Maria Da Costa

CRISPR-Cas12a (Cpf1) proteins are RNA-guided enzymes that bind and cut DNA as components of bacterial adaptive immune systems. Like CRISPR-Cas9, Cas12a has been harnessed for genome editing on the basis of its ability to generate targeted, double-stranded DNA breaks. Here we show that RNA-guided DNA binding unleashes indiscriminate single-stranded DNA (ssDNA) cleavage activity by Cas12a that completely degrades ssDNA molecules. We find that target-activated, nonspecific single-stranded deoxyribonuclease (ssDNase) cleavage is also a property of other type V CRISPR-Cas12 enzymes. By combining Cas12a ssDNase activation with isothermal amplification, we create a method termed DNA endonuclease-targeted CRISPR trans reporter (DETECTR), which achieves attomolar sensitivity for DNA detection. DETECTR enables rapid and specific detection of human papillomavirus in patient samples, thereby providing a simple platform for molecular diagnostics.

OBJECTIVES: The incidence of anal cancer among men who have sex with men (MSM) has continued to increase since the introduction of highly active antiretroviral therapy (HAART). The prevalence of the putative anal cancer precursor, anal intraepithelial neoplasia (AIN) was high among HIV-positive MSM prior to the availability of HAART but little is known about AIN since HAART was introduced. We characterized the prevalence of AIN among HIV-positive MSM and examined the association between AIN and various factors including use of HAART. DESIGN AND METHODS: A baseline point-prevalence analyses in a prospective cohort study of AIN was performed at a university-based research clinic. A total of 357 HIV-positive MSM with no history of anal cancer completed a questionnaire detailing behaviors and medical history, anal cytology and human papillomavirus (HPV) testing, and high-resolution anoscopy with biopsy for detection of AIN. RESULTS: Eighty-one percent of participants with available CD4+ cell counts at baseline had AIN of any grade; 52% had AIN 2 or 3; and 95% had anal HPV infection. In multivariate analysis, detection of > or = 6 HPV types [odds ratio (OR), 36; 95% confidence interval (CI), 7.4-171) and use of HAART (OR, 10; 95% CI, 2.6-38) were associated with AIN after adjustment for length of time participants were HIV-positive, CD4+ cell count and HIV viral load. CONCLUSIONS: The prevalence of AIN has remained high among HIV-positive MSM after the introduction of HAART. Our data indicate that HAART is not associated with a reduced prevalence of AIN and support measures to prevent anal cancer among HIV-positive MSM whether or not they are using HAART.

Little is known about the epidemiology of anal human papillomavirus (HPV) infection in women. We studied 251 human immunodeficiency virus (HIV)-positive and 68 HIV-negative women for the presence of anal HPV by use of polymerase chain reaction (PCR) and hybrid capture. Medical and behavioral risk factors were evaluated; 76% of HIV-positive and 42% of HIV-negative women were found to have anal HPV DNA via analysis by PCR (relative risk [RR], 1.8; 95% confidence interval [CI], 1.3-2.5). Among 200 women for whom there were concurrent anal and cervical HPV data, anal HPV was more common than cervical HPV in both HIV-positive (79% vs. 53%) and HIV-negative women (43% vs. 24%). By multivariate analysis of HIV-positive women, CD4(+) cell counts </=200 cells/mm(3), compared with counts >500 cells/mm(3) (RR, 1.4; 95% CI, 1.1-1.5), and cervical HPV infection (RR, 1.3; 95% CI, 1.1-1.4) were associated with anal HPV infection. Women >45 years old had reduced risk, compared with women <36 years old (RR, 0.80; 95% CI, 0.50-0.99), as did African American women (RR, 0.86; 95% CI, 0.72-1.0), compared with white women. Anal HPV infection is underrecognized in HIV-positive and high-risk HIV-negative women.