Engineering microdeletions and microduplications by targeting segmental duplications with CRISPR.

Area of Science:

• Genetics
• Genomic Medicine
• Molecular Biology

Background:

• Recurrent genomic disorders arise from non-allelic homologous recombination (NAHR) between segmental duplications (SDs).
• These disorders cause distinct human diseases, but modeling them is challenging due to complex genomic architecture.
• Developing tools to generate reciprocal copy number variants (CNVs) is crucial for studying these conditions.

Purpose of the Study:

• To introduce a novel CRISPR/Cas9 genome engineering method, SCORE, for modeling reciprocal genomic disorders.
• To demonstrate SCORE's capability in generating reciprocal CNVs that mimic NAHR.
• To validate the method's reproducibility and the transcriptional similarity between in vivo and in vitro models.

Main Methods:

• Utilized CRISPR/Cas9 genome editing with single-guide CRISPR/Cas targeting of repetitive elements (SCORE).
• Applied SCORE to generate reciprocal CNVs at specific genomic loci (16p11.2 and 15q13.3).
• Employed RNA sequencing to compare transcriptional signatures between human subjects and engineered models.

Main Results:

• Successfully generated reciprocal CNVs at 16p11.2 and 15q13.3 using the SCORE method.
• Demonstrated in vivo alteration of SDs mediating NAHR.
• Confirmed reproducible results and reliable clustering of transcriptional signatures between models and subjects.

Conclusions:

• SCORE is a reproducible and effective method for modeling reciprocal genomic disorders.
• This approach facilitates the study of genomic disorders by creating accurate in vivo and in vitro models.
• Further development may enable the use of SCORE for correcting genetic defects.