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通过长读长测序对CRISPR RNA引导的转座产物进行无偏分析。

Unbiased profiling of CRISPR RNA-guided transposition products by long-read sequencing.

作者信息

Vo Phuc Leo H, Acree Christopher, Smith Melissa L, Sternberg Samuel H

机构信息

Department of Molecular Pharmacology and Therapeutics, Columbia University, New York, NY, USA.

Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA.

出版信息

Mob DNA. 2021 Jun 8;12(1):13. doi: 10.1186/s13100-021-00242-2.

Abstract

Bacterial transposons propagate through either non-replicative (cut-and-paste) or replicative (copy-and-paste) pathways, depending on how the mobile element is excised from its donor source. In the well-characterized E. coli transposon Tn7, a heteromeric TnsA-TnsB transposase directs cut-and-paste transposition by cleaving both strands at each transposon end during the excision step. Whether a similar pathway is involved for RNA-guided transposons, in which CRISPR-Cas systems confer DNA target specificity, has not been determined. Here, we apply long-read, population-based whole-genome sequencing (WGS) to unambiguously resolve transposition products for two evolutionarily distinct transposon types that employ either Cascade or Cas12k for RNA-guided DNA integration. Our results show that RNA-guided transposon systems lacking functional TnsA primarily undergo copy-and-paste transposition, generating cointegrate products that comprise duplicated transposon copies and genomic insertion of the vector backbone. Finally, we report natural and engineered transposon variants encoding a TnsAB fusion protein, revealing a novel strategy for achieving RNA-guided transposition with fewer molecular components.

摘要

细菌转座子通过非复制性(剪切粘贴)或复制性(复制粘贴)途径进行传播,这取决于移动元件如何从其供体来源中切除。在特征明确的大肠杆菌转座子Tn7中,异源三聚体TnsA-TnsB转座酶在切除步骤中通过在每个转座子末端切割两条链来指导剪切粘贴转座。对于RNA引导的转座子(其中CRISPR-Cas系统赋予DNA靶标特异性)是否涉及类似途径尚未确定。在这里,我们应用基于群体的长读长全基因组测序(WGS)来明确解析两种进化上不同的转座子类型的转座产物,这两种转座子类型利用Cascade或Cas12k进行RNA引导的DNA整合。我们的结果表明,缺乏功能性TnsA的RNA引导转座子系统主要进行复制粘贴转座,产生共整合产物,该产物包含重复的转座子拷贝和载体骨架的基因组插入。最后,我们报告了编码TnsAB融合蛋白的天然和工程化转座子变体,揭示了一种用更少分子成分实现RNA引导转座的新策略。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1e3a/8188705/9d007541f146/13100_2021_242_Fig1_HTML.jpg

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