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Nuclease-mediated DNA assembly

A technology of nuclease and exonuclease, which is applied in the direction of DNA preparation, recombinant DNA technology, and the introduction of foreign genetic material using vectors, etc. It can solve problems such as limited accuracy, time-consuming multi-step reactions, and unpredictable off-target effects.

Active Publication Date: 2017-05-24
REGENERON PHARM INC
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, existing techniques suffer from limited precision, which can lead to unpredictable off-target effects and time-consuming multi-step reactions

Method used

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  • Nuclease-mediated DNA assembly
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Examples

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example

[0178] The following examples are given to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are All or only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (eg amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

example 1

[0179] Example 1: BAC digestion with CAS9 and assembly with selection cassette

[0180] Artificial crRNA and artificial tracrRNA were designed to target specific sequences in MAID 6177 (116 kb LTVEC) for assembly with a 3 kb PCR product (UB-HYG). The PCR product contained a 50 bp region overlapping the vector. crRNA and tracrRNA were first dissolved in duplex buffer (30 mM HEPES, pH 7.5, 100 mM potassium acetate) to 100 μM. To anneal the RNA, add 10 µL of 100 µM crRNA and 10 µL of 100 µM tracrRNA to 80 µL of annealing buffer. Heat the RNA in a 90°C heating block, then remove the heating block from the heater and allow to cool on the bench. The final concentration of RNA was about 10 μM.

[0181] To digest BAC, use clean maxiprep BAC DNA and digest BAC according to the mixture below.

[0182]

[0183]

[0184] Digest at 37°C for 1 hour, then desalt for 30 minutes. The final reaction buffer contained: 20 mM Tris 7.5; 100-150 mM NaCl; 10 mM MgCl2; 1 mM DTT; 0.1 mM ED...

example 2

[0194] Example 2: Stitching together two overlapping BACs: humanization in the mouse MHC II locus (H2-A / H2-E) HLA-DQ+Humanized HLA-DR

[0195] Artificial crRNA and artificial tracrRNA were designed to target specific sequences in humanized HLA-DQ BAC for assembly with humanized HLA-DR BAC. These vectors contain approximately 70 bp overlapping regions formed by Cas9 cleavage at two sites on each vector (see figure 2 ). Dissolve crRNA and tracrRNA in Hybe buffer to 100 μM. To anneal the RNA, add 10 µL of 100 µM crRNA and 10 µL of 100 µM tracrRNA to 80 µL of annealing buffer. Place the RNA in a 90°C heating block, then remove the heating block from the heater and allow to cool on the bench. The final concentration of RNA was about 10 μM.

[0196] For digestion of BAC, clean maxiprep BAC DNA can be used. Digest each BAC individually according to the following mix:

[0197]

[0198] The BAC vector should be digested at 37°C for 1 hour and then heat inactivated at 65°C...

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Abstract

Methods are provided herein for assembling at least two nucleic acids using a sequence specific nuclease agent (e.g., a gRNA-Cas complex) to create end sequences having complementarity and subsequently assembling the overlapping complementary sequences. The nuclease agent (e.g., a gRNA-Cas complex) can create double strand breaks in dsDNA in order to create overlapping end sequences or can create nicks on each strand to produce complementary overhanging end sequences. Assembly using the method described herein can assemble any nucleic acids having overlapping sequences or can use a joiner oligo to assemble sequences without complementary ends.

Description

[0001] Cross References to Related Applications [0002] This application claims U.S. Provisional Application No. 62 / 015,809, filed June 23, 2014, U.S. Provisional Application No. 62 / 016,400, filed June 24, 2014, and U.S. Provisional Application No. 62 / 016,400, filed August 13, 2014. 62 / 036,983, each of these US provisional applications is hereby incorporated by reference in its entirety. [0003] As a text file submitted via EFS WEB [0004] Electronically submit the official text of the sequence listing as a sequence listing in ASCII format via EFS-Web, the file name is 461002SEQLIST.TXT, the creation date is June 23, 2015, the file size is 66KB, and the file is consistent with this manual Commit at the same time. The Sequence Listing contained in this document in ASCII format is part of this specification and is hereby incorporated by reference in its entirety. Background technique [0005] In the past, overlap extension has been used as a means of synthesizing larger d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12N15/64C12N15/66
CPCC12N15/1031C12N15/64C12N15/66C12N15/10
Inventor 克里斯·舒恩赫约翰·麦克沃特科里·莫蒙林恩·麦克唐纳安德鲁·J.·墨菲格雷格·S.·沃肖约瑟·F.·罗哈斯卡曼·维纳斯·莱大卫·M.·巴伦苏埃拉凯特琳·蒙塔尼亚
Owner REGENERON PHARM INC
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