A crucial and often decisive test of a nuclear gene being involved in a given process is the complementation of mutants. Restoring the wild type phenotype by the wild type gene introduced into the mutant is a major piece of evidence for the function of this gene. We have developed a rapid and reliable method to complement protoplasts from plants with mutations in mitochondrial RNA editing with the respective wild type genes. The method furthermore allows testing the functionality of modified protein sequences without the need to make and grow transgenic plants, which is very time-consuming. We successfully employed this method for several nuclear-encoded genes involved in RNA editing at specific sites in mitochondria of Arabidopsis thaliana.
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http://dx.doi.org/10.1007/978-1-61779-018-8_10 | DOI Listing |
Kardiol Pol
January 2025
1st Department of Cardiology, Poznan University of Medical Sciences, Poznań, Poland.
PLoS One
December 2024
Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada.
The ability to determine the essentiality of a gene in the protozoan parasite Leishmania is important to identify potential targets for intervention and understanding the parasite biology. CRISPR gene editing technology has significantly improved gene targeting efficiency in Leishmania. There are two commonly used CRISPR gene targeting methods in Leishmania; the stable expression of the gRNA and Cas9 using a plasmid containing a Leishmania ribosomal RNA gene promoter (rRNA-P stable protocol) and the T7 RNA polymerase based transient gRNA expression system in promastigotes stably expressing Cas9 (T7 transient protocol).
View Article and Find Full Text PDFNat Genet
January 2025
Institute of Molecular Oncology, Philipps-University, Marburg, Germany.
The mutational landscape of TP53, a tumor suppressor mutated in about half of all cancers, includes over 2,000 known missense mutations. To fully leverage TP53 mutation status for personalized medicine, a thorough understanding of the functional diversity of these mutations is essential. We conducted a deep mutational scan using saturation genome editing with CRISPR-mediated homology-directed repair to engineer 9,225 TP53 variants in cancer cells.
View Article and Find Full Text PDFNat Commun
January 2025
Interdisciplinary Life Sciences Graduate Programs, University of Texas at Austin, Austin, TX, 78712, USA.
Type II CRISPR endonucleases are widely used programmable genome editing tools. Recently, CRISPR-Cas systems with highly compact nucleases have been discovered, including Cas9d (a type II-D nuclease). Here, we report the cryo-EM structures of a Cas9d nuclease (747 amino acids in length) in multiple functional states, revealing a stepwise process of DNA targeting involving a conformational switch in a REC2 domain insertion.
View Article and Find Full Text PDFSci Data
January 2025
Gakushuin University, Faculty of Science, Department of Life Science, Mejiro 1-5-1, Toshima-ku, Tokyo, 171-8588, Japan.
The wild silk moth, Bombyx mandarina, is the closest relative of the domesticated silk moth, Bombyx mori. National BioResource Project of Japan (NBRP) maintains a B. mandarina strain derived from individuals captured at Sakado (Saitama, Japan) in 1982.
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