Aug 2026· Journal of Visualized Experiments· Vol 234· 0 citations
Medicine
TL;DR
Cas9 ablation enabled restoration of CUL4B expression and function following introduction of a CUL4B expression plasmid, providing proof of concept for a broadly applicable approach to studying protein function through rescue experiments.
Abstract
CRISPR-Cas9 gene editing technology has revolutionized molecular biology. Often, this technology is employed to delete a gene encoding a protein of interest. The resulting phenotype provides valuable insight into the protein's function. The functional importance of the target protein can be confirmed by reintroducing the protein to restore the lost function (rescue). This is typically accomplished by introducing the protein-coding cDNA in trans using an expression vector. However, in knockout cell lines that stably express the CRISPR-Cas9 system, the newly introduced expression plasmid may also be cleaved by Cas9. The protocol presented here provides a strategy to circumvent this potential barrier to rescue experiments. This approach is demonstrated using HEK293 cells in which the gene encoding the E3 ubiquitin ligase scaffold protein CUL4B was disrupted by CRISPR-Cas9. Transduction of these cells with a guide RNA (gRNA) targeting the integrated Cas9 transgene resulted in the loss of detectable Cas9 protein. Cas9 ablation enabled restoration of CUL4B expression and function following introduction of a CUL4B expression plasmid. These results provide proof of concept for a broadly applicable approach to studying protein function through rescue experiments.
The application of genome editing, CRISPR/Cas9 has revolutionized plant breeding by enabling precise, efficient, and targeted modification of native genes, significantly accelerating the development of improved agronomic traits of crops. Therefore, CRISPR/Cas9 technology currently the most extensively used genome editing technique worldwide because of its simple design, cost-effectiveness, high efficiency, good reproducibility, high engineering feasibility, ability to create gene knockout, RNA editing, and quick cycle. It is used to knock in or knock out genes of interest and for generating models for genetic studies. The main components of the CRISPR/Cas9 system are an RNA-guided Cas9 endonuclease and a single-guide RNA (sgRNA). The workflow of CRISPR/Cas9 gene editing comprises selecting target sites, designing and synthesizing sgRNA, introducing transformation constructs or ribonucleoprotein (RNP) in plant cells, followed by transformation and identification of edited lines. This approach bypasses the formal regulations on GMOs, thus encouraging the widespread adoption RNA-guided gene editing in agricultural sciences and biotechnology. The system is now being utilized in the biofortification of cereal crops such as rice, wheat, barley, and maize, including vegetable crops such as potato and tomato. The world's first genome-edited rice varieties are DRR Dhan 100 (Kamala) and Pusa DST Rice 1 developed by the Indian Council of Agricultural Research (ICAR), New Delhi, India in 2025 with the objective of bringing about revolutionary changes in terms of higher production, climate adaptability, and water conservation. The CRISPR/Cas9-based crop genome editing has been utilized in imparting/producing qualitative enhancement in aroma, shelf life, sweetness, and quantitative improvement in starch, protein, gamma-aminobutyric acid (GABA), oleic acid, anthocyanin, phytic acid, gluten, and steroidal glycoalkaloid contents. Some varieties have even been modified to become disease and stress-resistant. Therefore, CRISPR/Cas9 is aiding in developing climate-ready crops and improving crop quality parameters such as appearance, palatability, nutritional components, and other preferred traits. Gene editing tools are used to generate changes to the native genetic material. Unlike GMOs, which introduce novel configurations of genetic materials typically derived from other organisms, gene editing methods modify existing genetic material in ways that can yield beneficial outcomes.
Ravindra B. Malabadi, Raju K. Chalannavar· World Journal of Advanced Re...· 0 citations
REPLACE is established as a versatile platform for homology-independent sequence replacement and as a complementary approach for genetic correction in settings where homology-directed repair (HDR), base editing (BE) or prime editing (PE) are inefficient or not applicable.
Tu Dang, Alexandra Roman, Anja Zimmer et al.· bioRxiv· 0 citations
It is reported that the choice of sgRNA target site affects its targeted cleavage activity and show that the combination of guideRNA with SpCas9 can effectively delete the exonic fragment of the SLC25A38 gene in HEK293T cells.
S. Yazdanparast, Hamid Galehdari, S. Khatami et al.· Journal of Applied Genetics· 0 citations
The generation of a WSL-Cas9 monoclonal cell line with high CRISPR/Cas9 editing efficiency, stable proliferation, and permissiveness for robust ASFV replication is reported, providing a reliable platform for future genome-wide functional screening to systematically identify host factors governing ASFV entry.
Meng Yao, Tianlong Li, Bin Sun et al.· Sheng wu gong cheng xue bao...· 0 citations
Precise knock-in of fluorescent reporters is a powerful tool for studying the dynamic cellular and molecular processes of embryogenesis. However, conventional CRISPR-Cas9 knock-in of large inserts, such as full-length fluorescent proteins, is inefficient. This has limited its application in many emerging model systems, including sea urchins. Here, we overcome this barrier using a transgenic Lytechinus pictus line that constitutively and ubiquitously expresses a large fragment of mNeonGreen (mNG3K1-10). In this line, fluorescence is only reconstituted when CRISPR-mediated knock-in delivers mNG211, the 11th beta strand of the fluorescent protein, to complement the constitutively expressed fragment. Because this strategy requires integrating only the short 11th-strand, together with short homology arms (∼130 nt total), by homology directed repair, it circumvents the size constraints that limit conventional full-length reporter knock-ins using CRISPR. Using this approach, we achieved integration efficiencies of 14-22%, roughly an order of magnitude higher than those obtained with full-length fluorescent protein knock-ins. This provides a streamlined, scalable method for endogenous protein visualization in echinoderm embryos and a valuable resource for studying gene function, morphogenesis, and toxicant response in this classic developmental model.
This chapter outlines a comprehensive methodology for the design, assembly, and functional assessment of CRISPR/dCas9 systems optimized for tomato to investigate pathogen-associated responses.
Ananya Mukherjee, Shrabani Basak, Raghuvir Singh et al.· Methods in molecular biology· 0 citations
