CRISPR‐Cas9‐Targeted Nanopore Sequencing for STR Typing

CRISPR-Cas9-targeted sequencing can enrich DNA regions of interest by directing the Cas9 protein to bind and cleave specific DNA sequences via single-guide RNA (sgRNA). It is interesting to explore the efficacy of using CRISPR-Cas9-targeted nanopore sequencing (referred to as Cas9-seq), a polymerase chain reaction (PCR)-free workflow, for forensic short tandem repeats (STR) profiling, and to compare it with the amplification-based approach. In this pilot study, we constructed a Cas9-seq method for profiling seven STR loci, including D18S51, FGA, TPOX, D16S539, vWA, CSF1PO, and TH01. With 3 µg DNA inputs from human NA12878 and 293T cell lines, we achieved 643.45- and 468.34-fold enrichment ratios of the sgRNA-targeted regions by using Cas9-seq, respectively. Compared to nanopore sequencing of PCR amplicon products (amplicon-seq) of the ForenSeq DNA Signature Prep kit, the Cas9-seq reads had an ultralow strand bias. However, surprisingly, Cas9-seq did not show advantages in allele balance and had higher noise in the reads. At the seven STR loci for the two samples, both Cas9-seq and amplicon-seq had three genotyping errors. Additionally, there were no false-positive single-nucleotide polymorphisms (SNPs) introduced by Cas9-seq, whereas amplicon-seq produced three. In sum, we conclude that the PCR-free Cas9-seq might not be favorable for forensic STR genotyping.