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Methods and reagents for characterizing genomic editing, clonal expansion, and associated applications

A genome editing, genome technology, applied in the fields of genomics, biochemical equipment and methods, proteomics, etc., can solve problems such as sensitivity that cannot provide early selection

Pending Publication Date: 2021-04-16
TWINSTRAND BIOSCI INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, currently used assays do not provide the sensitivity to detect such early selection

Method used

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  • Methods and reagents for characterizing genomic editing, clonal expansion, and associated applications
  • Methods and reagents for characterizing genomic editing, clonal expansion, and associated applications
  • Methods and reagents for characterizing genomic editing, clonal expansion, and associated applications

Examples

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example 1

[0246] In one example, an initial cell population is subjected to targeted genome editing using a CRISPR / Cas9 endonuclease system. Cas9 causes double-strand breaks in genomic DNA, which has been shown in some applications to induce cell cycle arrest through the TP53 pathway (ie, PMID 29892062, PMID 29892067). Theoretically, genome editing has lower efficiency in some cases, such as in embryonic stem cells, due to cell cycle arrest induced by double-strand break reactions, which are mediated by TP53. Those cells that did not stall after the genome editing process likely contained inactivating mutations or deletions or loss of TP53 (or other cancer driver genes) that disrupted the corresponding function and resulted in less restricted cell growth. Clonal expansion of cells carrying this mutation will outcompete neighboring cells. In this example, double-sequencing will be used to generate error-corrected consensus sequence reads of targeted double-stranded DNA molecules and det...

example 2

[0248] This example describes the use of dual sequencing to determine whether early mutations in cancer driver genes reflect the tumorigenic potential of a test carcinogen. Using model mutation inducers, this example demonstrates that dual sequencing is capable of resolving mutations in a single DNA molecule in a population. This example demonstrates that the methods of the present disclosure provide the necessary sensitivity to detect such early neoplastic clonal selection of cells in a cell population and the subsequent clonal expansion of such cells.

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Abstract

Methods for characterizing genome editing, clonal expansion and associated reagents for use in such methods are disclosed herein. Some embodiments of the technology are directed to characterizing a population of cells following an engineered genomic editing event, that includes in some embodiments characterizing genomic alterations occurring at both intended and unintended genomic loci within the genome of the populations of cells. Other embodiments are directed to utilizing Duplex Sequencing for assessing a clonal selection in mixed cell populations and / or cell populations following a genomic editing event. Further examples of the present technology are directed to methods for detecting and assessing clonal expansion of cells following a genomic editing event.

Description

[0001] related application [0002] This application claims priority and benefit to U.S. Provisional Patent Application No. 62 / 697,397, filed July 12, 2018, the disclosure of which is hereby incorporated by reference in its entirety. Background technique [0003] For a variety of applications, the potential for clonal expansion and early neoplastic clonal selection in mixed cell populations, such as cells harboring mutations in cancer drivers, needs to be assessed. However, currently used assays do not provide the sensitivity to detect such early selection. Furthermore, in the field of targeted genome editing, tools are needed to assess the successful application of genome editing without further non-targeted nucleic acid alterations. Contents of the invention [0004] The present technology generally relates to methods for detecting and evaluating clonal selection and / or amplification, and related reagents for such methods. In particular, some embodiments of the technolog...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N15/10C12N15/11C12Q1/02C12Q1/6844C12Q1/6809
CPCC12Q1/6869C12Q1/6806G16B20/20G16B30/10C12Q2525/191C12Q2565/514C12Q2537/165C12Q2523/109C12Q2600/156
Inventor J·J·索尔克C·C·瓦伦丁三世
Owner TWINSTRAND BIOSCI INC