Biology subjects

Kasprzyk, M. E.

Publications and source records attributed to Kasprzyk, M. E..

3 recordsLinked to original sources

bioRxiv2022

TransCRISPR - sgRNA design tool for CRISPR/Cas9 experiments targeting transcription factor motifs

Eukaryotic genomes contain several types of recurrent DNA motifs, e.g. transcription factor motifs, miRNA binding sites, repetitive elements. CRISPR/Cas9 can facilitate identification and study of crucial DNA motifs. We present transCRISPR, the first online tool dedicated to search for DNA sequence motifs in the user-provided genomic regions and design optimal sgRNAs targeting them. Users can obtain sgRNAs for chosen DNA motifs, for up to tens of thousands of target regions in 30 genomes, either for the Cas9 or dCas9 system. TransCRISPR provides user-friendly tables and visualizations, summarizing features of identified motifs and designed sgRNAs such as genomic localization, quality scores, closest transcription start sites, and others. Experimental validation of sgRNAs for MYC binding sites designed with transCRISPR confirmed efficient disruption of the targeted motifs and effect on expression of MYC-regulated genes. TransCRISPR is available from https://transcrispr.igcz.poznan.pl/transcrispr/

molecular biology↗

bioRxiv2021

Adar-mediated A-to-I editing is required for establishment of embryonic body axes in zebrafish

Adenosine deaminases (ADARs) catalyze the deamination of adenosine to inosine, also known as A-to-I editing, in RNA. Although A-to-I editing occurs widely across animals, and is well studied, new biological roles are still being discovered. Here, we study the role of A-to-I editing in early zebrafish development. We demonstrate that Adar, the zebrafish orthologue of mammalian ADAR1, is essential for establishing the antero-posterior and dorso-ventral axes and patterning. Genome-wide editing discovery revealed pervasive editing in maternal and the earliest zygotic transcripts, the majority of which occurred in the 3-UTR. Interestingly, transcripts implicated in gastrulation as well as dorso-ventral and antero-posterior patterning were found to contain multiple editing sites. Adar knockdown or overexpression affected gene expression and global editing patterns at 12 hpf, but not earlier. Our study established that RNA editing by Adar is necessary for the earliest steps of embryonic patterning along the zebrafish antero-posterior and dorso-ventral axes.

developmental biology↗

bioRxiv2021

CRISPR/Cas9 screen for functional MYC binding sites reveals MYC-dependent vulnerabilities in K562 cells

The transcription factor MYC is a proto-oncogene with a well-documented essential role in the pathogenesis and maintenance of several types of cancer. MYC binds to specific E-box sequences in the genome to regulate gene expression in a cell type- and developmental stage-specific manner. To date, a comprehensive analysis of direct MYC targets with essential roles in different types of cancer is missing. To enable identification of functional MYC binding sites and corresponding target genes, we designed a CRISPR/Cas9 library to destroy E-box sequences in a genome-wide fashion. In parallel, we used the Brunello library to knockout protein-coding genes. We performed high-throughput screens with these libraries in four MYC-dependent cancer cell lines: K562, ST486, HepG2 and MCF7, which revealed several essential E-boxes and genes. Among them we pinpointed crucial known and novel MYC-regulated genes involved in pathways associated with cancer development. Extensive validation of our approach in K562 cells confirmed that E-box disruption affects MYC binding, target genes expression and cell proliferation. Our unique, well-validated tool opens new possibilities to gain novel insights into MYC-dependent vulnerabilities in cancer cells.

molecular biology↗