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Modified bacterial reverse transcription elements with enhanced DNA production

A transcribed, purposeful technology in the field of modified bacterial reverse transcription elements with enhanced DNA production that addresses inefficiencies and background limitations

Pending Publication Date: 2022-06-24
THE J DAVID GLADSTONE INST A TESTAMENTARY TRUST ESTABLISHED UNDER THE WILL OF J DAVID GLADS +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although promising, these applications suffer from lower-than-anticipated efficiency and background limitations, which may arise from elements in the endogenous form of retrotranscription

Method used

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  • Modified bacterial reverse transcription elements with enhanced DNA production
  • Modified bacterial reverse transcription elements with enhanced DNA production
  • Modified bacterial reverse transcription elements with enhanced DNA production

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0261] Example 1: Materials and Methods

[0262] This example illustrates some of the material methods used to develop the present invention.

[0263] Bacterial strains, plasmids and culture conditions

[0264] Experiments were performed in BL21-AI E. coli (Thermo Fisher), which contains integrated arabinose-inducible T7 polymerase, endogenous CRISPR array, endogenous reverse transcriptase, but no endogenous Cas1+2 or bMS .346 (which is a variant of MG1655 with integrated arabinose-inducible T7 polymerase but no endogenous retrotranscriptase).

[0265] exist Figure 3B , 3C In the reverse transcriptase-based experiments described in , 4D, 7B, 7C-2, 8B, 9B, and 10B to D, the reverse transcriptase was expressed from a plasmid with an erythromycin-inducible promoter (mphR-ec86RT) (see Rogers et al., Nucleic Acids Res. 2015 Sep3;43(15):7648-60.doi:10.1093 / nar / gkv616.Epub 2015 Jul 7, which is hereby incorporated by reference in its entirety). The msd and msr elements were ex...

Embodiment 2

[0274] Example 2: Bacterial reverse transcription elements (retrotranscripts) for enhancing DNA production in cells

[0275] Rewriting the genome of living cells requires new DNA. Currently, workers synthesize this DNA exogenously and deliver it to cells, where it serves as a template for genome engineering or as a barcode to mark cells or cellular events. However, delivering sufficiently abundant exogenous DNA to overcome inefficiencies in homology-directed repair (HDR) and integration processes, especially in complex tissues, remains extremely challenging. Furthermore, there is no way to gate delivery by cell type or cell state to enable targeting of subpopulations of cells with specific DNA sequences.

[0276] If we can produce engineered DNA sequences abundantly and on-demand within the cells of our choice - just as we produce RNA and proteins - we can overcome the inefficiencies of HDR and unlock the ability to deliver different templates to different cells. We can us...

Embodiment 3

[0282] Example 3: Screening of engineered retrotran variants

[0283] The inventors expressed Retrocon-Eco1 (also known as ec86 or Retro-Eco1 ncRNA) in E. coli. The sequence of this wild-type retro-Eco1 ncRNA is shown below as SEQ ID NO: 15.

[0284]

[0285] Quantitative PCR (qPCR) showed that expression of the expressed reverse transcript-Eco1 in E. coli yielded approximately 800 to 1,000 ssDNA copies / cell ( Figure 3B ). like Figure 3C As shown, the ssDNA thus produced can be visualized, quantified and purified on denaturing gels.

[0286] The inventors also produced constructs encoding various retroconductive elements. For example, reverse transcriptase is separated from msr / msd (primer-template), allowing msr and msd to be provided to reverse transcriptase in trans (rather than the typical cis arrangement) ( Figure 4A , 4B). This trans arrangement eliminates the cryptic termination signal of reverse transcriptase. The sequence of the retrotran-Eco1 msr-only ...

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Abstract

Engineered reverse transcripters that are modified to enhance the production of multi-copy single-stranded DNA (msDNA) are provided. In addition, vector systems encoding such engineered reverse transcripters and methods of using engineered reverse transcripters and vector systems encoding same in a variety of applications such as CRISPR / Cas mediated genome editing, recombinant engineering, cell barcoding, and molecular recording are also disclosed.

Description

[0001] priority application [0002] This application claims the benefit of priority from the filing date of US Provisional Application Serial No. 62 / 899,625, filed September 12, 2019, the contents of which are specifically incorporated herein by reference in their entirety. [0003] Incorporated by reference to the Sequence Listing provided as a text file [0004] The sequence listing is provided as a text file "2072305.txt", which was created on September 10, 2020, and whose size is 12,288 bytes. The contents of this text document are incorporated herein by reference in their entirety. Background technique [0005] Retroconverters are retroelements present in almost all myxobacteria (Dhundale et al. Journal of Bacteriology 164, 914-917 (1985)), in E. coli (Lampson et al. Science 243, 1033- 1038 (1989)), V. cholerae (Inouye et al. Microbiology and Immunology 55, 510-513) and rare in other bacteria. The reverse transcript operon encodes, in order, the RNA primer (multi-copy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/10
CPCC12N15/102C12N2310/20C12N15/63C12Y207/07049C12N9/1276C12Q2563/179C12N9/22C12N2800/80C12Q1/48
Inventor 塞特·希普曼
Owner THE J DAVID GLADSTONE INST A TESTAMENTARY TRUST ESTABLISHED UNDER THE WILL OF J DAVID GLADS