CRISPR/Cas-Based Prenatal Screening for Aneuploidy: Challenges and Opportunities for Early Diagnosis

Abstract

Aneuploidy is increasingly recognized globally as a common cause of miscarriage among expectant mothers. The existing prenatal screening techniques for detecting aneuploidy have several limitations. The ability to diagnose aneuploidy early in a non-invasive manner is not feasible with the current screening methods, as they may produce false positive or false negative results. Recently, the widely used gene editing tool CRISPR/Cas has shown great promise in diagnostics. This review summarizes the prenatal screening tests used in the first trimester to assess aneuploidy conditions. Additionally, we discuss the advantages and disadvantages of molecular diagnostic tests, including the benefits and challenges of CRISPR/Cas-based trisomy detection. Thus, the proposed prenatal screening using CRISPR/Cas could provide significant benefits to expectant mothers by potentially enabling the early diagnosis of trisomy, helping to prevent miscarriage and birth defects. Furthermore, it opens new avenues for research, allowing clinicians and researchers to develop, optimize, and implement CRISPR/Cas-based prenatal screening assays in the future.

Keywords: CRISPR/Cas; aneuploidy; cell-free fetal DNA; molecular diagnostics; prenatal screening.

Figure 1

Various techniques are used in prenatal screening to detect aneuploidy. 1. Karyotyping 2. Fluorescent in situ hybridization (FISH) 3. Real-time quantitative polymerase chain reaction (RT-qPCR) 4. Microarray 5. Next generation sequencing (NGS) and 6. A proposed advanced technique using CRISPR/Cas system.

Figure 2

Proposed non-invasive prenatal (NIPT) screening by a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-based assay. 1. Maternal blood samples will be collected from the gestational mother and processed to isolate the cell-free fetal DNA (cffDNA) using the kit method. 2. The cffDNA will be amplified using the fluorophore-labeled primers designed for recombinase polymerase amplification (RPA). 3. The RPA mixture will contain single guide (sg) RNA and Cas nucleases designed to target the chromosomal regions to detect aneuploidies by CRISPR/Cas assay. 4. For multiplex detection, various sgRNAs will be programmed to target trisomies such as Trisomy 13, Trisomy 18, and Trisomy 21. The detection of 3 targets will be favored by adding reporters for Cas12f, Cas12b, and Cas12a in HEX (hexachlorofluorescein), TEX (TEX615), and FAM channels. The sgRNAs will recognize and bind to the fluorophore-targeted cffDNA resulting in the formation of the fluorophore-DNA-Cas-sgRNA complex. This will result in the generation of fluorescence by the separation of fluorophore and quencher. 5. The fluorescence generated will be measured using a spectrofluorometer or plate reader with distinct absorbance and emission wavelengths.