Mapping genetic interactions (GIs) is crucial for understanding genetic network complexity. In this study, we investigate the utility of Cas13d, a CRISPR system targeting RNA, for GI mapping and compare it to Cas9 and Cas12a, two DNA nucleases commonly used for GI mapping. We find that Cas13d induces faster target gene perturbation and generates more uniform cell populations with double perturbations than Cas9 or Cas12a. We then encounter Cas13d gRNA-gRNA interference when concatenating gRNAs targeting different genes into one gRNA array, which we overcome by a dual promoter gRNA expression strategy. Moreover, by concatenating three gRNAs targeting the same gene into one array, we are able to maximize the Cas13d-mediated knockdown effects. Combining these strategies enhances proliferation phenotypes while reducing library size and facilitates reproducible quantification of GIs in oncogenic signaling pathways. Our study highlights the potential of Cas13d for GI mapping, promising advancements in understanding therapeutically relevant drug response pathways.
Download full-text PDF |
Source |
|---|---|
| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC11828948 | PMC |
| http://dx.doi.org/10.1038/s41467-025-56747-4 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated proteins (Cas) systems have revolutionized genome editing by providing high precision and versatility. However, most genome editing applications rely on a limited number of well-characterized Cas9 and Cas12 variants, constraining the potential for broader genome engineering applications. In this study, we extensively explored Cas9 and Cas12 proteins and developed CasGen, a novel transformer-based deep generative model with margin-based latent space regularization to enhance the quality of newly generative Cas9 and Cas12 proteins.
View Article and Find Full Text PDFPeptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) in primary human immune cells and hematopoietic stem cells.
Nat Protoc
March 2025
Innovative Genomics Institute, University of California Berkeley, Berkeley, CA, USA.
Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) is a new approach for ex vivo genome editing of primary human cells. PERC uses a single amphiphilic peptide reagent to mediate intracellular delivery of the same pre-formed CRISPR ribonucleoprotein enzymes that are broadly used in research and therapeutics, resulting in high-efficiency editing of stimulated immune cells and cultured hematopoietic stem and progenitor cells (HSPCs). PERC facilitates nuclease-mediated gene knockout, precise transgene knock-in and base editing.
View Article and Find Full Text PDFThe mosaicism of Cas-induced mutations and pleiotropic effects of scarlet gene in an emerging model system.
Heredity (Edinb)
February 2025
Division of Biological Sciences, University of Missouri, Columbia, MO, 65211, USA.
The effective use of CRISPR technologies in emerging model organisms faces significant challenges in efficiently generating heritable mutations and in understanding the genomic consequences of induced DNA damages and the inheritance patterns of induced mutations. This study addresses these issues by 1) developing an efficient microinjection delivery method for gene editing in the microcrustacean Daphnia pulex; 2) assessing the editing dynamics of Cas9 and Cas12a nucleases in the scarlet knock-out mutants; and 3) investigating the transcriptomes of scarlet mutants to understand the pleiotropic effects of scarlet gene. Our reengineered microinjection method results in efficient biallelic editing with both nucleases.
View Article and Find Full Text PDFEnhancing genome editing efficiency in goldfish (Carassius auratus) through utilization of CRISPR-Cas12a (Cpf1) temperature dependency.
Int J Biol Macromol
February 2025
Beijing Key Laboratory of Fishery Biotechnology, Fisheries Science Institute, Beijing Academy of Agriculture and Forestry Sciences, Beijing, China. Electronic address:
The CRISPR/Cas technology has demonstrated revolutionary potential across various fields, including agriculture, medicine, and food safety detection. However, the utility of CRISPR/Cas12a, a particularly promising gene-editing tool, is constrained by its temperature sensitivity, limiting its application in low-temperature environments. In this study, we developed a gene-editing technique based on the CRISPR/Cas12a system in the poikilothermic species goldfish Carassius auratus.
View Article and Find Full Text PDFEvaluation of Cas13d as a tool for genetic interaction mapping.
Nat Commun
February 2025
Universitätsmedizin Halle, Martin Luther University Halle-Wittenberg, Halle (Saale), 06120, Halle, Germany.
Mapping genetic interactions (GIs) is crucial for understanding genetic network complexity. In this study, we investigate the utility of Cas13d, a CRISPR system targeting RNA, for GI mapping and compare it to Cas9 and Cas12a, two DNA nucleases commonly used for GI mapping. We find that Cas13d induces faster target gene perturbation and generates more uniform cell populations with double perturbations than Cas9 or Cas12a.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!