活细胞记录器在细胞谱系追踪中的应用和前景

doi:10.12211/2096-8280.2024-082

摘要/Abstract

摘要：

基于DNA的活细胞分子记录器技术，通过诱导可遗传的DNA变异，为细胞历史的追溯提供了一种创新手段。作为新一代细胞谱系追踪方法的代表，该技术能够与单细胞测序、多组学测序等技术相集成，帮助科研人员重构细胞发育分化路径及肿瘤起源的谱系发生树，是探究这些核心生物学议题的有效平台。本综述系统性回顾了自2016年以来基于Cas9的分子记录器在谱系追踪领域的技术演变轨迹与应用进展，同时综合分析了一些新型分子记录器的研究动态，并对其优势与局限性进行了评估。自2016年以来，以CRISPR-Cas9系统为核心的分子记录器取得了显著进展，并逐渐成为该领域的主流技术，研究人员在优化编辑效率和增加记录位点等方面进行了充分的探索。尽管如此，以Cas9为基础的分子记录器仍面临CRISPR-Cas9系统固有的限制与挑战，例如DNA双链断裂带来碱基缺失，进而引起记录信息丢失。这促使研究者们探索开发新型分子记录器，以期作为谱系追踪的更高效精准的工具。先导编辑器、DNA结合蛋白融合碱基编辑器以及T7 转录聚合酶融合碱基编辑器等基于新原理的分子记录器能够避免DNA双链断裂，以碱基替换而非碱基缺失的形式写入信息。相较于Cas9系统，它们展现出独特优势，同时也伴随着潜在的风险与挑战。先导编辑器可以以时间顺序的方式记录信息，但脱靶效应仍然是一个问题。DNA结合蛋白融合碱基编辑器提高了编辑效率和特异性，但它们在不同细胞类型中的有效性需要进一步探索。T7 RNA聚合酶融合碱基编辑器已经在体内定向进化系统中取得了成功，但它们目前在哺乳动物系统中的应用仍然有限。未来，基于DNA的分子记录器的研究应着重于优化编辑效率、降低信息丢失率、提高谱系恢复效率，并探索其在复杂生物系统中的应用潜力。

关键词: 分子记录器, 细胞谱系追踪, CRISPR相关蛋白, 基因突变, 碱基编辑

Abstract:

Tracing the division and differentiation history of cells is a critical issue in organismal development and cancer research. Live cell DNA-based molecular recorders, a synthetic biotechnology that induces heritable DNA variations, offers an innovative approach for reconstructing cell lineage histories. As a representative of the new generation of cell lineage tracing methods, this technology can be integrated with high-throughput single-cell sequencing and multi-omics sequencing, enabling the reconstruction of developmental differentiation pathways of cells and the phylogenetic trees of tumorigenesis. Live cell DNA-based molecular recorders serve as an effective platform for exploring these core biological questions. This review systematically analyzes the technological evolution of Cas9-based molecular recorders in lineage tracing since 2016 and its demonstrated applications, while also analyzing the research trends of some novel molecular recorders and evaluating their advantages and limitations. Since 2016, molecular recorders based on the CRISPR-Cas9 system have made significant progress and have gradually become the mainstream technology in this field. However, Cas9-based molecular recorders still suffer from several inherent limitations, such as the low lineage resolution due to insufficient editing efficiencies, the loss of recorded information caused by DNA double-strand breaks, and potential lineage merging due to barcode homoplasy. These limitations pose challenges to prompt researchers to explore and develop new types of molecular recorders as more efficient and precise tools for cell lineage tracing. Novel molecular recorders based on new principles, such as prime editors, DNA-binding protein-fused base editors, and T7 RNA polymerase-fused base editors, can avoid DNA double-strand breaks and record information through base substitutions rather than deletions. Compared to the Cas9 system, they exhibit unique advantages but also come with potential risks and challenges. Prime editors can record information in a temporal sequential manner, but off-target effects remain a concern. DNA-binding protein-fused base editors offer high editing efficiencies and specificities, but their effectiveness across different cell types requires further exploration. T7 RNA polymerase-fused base editors have achieved success in in vivo directed evolution systems, but their application in mammalian systems is still limited. In the future, the research of DNA-based molecular recorders should focus on optimizing editing efficiency, reducing information loss rate, improving lineage recovery efficiency, and exploring their application potential in complex biological systems.

Key words: molecular recorder, cell lineage tracing, CRISPR-related protein, gene mutation, base editing

中图分类号:

Q816

姜百翼, 钱珑. 活细胞记录器在细胞谱系追踪中的应用和前景[J]. 合成生物学, DOI: 10.12211/2096-8280.2024-082.

Baiyi Jiang, Long Qian. Application and prospect of live cell molecular recorder in cell lineage tracing[J]. Synthetic Biology Journal, DOI: 10.12211/2096-8280.2024-082.

图/表 5

图1 理想的分子记录器［42-44］和Cas9分子记录器的代表性研究［22，38-40，42-43，48-49］（Rosa26 Col1a1：基因组上landing pad位点，用于外源基因的定点整合。R26， pU6， CAG， EF1A：组成型启动子。TetO：诱导型启动子，由rtTA激活。M2-rtTA：诱导系统，由诱导剂doxycycline（dox）激活。）

Fig. 1 Ideal molecular recorder[42-44] and representative studies of Cas9-based molecular recorders[41-42,45-47,49-51](Rosa26, Col1a1: landing pad sites on genome, for site-specific integration of exogenous genes. R26, pU6, CAG, EFIA: constitutive promoters. TetO: inducible promoter, activated by rtTA. M2-rtTA: inducible system, activated by doxycycline(dox) inducer. )

表1 Cas9分子记录器的特点总结（追踪时间：参考文献中的最长实验时间。是否稳定整合：即是否将分子记录器整合到细胞基因组上。）

Table 1 A summary of the characteristics of Cas9-based molecular recorders (Tracing time: Maximal experimental time in the cited study. Stable integration: whether the molecular recorder is integrated into the cell genome.)

名称及参考文献	分子记录器	记录窗口大小/bp	靶点数量/site	主要突变类型	追踪时间/days	物种	是否稳定整合	研究领域
GESTALT^[41]	Cas9	266	10	碱基删除	3	斑马鱼	否	发育
scGESTALT^[47]	Cas9	363	10	碱基删除	23-25	斑马鱼	否	发育
MARC1^[46]	Cas9	240	~20	碱基删除	19-21	小鼠	是	发育
LINNAEUS^[45]	Cas9	75	16-32	碱基删除	5	斑马鱼	否	发育
CHYRON^[48]	Cas9+TdT	100	5	碱基插入	22	HEK293T细胞系	是	—
CARLIN^[49]	Cas9	276	10	碱基删除	7	小鼠	是	发育
DARLIN^[42]	Cas9+TdT	828	30	碱基插入	7	小鼠	是	发育
2021年的一项研究^[50]	Cas9	—	~30	碱基删除	54	小鼠	是（仅肿瘤）	肿瘤
KP-TRACER^[51]	Cas9	—	30-90	碱基删除	150-180	小鼠	是（嵌合体）	肿瘤

图2 重建谱系发生树与整合组学数据分析［43，51］、Cas9分子记录器的缺点与错误构建的谱系发生树［3，42，51］

Fig. 2 Reconstructed phylogeny trees and integrated omics data analysis[43,51], shortcomings of Cas9-based molecular recorders and errors in reconstructing phylogeny trees [3,42,51]

图3 三种新型分子记录器的工作原理［64，79，94，98］（pegRNA：先导编辑引导RNA。HsAID：碱基编辑器。iSceI：DNA结合蛋白。rtTA：诱导系统。本图图片均来自参考文献并被翻译为中文。）

Fig. 3 The working principles of three novel molecular recorders[64,79,94,98](pegRNA: prime editing guide RNA. HsAID: base-editor. iSceI: DNA binding protein. rtTA: inducible system. Images are all from references and translated into Chinese.)

表2 新型分子记录器与Cas9分子记录器对比分析

Table 2 Comparative analysis of novel molecular recorders and Cas9-based molecular recorder

分子记录器	稳定性	时序写入	特异性	编辑窗口大小	编辑效率	技术发展程度
Cas9^{[41-42,45-51]}	易丢失	否	易脱靶	窄	——	高
Prime Editing^[64-66]	高	是	易脱靶	窄	低于Cas9	中
DNA结合蛋白^[77-79]	高	否	强	宽	高于Cas9	中
Muta-T7^[94-98]	高	否	强	可调节	未知	低

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