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Genome editing technologies have provided opportunities to manipulate literally any genomic location, opening new avenues for reverse genetics-based improvements. Among them, CRISPR/Cas9 is the most versatile tool for genome editing applications in prokaryotes and eukaryotes. Here, we provide a guide to successfully carry out high-efficiency genome editing in Chlamydomonas reinhardtii using preassembled CRISPR/Cas9-gRNA ribonucleoprotein (RNP) complexes., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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.

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.

Immunolocalization analysis is a principal tool to study protein expression and subcellular distribution in plant cells or tissues. In this chapter, we present the method of the preparation of lightly fixed fresh rice leaf tissue for immunolocalization analysis and detection of the protein of interest using fluorescent probes by fluorescent microscopy. This method especially does not need the process of embedding plant materials that saves time and prevents alterations of cellular compounds and structure during sample preparation. Using this method, the C 4 rice project compared the expressions of the proteins of interest among C 4 model plants, wild-type rice, and transgenic or mutant plants and successfully selected the transgenic plants with the correct location of each protein to create a C 4 rice prototype.

With continued economic development in Asia the demand for high yielding varieties with premium grain quality traits is set to increase. This presents a significant challenge to plant breeders because varieties must be tailored to meet regional preferences. It is already apparent that traditional breeding techniques cannot meet this challenge and so emerging genomics technologies will have to be utilized. Genome editing tools afford the ability to efficiently and precisely manipulate the genome. Among these, the bacterial clustered, regularly interspaced, short palindromic repeat (CRISPR) associated protein 9 (Cas9) or CRISPR-Cas9 has emerged as the easiest, most economic, and efficient technology to undertake genome editing in rice. This technique allows precise site-specific gene modification or integration. In this chapter we present a method for utilizing CRISPR-Cas9 for improving grain quality traits in rice; this should enable molecular breeders to quickly and efficiently produce high yielding rice varieties tailored to meet specific cultural and regional requirements for grain quality.

CRISPR has arguably been the fastest growing genome editing tool so far. CRISPR/Cas9 (Cas9) has been proved to be efficient and precise in genome editing. However Cas9 has certain limitations. CRISPR/Cpf1 (Cpf1) has been discovered as an alternate approach that can overcome some of those limitations. Cpf1 allows targeting in AT-rich region, creating a staggered cleavage, and cutting at the distal end to the PAM (Protospacer Adjacent Motif) regions. We have successfully tested the efficiency of Cpf1 system in rice using OsEPFL9 which is a developmental gene known to regulate the stomatal density in leaf. Regulation of stomatal density and patterning is an important factor in regulating plant physiology, especially in improving the plant water use efficiency. We targeted the Exon1 of OsEPFL9 and the knockout lines were studied for several generations for establishment of stabilized editing, as well as transmission and segregation of edits through generations. The usage of Cpf1 as a genome editing tool to manipulate stomatal patterning may further help us gain more insight of the physiology of rice in stress conditions.