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PgmNr 205: CRISPR-capture: A novel, low-cost, and scalable method for targeted sequencing.

T.L. Mighell 1,2; C.A. Thornton 2; B.L. O'Connell 2; R.M. Mulqueen 2; C.V. Miller 3; A.C. Adey 2; D. Doherty 3; B.J. O'Roak 2

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1) Neuroscience Graduate Program, Oregon Health & Science Univ, Portland, Oregon.; 2) Department of Molecular & Medical Genetics, Oregon Health & Science Univ, Portland, Oregon.; 3) Department of Pediatrics, University of Washington, Seattle, Washington.

Despite reductions in genome sequencing costs, targeted sequencing methods still have high utility for research and clinical applications; e.g., screening for off-target genome editing or identifying pathogenic mutations in Mendelian disorders. Current target enrichment strategies, whether PCR or hybridization based, still suffer from issues with scalability, bias, and cost (especially for custom targets). Moreover, sequencing a complete gene body remains challenging as non-exonic regions are not typically part of commercial gene panels. To address these challenges, we have developed a novel, low-cost, and scalable method that enables efficient and uniform capture of any set of genomic loci.

Our approach, CRISPR-Capture, is built around the CRISPR-Cas12a system, in which a guide RNA (gRNA) directs the Cas12a endonuclease to a target. Critically, Cas12a double stranded cleavage occurs in a staggered fashion, leaving 4-5 nucleotide overhangs. We reasoned that in vitro incubation of genomic DNA with a pool of gRNAs and Cas12a would result in enrichment of “sticky” ligatable ends at programmed sites. Ligation of a biotinylated sequencing adapter is followed by traditional library preparation methods. To further reduce cost, our method supports use of gRNAs created by in vitro transcription of low-cost DNA templates. Optimizations led to a single-day capture protocol requiring only 100 ng input DNA, and standard molecular biology equipment and reagents.

To validate this method, we designed a pilot gRNA set targeting 47 known and candidate genes (~3.5Mb) associated with Joubert Syndrome (JS), a genetically heterogeneous, recessive disorder. We tiled 7,176 gRNAs at 500bp intervals, without any design criteria except the presence of a protospacer adjacent motif (PAM). gRNA performance ranged over 1,000-fold and 30% of reads aligned to gRNA specified loci. We next identified critical sequence features governing performance, including GC imbalance and PAM sequence. A linear regression model strongly predicts gRNA performance (r= 0.75). We are currently applying an optimized gRNA set to a large JS cohort (n=578), where ~30% are genetically unsolved, i.e. they lack 2 coding rare variants in the same known gene. With these data, we aim to identify or rule out non-coding mutations in known genes and prioritize families for novel gene discovery. Overall, our CRISPR-Capture platform provides a low-cost and simple workflow for any highly multiplexed sequencing application.