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Method for editing prokaryotic genomes using endogenic CRISPR-Cas (CRISPR-associated) system

A genome editing and prokaryotic technology, applied in the field of prokaryotic genome editing, can solve the problems of limiting the application of CRISPR/Cas9 system, affecting the activity of Cas9 protein, affecting the specificity of gene editing, etc., achieving short time period, reducing workload and screening The effect of high positive rate

Active Publication Date: 2016-02-17
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

First, CRISPR / Cas9 may have off-target effects. It has been found that Cas9 protein may allow a certain degree of mismatch between crRNA and target sequence, and the number and position of these mismatches will inevitably affect the specificity of gene editing.
Secondly, to use the exogenous CRISPR / Cas9 system, it is necessary to optimize the Cas9 protein suitable for host cells, and to express Cas9 protein and sgRNA (guide RNA) in the cell at the same time to play a role, and the procedure is relatively complicated.
In addition, in some organisms with extreme growth conditions, the intracellular environment may affect the activity of Cas9 protein, thus limiting the application of CRISPR / Cas9 system

Method used

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  • Method for editing prokaryotic genomes using endogenic CRISPR-Cas (CRISPR-associated) system
  • Method for editing prokaryotic genomes using endogenic CRISPR-Cas (CRISPR-associated) system
  • Method for editing prokaryotic genomes using endogenic CRISPR-Cas (CRISPR-associated) system

Examples

Experimental program
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Effect test

Embodiment 1

[0038] A method for editing a prokaryotic genome using an endogenous CRISPR-Cas system, comprising the following steps:

[0039] This example uses the endogenous (Type I-A CRISPR system and Type III-B CRISPR system) CRISPR system of S. islandicus REY15A to carry out precise deletion mutation of its lacS gene as an example to illustrate:

[0040] 1. Construction of Editing Plasmids

[0041] (1) A total of 40 bases from +933 to +972 were selected from the S. islandicus REY15Aβ-galactosidase gene lacS (SIRE_RS11295) as the protospacer, and its reverse complementary sequence was 5'-AGTGTAGTAATTAACACCAATCCAGTCTAACCTACCCTT-3', It is next to a CCT-PAM (Protospacer Adjacent Motif), so it can be targeted by the Type I-A CRISPR system; at the same time, because the 5' end sequence of crRNA is mismatched with the corresponding target site sequence, it can also be targeted by the Type III-B CRISPR system at the same time. Based on this protospacer, two primers (LacS-E-SpF / LacS-E-SpR) (Ta...

Embodiment 2

[0054] A method for editing a prokaryotic genome using an endogenous CRISPR-Cas system, comprising the following steps:

[0055] In the embodiment of the present invention, the C-terminus of the Cmr-2α protein of S. islandicus REY15A endogenous III-B type CRISPR system is used as an example to illustrate the 6×His tag insertion mutation.

[0056] 1. Construction of Editing Plasmids

[0057] (1) Select the last 25 bases of the cmr-2α gene (SIRE_RS04505) and the first 7 bases of the cmr-3α gene on the S. islandicus REY15A genome as a protospacer, and its reverse complement The sequence is 5'-AGTGTAGTAATTAACACCACAATCCAGTCTAACCTACCCCT-3', without CCN-PAM in front of it, so it only has the DNA interference activity of type III-B CRISPR. Based on this protospacer, two primers (2α-His-SpF / 2α-His-SpR) (Table 1) were designed, and the two primers were annealed to generate spacer fragments with sticky ends at both ends;

[0058] (2) The artificial CRISPR vector pSe-Rp is digested with...

Embodiment 3

[0070] A method for editing a prokaryotic genome using an endogenous CRISPR-Cas system, comprising the following steps:

[0071] This example uses the endogenous Type I-A CRISPR system of Sulfolobus Icelandicus REY15A to carry out multi-site mutation of its Cmr-2α protein HDdomain as an example to illustrate.

[0072] 1. Construction of Editing Plasmids

[0073] (1) On the S. islandicus REY15A genome, select 40 bases at the four conserved amino acids H / D / K / I of the N-terminal HDdomain of the cmr-2α gene as the protospacer, and its sequence is 5'-CGACCCTCCTTGGAAGGCATGGGTAATTACAAGGAATATT-3 ', preceded by CCA-PAM. Based on this protospacer, two primers (2α-HDmut-SpF / 2α-HDmut-SpR) were designed, and the two primers were annealed to generate a spacer fragment with sticky ends at both ends;

[0074] (2) The artificial CRISPR vector pSe-Rp is digested with BspMI, and the digested product is enzyme-ligated with the above spacer fragment to obtain an artificial CRISPR plasmid;

[0075...

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Abstract

The invention relates to a method for editing prokaryotic genomes using an endogenic CRISPR-Cas (CRISPR-associated) system. An editor plasmid carrying both an artificial CRISPR cluster and a donor DNA is required to be established for the genome editing of a prokaryote containing the endogenic CRISPR-Cas system, after NDA interference is caused by the endogenic CRISPR-Cas to the genome, and the genome is edited through homologous recombination. The method has the advantages that a range of applicable hosts is wide, all bacteria and archaea containing the endogenic CRISPR-Cas being available for operation; the method is applicable to operations in various editing manners, such as deletion, insertion and point mutation; the editing efficiency is higher, screening positive rage is high, and the background is low; a flow is simple, a short period of time is required, and the workload of prokaryotic genome editing is greatly reduced.

Description

technical field [0001] The invention belongs to the fields of genomics, genetic engineering and biotechnology, and specifically relates to a method for editing prokaryotic genomes by using an endogenous CRISPR-Cas system. Background technique [0002] CRISPR-Cas system, as an acquired immune system for prokaryotes to resist viruses and other foreign invading nucleic acids, widely exists in about 90% of archaea and 40% of bacteria (VanderOostJetal., 2014; BarrangouRetal., 2014). CRISPR-Cas systems are divided into three main types: type I, type II, and type III; they each have a signature protein: Cas3, Cas9, and Cas10 (MakarovaK Setal., 2011). The type II CRISPR system only requires a Cas9 protein, a crRNA and a trans-acting RNA to perform DNA interference activity (DeltchevaE etal., 2011; Gasiunas Getal., 2012). The simple type II CRISPR system has therefore been developed into a eukaryotic genome editing tool (Jinek Metal., 2012; Wang Hetal., 2013), and is widely used in ...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N15/70C12N1/21
Inventor 佘群新梁运祥李英俊潘赛夫任敏冯明霞彭楠
Owner HUAZHONG AGRI UNIV
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