Journal: Nature neuroscience

Article Title: Engineering microdeletions and microduplications by targeting segmental duplications with CRISPR

doi: 10.1038/nn.4235

Figure Lengend Snippet: ( a ) Overview and design of the experimental procedures. ( b ) Illustration of targeted 16p11.2 rMDS segment and/or SDs. For simplicity, only protein coding genes are shown from the Ensembl GRCh37 V.71 annotation . The targeted unique genomic segment for the dual-guide 575 kb deletion is indicated in yellow, and the single-guide RNA targeting the SDs to promote a model of NAHR-mediated CNV is indicated in red (Target sequence: 5′-ACATGCCTATATCGCATAG -3′, chromosome 16: 29,487,572–29,487,590 and 30,226,917–30,226,935). ( c ) Efficiency of CRISPR/Cas9 generation of the 740 kb rMDS using the single-guide SCORE method that targets the SDs is shown, as determined by copy number screening assay for six genes. Further characterization by microarray and RNAseq was performed for a subset of microdeletion clones (6), and all microduplication clones (5). ( d ) Microarray analyses showing deletion (CRISPR Del) and duplication (CRISPR Dup) of the 16p11.2 region in CRISPR-treated lines is shown. Gains or losses of 16p11.2 region were determined by normalized log2 ratios. No off-target CNVs were observed in CRISPR-generated clones.

Article Snippet: Array-based comparative genomic hybridization (aCGH) was performed on the Agilent 4×180K SurePrint G3 Human CGH Microarray (Design #022060) according to the protocol provided by the manufacturer.

Techniques: Sequencing, CRISPR, Screening Assay, Microarray, Clone Assay, Generated