Abstract
The genome is the blueprint for an organism. Interrogating the genome, especially locating critical cis-regulatory elements, requires deletion analysis. This is conventionally performed using synthetic constructs, making it cumbersome and non-physiological. Thus, we created Cas9-mediated Arrayed Mutagenesis of Individual Offspring (CAMIO) to achieve comprehensive analysis of a targeted region of native DNA. CAMIO utilizes CRISPR that is spatially restricted to generate independent deletions in the intact Drosophila genome. Controlled by recombination, a single guide RNA is stochastically chosen from a set targeting a specific DNA region. Combining two sets increases variability, leading to either indels at 1–2 target sites or inter-target deletions. Cas9 restriction to male germ cells elicits autonomous double-strand-break repair, consequently creating offspring with diverse mutations. Thus, from a single population cross, we can obtain a deletion matrix covering a large expanse of DNA at both coarse and fine resolution. We demonstrate the ease and power of CAMIO by mapping 5′UTR sequences crucial for chinmo's post-transcriptional regulation.
Highlights
Understanding how complex biology unfolds, advances, and evolves from genomic sequences requires development of precise and efficient tools to interrogate the genome
To efficiently and comprehensively perform deletion analysis of a genomic region, we envision a mutagenesis pipeline where multiple mutations are produced from a single founder animal
This could be best achieved by inducing mutagenesis in individual germ cell rather than the commonly used germline stem cells (GSC)
Summary
Understanding how complex biology unfolds, advances, and evolves from genomic sequences requires development of precise and efficient tools to interrogate the genome. There are bioinformatics resources for predicting cis-regulatory elements (CREs) [1,2]. Locations of CREs can be inferred from chromatin state [3–6]. Such methods are restricted to canonical genomic signatures. These putative CREs need to be assessed molecularly to determine whether they are functional. Thanks to the advancement in DNA synthesis and sequencing capacity, high-throughput platforms have been designed to survey CREs on a genome-wide scale [12–15]. These approaches are intrinsically artificial and indirect, and most unsatisfactorily preclude studying regulatory elements in their native environment
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