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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.

Background Since circulating tumor DNA (ctDNA) sequencing is increasingly being applied in clinical management of patients with cancer, its testing accuracy has become a matter of serious concern. To address this issue, a long-term ctDNA analysis proficiency testing (PT) scheme for next-generation sequencing (NGS) was launched in China in 2018, serving as an educational tool for assessing and improving the testing quality of NGS-based ctDNA detection. Methods Feedback from participating laboratories across 23 different PT samples containing different variants with varying variant allele frequency was collected between 2018 and 2021. To further show the landscape of changing conditions in accuracy and reliability of NGS-based ctDNA testing, performance was analyzed by evaluating the cfDNA extraction kits, testing panels, target enrichment strategies, and sequencing platforms. Results During the 4 years, 2745 results reported from 504 laboratories were evaluated. Only 66.3% of results from laboratories were entirely in concordance with the expected results. Nonetheless, along with an increasing number of participating laboratories, the number of errors occurring in laboratories, and the proportion of laboratories that experienced errors both showed a significant downward trend. No obvious differences in the error rates were found regarding the kit manufacturers or sequencing platform. Moreover, the individual performances of the laboratories improved when they participated in more PT scheme rounds. Conclusions These data demonstrated that the performance of individual Chinese laboratories for NGS-based ctDNA analysis continuously improved over time with participation in PT schemes. However, further care must also be taken in standardized operations and validations.

MicroRNAs are related to the development of hepatocellular carcinoma and can serve as potential therapeutic targets. Therapeutic strategies increasing tumor-suppressive microRNAs and reducing oncogenic microRNAs have been developed. Herein, the effects of simultaneously altering two microRNAs using MS2 virus-like particles were studied. The sequences of microRNA-21-sponge and pre-microRNA-122 were connected and cloned into a virus-like particle expression vector. Virus-like particles containing microRNA-21-sponge and pre-microRNA-122 sequences were prepared and crosslinked with a cell-specific peptide targeting hepatocellular carcinoma cells. Delivery effects were studied using RT-qPCR and functional assays to investigate the level of target mRNAs, cell toxicity, and the effects of proliferation, invasion, and migration. Virus-like particles delivered miR-21-sponge into cells, with the Ct value reaching 10 at most. The linked pre-miR-122 was processed into mature miR-122. The mRNA targets of miR-21 were derepressed as predicted and upregulated 1.2–2.8-fold, and the expression of proteins was elevated correspondingly. Proliferation, migration, and invasion of HCC cells were inhibited by miR-21-sponge. Simultaneous delivery of miR-21-sponge and miR-122 further decreased proliferation, migration, and invasion by up to 34%, 63%, and 65%, respectively. And the combination promoted the apoptosis of HCC cells. In conclusion, delivering miR-21-sponge and miR-122 using virus-like particles modified by cell-specific peptides is an effective and convenient strategy to correct microRNA dysregulation in hepatocellular carcinoma cells and is a promising therapeutic strategy for hepatocellular carcinoma.

Background To meet the requirements of the rapidly progressing genetic testing technologies in clinical laboratories, assuring the quality of genetic tests by utilizing appropriate quality control materials is of paramount importance. The CRISPR/Cas9 technology was used to prepare quality control materials because genome‐edited human cell lines are one of the major resources for quality control materials. Methods In this study, in vitro transcribed sgRNA were transfected into a Cas9‐expressing lymphoblastoid cell line (LCL)—by electroporation—to simulate the SEA‐type deletion observed in α‐thalassemia. The edited positive cell line was screened and identified by polymerase chain reaction (PCR) followed by Sanger sequencing. The whole‐genome sequencing was also performed to show evidence of predicted mutation. Results The results showed that electroporation of the in vitro transcribed gRNAs into stable Cas9‐expressing LCL was a more efficient gene‐editing technique as compared to plasmid‐mediated transfection, and that the positive rates could reach up to 35.9%. The predominance of indel sizes relative to the predicted deletion length was clustered between 10 and 0 bp. The results of whole‐genome sequencing also demonstrated the existence of SEA‐type deletion of α‐thalassemia. Conclusions Gene‐editing based on Cas9‐expressing LCL by electroporation of sgRNA was a more efficient approach to introduce mutations for generating quality control materials for genetic testing. The edited lymphoblastoid cell lines were feasible to serve as quality control materials in 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.

The echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase (ALK) receptor tyrosine kinase (EML4-ALK) rearrangement is an important biomarker that plays a pivotal role in therapeutic decision making for non-small-cell lung cancer (NSCLC) patients. Ensuring accuracy and reproducibility of EML4-ALK testing by fluorescence in situ hybridization, immunohistochemistry, RT-PCR, and next-generation sequencing requires reliable reference materials for monitoring assay sensitivity and specificity. Herein, we developed novel reference materials for various kinds of EML4-ALK testing. CRISPR/Cas9 was used to edit various NSCLC cell lines containing EML4-ALK rearrangement variants 1, 2, and 3a/b. After s.c. inoculation, the formalin-fixed, paraffin-embedded (FFPE) samples from xenografts were prepared and tested for suitability as candidate reference materials by fluorescence in situ hybridization, immunohistochemistry, RT-PCR, and next-generation sequencing. Sample validation and commutability assessments showed that all types of FFPE samples derived from xenograft tumors have typical histological structures, and EML4-ALK testing results were similar to the clinical ALK-positive NSCLC specimens. Among the four methods for EML4-ALK detection, the validation test showed 100% concordance. Furthermore, these novel FFPE reference materials showed good stability and homogeneity. Without limitations on variant types and production, our novel FFPE samples based on CRISPR/Cas9 editing and xenografts are suitable as candidate reference materials for the validation, verification, internal quality control, and proficiency testing of EML4-ALK detection.

Next-generation sequencing (NGS) is commonly used in a clinical setting for diagnostic and prognostic testing of genetic mutations to select optimal targeted therapies. Herein, we describe the development of a custom NGS assay for detecting single-nucleotide variants (SNVs) and copy number variations (CNVs) in a panel of 51 genes related to breast cancer. We designed and implemented a validation strategy in accordance with principles and guidelines developed by the Next-Generation Sequencing: Standardization of Clinical Testing work group using artificial, cell-free DNA (cfDNA) with mutant fragments prepared in a simple, rapid, and cost-effective manner. For SNV detection, our test had 96.30% sensitivity at mutant allele frequency ≥0.5% with high specificity (99.9997%) and accuracy (99.9996%). For CNV detection, the approach had 95.83% sensitivity for copy numbers at 1.25× (25.6% extra copies) with high specificity (99.77%) and accuracy (99.76%). In addition, our NGS-based assay demonstrated high intrarun and interrun reproducibility, high consistency compared to digital PCR, and a low cross-contamination rate. An overall assessment using cfDNA and plasma cfDNA samples demonstrated our custom NGS assay yields a reliable and robust detection sensitivity with a mutant allele frequency as low as 0.5% for SNVs and copy number of 1.25× for CNVs.

Effective characterization and imaging of endogenous RNA transcripts have important value in the diagnosis, treatment, and prognosis of diseases. Traditional qRT-PCR as a liquid-based RNA detection method might lead to false-negative results due to the admixture of too many nontarget cells. Also, many in situ RNA imaging methods were hindered by long turnaround time and insufficient signals. Here, we describe and evaluate a CRISPR/dCas9-MS2-based RNA fluorescence in situ hybridization assay (RCasFISH) for in situ amplified imaging and quantification of RNA transcripts in fixed cells as well as formalin-fixed, paraffin-embedded (FFPE) tissue sections at a single-molecular level in individual cells. Compared to single molecular FISH (smFISH), RCasFISH yields brighter dot signals and a better signal-to-noise ratio (SNR) with lower costs and less than 1.5 h of hybridization. In addition, by using human epidermal growth factor receptor 2 (HER2) as a model, we quantified individual HER2 mRNA molecules in clinical breast cancer FFPE tissue sections and demonstrated its potential to resolve FISH-equivocal cases. Therefore, RCasFISH may provide a new approach for gene expression studies in basic research and hold the potential for molecular diagnostic applications.

For patients with chronic myeloid leukemia, reverse transcription quantitative polymerase chain reaction is widely used in laboratories to quantify BCR-ABL1 fusion gene transcripts for disease management. Many efforts have been made to standardize the BCR-ABL1 testing assay, including the primary and secondary reference reagents, but the secondary standards have not been developed and used in the standardization program in China. With the use of armored RNA technology, armored RNA of BCR-ABL1 and control genes was manufactured to prepare the secondary reference material anchored to the World Health Organization primary reference calibrators for standardization of BCR-ABL1 testing assays. The secondary reference was sent to 30 laboratories in China for validation. Data from an external quality assessment after the standardization process were collected and analyzed as well. The assigned %BCR-ABL1/ABL1IS values of the four levels of the secondary material panels were 0.0118, 0.1345, 1.3808, and 19.4266, respectively. In validation trials, 70.0% (21/30) of laboratories obtained valid conversion factors for the BCR-ABL1 assay. All valid conversion factors from 11 international scale laboratories were equivalent to their respective previous values. External quality assessment data indicated that the accuracy and precision between laboratories were improved. Moreover, the quantity of the panels is abundant to be used as quality control samples for monitoring the shift of data. In this study, we established a secondary genetic reference panel for BCR-ABL1 quantification. This study will play a role in facilitating the worldwide dissemination of the international scale, especially in promoting the standardization of molecular monitoring 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.

Liquid biopsy testing is rapidly emerging as a diagnostic means of identifying circulating free DNA (cfDNA) disease-associated variants. However, the reporting of cfDNA variants remains inconsistent due in part to the application of multiple testing pipelines which raise uncertainty about current cfDNA detection efficiency. External quality assurance (EQA) programs are required to monitor, evaluate and help improve laboratory performance for cfDNA variant detection and in clinical interpretation. This study therefore evaluated the performance of diagnostic laboratories currently performing cfDNA testing in China, Australia and New Zealand. A total of 89 laboratories participated in this EQA program. Reference testing material comprised of cfDNA manufactured to contain six different genotypes in four different genes (EGFR, KRAS, BRAF, NRAS). The predicted genotypic variant allelic frequencies ranged between 0.5% - 2.5%. Proficiency testing used a z-score on the laboratory consensus allelic frequency data to compare laboratory performance for the detection of the different genotypes. Allelic frequency genotyping data were received from 88 of the 89 laboratories. Next generation sequencing and digital PCR testing platforms were primarily used by participants in this pilot EQA. The average consensus data for each cfDNA genotype identified allelic frequencies ranging between 0.39% - 4.4%. Z-score proficiency testing found that >92% of clinical laboratories were concordant for detecting the cfDNA variants. The data from this pilot study suggest that current cfDNA testing platforms can detect cfDNA allelic frequency variants from 0.39% and above with high levels of confidence. In addition, these data highlight the importance of laboratories enrolling on EQA programs so that proficiency in cfDNA diagnostic testing can be determined and potential sources of error identified and addressed.

Molecular measurable residual disease (MRD) monitoring based on real-time quantitative reverse transcription PCR (RT-qPCR) plays an important role in acute promyelocytic leukemia (APL) management, but the performance status of clinical reports is unknown. This study focuses on the specific elements in molecular MRD monitoring report and their impact on clinical decision-making. The participating laboratories were asked to submit real and formal clinical reports for mock samples panel with APL clinical case. The MRD-specific elements were analyzed and summarized. The significance of longitudinal MRD monitoring curve and the missing MRD-specific elements for clinical decision-making were assessed. MRD-specific elements were significantly missing in clinical reports. The element “testing results” existed great inconsistencies in the written form of testing items and data. The longitudinal MRD monitoring curve of false-negative or false-positive MRD result was obviously different from all-correct. It not only identified MRD time point of tissue sampling relative to treatment and ensured the reliability of the negative MRD results, but also gave MRD diagnosis, clinical interpretation, and further recommendation. Clinician-friendly reports with MRD-specific elements can better serve clinical practice. The correctly intuitive results and clinically important MRD-specific elements can provide a good description of the reliability and clinical significance of MRD results.

Recently, a novel technique named the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas)9 system has been rapidly developed. This genome editing tool has improved our ability tremendously with respect to exploring the pathogenesis of diseases and correcting disease mutations, as well as phenotypes. With a short guide RNA, Cas9 can be precisely directed to target sites, and functions as an endonuclease to efficiently produce breaks in DNA double strands. Over the past 30 years, CRISPR has evolved from the 'curious sequences of unknown biological function' into a promising genome editing tool. As a result of the incessant development in the CRISPR/Cas9 system, Cas9 co-expressed with custom guide RNAs has been successfully used in a variety of cells and organisms. This genome editing technology can also be applied to synthetic biology, functional genomic screening, transcriptional modulation and gene therapy. However, although CRISPR/Cas9 has a broad range of action in science, there are several aspects that affect its efficiency and specificity, including Cas9 activity, target site selection and short guide RNA design, delivery methods, off-target effects and the incidence of homology-directed repair. In the present review, we highlight the factors that affect the utilization of CRISPR/Cas9, as well as possible strategies for handling any problems. Addressing these issues will allow us to take better advantage of this technique. In addition, we also review the history and rapid development of the CRISPR/Cas system from the time of its initial discovery in 2012.

In 2014, Ebola hemorrhagic fever broke out in West Africa. As contact between China and West Africa is frequent, the possibility that Ebola virus would enter China was high. Thus, an external assessment of the quality of Ebola virus detection was organized by the National Center for Clinical Laboratories in China. Virus-like particles encapsulating known sequences of epidemic strains of Ebola virus from 2014 were prepared as positive quality controls. The sample panel, which was composed of seven positive and three negative samples, was dispatched to 19 laboratories participating in this assessment of Ebola virus detection. Accurate detection was reported at 14 of the 19 participating laboratories, with a sensitivity of 91.43% and a specificity of 100%. Four participants (21.05%) reported false-negative results and were classified as "acceptable." One participant (5.26%) did not detect any positive samples and was thus classified as "improvable." Based on the results returned, the ability to detect weakly positive Ebola specimens should be improved. Furthermore, commercial assays and the standard primers offered by the Chinese Centers for Disease Control and Prevention were found to be most accurate and dependable for Ebola detection. A two-target detection approach is recommended for Ebola screening; this approach could reduce the probability of false-negative results. Additionally, standardization of operations and punctual adjustment of instruments are necessary for the control and prevention of Ebola virus.

Introduction Currently, several approaches are being used to detect echinoderm microtubule associated protein like 4 gene ( EML4 )–anaplastic lymphoma receptor tyrosine kinase gene ( ALK ) rearrangement, but the performance of laboratories in China is unknown. To evaluate the proficiency of different laboratories in detecting EML4-ALK rearrangement, we organized a proficiency test (PT). Methods We prepared formalin-fixed, paraffin-embedded samples derived from the xenograft tumor tissue of three non–small cell lung cancer cell lines with different EML4-ALK rearrangements and used PTs to evaluate the detection performance of laboratories in China. Results We received results from 94 laboratories that used different methods. Of the participants, 75.53% correctly identified all samples in the PT panel. Among the errors made by participants, false-negative errors were likely to occur. According to the methodology applied, 82.86%, 76.67%, 77.78%, and 66.67% of laboratories using reverse transcriptase polymerase chain reaction, fluorescence in situ hybridization, next-generation sequencing, and immunohistochemical analysis, respectively, could analyze all the samples correctly. Moreover, we have found that the laboratories' genotyping capacity is high, especially for variant 3. Conclusion Our PT survey revealed that the performance and methodological problems of laboratories must be addressed to further increase the reproducibility and accuracy of detection of EML4 - ALK rearrangement to ensure reliable results for selection of appropriate patients.

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.