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CRISPR/Cas9-Mediated Gene Knockout in Cereal Crops.
High-precision genome editing tools, such as programmable nucleases, are poised to transform crop breeding and significantly impact fundamental plant research. Among these tools, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR-associated 9) system is a programmable, RNA-guided nuclease that introduces targeted, site-specific double-stranded breaks in the target DNA loci. When these breaks are repaired, it often results in a frame-shift mutation via short insertion/deletion (indel), leading to gene knockout. Since its first successful use in plants, CRISPR/Cas9 has been widely adopted for targeting genes of agronomic and scientific importance in multiple crops, including rice, maize, wheat, and sorghum. These cereal crops ensure global food security, provide essential nutrition, and support economic stability. Additionally, such crops support biofuel production, livestock feed, and sustainable farming practices through crop rotation. This article outlines the strategies for implementing CRISPR/Cas9 genome editing in plants, including a step-by-step process of guide RNA target selection, oligonucleotide design, construct development, assembly, and analysis of genome edits. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: CRISPR/Cas9 guide RNA target selection Support Protocol 1: Genomic DNA extraction in-house protocol Basic Protocol 2: Construction of a binary plasmid vector Support Protocol 2: Agrobacterium transformation with a binary vector construct and stability check Support Protocol 3: Plant transformation Basic Protocol 3: Genotyping of edited events.
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Knock-In Strategy for Editing Human and Zebrafish Mitochondrial DNA Using Mito-CRISPR/Cas9 System.
The mitochondria DNA (mtDNA) editing tool, zinc finger nucleases (ZFNs), transcription activator-like effector nuclease (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system, is a promising approach for the treatment of mtDNA diseases by eliminating mutant mitochondrial genomes. However, there have been no reports of repairing the mutant mtDNA with homologous recombination strategy to date. Here, we show a mito-CRISPR/Cas9 system that mito-Cas9 protein can specifically target mtDNA and reduce mtDNA copy number in both human cells and zebrafish. An exogenous single-stranded DNA with short homologous arm was knocked into the targeting loci accurately, and this mutagenesis could be steadily transmitted to F1 generation of zebrafish. Moreover, we found some major factors involved in nuclear DNA repair were upregulated significantly by the mito-CRISPR/Cas9 system. Taken together, our data suggested that the mito-CRISPR/Cas9 system could be a useful method to edit mtDNA by knock-in strategy, providing a potential therapy for the treatment of inherited mitochondrial diseases.
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Simple and Rapid Assembly of TALE Modules Based on the Degeneracy of the Codons and Trimer Repeats.
Transcription activator-like effectors (TALEs) have been effectively used for targeted genome editing, transcriptional regulation, epigenetic modification, and locus-specific DNA imaging. However, with the advent of the clustered regularly interspaced short palindromic repeat/Cas9 system, an easy-to-use tool with the same function as TALEs, TALEs have recently been abandoned because of their complexity, time consumption, and difficult handling in common labs. Here, we described a degenerated codon-based TALE assembly system for simple, rapid, and efficient TALE assembly. TALE trimers with nonrepetitive DNA sequences were amplified by PCR and sequentially assembled via Gibson assembly. Our method is cost-effective, requires only commonly used basic molecular biology reagents, and takes only 2 h from target sequence analysis to completion. This simple, rapid, and lab-friendly TALE assembly method will restore the value of TALEs in DNA targeting.
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DNA and RNA editing for the therapy of human diseases: current status, challenges, and future prospects.
The rapid development of DNA- and RNA-editing tools (collectively referred to as gene editing technologies) has caused a paradigm shift in the treatment of human diseases from symptomatic treatment to precision-based medicine. Both DNA-based and RNA-based editing systems, including Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-derived technologies and newly developed RNA editing tools, have pushed technological frontiers in terms of editing precision, hierarchical control, and reversibility; they have accumulated a growing body of preclinical and clinical evidence across diverse diseases ranging from inherited disorders to cancer, infectious diseases, and neurodegenerative diseases (ND). This review systematically summarizes the core principles and representative advances of DNA-based genome editing and RNA-based transcriptome editing technologies, comprehensively compares the two categories of technical strategies in terms of therapeutic potential, durability of effects, and risk profiles, and further explores the key challenges for achieving long-term safe and efficient in vivo applications, covering core bottlenecks such as delivery efficiency, tissue specificity, genotoxicity, and immunogenicity. Safety assessment has broadened to include tracking genotoxicity and genomic structural variations, whereas delivery systems and tissue specificity are determinant factors for in vivo therapeutic applications. Through the employment of both permanent and reversible editing strategies with high cargo-writing capacity and low integration risk, combined with programmable delivery systems, the therapeutic potential of hard-to-transfect tissues and complex diseases is anticipated to be broadened, opening new paths for clinical translation.
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Genome Editing in Cotton Using CRISPR/Cas9 System.
The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system is emerging as effective strategy for generating site-specific mutations. Recently, CRISPR/Cas9-mediated genome editing system have been rapidly optimized and applied in crop genetic improvement. In this chapter, we present a simple and high-efficiency method for crating targeted gene mutation in allotetraploid cotton genome using CRISPR/Cas9 system. This chapter will employ GhMYB25-like A and GhMYB25-like D that derived from upland cotton (Gossypium hirsutum) A subgenome and the D subgenome, respectively, as an example to introduce the procedure of how to generate effective mutations in cotton genome using CRISPR/Cas9-based biotechnology. Based on our previous results, this CRISPR/Cas9 system can induce a proportion of 14.2-21.4% fragment truncation events in GhMYB25-like A and GhMYB25-like D genome sites. In addition, PCR product sequencing results suggest that the mutation frequencies that occurred in GhMYB25-like A and GhMYB25-like D DNA sites are 100% and 98.8%, respectively. More important, the off-target-caused mutation events have not been detected in our transgenic plants, even one of the putative off-target site only have one nucleotide mismatch with the designed GhMYB25 sgRNA. Thus, this CRISPR/Cas9 method might be an effective approach for targeted mutagenesis in cotton genome.
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RNA-Targeting CRISPR-Cas Systems and Their Applications.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) systems have revolutionized modern molecular biology. Numerous types of these systems have been discovered to date. Many CRISPR-Cas systems have been used as a backbone for the development of potent research tools, with Cas9 being the most widespread. While most of the utilized systems are DNA-targeting, recently more and more attention is being gained by those that target RNA. Their ability to specifically recognize a given RNA sequence in an easily programmable way makes them ideal candidates for developing new research tools. In this review we summarize current knowledge on CRISPR-Cas systems which have been shown to target RNA molecules, that is type III (Csm/Cmr), type VI (Cas13), and type II (Cas9). We also present a list of available technologies based on these systems.
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Recent Advances in Preclinical Developments Using Adenovirus Hybrid Vectors.
Adenovirus (Ad)-based vectors are efficient gene-transfer vehicles to deliver foreign DNA into living organisms, offering large cargo capacity and low immunogenicity and genotoxicity. As Ad shows low integration rates of their genomes into host chromosomes, vector-derived gene expression decreases due to continuous cell cycling in regenerating tissues and dividing cell populations. To overcome this hurdle, adenoviral delivery can be combined with mechanisms leading to maintenance of therapeutic DNA and long-term effects of the desired treatment. Several hybrid Ad vectors (AdV) exploiting various strategies for long-term treatment have been developed and characterized. This review summarizes recent developments of preclinical approaches using hybrid AdVs utilizing either the Sleeping Beauty transposase system for somatic integration into host chromosomes or designer nucleases, including transcription activator-like effector nucleases and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease for permanent gene editing. Further options on how to optimize these vectors further are discussed, which may lead to future clinical applications of these versatile gene-therapy tools.
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Therapeutic Genome Editing and In Vivo Delivery.
Improvements in the understanding of human genetics and its roles in disease development and prevention have led to an increased interest in therapeutic genome editing via the use of engineered nucleases. Various approaches have been explored in the past focusing on the development of an effective and safe system for sequence-specific editing. Compared to earlier nucleases such as zinc finger nuclease and transcription activator-like effector nuclease, the relatively low cost and ease of producing clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR/Cas9) systems have made therapeutic genome editing significantly more feasible. CRISPR/Cas9 genome editing has shown great potential to correct genetic mutations implicated in monogenic diseases and to eradicate latent or chronic viral infections in preclinical studies. Several CRISPR/Cas9-based therapeutics have reached the clinical stage, including treatments for inherited red blood cell disorders and Leber Congenital Amaurosis 10, as well as CRISPR/Cas9-edited T cells designed to target and destroy cancer cells. Further advances in therapeutic genome editing will rely on a safe and more efficient method of in vivo CRISPR/Cas9 delivery and improved efficiency of homology-directed repair for site-specific gene insertion or replacement. While other reviews have focused on one or two aspects of CRISPR/Cas9 genome editing, this review aims to provide a summary of the mechanisms of genome editing, the reasons for the emerging interest in CRISPR/Cas9 compared to other engineered nucleases, the current progress in developing CRISPR/Cas9 delivery systems, and the current preclinical and clinical applications of CRISPR/Cas9 genome editing.
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Synthesis-dependent repair of Cpf1-induced double strand DNA breaks enables targeted gene replacement in rice.
The recently developed CRISPR (clustered regularly interspaced short palindromic repeats)/Cpf1 system expands the range of genome editing and is emerging as an alternative powerful tool for both plant functional genomics and crop improvement. Cpf1-CRISPR RNA (crRNA) produces double strand DNA breaks (DSBs) with long 5'-protruding ends, which may facilitate the pairing and insertion of repair templates through homology-directed repair (HDR) for targeted gene replacement and introduction of the desired DNA elements at specific gene loci for crop improvement. However, the potential mechanism underlying HDR of DSBs generated by Cpf1-crRNA remains to be investigated, and the inherent low efficiency of HDR and poor availability of exogenous donor DNA as repair templates strongly impede the use of HDR for precise genome editing in crop plants. Here, we provide evidence of synthesis-dependent repair of Cpf1-induced DSBs, which enables us precisely to replace the wild-type ALS gene with the intended mutant version that carries two discrete point mutations conferring herbicide resistance to rice plants. Our observation that the donor repair template (DRT) with only the left homologous arm is sufficient for precise targeted allele replacement offers a better understanding of the mechanism underlying HDR in plants, and greatly simplifies the design of DRTs for precision genome editing in crop improvement.
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Illegitimate translation causes unexpected gene expression from on-target out-of-frame alleles created by CRISPR-Cas9.
CRISPR-Cas9 is efficient enough to knock out both alleles directly by introducing out-of-frame mutations. We succeeded in making biallelic on-target frameshift mutations of the endogenous Gli3 gene; however, the GLI3 protein was expressed in all six of the established cell lines carrying homozygous out-of-frame mutations. We developed a dual-tagged expression vector and proved that illegitimate translation (ITL) was the cause of the unexpected Gli3 expression. Thus, gene expression must be examined even if designed on-target out-of-frame sequences are introduced by genome editing. In addition, it is highly recommended to pre-examine the occurrence of ITL in vitro prior to the design and construction of any genome-editing vectors. In vitro assay systems such as the dual-tagged ITL assay system developed in this study should aid the identification and elucidation of ITL-based human diseases and gene expression.
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