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Neonatal hypotonia (NH) is a common, non-specific symptom manifested in numerous and disparate disorders of genetic origin. These disorders can be caused by diverse genetic changes including chromosomal aberrations, copy number variations (CNVs), methylation changes, trinucleotide repeat expansions, and single nucleotide variants (SNVs). Currently, no single laboratory testing modality is available to test these disparate etiologies. We have designed a targeted sequencing approach to identify and prioritize genetic changes in loci related to NH phenotypes in a single test, thus reducing the number and types of tests required to make a diagnosis. This amplification-free approach uses CRISPR/Cas9 DNA cutting technology to produce targeted sequencing libraries of NH related genomic loci for sequencing using the PacBio Sequel IIe system, which sequences native DNA in long reads, allowing for the detection of methylation changes, trinucleotide repeat expansions and CNVs that can be missed by typical short read sequencing. We designed a set of guide RNAs (gRNAs) targeting a panel of 116 NH loci of interest. To maximize Cas9 cutting at each locus, gRNAs were chosen such that 3 gRNAs cut upstream and 3 downstream of each target. Size-based sequencing bias was avoided by constraining all target loci to 11-14 kilobases in length; thus, most genic loci required division into multiple fragments for sequencing, for a total of 1050 fragments in the panel. We in vitro transcribed the resulting library of 6300 guide RNAs (1050 fragments x 6 gRNAs) from DNA oligonucleotides. Remarkably, when complexed with Cas9, this highly multiplexed gRNA library successfully cut NH loci from patient genomic DNA isolated from blood as evidenced by the inability to produce polymerase chain reaction amplicons across the targeted Cas9 cutting sites. Additionally, no off-target cutting was identified at several nearby regions tested. This specific cutting allows for production of targeted sequencing libraries of native DNA containing only the NH loci of interest. We anticipate that optimization of this method will reduce costs and turnaround time for hypotonia diagnoses in the future. Additionally, this approach can be applied for testing in other complex disorders, or to produce any targeted sequencing panel containing native genomic regions of interest in a cost-effective and multiplexed manner.

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A novel and comprehensive testing strategy to identify the genetic etiology of neonatal hypotonia phenotypes