DNA methylation is an important promising biomarker for cancer diagnosis and monitoring.Therefore,the assessment of DNA methylation levels is helpful for the prognosis and diagnosis of cancer.However,it is still a hug...DNA methylation is an important promising biomarker for cancer diagnosis and monitoring.Therefore,the assessment of DNA methylation levels is helpful for the prognosis and diagnosis of cancer.However,it is still a huge challenge to sensitively and accurately quantify the levels of DNA methylation in clinical sample.In this work,we proposed a protospacer adjacent motif(PAM)-free mediated CRISPR-Cas12a ultra-sensitive and quantitative DNA methylation detection method.Through recognizing the ds DNA with toehold region,CRISPR-Cas12a not only got rid of the limitation of PAM,but also improved its distinction ability for single Cp G site methylation,nearly 5-fold that of conventional PAM-containing ds DNA.We further introduced assist-strand and design an artificial mismatch to greatly improve the ability to distinguish single Cp G methylation site.Our results showed that the discrimination factor was >200.Then,we constructed toe-ds DNA by using “heating and freezing”,which made our method universally applicable and feasible.In addition,we greatly simplified the difficulty of primer design.Our method detected four highly methylated genes acyl carrier protein(ACP),CLV3/ESR-related(CLE),Disabled(DAB) and Homeobox(HOX) with a detection limit of 0.01 % and excellent linearity in DNA methylation standards.Then,we verified the clinical utility of this method in 29 hepatocellular carcinomas,11 ovarian cancers and4 health people.In conclusion,we have successfully constructed a PAM-free CRISPR-Cas12a DNA methylation quantification method,which achieves high congruence in sensitivity,specificity and universality,fully demonstrating its significant clinical application value.展开更多
基金financially supported by the Natural Science Foundation of Wuhan City (Chenguang Project) (No.2024040801020331)the Natural Science Foundation of Hubei Province of China (No.2023AFB402)+1 种基金the National Key Research and Development Program of China (No.2023YFE0210200)Interdisciplinary Research Program of HUST。
文摘DNA methylation is an important promising biomarker for cancer diagnosis and monitoring.Therefore,the assessment of DNA methylation levels is helpful for the prognosis and diagnosis of cancer.However,it is still a huge challenge to sensitively and accurately quantify the levels of DNA methylation in clinical sample.In this work,we proposed a protospacer adjacent motif(PAM)-free mediated CRISPR-Cas12a ultra-sensitive and quantitative DNA methylation detection method.Through recognizing the ds DNA with toehold region,CRISPR-Cas12a not only got rid of the limitation of PAM,but also improved its distinction ability for single Cp G site methylation,nearly 5-fold that of conventional PAM-containing ds DNA.We further introduced assist-strand and design an artificial mismatch to greatly improve the ability to distinguish single Cp G methylation site.Our results showed that the discrimination factor was >200.Then,we constructed toe-ds DNA by using “heating and freezing”,which made our method universally applicable and feasible.In addition,we greatly simplified the difficulty of primer design.Our method detected four highly methylated genes acyl carrier protein(ACP),CLV3/ESR-related(CLE),Disabled(DAB) and Homeobox(HOX) with a detection limit of 0.01 % and excellent linearity in DNA methylation standards.Then,we verified the clinical utility of this method in 29 hepatocellular carcinomas,11 ovarian cancers and4 health people.In conclusion,we have successfully constructed a PAM-free CRISPR-Cas12a DNA methylation quantification method,which achieves high congruence in sensitivity,specificity and universality,fully demonstrating its significant clinical application value.
