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CN-121975807-A - Self-guiding stpegRNA, recombinant plasmid containing same and CRISPR editing method for cell lineage tracking

CN121975807ACN 121975807 ACN121975807 ACN 121975807ACN-121975807-A

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to a self-guiding stpegRNA, a recombinant plasmid containing the self-guiding stpegRNA and a CRISPR editing method for cell lineage tracking. The core of the invention is a DNA sequence, which can be re-identified and mediated by editing and self-extension of the DNA after being transcribed into pegRNA, and the sequence is characterized in that the 5'-3' sequences at the head end and the tail end of pegRNA are basically consistent, and a pegRNA skeleton (scaffold) is subjected to self-oriented (home) transformation. INFINITE PRINTER development marks the era of the transfer of CRISPR lineage tracking technology from "limited records" to "unlimited resolution". The method realizes the collaborative breakthrough of capacity, resolution and safety through the fundamental reconstruction of the editing mode, and provides a brand new paradigm for research of developmental biology, oncology and regenerative medicine. In the future, with the improvement of editing efficiency and the innovation of calculation algorithms, the system is expected to become a standard tool for analyzing complex biological systems and plays a revolutionary role in the fields of personalized medicine, early diagnosis of diseases and the like.

Inventors

  • CHEN DEMENG
  • ZHANG ZHIHUI
  • LI YINGYI
  • WANG CHENG
  • PENG LIANG

Assignees

  • 中山大学附属第一医院

Dates

Publication Date
20260505
Application Date
20260119

Claims (9)

  1. 1. A self-directing type stpegRNA, which is pegRNA formed by transcription of a DNA sequence, wherein the 5'-3' sequences at the head and tail ends of pegRNA are substantially identical, and the framework sequence is self-directing engineered so that pegRNA can re-recognize and mediate editing and self-extension of its own coding DNA sequence.
  2. 2. The self-directing device stpegRNA of claim 1, wherein the 5' end of the "GTT" trinucleotide in the pegRNA framework sequence is replaced with "GGG".
  3. 3. The self-directing stpegRNA of claim 1 or 2, wherein the protospacer region of pegRNA comprises repeat units that are initiated by an 'AC' dinucleotide.
  4. 4. The self-directing stpegRNA as described in claim 3, wherein the total length of the protospacer region is 16 bp.
  5. 5. A recombinant plasmid comprising the coding sequence of self-directing stpegRNA of any one of claims 1 to 4.
  6. 6. The recombinant plasmid of claim 5, further comprising a Prime Editor expression element and/or a Base Editor expression element.
  7. 7. The recombinant plasmid of claim 6, wherein the Prime Editor expression element comprises a PEmax coding sequence driven by a CMV promoter and the Base Editor expression element comprises a AncBE max coding sequence driven by a CMV promoter.
  8. 8. A CRISPR editing method for lineage tracking, comprising the steps of: (1) Introducing the recombinant plasmid of any one of claims 5 to 7 into a target cell; (2) Under the action of Prime Editor and/or Base Editor, the self-directed stpegRNA mediates editing and extension of the target DNA sequence; (3) The edited DNA sequence was analyzed by sequencing to construct cell lineages.
  9. 9. The method of claim 8, wherein the editing mode comprises two modes: the Base Editor mediates C-T mutation in a protospacer near-end editing window, and then the Prime Editor recognizes mutation sites and guides sequence extension; mode two, prime Editor directly binds to original repeat sequence to mediate sequence extension when protospacer is not edited by Base Editor.

Description

Self-guiding stpegRNA, recombinant plasmid containing same and CRISPR editing method for cell lineage tracking Technical Field The invention belongs to the technical field of molecular biology, and particularly relates to a self-guiding stpegRNA, a recombinant plasmid containing the self-guiding stpegRNA and a CRISPR editing method for cell lineage tracking. Background Cell lineage tracking is a gold standard for understanding cell fate, and cell barcode labeling (Cellular barcoding) technology enables characterization of cell lineages by introducing variable DNA markers into cells in combination with high throughput sequencing. As an emerging technology, cell bar code marking provides new possibility for recording and analyzing molecular event sequence, SCGESTALT is an important breakthrough in the process, DNA bar codes are edited by using a CRISPR base editing system to increase diversity of the bar codes, relatively accurate interpretation （Raj B, Wagner DE, McKenna A, Pandey S, Klein AM, Shendure J, Gagnon JA, Schier AF: Simultaneous single-cell profiling of lineages and cell types in the vertebrate brain.Nat Biotechnol 2018, 36(5):442-450.）. of cell fate under single cell resolution is realized by combining a single cell transcriptome sequencing technology, and provides an important solution （Wang R, Dang M, Harada K, Han G, Wang F, Pool Pizzi M, Zhao M, Tatlonghari G, Zhang S, Hao Det al: Single-cell dissection of intratumoral heterogeneity and lineage diversity in metastatic gastric adenocarcinoma.Nat Med 2021, 27(1):141-151. for solving sources of tumor heterogeneity, functions of dominant clone in tumor metastasis process, critical molecular mechanism and other serious medical problems when tumor drug resistance occurs and develops, and the method for preparing the DNA bar codes by using the CRISPR base editing system Simeonov KP, Byrns CN, Clark ML, Norgard RJ, Martin B, Stanger BZ, Shendure J, McKenna A, Lengner CJ: Single-cell lineage tracing of metastatic cancer reveals selection of hybrid EMT states.Cancer Cell 2021, 39(8):1150-1162 e1159.）. Currently, by expressing different sgrnas and introducing target barcodes corresponding to the different sgrnas, the CRISPR-Cas9 system can edit a plurality of different target barcodes to increase the number of cells （Bowling S, Sritharan D, Osorio FG, Nguyen M, Cheung P, Rodriguez-Fraticelli A, Patel S, Yuan WC, Fujiwara Y, Li BEet al: An Engineered CRISPR-Cas9 Mouse Line for Simultaneous Readout of Lineage Histories and Gene Expression Profiles in Single Cells.Cell 2020, 181(7):1693-1694., Li L, Bowling S, McGeary SE, Yu Q, Lemke B, Alcedo K, Jia Y, Liu X, Ferreira M, Klein AMet al: A mouse model with high clonal barcode diversity for joint lineage, transcriptomic, and epigenomic profiling in single cells.Cell 2023, 186(23):5183-5199 e5122. and Xie L, Liu H, You Z, Wang L, Li Y, Zhang X, Ji X, He H, Yuan T, Zheng Wet al: Comprehensive spatiotemporal mapping of single-cell lineages in developing mouse brain by CRISPR-based barcoding.Nat Methods 2023, 20(8):1244-1255.）. which can be eventually tracked, however, increasing the sgrnas brings more off-target events, possibly causing damage to key genes, has higher genotoxicity (Genetic Toxin), means that the number of the sgrnas and the target barcodes which can be introduced is limited, is exhausted within a certain period, and can not reach the requirement of describing mammalian cell lineages with a large number of cells and a long life cycle without sacrificing resolution. On the basis, nicholas and the like develop a target bar code （Hughes NW, Qu Y, Zhang J, Tang W, Pierce J, Wang C, Agrawal A, Morri M, Neff N, Winslow MMet al: Machine-learning-optimized Cas12a barcoding enables the recovery of single-cell lineages and transcriptional profiles.Mol Cell 2022, 82(16):3103-3118 e3108.）. which can be repeatedly edited in a machine learning mode, and the target bar code can be repeatedly edited through the combination of two pairs of crRNA and the target bar code, so that a large number of unique DNA bar codes can be generated, and lineage tracking is performed on a large number of cells. However, this approach still relies on double strand breaks and non-homologous end joining. Furthermore, due to its repeated editing of the target bar code, it may result in the loss or corruption of existing lineage information, thereby affecting the integrity of the record. The lineage tree built by the method is highly dependent on deduction and reproduction of editing events by an algorithm, and cannot reflect the actual development process of organisms. And repeated editing of the same target site guided by a single crRNA suggests that the system allows off-target events to occur, has potential genotoxicity, and may be more difficult to achieve in vivo application to model animals. These techniques significantly improve the resolution of cell dynamics observed by humans, but still face many challenges, from the root,