Abstract
The development of new techniques to create gene knockouts and knock-ins is essential for successful investigation of gene functions and elucidation of the causes of diseases and their associated fundamental cellular processes. In the biomedical model organism Dictyostelium discoideum, the methodology for gene targeting with homologous recombination to generate mutants is well-established. Recently, we have applied CRISPR/Cas9-mediated approaches in Dictyostelium, allowing the rapid generation of mutants by transiently expressing sgRNA and Cas9 using an all-in-one vector. CRISPR/Cas9 techniques not only provide an alternative to homologous recombination-based gene knockouts but also enable the creation of mutants that were technically unfeasible previously. Herein, we provide a detailed protocol for the CRISPR/Cas9-based method in Dictyostelium. We also describe new tools, including double knockouts using a single CRISPR vector, drug-inducible knockouts, and gene knockdown using CRISPR interference (CRISPRi). We demonstrate the use of these tools for some candidate genes. Our data indicate that more suitable mutants can be rapidly generated using CRISPR/Cas9-based techniques to study gene function in Dictyostelium.
Highlights
Many human diseases, such as cancer, heart disease, and diabetes, are characterized by multifactorial genetic inheritance (Khera et al, 2018; Ishigaki et al, 2020)
We investigated RNA levels in the CRISPR interference (CRISPRi) cells expressing T1, 2, 3, and 4 sgRNA and found that RNA levels were less than 10% of what was observed for controls
This method is still widely used in Dictyostelium, but CRISPR/Cas9mediated gene manipulation is just emerging
Summary
Many human diseases, such as cancer, heart disease, and diabetes, are characterized by multifactorial genetic inheritance (Khera et al, 2018; Ishigaki et al, 2020). Different versions of Cas[9], such as xCas[9], SpCas9-NG and SpRY, recognize a broader variety of PAM recognition sequences with less stringent motif requirements (Hu et al, 2018; Nishimasu et al, 2018; Walton et al, 2020) Using these various types of Cas[9], a wide range of genome editing applications, including knockouts, inducible knockouts, knockdowns, knock-ins, point mutations and deletions, have been established in Dictyostelium (Table 1; Muramoto et al, 2019; Asano et al, 2021). We developed a straightforward CRISPRi system to reduce mRNA and protein levels in the cells In this methodological paper, we summarize these applications and focus on detailing the selection of appropriate CRISPR/Cas[9] vectors and procedures for each technology to manipulate the Dictyostelium genome for the study of human disease-related genes. Cas9 * Cas[9] Cas9 * Cas[9] Cas[9] Cas9 * Cas[9] Cas[9] Cas[9] Cas[9] Cas[9] nickase * Cas[9] nickase * dCas[9] dCas[9] dCas[9] dCas[9] dCas[9] dCas[9] Cas9-NG Cas9-NG Cas9-NG Cas9-NG nickase SpRY SpRY SpRY SpRY
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
