Gene editing technology application - RocGene (Beijing) Technology Co., Ltd.

Application Requirements

In gene editing research, accurately assessing the editing efficiency of target genes by gene editing tools such as CRISPR-Cas9 is critical. Whether it is gene knockout, knock-in or base editing, it is necessary to accurately quantify the degree and efficiency of gene editing to judge the effectiveness of gene editing strategies, and provide data support for subsequent optimization of experimental conditions and in-depth study of gene function. Therefore, research institutions need a set of cost-effective detection solutions with high temperature control accuracy, accurate quantification, strong sensitivity/specificity, and compatibility with trace samples.

Solutions

High-Throughput Editing Screening Solution - ArchiPure Series + Archimed R Series + qPCR Premix

Sample Processing: High-purity nucleic acid is extracted from samples using 16/32/96-throughput fully automated nucleic acid extractor, improving the sensitivity and reliability of subsequent detection.
Supporting Reagents: High-sensitivity reverse transcription and PCR reaction Mix, improving reverse transcription efficiency, reducing mismatch probability, and enhancing detection result accuracy.
Detection Platform: ΔΔCq method/multiplex probe method/double standard curve method for relative quantification can efficiently determine editing efficiency; 96/384-well instruments achieve ultra-high detection throughput.

Efficiency Enhancement Solution - ArchiCycler Series + ArchiPure Series + Archimed R Series + qPCR Premix

Sample Processing: High-purity nucleic acid is extracted from samples using 16/32/96-throughput fully automated nucleic acid extractor, improving the sensitivity, reliability and detection throughput of subsequent detection.
Primer Validation: ArchiCycler gradient PCR supports user-defined annealing temperature with physical isolation and ±0.1℃ high-precision temperature control, enabling efficient confirmation of the optimal annealing temperature for primers and effectively improving the efficiency of Prime Editing, multi-site simultaneous editing, etc.
Supporting Reagents: High-sensitivity reverse transcription and PCR reaction Mix, improving reverse transcription efficiency, reducing mismatch probability, and enhancing detection result accuracy.
Detection Platform: 1.33× higher sensitivity; Tube heating mode ensures more accurate detection results for trace samples; ΔΔCq method/multiplex probe method/double standard curve method for relative quantification; 96/384-well instruments achieve ultra-high detection efficiency.

Application Scenarios

Editing Efficiency Validation

After CRISPR-Cas9-mediated gene knockout or knock-in experiments, it is necessary to accurately evaluate the editing efficiency of target genes to judge the performance of the gene editing system. For example, after knocking out a specific oncogene in tumor cells, it is required to detect the residual expression of the gene in cells.

Off-Target Effect Detection after Base Editing

Base editors (e.g., BE3, ABE) may cause unintended base mutations at other genomic loci. Real-time fluorescent quantitative PCR is required to detect the mutation frequency of potential off-target sites to evaluate the safety of gene editing. For example, when using therapeutic base editing to repair disease-related gene mutations, it is necessary to screen for off-target mutations across the whole genome.

Dynamic Monitoring of Target Gene Expression after Gene Editing

Gene editing (e.g., promoter editing, enhancer modification) may alter the transcriptional activity of target genes. It is necessary to dynamically monitor the mRNA expression levels of target genes at different time points after editing to analyze the effect of gene expression regulation. For example, after editing the promoter of pluripotency genes in stem cells, monitor the expression changes of the gene during differentiation.

Allele-Specific Expression Analysis of Single-Base Editing

When performing single-base editing in heterozygous cells, only one allele may be edited. Real-time fluorescent quantitative PCR is required to distinguish the expression differences between the two alleles to determine the allele specificity of editing. For example, when repairing heterozygous mutations in dominant genetic diseases, it is necessary to confirm that only the mutant allele is edited and the expression of the normal allele is not affected.

Integration Efficiency Detection of Gene Editing Vectors

In virus vector-mediated gene editing, it is necessary to detect the integration efficiency of exogenous gene editing vectors in the host genome to evaluate the safety and effectiveness of the vectors. For example, in gene therapy, when using adeno-associated virus (AAV) vectors to carry CRISPR components, it is necessary to clarify the integration copy number of the vector in the genome of target cells.

Detection Workflow

High-Throughput Editing Screening Solution - ArchiPure Series + Archimed R Series + qPCR Premix

Our Advantages

High-Throughput Detection

All solutions support 96/384 throughput detection, standard with ΔΔCq/double standard curve methods; the independently developed probe-based relative quantification analysis method can determine the expression levels of multiple genes in an ultra-efficient and rapid manner, and adapt to detection needs of different throughputs.

Excellent Sensitivity and Specificity

High-performance self-developed enzyme system, combined with ultra-fast temperature ramping rate and 1.33× resolution instrument sensitivity, ensures high sensitivity and specificity of results.

Wide Applicability

Gradient temperature can be edited independently; physical isolation design ensures the performance of optimizing primer reaction conditions; ±0.1℃ temperature accuracy + Tube temperature control mode is more suitable for the needs of micro-volume reactions.

Success Cases

Scenario: Researchers performed gene editing on mouse embryonic stem cells by knocking out a specific gene to investigate its regulatory role in the expression of embryonic stem cell differentiation-related genes. Through real-time fluorescent quantitative PCR technology, the expression level changes of multiple genes related to embryonic stem cell differentiation (e.g., Oct4, Sox2, Nanog, etc.) before and after gene editing were detected.

Requirements: After verifying gene knockout, it is necessary to determine the expression level changes of multiple genes related to embryonic stem cell differentiation (e.g., Oct4, Sox2, Nanog, etc.) before and after editing. A platform that can complete the detection of relative expression values of multiple target genes in a single experiment is required to improve detection efficiency.

Solution:

Researchers used Archimed R6 with probe-based relative quantification method, labeling with different fluorescence channels to detect the expression of three different target genes in one experiment.

Results:

The results showed that after gene knockout, the expression levels of pluripotency genes such as Oct4, Sox2, and Nanog decreased significantly, while the expression of differentiation-related genes was upregulated. Further analysis revealed that the knocked-out gene may affect the differentiation process of embryonic stem cells by regulating the expression and binding activity of related transcription factors. It also demonstrated the important value of Archimed R series instruments in the simultaneous expression validation of multiple genes in gene editing research.

On-site Usage Photos

Detection Results