CRISPR-Cas介导的基因编辑和慢病毒载体（Lentiviral vector，LV）介导的基因转导是当前细胞与基因治疗领域的两项核心技术，广泛应用于治疗多种遗传性和获得性疾病。然而，脱靶效应和随机整合引发的潜在安全性问题，依然是限制这些技术临床转化的重大障碍。

现有脱靶检测方法，如GUIDE-seq、OliTag-seq和DISCOVER-seq+，虽然推动了该领域的进展，但仍存在灵敏度不足、对不同断裂末端类型适应性差、标签依赖性强等局限，难以全面覆盖钝末端（Blunt end）和黏性末端（Staggered end）断裂，同时也无法高效监测病毒载体整合事件。因此，亟需开发一种灵敏度高、适用范围广、操作简便的新型检测平台，以提升基因编辑和病毒载体转导的安全性评估水平。针对上述问题，本研究在优化的OliTag-seq基础上，开发了新一代脱靶检测平台——AviTag-seq。该方法基于AAV-ds39载体递送修饰ds39标签，能够在体内外精准捕获双链断裂（Double-stranded break，DSB）位点，特别在钝末端与黏性末端类型下，表现出更高的检测灵敏度。AviTag-seq结合反向末端重复序列（Inverted terminal repeat，ITR）引物和12nt唯一分子标识符（Unique molecular identifier，UMI）策略，突破了传统方法中的引物偏倚问题，实现了双端无方向性扩增，从而大幅度提升了脱靶事件的检测精度与捕获效率。ITR引物的应用不仅打破了对特定标签序列的依赖，使AviTag-seq具备更广泛的适用性，可直接应用于所有基于腺相关病毒（Adeno-associated virus，AAV）载体的基因编辑体系，同时支持同步监测AAV整合位点，为AAV基因治疗产品的安全性监控提供了更加全面且高效的技术方案。

实验结果表明，AviTag-seq在SpCas9系统中，脱靶检测精度略优于OliTag-seq；而在Cas12a系统中，检测到的reads数量提升了最多20倍，显著增强了对黏性末端断裂的检测能力。与传统ds39引物相比，ITR-UMI策略使得检测到的reads数量提升了约25倍，独特脱靶位点数量增加了1.9倍，极大提高了脱靶事件的捕获效率与准确性。此外，AviTag-seq在小鼠体内实验中表现出优异性能，显示其在脱靶检测的灵敏度、准确性和成本控制方面，明显优于目前常用的DISCOVER-seq+方法。

在慢病毒整合位点解析方面，传统基于PCR的方法往往存在扩增偏倚，容易对高丰度插入事件产生偏好，导致低丰度、罕见插入位点被忽略，进而影响整合谱图的准确性。本研究借鉴INSPIIRED策略，引入阻断型寡核苷酸，并系统优化PCR扩增条件，结合LTR-UMI标记，实现了单拷贝分辨率的整合位点精准定量分析。在群体细胞水平，我们共检测到约40万个整合位点；在单克隆细胞中，通过LTR-UMI策略实现了百万分之一的丰度分辨率，成功识别出若干具有长期稳定表达潜力的优势整合位点。此外，我们还通过慢病毒转导293T、K562、iPSC和T细胞，系统评估了不同细胞类型中慢病毒整合后的稳定表达特性，筛选出适用于后续载体优化与安全港验证的潜在优选整合位点。

综上所述，本研究通过“AAV-标签+ITR-UMI”双重优化，在体内外体系中实现了对钝末端与黏性末端DSB的高灵敏捕获，并同步输出AAV整合图谱；结合LTR-UMI单拷贝精度的慢病毒整合位点解析，构建了覆盖CRISPR基因编辑与病毒载体转导全流程的安全性评估平台。AviTag-seq及相关整合分析策略具有高通量、低偏倚、适用性广和易于复用的优势，为基因治疗和癌症免疫治疗等临床应用提供了坚实的技术支撑。我们期待本研究成果能够加速基因治疗技术的临床转化，推动基因编辑疗法向更高安全性与更高有效性方向迈进。

CRISPR-Cas-mediated gene editing and lentiviral vector (LV)-mediated gene transduction are two core technologies in the current field of cell and gene therapy, widely applied in treating various inherited and acquired diseases. However, potential safety concerns arising from off-target effects and random integration remain significant obstacles limiting the clinical translation of these technologies.

Existing off-target detection methods, such as GUIDE-seq, OliTag-seq, and DISCOVER-seq+, have advanced the field but still suffer from limitations including insufficient sensitivity, poor adaptability to different break end types, and strong tag dependency. These methods struggle to comprehensively cover blunt end and staggered end breaks and cannot efficiently monitor viral vector integration events. Therefore, there is an urgent need to develop a novel detection platform with high sensitivity, broad applicability, and simple operation to enhance the safety assessment of gene editing and viral vector transduction.

Addressing these challenges, this study developed a next-generation off-target detection platform, AviTag-seq, based on an optimized OliTag-seq framework. This method utilizes an AAV-ds39 vector to deliver modified ds39 tags, enabling precise capture of double-stranded break (DSB) sites both in vitro and in vivo. It demonstrates significantly higher detection sensitivity, particularly for blunt and staggered ends. AviTag-seq incorporates inverted terminal repeat (ITR) primers and a 12nt unique molecular identifier (UMI) strategy, overcoming primer bias inherent in traditional methods. This enables bidirectional amplification, substantially improving the detection accuracy and capture efficiency of off-target events. The use of ITR primers eliminates dependency on specific tag sequences, granting AviTag-seq broader applicability. It can be directly applied to all adeno-associated virus (AAV) vector-based gene editing systems and simultaneously supports monitoring AAV integration sites, providing a more comprehensive and efficient technical solution for the safety monitoring of AAV gene therapy products.

Experimental results showed that AviTag-seq achieved slightly superior off-target detection accuracy compared to OliTag-seq in the SpCas9 system. In the Cas12a system, the number of detected reads increased by up to 20-fold, significantly enhancing the detection capability for staggered end breaks. Compared to traditional ds39 primers, the ITR-UMI strategy increased the number of detected reads by approximately 25-fold and the number of unique off-target sites by 1.9-fold, greatly improving off-target event capture efficiency and accuracy. Furthermore, AviTag-seq exhibited excellent performance in mouse in vivo experiments, demonstrating clear advantages over the commonly used DISCOVER-seq+ method in terms of sensitivity, accuracy, and cost-effectiveness for off-target detection.

Regarding lentiviral integration site analysis, traditional PCR-based methods often suffer from amplification bias, favoring high-abundance insertion events and overlooking low-abundance, rare integration sites, thereby compromising the accuracy of integration profiles. Drawing inspiration from the INSPIIRED strategy, this study introduced blocking oligonucleotides, systematically optimized PCR amplification conditions, and combined them with LTR-UMI labeling to achieve precise quantitative analysis of integration sites at single-copy resolution. At the population cell level, we detected approximately 400,000 integration sites. In monoclonal cells, the LTR-UMI strategy enabled a resolution of one part per million, successfully identifying several preferential integration sites with potential for long-term stable expression. Additionally, by transducing 293T, K562, iPSC, and T cells with lentivirus, we systematically evaluated the stable expression characteristics post-integration in different cell types, screening potential preferential integration sites suitable for subsequent vector optimization and safe harbor validation.

In summary, this study achieved high-sensitivity capture of both blunt and staggered end DSBs in in vitro and in vivo systems through the dual optimization of "AAV-tag + ITR-UMI", simultaneously generating AAV integration profiles. Combined with LTR-UMI-based lentiviral integration site analysis at single-copy resolution, we constructed a comprehensive safety assessment platform covering the entire workflow of CRISPR gene editing and viral vector transduction. AviTag-seq and the associated integration analysis strategies offer advantages of high throughput, low bias, broad applicability, and easy reusability, providing robust technical support for clinical applications such as gene therapy and cancer immunotherapy. We anticipate that this research will accelerate the clinical translation of gene therapy technologies and advance gene editing therapies towards higher safety and efficacy.