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Large genomic DNA knock-in and uses thereof

A genomic, large-scale technology applied in the field of large-scale genomic DNA knock-in and its application

Inactive Publication Date: 2018-08-31
JACKSON LAB THE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite advances in CRISPR technology, however, to the best of Applicant's knowledge, HDR (eg, HR)-based recombination is currently limited to editing relatively small regions of genomic DNA

Method used

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  • Large genomic DNA knock-in and uses thereof
  • Large genomic DNA knock-in and uses thereof
  • Large genomic DNA knock-in and uses thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0203] Example 1: Knock-in of 25000 base pair BAC-derived genomic DNA by CRISPR / Cas9-stimulated homologous recombination

[0204] This example describes the invention described herein for the humanization of specific regions of the mouse genome on the scale of 10,000 to 100,000 base pairs (10 to 100 kb) using large exogenous genomic DNA from human genomic DNA fragments use in. More specifically, this example provides a CRISPR-driven replacement (eg, humanization) of approximately 17,000 bases of the mouse tumor suppressor gene Bcl2l11 with an orthologous, disease-associated 25-kb fragment of the corresponding human gene BCL2L11 Base pair (17kb) fragment.

[0205] a) Large exogenous genomic DNA from humans

[0206]In this case, BAC DNA was purified from a BAC clone (human: library RP11, clone 695-B-23) containing a gene of interest such as the human BCL2L11 gene. The purified DNA was then electroporated into recombinant E. coli strain SW102. see figure 1 The third row fro...

Embodiment 2

[0286] Example 2: Knock-in of a 25,000 base pair BAC-derived donor molecule by traditional ES cell-based homologous recombination

[0287] In contrast to the CRISPR / Cas9-driven approach described in Example 1, we also used a traditional approach involving classical ES cell targeting, dual selection, and recombinase-driven cassette removal.

[0288] The general steps in this conventional method are substantially the same as those in Example 1 except for the following specific steps.

[0289] b') Preparation of targeting vectors using bacterial artificial chromosomes (BACs)

[0290] In obtaining the pTLD14 plasmid (see Example 1), we encountered some difficulties with blasticidin-based selection of embryonic stem (ES) cells. Therefore, we negatively select the rpsL intermediate with Puro R The open reading frame (ORF) of Bsd replaced R open reading frame.

[0291] The resulting vector, pTLD39, contains a 27,282-bp central fragment of the human BCL2L11 gene flanked by 12,773-...

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Abstract

The present invention provides compositions and methods for utilizing a large capacity cloning vector (e.g., BAC) to carry a large exogenous genomic DNA (about 10-300 kb) flanked by a proximal and distal regions (10 kb) to efficiently insert into the genome of a cell in a CRISPR / Cas9-stimulated homologous recombination. Methods and compositions for microinjecting a large human gene into a mouse zygote to prepare a genetically modified mouse are also provided.

Description

[0001] Citations to Related Applications [0002] This application claims the benefit of priority under 35 U.S.C. § 119(e) to US Provisional Application No. 62 / 252,080, filed November 6, 2015, the entire contents of which are incorporated herein by reference in their entirety. [0003] governmental support [0004] This invention was made with US Government support under Grant No. P30CA034196 awarded by the National Cancer Institute (NCI) of the National Institutes of Health (NIH). The United States Government has certain rights in this invention. Background technique [0005] Genome editing is a type of genetic engineering in which DNA is inserted, replaced, or removed from the genome. In recent years, genome editing has been achieved using artificially engineered nucleases (also known as "molecular scissors"). The nucleases create double-strand breaks (DSBs) at desired locations in the genome and utilize the cell's endogenous machinery to repair through homology-direct...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/027C07K14/47C12N15/90C12N15/113C12N9/22C12N15/85
CPCA01K67/0278A01K2207/15A01K2217/072A01K2227/105C07K14/4747C12N9/22C12N15/907C12N2015/8527C12N2740/16043C12N15/11C12N2800/80C12N2310/20
Inventor D·贝格斯特罗姆T·莱迪-戴维斯
Owner JACKSON LAB THE