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APPLICATIONS OF CRISPRi IN HIGH THROUGHPUT METABOLIC ENGINEERING

A modular and constructive technology, applied in the high-throughput field, can solve problems such as expensive, low-efficiency effects, and off-target mutations

Pending Publication Date: 2021-04-23
ZYMERGEN INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

De novo CRISPR guide RNA design and gene targeting can be time-consuming and expensive and also susceptible to inefficiencies and possible off-target mutations

Method used

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  • APPLICATIONS OF CRISPRi IN HIGH THROUGHPUT METABOLIC ENGINEERING

Examples

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example

[0380] The following examples are given for the purpose of illustrating various embodiments of the present disclosure and are not meant to limit the present disclosure in any way. As defined by the scope of the claims, example variations and other uses will occur to those skilled in the art that are encompassed within the spirit of the present disclosure.

example 1

[0381] Example 1: One-pot in vitro modular CRISPR cloning

[0382] This example describes the generation of plasmid 13001009086 (SEQ ID NO: 82) by transferring an insert from one plasmid to another in a one-pot reaction. see Figure 4 .

[0383] Both plasmids carry cloning tags (cTAG K[SEQ ID NO:78] / cTAGL[SEQ ID NO:79] and cTAGK'[SEQ ID NO:80] / cTAGL'[SEQ ID NO:80] ID NO:81]). To drive cloning responses to edited plasmids, the Cpf1 spacer was in opposite orientation (K / K' and L / L', respectively) on the acceptor and donor plasmids. This inside-out / outside-in digestion removes the Cpf1 spacer in the final product, thereby eliminating re-cleavage of the desired product (see Figure 4 Curved arrows in , which depict inside-out digestion in the '485 plasmid and outside-in digestion in the '784 plasmid). The Cas9 spacer is retained, enabling iterative editing at this site. Thus, large modular constructs allow for a fast one-pot reaction scheme for iterative editing.

[0384] T...

example 2

[0387] Example 2: In vitro modular CRISPR cloning

[0388] This example is designed to demonstrate the flexibility of CRISPR cloning. As an initial step, several resistance plasmids encoding kanamycin or chloramphenicol resistance genes were generated from source vectors pzHR039 (SEQ ID No: 100) and 13000223370 (SEQ ID No: 101 ), respectively. The kanamycin resistance plasmids were each designed to contain various Cpf1 landing sites flanking the GFP gene (when digested, these plasmids produced a "kanamycin-resistant plasmid backbone"). The chloramphenicol resistance plasmids were each designed to contain various Cpf1 landing sites flanking the chloramphenicol resistance gene (when digested, these plasmids produced "chloramphenicol-resistant inserts"). The sequence and vector map of each plasmid used in this example is disclosed in Table 6.

[0389] Each kanamycin- and chloramphenicol-resistant plasmid was initially linearized with type II restriction enzymes KpnI-HF and PvuI...

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Abstract

The disclosure describes methods, compositions, and kits for high throughput DNA assembly reactions in vitro. The disclosure further describes modular CRISPR DNA constructs comprising modular insert DNA parts flanked by cloning tag segments comprising pre-validated CRISPR protospacer / protospacer adjacent motif sequence combinations. High throughput methods of CRISPRi and CRISPRa are also disclosed.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS [0002] This application claims the benefit of priority from US Provisional Application No. 62 / 764,672, filed August 15, 2018, which is hereby incorporated by reference in its entirety for all purposes, including all descriptions, references Literature, drawings and claims. [0003] Government funding [0004] This invention was made with government support under Agreement No. HR0011-15-9-0014 awarded by DARPA. The government has certain rights in this invention. [0005] Statement Regarding Sequence Listing [0006] The Sequence Listing related to this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into this specification. The name of the text file containing the sequence listing is ZYMR_030_01WO_SeqList_ST25.txt. The text file is 799kb, created on August 14, 2019 and submitted electronically via EFS-Web. technical field [0007] The present disclosure relates to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/62C12N15/64C12N15/66C07K19/00C12K15/113
CPCC12N9/22C12N15/63C12N15/102C12N2310/20C12N15/64C12N15/113
Inventor B·柴金德H·M·范罗苏姆A·米勒P·珀科维奇S·希捷卡K·帕特尔
Owner ZYMERGEN INC
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