Engineering microdeletions and microduplications by targeting segmental duplications with CRISPR

Abstract

Recurrent, reciprocal genomic disorders resulting from non-allelic homologous recombination (NAHR) between near-identical segmental duplications (SDs) are a major cause of human disease, often producing phenotypically distinct syndromes. The genomic architecture of flanking SDs presents a significant challenge for modeling these syndromes; however, the capability to efficiently generate reciprocal copy number variants (CNVs) that mimic NAHR would represent an invaluable modeling tool. We describe here a CRISPR/Cas9 genome engineering method, Single-guide-CRISPR/Cas-targeting-Of-Repetitive-Elements (SCORE), to model reciprocal genomic disorders and demonstrate its capabilities by generating reciprocal CNVs of 16p11.2 and 15q13.3, including alteration of one copy-equivalent of the SDs that mediate NAHR in vivo. The method is reproducible and RNAseq reliably clusters transcriptional signatures from human subjects with in vivo CNV and their corresponding in vitro models. This new approach will provide broad applicability for the study of genomic disorders and, with further development, may also permit efficient correction of these defects.

Variant Interpretation: Functional Assays to the Rescue

“…Cas9 can also be used to delete larger regions, leading to a better understanding of the effects of copy-number variation. 78,79 However, breakpoints will have to be carefully chosen given that an effectively infinite number of copy-number variants are possible.…”

Human adaptation and evolution by segmental duplication

“…Finally, functional studies assaying duplications are not well established. While CRISPR/Cas9 technology has facilitated knockouts within human induced pluripotent stem cells (iPSCs), the high degree of sequence identity between paralogs makes such an undertaking nontrivial often promoting the formation of large-scale rearrangements [64]. Though less straightforward due to the megabase pairs of highly rearranged sequence frequently associated with HSDs, genome editing can also be used to “knock in” HSDs in species where these genes do not exist.…”

Cellular Phenotypes in Human iPSC-Derived Neurons from a Genetic Model of Autism Spectrum Disorder

“…Levels of mRNA expression of 16p11.2 genes correspond to copy number in human lymphoblastoid cell lines (LCLs), in induced pluripotent stem cells (iPSCs) and in the mouse brain, i.e. reduced in deletion carriers (16pdel) and increased in duplication carriers (16pdup)(Blumenthal et al, 2014; Tai et al, 2016). Studies in animal models and transcriptome analyses in LCLs have identified genes in the region involved in synaptogenesis, chromatin modulation and ciliary function (Blumenthal et al, 2014; Luo et al, 2012; Migliavacca et al, 2015).…”