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System for multi-round editing of fungal genome by CRISPR system and method thereof

A gene editing and genome technology, applied in the field of genetic engineering and biology, can solve the problems of fewer types of resistance markers, lower positive rate, and difficult gene editing tasks

Active Publication Date: 2019-09-06
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the CRISPR-Cas editing system of filamentous fungi still has certain defects. First, because it is difficult to obtain suitable auxotrophic strains in most filamentous fungi, and they are only sensitive to a few antibiotics, there are very few types of resistance markers that can be selected. ;Secondly, if the traditional CRISPR-Cas9 system is used for editing, since the genome-edited cells are transferred into Cas9, gRNA expression vectors, and donor DNA, the cells cannot perform multiple rounds of genome editing due to the presence of these selection markers ; and when the genome sequence of more than 5 sites is edited at the same time, the positive rate will decrease; therefore, in the case where multiple genes (more than 10) need to be destroyed, deleted or inserted at the same time, the task of gene editing becomes quite difficult and time-consuming

Method used

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  • System for multi-round editing of fungal genome by CRISPR system and method thereof
  • System for multi-round editing of fungal genome by CRISPR system and method thereof
  • System for multi-round editing of fungal genome by CRISPR system and method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045]Example 1. Construction of the Myceliophthora thermophila genome editing system mediated by CRISPR-Cas12a (AsCpf1)

[0046] 1. Construction of Cas12a (AsCpf1) expression cassette vector

[0047] With plasmid p0380-bar (Liu Q, Gao RR, Li JG, Lin LC, Zhao JQ, Sun WL, TianCG. Development of a genome-editing CRISPR / Cas9system in thermophilic fungal Myceliophthora species and its application to hyper-cellulase productionstrain engineering. Biotechnology for Biofuels.2017, 10:1.) to construct an expression vector for the backbone. With reference to the genome of Myceliophthora thermophila, Cas12a (AsCpf1) is selected from the AsCpf1 protein of Acidaminococcussp.BV3L6 to carry out codon bias optimization (codons are optimized according to the expression preference of Myceliophthora thermophila), in the N of AsCpf1 protein The nuclear localization signal sequence (PPRKRAKTEDE) of Myceliophthora thermophila transcription factor hacI (MYCTH_2310995) was added to the C-terminal an...

Embodiment 2

[0082] Example 2, CRISPR-Cas12a (AsCpf1) system for simultaneous editing of multiple genes in the Myceliophthora thermophila genome

[0083] 1. The crRNA single-plasmid system realizes editing of multiple genes in the genome

[0084] The first group of editing system is to mix Cas enzyme expression box Ptef1-AsCpf1-TtprC, crRNA expression elements U6p-crRNA-cre1, U6p-crRNA-res1 and U6p-crRNA-gh1-1 in molar ratio, and its homologous supply The body DNA fragments donor-2-cre1, donor-res1 and donor-1-gh1-1 are mixed in equimolecular molar ratios, the above three are the total amount of Cas enzyme expression box, crRNA and homologous donor DNA in molecular After co-transformation into the protoplast cells of Myceliophthora thermophila wild-type strain ATCC 42464 at a molar ratio of 1:1:1, Cas12a (AsCpf1) was mediated by crRNA through the protospacer and the DNA of the target gene on the genome of the host cell The strands are paired to recognize the target site for cleavage, and ...

Embodiment 3

[0097] Example 3, CRISPR-Cas12a (AsCpf1) system multiple rounds of genome editing of Myceliophthora thermophila

[0098] 1. The crRNA tandem plasmid system realizes the second round of genome editing

[0099] The second group of editing system is the Cas enzyme expression cassette Ptef1-AsCpf1-TtprC, the tandem expression element U6p-array2-neo-alp1-rca1-hcr1, and its donor DNA fragments donor-1-alp1, donor-2-gh1-1 , donor-rca1 and donor-hcr1 were mixed at equal molecular molar ratios, and the above three were co-transformed into Myceliophthora thermophila cre1 at a molecular molar ratio of 1:1:1, and the three genes res1 and gh1-1 had been edited at the same time In the first round of mutant strain 3M protoplast cells, Cas12a (AsCpf1) is mediated by crRNA, through protospacer pairing with the DNA strand of the target gene on the host cell genome to recognize the target site for cutting, and then the donor DNA fragment is combined with Homologous recombination occurs between ...

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Abstract

The invention discloses a system for multi-round genome editing in fungal cell by a CRISPR system, and a method for multi-round genome editing in fungal cells. The system for multi-round genome editing performs multi-round editing operations on the target fungal genome, wherein the homologous donor DNA in each round of editing operations contains one or more corresponding homologous donor DNA sequences for editing by aiming at different genes, only one donor homologous donor DNA contains a marker gene, and the marker genes of two adjacent rounds are different. The system successfully realizesconvenient, high-efficiency, multi-round (unrestricted) editing of the filamentous fungal genome, and has important significance for genome-directed editing and metabolic engineering of filamentous fungi.

Description

technical field [0001] The invention belongs to the field of genetic engineering and biotechnology, and specifically relates to the transformation of fungal genomes, more specifically to a filamentous fungal genome editing system and a method for editing the filamentous fungal genome. Background technique [0002] Filamentous fungi are not only important model strains for the study of eukaryotic life, but also major producers of antibiotics, enzymes and organic acids. Filamentous fungi are widely used to produce commercially valuable biological substances, so research on filamentous fungi has become particularly important, among which research on gene function and genetic modification of filamentous fungi is currently the main focus, and genome editing technology It is currently the most potential technical means for analyzing gene functions and genetically improving strains. Among them, clustered regularly interspaced short palindromic repeats and related systems (CRISPR-C...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N9/22C12N15/80C12N15/113C12N15/90
CPCC12N9/22C12N15/80C12N15/113C12N15/902C12N2800/22C12N2310/10C12N2310/20
Inventor 田朝光刘倩张永利李芳雅
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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