基于 CRISPR 的穿梭克隆：一种高通量克隆方法
June 13th, 2025
我们描述了一种高通量克隆方法的方案，即基于 CRISPR 的穿梭克隆（CRISPR穿梭克隆），它允许在载体之间转移感兴趣的 DNA 片段，而无需对 DNA 片段进行 PCR 扩增。
我们描述了高通量克隆方法CRISPRshuttle的方案。这涉及在载体之间转移目标 DNA 片段，而无需对 DNA 片段进行 PCR 扩增。现有技术，如门户输注和单载体克隆 PCR 扩增。这种方法需要特定片段的程序，包括初级设计和分泌验证。这些标签是劳动密集型且耗时的。CRISPR 穿梭消除了 PCR，同时将载体之间的目标 DNA 片段转移到连续的试管反应中。该方法超越了对特定片段处理的需求，从而加快了样品的构建。
[旁白]首先，通过组合屏幕上显示的试剂，为给定数量的消化反应制备预混液。将正确标记的 0.2 毫升管放在冰上的铝制冷却块中。通过混合适量的 CAS9、sgRNA、10、CAS9 缓冲液和 DEPC 处理水，制备用于 N 次反应的预混液。将预混液彻底混合并旋转。将 3.75 微升预混液等分到每个反应管中。将 0.75 微升 0.03 微摩尔 PLX 304 ORF 质粒加入每个彻底混合的试管中，并将试管在 37 摄氏度下孵育一小时。通过混合 0.14 微升 3.36 微摩尔线性化 pBIDC-UASC-pLXvect 和 1.8 微升 Gibson 组装预混液来制备预混液。将预混液彻底混合并旋转。现在将 1.94 微升预混液等分到每个试管中。将 1.66 微升 CAS9 切割质粒加入每个试管中。充分混合并在 50 摄氏度下孵育一小时。在冰上解冻细菌感受态细胞。将 10 微升解冻的细胞等分到每个预冷的 1.5 毫升管中。轻轻地将 10 微升感受态细胞与 1 微升 Gibson 组装产物混合。将试管放在冰上 30 分钟。在 42 摄氏度下对试管进行热电击一分钟，然后在冰上冷却两分钟。将 100 微升预热的 SOC 培养基加入每个试管中，并在 37 摄氏度下以 250 RPM 摇动一小时。最后，将细胞置于含有每毫升氯霉素15微克的LB琼脂平板上，并在37摄氏度下孵育过夜。该图显示了使用 CRISPRshuttle 系统生成的 UAS-cDNA/ORF 质粒限制性分析的代表性结果。在该分析中，对 15 个 UAS-cDNA/ORF 构建体的 PVU2 限制性酶切表明，所有样品在大约 1072 个碱基对和 1,820 个碱基对处表现出预期的片段模式。
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概述
本文介绍了一种称为CRISPR基因梭克隆（CRISPRshuttle克隆）的高通量克隆方法的协议。这种创新技术无需PCR扩增即可在载体之间转移DNA片段。
关键研究组成部分
科学领域
- 基因工程
- 分子生物学
- CRISPR技术
背景
- 传统的克隆方法通常需要PCR扩增。
- CRISPR技术彻底改变了基因操作。
- 高通量方法对大规模研究至关重要。
- 高效的载体转移对各种应用至关重要。
研究目的
- 介绍一种新型克隆方法。
- 简化DNA片段转移过程。
- 消除克隆程序中对PCR的需求。
使用的方法
- CRISPRshuttle克隆协议的开发。
- DNA转移效率的评估。
- 与传统克隆方法的比较。
- 该方法在各种载体系统中的应用。
主要结果
- 成功地在载体之间转移了DNA片段。
- 与PCR方法相比，显著减少了时间和资源。
- 在多次测试中展示了高效率。
- 在基因研究中具有广泛应用的潜力。
结论
- CRISPRshuttle克隆是传统方法的可行替代方案。
- 该方法提高了基因工程的效率。
- 未来的研究可以利用这种技术进行复杂的基因操作。
常见问题
Executive Industry Relevance
High-throughput generation of genome-wide plasmid libraries is a critical enabler for systematic gene function studies and target validation in biopharma R&D. CRISPR-based shuttle cloning (CRISPRshuttle) eliminates PCR amplification, streamlining the transfer of DNA fragments between vectors and accelerating library construction. This capability enhances predictive confidence and operational efficiency at key discovery inflection points.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables rapid assembly of genome-scale plasmid libraries for functional genomics screens.
- Supports systematic interrogation of gene function across diverse biological pathways.
- Facilitates biological de-risking and robust target validation by enabling high-throughput construct generation.
- Improves predictive confidence for downstream target selection and triage.
Screening & Assay Development
- Prepares validated plasmid constructs for scalable screening workflows without PCR-induced sequence artifacts.
- Standardizes cloning outputs, supporting reproducibility and quantitative assay development.
- Accelerates readiness for high-throughput compound evaluation and phenotypic screening platforms.
- Enables efficient reuse of donor plasmid resources across multiple assay formats.
Translational & Preclinical Research
- Streamlines the generation of disease-relevant genetic constructs for preclinical model development.
- Supports continuity from discovery through preclinical validation by enabling rapid construct iteration.
- Reduces technical bottlenecks in translational biomarker and mechanistic studies.
- Enhances risk-adjusted advancement decisions by providing robust genetic tools.
Pipeline & Workflow Integration
CRISPRshuttle cloning integrates at the interface of early discovery and lead identification, enabling seamless transition from hypothesis-driven gene selection to high-throughput construct generation and screening.
- Discovery Biology: Accelerates hypothesis testing and pathway mapping by enabling rapid, scalable cloning of gene targets.
- Screening: Delivers standardized, PCR-free constructs for reproducible assay development and screening campaigns.
- Analytics: Provides quantitative outputs on cloning efficiency and construct integrity, supporting data-driven decision making.
- Translational Research: Facilitates the generation of constructs for disease-relevant models and biomarker studies.
- Enterprise Reuse: Establishes a reusable, high-throughput cloning platform for ongoing portfolio needs.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in gene function studies.
- Operational Value: Delivers high efficiency (>94%) and scalability, enabling rapid library generation by small teams.
- Strategic Value: Improves go/no-go decision quality and capital efficiency by accelerating construct availability.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of validated targets and assays.
Implementation Considerations
- Requires expertise in CRISPR/Cas9-mediated cleavage and Gibson assembly techniques.
- Needs access to compatible donor plasmids and CRISPRshuttle-ready vectors.
- Demands standardized protocols for cross-team reproducibility and data integrity.
- Adaptation may be needed for diverse vector backbones or organism-specific systems.
- Efficiency and throughput are dependent on reagent quality and workflow optimization.
