CRISPR-Cas9-Targeted Nanopore Sequencing for STR Typing.

Area of Science:

• Forensic genetics
• Molecular biology
• Genomics

Background:

• CRISPR-Cas9 technology enables targeted DNA enrichment via single-guide RNA (sgRNA).
• Polymerase chain reaction (PCR)-free workflows are desirable for forensic DNA analysis.
• Short tandem repeat (STR) profiling is a cornerstone of forensic genetics.

Purpose of the Study:

• To evaluate the efficacy of CRISPR-Cas9-targeted nanopore sequencing (Cas9-seq) for forensic STR profiling.
• To compare the performance of Cas9-seq with traditional amplification-based nanopore sequencing (amplicon-seq).

Main Methods:

• Developed a Cas9-seq method targeting seven forensic STR loci.
• Utilized human cell line DNA (NA12878 and 293T) with 3 µg input.
• Compared Cas9-seq results with amplicon-seq from the ForenSeq DNA Signature Prep kit.

Main Results:

• Achieved significant enrichment ratios (643.45x and 468.34x) for targeted regions.
• Cas9-seq exhibited ultralow strand bias but showed higher noise and no advantage in allele balance.
• Both methods yielded three genotyping errors per sample; Cas9-seq introduced no false-positive SNPs, unlike amplicon-seq.

Conclusions:

• PCR-free Cas9-seq demonstrated high enrichment and low strand bias.
• Cas9-seq did not outperform amplification-based methods for forensic STR genotyping accuracy.
• Further optimization is needed for Cas9-seq to be considered favorable for forensic applications.