools. https://www.selleckchem.com/products/gsk2830371.html CRISPR-Cas-based genome editing has opened a plethora of opportunities in agriculture as well as human health.CRISPR-Cas9 has revolutionized the field of genome engineering. Base editing, a new genome editing strategy, was recently developed to engineer nucleotide substitutions. DNA base editing systems use a catalytically impared Cas nuclease together with a nucleobase deaminase enzyme to specifically introduce point mutations without generating double-stranded breaks, which provide huge potential in crop improvement. Here, we describe fast and efficient preparation of user-friendly C to T base editors, BE3, and Target-AID. Presented are detailed protocols for T-DNA vector preparation with BE3 or modified Target-AID base editor based on Gateway assembly and efficiency assessment of base editing through a rice protoplast transient expression system.