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X-chromosome inactivation (XCI) analyses often assist in diagnostics of X-linked traits, however accurate assessment remains challenging with current methods. We developed a novel strategy using amplification-free Cas9 enrichment and Oxford nanopore technologies sequencing called XCI-ONT, to investigate and rigorously quantify XCI in human androgen receptor gene (AR) and human X-linked retinitis pigmentosa 2 gene (RP2). XCI-ONT measures methylation over 116 CpGs in AR and 58 CpGs in RP2, and separate parental X-chromosomes without PCR bias. We show the usefulness of the XCI-ONT strategy over the PCR-based golden standard XCI technique that only investigates one or two CpGs per gene. The results highlight the limitations of using the golden standard technique when the XCI pattern is partially skewed and the advantages of XCI-ONT to rigorously quantify XCI. This study provides a universal XCI-method on DNA, which is highly valuable in clinical and research framework of X-linked traits. [ABSTRACT FROM AUTHOR]