CRISPR-Cas system: A promising tool for rapid detection of SARS-CoV-2 variants.

COVID-19, caused by SARS-CoV-2, remains a global health crisis. The emergence of multiple variants with enhanced characteristics necessitates their detection and monitoring. Genome sequencing, the gold standard, faces implementation challenges due to complexity, cost, and limited throughput. The CRISPR-Cas system offers promising potential for rapid variant detection, with advantages such as speed, sensitivity, specificity, and programmability. This review provides an in-depth examination of the applications of CRISPR-Cas in mutation detection specifically for SARS-CoV-2. It begins by introducing SARS-CoV-2 and existing variant detection platforms. The principles of the CRISPR-Cas system are then clarified, followed by an exploration of three CRISPR-Cas-based mutation detection platforms, which are evaluated from different perspectives. The review discusses strategies for mutation site selection and the utilization of CRISPR-Cas, offering valuable insights for the development of mutation detection methods. Furthermore, a critical analysis of the clinical applications, advantages, disadvantages, challenges, and prospects of the CRISPR-Cas system is provided.

Analysis of Clinical Laboratory Detecting Challenging Variants from Exome Sequencing Using Simulated Patient-Parent Trio Sample: Pilot Study for Neurodevelopmental Disorder de Novo Variants.

To date, there has been no systematic analysis for the clinical laboratory in detecting technically challenging variants using the trio-based exome sequencing (ES) approach. Here, we present an interlaboratory pilot proficiency testing study that used synthetic patient-parent specimens to assess the detection of challenging variants with de novo dominant inheritance modes for neurodevelopmental disorders using various trio-based ES. In total, 27 clinical laboratories that performed diagnostic exome analyses participated in the survey. One of the 26 challenging variants was identified by all laboratories, whereas all 26 variants were identified by only nine laboratories. The lack of identification of mosaic variants was often due to the bioinformatics analysis that excluded the variant. For missing intended heterozygous variants, probable root causes were related to the technical bioinformatics pipeline and variant interpretation and reporting. For each missing variant, there may be more than one probable reason from the different laboratories. There was considerable variation in interlaboratory performance for detecting challenging variants using trio-based ES. This finding may have important implications for the design and validation of tests for different variant types in clinical laboratories, especially for technically challenging variants, and necessary workflow modification can potentially improve trio-based ES performance.

Circulating HPV DNA in HPV-associated cancers.

Human papillomavirus (HPV) infections are the primary cause of almost all cervical cancers, anal cancers, and a variable proportion of other anogenital tumors, as well as head and neck cancers. Circulating HPV DNA (cHPV-DNA) is emerging as a biomarker with extensive potential in the management of HPV-driven malignancies. There has been a rapid advancement in the development of techniques for analyzing cHPV-DNA for the detection, characterization, and monitoring of HPV-associated cancers. As clinical evidence accumulates, it is becoming evident that cHPV-DNA can be used as a diagnostic tool. By conducting clinical trials assessing the clinical utility of cHPV-DNA, the full potential of cHPV-DNA for the screening, diagnosis, and treatment of HPV-related malignancies can be corroborated. In this review, we examine the current landscape of applications for cHPV-DNA liquid biopsies throughout the cancer care continuum, highlighting future opportunities for research and integration into clinical practice.

A multi-laboratory assessment of clinical exome sequencing for detection of hereditary disease variants: 4441 ClinVar variants for clinical genomic test development and validation.

BACKGROUND AND AIMS: Whole-exome sequencing (WES) technology has become an essential tool in the clinical diagnostic for rare genetic disorders, however, the issues that reduce testing precision, sensitivity, and concordance are not clear under routine testing conditions. The study is to systematically evaluate the comparability of clinical WES testing results in laboratories under routine conditions. METHODS: We designed a multi-laboratory study across 24 participating laboratories in China. We assessed sequencing quality across capture methods and sequencing platforms, benchmarked the impact of coverage and callable regions on detecting single nucleotide variants (SNVs), small insertions and deletions (Indels) under the same computational approaches, and compared the sensitivity, precision and reproducibility on detecting mutations across laboratories. RESULTS: High inter-laboratory variability on variants detection were found across participating laboratories. Sample DNA concentration and sequencing evenness are two major variables that lead to the coverage variation. The difference in bioinformatics tools and computational settings affect the sensitivity and precision of the final output. Besides, copy-number variants (CNVs) identification is less reproducible than SNVs and Indels in the WES testing. We also compiled a list of 4441 low coverage ClinVar variants of 1176 genes from this study, which can be used as a source for creating in silico and synthetic DNA reference materials for clinical genetic disorder detection. CONCLUSIONS: The considerable inter-laboratory variability seen in both sequencing coverage evenness and variants detection highlights the urgent need to improve the precision, sensitivity and comparability of the results generated across different laboratories. The list of low coverage variants can have important implications for the development and validation of clinical genetic disorder tests by laboratories. This study also serves to best practice inform guidelines for detecting clinical genetic disorders by exome sequencing.

Carrier screening: An update.

Genetic carrier screening (CS) for reproductive decision making was introduced 50 years ago. Technological advances and improvements in knowledge of the human genome makes multi-disease, pan-ethnic CS possible. Such screening will identify most individuals as carriers of at least one autosomal recessive or X-linked recessive disorder. Past experiences and best practices have provided a framework for CS. Although its clinical utilization is increasing, some challenges remain. In this study, several aspects of CS panel implementation have been addressed including how to evaluate the quantitative gene inclusion criteria, how to classify the severity of genetic conditions, how to understand clinical validity at the level of gene-disease association and variant classification, and how to minimize residual risks.

Reprogramming of Human B Cells from Secreting IgG to IgM by Genome Editing.

B lymphocytes are activated and regulated by their interactions with T cells, a process that results in one-way class switching of immunoglobulins (ig) from IgM to IgG, IgE, or IgA. In this study, we show the application of clustered regularly interspaced short palindromic repeat-Cas9-induced nonhomologous end joining in B cells to achieve reverse-directional Ig class switching. By electroporating Cas9 and guide RNA and a Cµ encoding donor into cells, we engineered IgG-secreting human B cell lines to switch to express IgM antibody. This approach offers a new potential path for the production of IgM antibodies.

A national proficiency scheme for human leucocyte antigen typing by next-generation sequencing.

BACKGROUND: Accurate human leucocyte antigen (HLA) typing is essential for solid organ transplantation (SOT) and hematopoietic stem cell transplantation (HSCT). Next-generation sequencing (NGS) is increasingly adopted by clinical laboratories performing HLA typing. To ensure reliable NGS-based HLA typing throughout China, a HLA-NGS proficiency testing (PT) scheme was set up. METHODS: A panel of 10 DNA reference materials for 11 HLA loci were distributed to 24 participants. They were asked to submit NGS typing results for at least six loci including HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, and HLA-DPB1. Information regarding individual laboratory's sample preparation method, NGS platform, and genotyping software was also collected. RESULTS: Thirteen participants correctly identified HLA alleles in all samples. The total concordance rate for all HLA alleles typed was 99.2% (3390/3420). The overall discordance was 0.2% (3/1440) for Class I, and 1.4% (27/1980) for Class II loci. Further investigation of the typing mistakes pointed to poor sequencing coverage and wrong allele calls. The errors identified reflect the deficient aspect of NGS in HLA typing, which may have implications for quality improvement for relevant laboratories. CONCLUSIONS: The results of this PT underscore the necessity of regular participation in external quality assessment for clinical laboratories that perform HLA-NGS typing.

Development of a Novel Reference Material for Tumor Mutational Burden Measurement Based on CRISPR/Cas9 Technology.

As a biomarker that affects treatment decisions of immune checkpoint inhibitors, the accuracy, reliability, and comparability of tumor mutational burden (TMB) estimation is of paramount importance. To improve the consistency and reliability of these tests, qualified reference materials providing ground-truth data are crucial. In this study, we developed a set of formalin-fixed and paraffin-embedded (FFPE) samples with different TMB values as the novel reference materials for TMB estimation. By introducing several clinically relevant variants in MutS Homolog 2 (MSH2) gene and DNA polymerase epsilon (POLE) gene into human cell lines using CRISPR/Cas9 technology, we first constructed four typical cell lines which verified with hypermutator or ultramutator phenotype. Followed by cell mixing and paraffin embedding, the novel FFPE samples were prepared. It was confirmed that our novel FFPE samples have sufficient quantity of cells, high reproducibility, and they can provide matched wild type sample as the genetic background. The double-platform whole exome sequencing validation showed that our FFPE samples were also highly flexible as they containing different TMB values spanning a clinically relevant range (2.0-106.1 mut/Mb). Without limitations on production and TMB values, our novel FFPE samples based on CRISPR/Cas9 editing are suitable as candidate reference materials. From a practical point of view, these samples can be used for the validation, verification, internal quality control, and proficiency testing of TMB assessment.

Adaptation of ACMG-ClinGen Technical Standards for Copy Number Variant Interpretation Concordance.

This study aimed to evaluate inter-laboratory classification concordance for copy number variants (CNVs) with a semiquantitative point-based scoring metric recommended by the American College of Medical Genetics and Genomics (ACMG) and Clinical Genome Resources (ClinGen). A total of 234 CNVs distributed by the National Center of Clinical Laboratories (NCCLs), and 72 CNVs submitted by different laboratories, were distributed to nine clinical laboratories performing routine clinical CNV testing in China and independently classified across laboratories. The overall inter-laboratory complete classification concordance rate of the 234 distributed CNVs increased from 18% (41/234) to 76% (177/234) using the scoring metric compared to the laboratory's previous method. The overall inter-laboratory complete classification concordance rate of the 72 submitted CNVs was 65% (47/72) using the scoring metrics. The 82 variants that initially did not reach complete concordance classification and 1 additional CNV deletion were reviewed; 34 reached complete agreement, and the overall post-review complete concordance rate was 85% (260/306). Additionally, the overall percentage of classification discordance possibly impacting medical management [i.e., pathogenic (P) or likely pathogenic (LP) vs. variant of uncertain significance (VUS)] was 11% (35/306). The causes of initial and final discordance in the classification were identified. The ACMG-ClinGen framework has promoted consistency in interpreting the clinical significance of CNVs. Continuous training among laboratories, further criteria and additional clarification of the standards, sharing classifications and supporting evidence through public database, and ongoing work for dosage sensitive genes/regions curation will be beneficial for harmonization of CNVs classification.

Preparation of multiplexed control materials for cancer mutation analysis by genome editing in GM12878 cells.

BACKGROUND: Quality control materials are necessary for assay development, test validation, and proficiency testing in cancer mutation analysis. Most of the existing controls for somatic mutations only harbor a single variant and are derived from unstable cell lines. This study aimed to establish a method to create stable multianalyte controls in a defined background by genome editing in GM12878 cells, which also can be applied for the reference of next-generation sequencing. METHODS: GM12878 cells were electroporated with a donor plasmid containing a mutant DNA sequence and a Cas9/sgRNA expressing vector. The genome-edited GM12878 cell was validated with Sanger sequencing, amplification refractory mutation system (ARMS), and next-generation sequencing (NGS). RESULTS: We have successfully generated a mutant GM12878 cell line harboring the defined variants including single-nucleotide variants (SNVs), small insertions and deletions (indels), and structural variants (SVs). The introduction of intended mutations in GM12878 cell line was confirmed by both ARMS and sequencing methods. CONCLUSIONS: We developed a method for the preparation of the multiplexed controls for reference mutations in cancer gene by genome editing in GM12878 cells. This methodology can be used to generate other stable cancer reference materials with an unlimited supply.

An Initial Survey of the Performances of Exome Variant Analysis and Clinical Reporting Among Diagnostic Laboratories in China.

Exome sequencing has become an effective diagnostic method for Mendelian disorders. But the quality of services differs widely across laboratories in China, particularly in variant classification, even with the adoption of the ACMG guidelines. As an effort of quality control and improvement for better clinical utilization of exome sequencing, we assessed the exome data analysis and clinical reporting among Chinese laboratories. Five raw datasets of real clinical samples with associated phenotypes were sent to 53 laboratories. The participants independently performed secondary analysis, variant classification, and reporting. The first round of results was used for identifying problems associated with these aspects. Subsequently, we implemented several corrective actions and a training program was designed based on the identified issues. A second round of five datasets were sent to the same participants. We compared the performances in variant interpretation and reporting. A total of 85.7% (42/49) of participants correctly identified all the variants related with phenotype. Many lines of evidence using the ACMG guidelines were incorrectly utilized, which resulted in a large inter-laboratory discrepancy. After training, the evidence usage problems significantly improved, leading to a more consistent outcome. Participants improved their exome data analysis and clinical reporting capability. Targeted training and a deeper understanding of the ACMG guidelines helped to improve the clinical exome sequencing service in terms of consistency and accuracy in variant classification in China.

Continual Improvement of the Reliability of EML4-ALK Rearrangement Detection in Non-Small-Cell Lung Cancer: A Long-Term Comparison of ALK Detection in China.

The results of EML4-ALK testing are critical to manage ALK tyrosine kinase receptor inhibitor treatment. Thus, the accurate detection of ALK rearrangement is increasingly becoming a matter of serious concern. To address this issue, a long-term EML4-ALK proficiency testing (PT) scheme was launched in China in 2015, serving as an educational tool for assessing and improving the testing quality of EML4-ALK fusion detection. Responses across 20 different PT samples interrogating three different variants and wild-type samples were collected between 2015 and 2019. Performance was analyzed by evaluating the detection methods, kits, and pre-analytic practices used to further display the landscape of changing conditions of the reliability of EML4-ALK testing. During the 5 years, 3224 results reported from 988 laboratories were evaluated, with an overall error rate of 5.36%. Along with an increasing number of participating laboratories, the error rate within each of the different methods showed a significantly downward trend over the years. No obvious differences in the error rates were found regarding the testing methods or kit manufacturers. Moreover, the individual performance of the laboratories improved when they participated in more PT scheme rounds. The data demonstrated that the performance of individual Chinese laboratories for EML4-ALK testing continuously improved over time by participating PT schemes, regardless of their method. However, care must be taken in standardized operations and validations.

Molecular genetic testing and diagnosis strategies for dystrophinopathies in the era of next generation sequencing.

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive, inherited neuromuscular disorders, caused by pathogenic variants in the dystrophin gene that encodes the dystrophin protein. A number of mutations have been identified in the past years, producing dystrophin diversity and resulting in mild to severe phenotypes in patients. Mutations in the dystrophin gene can be characterized by laboratory testing to confirm a clinical diagnosis of DMD/BMD. Traditional genetic diagnostic strategy for DMD/BMD involves the initial detection of large mutations, followed by the detection of smaller mutations, where two or more analytical methods are employed. With the development of next generation sequencing (NGS) technology, comprehensive mutational screening for all variant types can be performed on a single platform in patients and carriers, although further optimization and validation are required. Furthermore, the discovery of cell-free fetal DNA (cffDNA) in maternal plasma provides basis for noninvasive prenatal diagnosis of DMD/BMD. Here, we discuss the correlation between genotype and phenotype, the current methods of molecular genetic testing and genetic diagnostic strategy for probands and female carriers of DMD/BMD, the diagnostic ability of a comprehensive targeted NGS strategy and the possibility of it replacing conventional methods.

Quantitative detection of hepatitis C virus RNA in urine of patients with chronic hepatitis C using a novel real-time PCR assay.

Hepatitis C virus (HCV) RNA can be detected in body fluids such as urine. However, because of deficiencies in established isolation and detection methods, the actual prevalence and form of HCV RNA in the urine of patients with hepatitis C remain unclear. To more sensitively and accurately measure urine HCV RNA levels, a novel real-time PCR assay with a modified isolation method and short amplicon designed for short HCV RNA fragments was developed in this study. The limit of detection, precision, linearity, and specificity of the assay was evaluated and demonstrated high-quality performance. The prevalence of HCV RNA in the urine of viremic patients infected with HCV was 60% (36/60), as determined by a 62-bp assay. The HCV RNA detection rate and concentration were much lower with a 157-bp assay, and were undetectable with 222- and 304-bp assays. With the 62-bp assay, patients with detectable urine HCV RNA had significantly higher plasma HCV RNA levels ( P < 0.001), and plasma and urine concentrations were significantly positively correlated ( R 2 = 0.708, P < 0.001). The method not only increased the detection rate of urine HCV RNA but also revealed the presence of short HCV RNA fragments in urine, indicating that urine from CHC patients with normal kidney function should not be infectious. In addition, it raised the possibility of urinary HCV RNA as a potential noninvasive marker for therapeutic monitoring of patients with hepatitis C.

Evaluation of tacrolimus-related CYP3A5 genotyping in China: Results from the First External Quality Assessment Exercise.

BACKGROUND: Tacrolimus is the most widely used immunosuppressant in solid organ transplant patients. The cytochrome P450 3A5 (CYP3A5) has been proved to be associated with tacrolimus dose requirement. Molecular detection for CYP3A5 genotyping is demanded for the optimization of treatments of tacrolimus. METHODS: To achieve the consistency and accuracy of the testing results, the Chinese National Center for Clinical Laboratories (NCCL) organized a national external quality assessment(EQA) program to evaluate the performance of laboratories providing CYP3A5 genotyping. Ten validated DNA samples covering the common genetic polymorphisms of CYP3A5 were delivered to 33 voluntary laboratories, and their detecting results and clinical written reports were evaluated. RESULTS: Thirty-three datasets were received. The corresponding analytical sensitivity was 95.9% (285/297 challenges; 95% confidence interval: 93.0%-97.9%), and the analytical specificity was 95.3% (346/363; 95% confidence interval: 92.6%-97.2%). Thirty of the participating laboratories correctly identified the CYP3A5 allele status for all EQA samples. Three laboratories made genotyping errors, and 2 of them failed to detect any of the homozygotes such as *1/*1 and *3/*3. Twenty-eight CYP3A5*3 tests reports were submitted, but many reports showed a shortage of essential information. No reports fulfilled all the consensus recommendations for pharmacogenetic test result reporting. CONCLUSION: The EQA program highlighted the necessity for an improvement in the accuracy of genotyping for some of the laboratories and a greater education on the reporting of CYP3A5 genotyping results.

Implications of and lessons learned from external assurance of eight influenza diagnostics in China.

This study evaluated the ability of laboratories in the Chinese mainland to conduct molecular detection of seasonal A(H1N1), A(H1N1)pdm09, A(H3N2), A(H5N1), A(H7N9), A(H9N2), B(Victoria), and B(Yamagata). Based on a genetically engineered system of virus-like particles (VLPs), the National Center for Clinical Laboratories of China (NCCLs) developed an external quality assessment (EQA) panel. The panel was distributed to 35 laboratories in mainland China to investigate the proficiency of the 16 assays for influenza molecular detection. Using genetic engineering technology, VLPs encapsulating the 37 target genes of 8 influenza viruses were generated. After verification and quantification, 26 influenza virus surrogates with different concentrations were prepared for EQA. Among the 35 participating laboratories, 319 datasets were returned to the NCCLs. Overall, 95.6% (305/319) of datasets correctly reported all 30 samples, while 2.2% (7/319) of datasets with more than one incorrect result were considered as "improvable". A total of 16 misdiagnosed and 18 undiagnosed results were reported. The data analyzed in this study showed good reproducibility in China, but improvements are needed to decrease misdiagnosed and undiagnosed cases, particularly for the A(H9N2) NA gene. Moreover, VLPs are a good alternative specimen type for assay training and proficiency testing purposes.

Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-Associated Endonuclease Cas9-Mediated Homology-Independent Integration for Generating Quality Control Materials for Clinical Molecular Genetic Testing.

Genome-edited human cell lines are important resources for producing quality control materials for clinical molecular genetic testing. Generating cell lines with defined mutations through homology-directed repair-based methods are inefficient and can lead to unwanted insertions and deletions in the target loci. Nonhomologous end joining in the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease Cas9 (Cas9) system was harnessed to generate genome-engineered cell lines harboring target mutations. Donor plasmids containing target sites for the single guide RNA (sgRNA) and homologous DNA fragments harboring important cancer gene mutations were cotransfected with the Cas9/sgRNA vector into wild-type human cells. The introduced mutations were validated in-house and in 44 laboratories using various techniques, including next-generation sequencing. Exogenous sequences containing the target mutations were efficiently integrated into the ALK receptor tyrosine kinase (ALK) locus in HEK293T and A549 cells. Successful introduction of artificial mutations was confirmed via both Sanger sequencing and the amplification refractory mutation system. Results of external pilot testing revealed that the DNA samples derived from genome-edited cell lines were widely applicable across multiple platforms and laboratories. This study demonstrates that CRISPR/Cas9-induced nonhomologous end joining is a valuable and novel method for generating artificial mutants for use in quality control applications in clinical molecular genetics.

Current progress of tacrolimus dosing in solid organ transplant recipients: Pharmacogenetic considerations.

Tacrolimus is effective for the prevention of acute rejection, but is also highly toxic and has great intra- and inter-individual variability in transplant patients. Genetic variation and other factors influence the response of an individual to tacrolimus treatment. Therefore, even if therapeutic drug monitoring is universally applied, rejection and toxicity still occur. Although the appropriate action on pharmacogenomic variability provides a cornerstone for the precise tacrolimus prescription, at present there are many obstacles to translating it into clinical practice. Pre-emptive genotyping is rarely performed because of practical and financial reasons. However, as the cost of sequencing continues to fall, it is feasible to span all clinically actionable genotypes and provide patients with relevant information throughout their lifetime, which would, therefore, optimize tacrolimus dosing by facilitating the structured dosing algorithms (for example, population pharmacokinetic models) and clinical decision support. In this review, we discuss the current challenges and opportunities for the translation of pharmacogenetic information of tacrolimus into clinical settings.

A novel cell line generated using the CRISPR/Cas9 technology as universal quality control material for KRAS G12V mutation testing.

BACKGROUND: KRAS mutations are the key indicator for EGFR monoclonal antibody-targeted therapy and acquired drug resistance, and their accurate detection is critical to the clinical decision-making of colorectal cancer. However, no proper quality control material is available for the current detection methods, particularly next-generation sequencing (NGS). The ideal quality control material for NGS needs to provide both the tumor mutation gene and the matched background genomic DNA, which is uncataloged in public databases, to accurately distinguish germline polymorphisms and somatic mutations. METHODS: We developed a novel KRAS G12V mutant cell line using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technique to make up for the deficiencies in existing quality control material and further validated the feasibility of the cell line as quality control material by amplification refractory mutation system (ARMS), Sanger sequencing, digital PCR (dPCR), and NGS. RESULTS: We verified that the edited cell line specifically had the G12V mutation, and the validation results presented a high consistency among the four methods of detection. The three cell lines screened contained the G12V mutation and the mutation allele fractions of G12V-1, G12V-2, and G12V-3 were 52.01%, 82.06%, and 17.29%, respectively. CONCLUSION: The novel KRAS G12V cell line generated using the CRISPR/Cas9 gene editing system is suitable as a quality control material for all current detection methods and provides a new direction in the development of quality control material.