From initial human papillomavirus (HPV) infection and precursor stages, the development of cervical cancer takes decades. High-sensitivity HPV DNA testing is currently recommended as primary screening method for cervical cancer, whereas better triage methodologies are encouraged to provide accurate risk management for HPV-positive women. Given that virus-driven genomic variation accumulates during cervical carcinogenesis, we designed a 39 Mb custom capture panel targeting 17 HPV types and 522 mutant genes related to cervical cancer. Using capture-based next-generation sequencing, HPV integration status, somatic mutation and copy number variation were analyzed on 34 paired samples, including 10 cases of HPV infection (HPV+), 10 cases of cervical intraepithelial neoplasia (CIN) grade and 14 cases of CIN2+ (CIN2: n = 1; CIN2-3: n = 3; CIN3: n = 9; squamous cell carcinoma: n = 1). Finally, the machine learning algorithm (Random Forest) was applied to build the risk stratification model for cervical precursor lesions based on CIN2+ enriched biomarkers. Generally, HPV integration events (11 in HPV+, 25 in CIN1 and 56 in CIN2+), non-synonymous mutations (2 in CIN1, 12 in CIN2+) and copy number variations (19.1 in HPV+, 29.4 in CIN1 and 127 in CIN2+) increased from HPV+ to CIN2+. Interestingly, ‘common’ deletion of mitochondrial chromosome was significantly observed in CIN2+ (P = 0.009). Together, CIN2+ enriched biomarkers, classified as HPV information, mutation, amplification, deletion and mitochondrial change, successfully predicted CIN2+ with average accuracy probability score of 0.814, and amplification and deletion ranked as the most important features. Our custom capture sequencing combined with machine learning method effectively stratified the risk of cervical lesions and provided valuable integrated triage strategies.
Genome editing technologies hold tremendous potential in biomedical research and drug development. Therefore, it is imperative to discover gene editing tools with superior cutting efficiency, good fidelity, and fewer genomic restrictions. Here, we report a CRISPR/Cas9 from Faecalibaculum rodentium, which is characterized by a simple PAM (5′-NNTA-3′) and a guide RNA length of 21–22 bp. We find that FrCas9 could achieve comparable efficiency and specificity to SpCas9. Interestingly, the PAM of FrCas9 presents a palindromic sequence, which greatly expands its targeting scope. Due to the PAM sequence, FrCas9 possesses double editing-windows for base editor and could directly target the TATA-box in eukaryotic promoters for TATA-box related diseases. Together, our results broaden the understanding of CRISPR/Cas-mediated genome engineering and establish FrCas9 as a safe and efficient platform for wide applications in research, biotechnology and therapeutics.
Zinc-finger nucleases (ZFNs), transcription activator-like endonucleases (TALENs), and CRISPR-associated Cas9 endonucleases are three major generations of genome editing tools. However, no parallel comparison about the efficiencies and off-target activity of the three nucleases has been reported, which is critical for the final clinical decision. We for the first time developed the genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) method in ZFNs and TALENs with novel bioinformatics algorithms to evaluate the off-targets. By targeting human papillomavirus 16 (HPV16), we compared the performance of ZFNs, TALENs, and SpCas9 in vivo. Our data showed that ZFNs with similar targets could generate distinct massive off-targets (287-1,856), and the specificity could be reversely correlated with the counts of middle "G" in zinc finger proteins (ZFPs). We also compared the TALENs with different N-terminal domains (wild-type [WT]/aN/bN) and G recognition modules (NN/NH) and found the design (aN or NN) to improve the efficiency of TALEN inevitably increased off-targets. Finally, our results showed that SpCas9 was more efficient and specific than ZFNs and TALENs. Specifically, SpCas9 had fewer off-target counts in URR (SpCas9, n = 0; TALEN, n = 1; ZFN, n = 287), E6 (SpCas9, n = 0; TALEN, n = 7), and E7 (SpCas9, n = 4; TALEN, n = 36). Taken together, we suggest that for HPV gene therapies, SpCas9 is a more efficient and safer genome editing tool. Our off-target data could be used to improve the design of ZFNs and TALENs, and the universal in vivo offtarget detection pipeline for three generations of artificial nu-cleases provided useful tools for genome engineering-based gene therapy.
A genome editing tool targeting the high-risk human papillomavirus (HPV) oncogene is a promising therapeutic strategy to treat HPV-related cervical cancer. To improve gene knockout efficiency, we developed a gene knockout chain reaction (GKCR) method for continually generating mutagenic disruptions and used this method to disrupt the HPV18 E6 and E7 genes. We verified that the GKCR Cas9/guide RNA (gRNA) cassettes could integrated into the targeted loci via homology-independent targeted insertion (HITI). The qPCR results revealed that the GKCR method enabled a relatively higher Cas9/gRNA cassette insertion rate than a control method (the common CRISPR-Cas9 strategy). Tracking of Indels by DEcomposition (TIDE) assay results showed that the GKCR method produced a significantly higher percentage of insertions or deletions (indels) in the HPV18 E6 and E7 genes. Furthermore, by targeting the HPV18 E6/E7 oncogenes, we found that the GKCR method significantly upregulated the P53/RB proteins and inhibited the proliferation and motility of HeLa cells. The GKCR method significantly improved the gene knockout efficiency of the HPV18 E6/E7 oncogenes, which might provide new insights into treatment of HPV infection and related cervical cancer.
Background Gene therapy has held promises for treating specific genetic diseases. However, the key to clinical application depends on effective gene delivery. Methods Using a large animal model, we developed two pharmaceutical formulations for gene delivery in the pigs’ vagina, which were made up of poly (β-amino ester) (PBAE)-plasmid polyplex nanoparticles (NPs) based two gel materials, modified montmorillonite (mMMT) and hectorite (HTT). Findings By conducting flow cytometry of the cervical cells, we found that PBAE-GFP-NPs-mMMT gel was more efficient than PBAE-GFP-NPs-HTT gel in delivering exogenous DNA intravaginally. Next, we designed specific CRISPR/SpCas9 sgRNAs targeting porcine endogenous retroviruses (PERVs) and evaluated the genome editing efficacy in vivo . We discovered that PERV copy number in vaginal epithelium could be significantly reduced by the local delivery of the PBAE-SpCas9/sgRNA NPs-mMMT gel. Comparable genome editing results were also obtained by high-fidelity version of SpCas9, SpCas9-HF1 and eSpCas9, in the mMMT gel. Further, we confirmed that the expression of topically delivered SpCas9 was limited to the vagina/cervix and did not diffuse to nearby organs, which was relatively safe with low toxicity. Interpretation Our data suggested that the PBAE-NPs mMMT vaginal gel is an effective preparation for local gene therapy, yielding insights into novel therapeutic approaches to sexually transmitted disease in the genital tract. Funding This work was supported by the National Science and Technology Major Project of the Ministry of science and technology of China (No. 2018ZX10301402); the National Natural Science Foundation of China (81761148025, 81871473 and 81402158); Guangzhou Science and Technology Programme (No. 201704020093); National Ten Thousand Plan-Young Top Talents of China, Fundamental Research Funds for the Central Universities (17ykzd15 and 19ykyjs07); Three Big Constructions—Supercomputing Application Cultivation Projects sponsored by National Supercomputer Center In Guangzhou; the National Research FFoundation (NRF) South Africa under BRICS Multilateral Joint Call for Proposals; grant 17–54–80078 from the Russian Foundation for Basic Research.
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