RT Journal Article SR Electronic T1 CRISPR–Cas9/long-read sequencing approach to identify cryptic mutations in BRCA1 and other tumour suppressor genes JF Journal of Medical Genetics JO J Med Genet FD BMJ Publishing Group Ltd SP 850 OP 852 DO 10.1136/jmedgenet-2020-107320 VO 58 IS 12 A1 Tom Walsh A1 Silvia Casadei A1 Katherine M Munson A1 Mary Eng A1 Jessica B Mandell A1 Suleyman Gulsuner A1 Mary-Claire King YR 2021 UL http://jmg.bmj.com/content/58/12/850.abstract AB Current clinical approaches for mutation discovery are based on short sequence reads (100–300 bp) of exons and flanking splice sites targeted by multigene panels or whole exomes. Short-read sequencing is highly accurate for detection of single nucleotide variants, small indels and simple copy number differences but is of limited use for identifying complex insertions and deletions and other structural rearrangements. We used CRISPR-Cas9 to excise complete BRCA1 and BRCA2 genomic regions from lymphoblast cells of patients with breast cancer, then sequenced these regions with long reads (>10 000 bp) to fully characterise all non-coding regions for structural variation. In a family severely affected with early-onset bilateral breast cancer and with negative (normal) results by gene panel and exome sequencing, we identified an intronic SINE-VNTR-Alu retrotransposon insertion that led to the creation of a pseudoexon in the BRCA1 message and introduced a premature truncation. This combination of CRISPR–Cas9 excision and long-read sequencing reveals a class of complex, damaging and otherwise cryptic mutations that may be particularly frequent in tumour suppressor genes replete with intronic repeats.