| Popis: |
CD-46 plays a crucial role in the human immune system. We wish to illuminate the role of zebrafish rca2.1, a CD-46 ortholog, in development and health. The sequence and expression of rca2.1 suggests it is a better orthologue to human CD-46 than murine candidate genes. Located on chromosome 23, rca2.1 spans 25 kb and the latest genome assembly predicts three alternative splice forms. Our initial goal is to generate rca2.1 null zebrafish using CRISPR/Cas9. Based on concerns that novel translational start sites and/or decay of otherwise non-functional RNAs can elicit genetic compensation, we sought to disrupt all rca2.1 transcription. To identify the transcriptional start site (TSS) and to determine which predicted alternate transcripts are expressed during early development, we established a private UCSC track featuring valuable CAGE and RNAseq data from public sources. We used this track to select gRNA combinations aimed at cooperatively deleting the TSS target and downstream spans of rca2.1. Each candidate gRNA was pre-tested for cutting efficiency. Fertilized embryos were then co-injected with either (#1) three gRNAs targeting the TSS, start codon and termination codon, or (#2) two gRNAs targeting the TSS and termination codon. With the intention of stimulating precise joining of the deletion gap, a 100 nt oligonucleotide was included in the second co-injection, with hybrid sequence from upstream of the 5’ cut site and downstream of the 3’ cut site. Fragment analysis indicated the presence of diagnostic peaks arising from the desired 20 Kb deletion in close to 20% of F0 embryos injected by either approach. Germ-line transmission from 7 out of 8 F0 adults representing both approaches has subsequently been achieved. Thus, using multiple gRNAs with or without a facilitating oligo, we have efficiently recovered large genomic rca2.1 deletion alleles. Unlike classic CRISPR/Cas9 in-del alleles, interpretation of phenotypes observed should be uncomplicated by concerns of extant or novel alternative transcripts or genetic compensation linked to nonsense-mediated decay. |
