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Genome editing by guided endonuclease and single-stranded oligonucleotide

An endonuclease, oligonucleotide technology, applied in the field of genome editing by guided endonuclease and single-stranded oligonucleotide, can solve the problem of difficulty in finding PAM site protospacer sequences, undesired, etc. question

Pending Publication Date: 2021-06-25
NOVOZYMES AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] A limitation of common genome editing methods is that it may be difficult to find suitable PAM sites and good protospacer sequences near where you intend to modify the genome, and / or between the target site and the point mutation in the open reading frame The incorporation of multiple silent changes in codons of , may lead to undesired effects such as alternative splicing in eukaryotes

Method used

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  • Genome editing by guided endonuclease and single-stranded oligonucleotide
  • Genome editing by guided endonuclease and single-stranded oligonucleotide
  • Genome editing by guided endonuclease and single-stranded oligonucleotide

Examples

Experimental program
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Effect test

example

[0209] The purpose of these examples was to demonstrate that directed mutagenesis or genome editing using polynucleotide-directed endonucleases such as Cas9 or MAD7 is possible using single-stranded oligonucleotides as donor DNA.

[0210] strain

[0211] Trichoderma reesei BTR213 is described in WO 2013 / 086633. Trichoderma reesei strain TrGMEr62-24a2-1 is a ku70 disrupted strain of Trichoderma reesei BTR213.

[0212] A. oryzae AT526 is a ligD disrupted strain derived from JaL1903 described in WO 18167153 (Example 4).

[0213] Media and Solutions

[0214] LB+Amp medium consists of: 10 g of Bacto TM Tryptone, 5g of Bacto TM Yeast extract, 5 g of sodium chloride, 1 ml of 100 mg / ml ampicillin (filter sterilized and added after autoclaving) and deionized water made up to 1 liter. Sterilize the solution by autoclaving.

[0215] PDA board by 39g Difco TM Consisting of potato dextrose agar and deionized water made up to 1 liter. Sterilize the solution by autoclaving.

[0216]...

example 1

[0227] Example 1: Trichoderma reesei protoplast production

[0228] Protoplast preparation and transformation of Trichoderma reesei was performed using a similar protocol to Penttila et al., 1987, Gene [Gene] 61:155-164. Briefly, Trichoderma reesei was grown for 17 hours at 27°C in two shake flasks containing 25 ml of YPD medium each with gentle agitation at 90 rpm. Mycelia were collected by filtration using a vacuum driven disposable filtration system (Millipore) and washed twice with deionized water and twice with 1.2M sorbitol. The washed mycelia were suspended in 30 ml of Yatalase containing 5 mg / ml by gently shaking at 75-90 rpm at 34 °C TM (Takara Bio USA, Inc.) and 0.5 mg / ml chitinase (Sigma Chemical Co.) in 1.2 M sorbitol for 60-75 minutes to generate protoplasts body. Protoplasts were collected by centrifugation at 834xg for 6 minutes and washed twice with cold 1.2M sorbitol. Protoplasts were counted using a hemocytometer and resuspended to a final concentration o...

example 2

[0229] Example 2: CRISPR / Cas9 Backbone Vector pSMAI290

[0230] Plasmid pSMAI290 (SEQ ID NO: 1, figure 1 ) is a CRISPR / Cas9 expression plasmid for use with The HiFi DNA Assembly Cloning Kit (New England Biolabs Inc.) clones the protospacer into BglII digested pSMAI290. Plasmid pSMAI290 contains the S. pyogenes Cas9 protein coding sequence (nucleotides 9968-14,098 in pSMAI290) and is codon-optimized for use in Aspergillus niger, and at the 3' end of the S. pyogenes Cas9 open reading frame. SV40 nuclear localization signal (NLS; nucleotides 14,072-14,095) to ensure that Cas9 will be localized to the nucleus. Expression of S. pyogenes Cas9 was under the control of the A. nidulans tef1 promoter (nucleotides 9082-9967) and terminator (nucleotides 14,099-14,297) from pFC330-333 ( et al, 2015, PLoS One [PLOS ONE] 10(7):1-18). Plasmid pSMAI290 also has all elements for single guide RNA (sgRNA) expression and consists of: Magnaporthe oryzae U6-2 promoter (nucleotides 8186-8685), s...

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Abstract

The present invention relates to methods for introducing one or more desired nucleotide modification(s) in a target sequence in the genome of a microorganism cell using a polynucleotide-guided endonuclease, e.g., the MAD7 enzyme isolated and described by InscriptaTM or the well-known Streptococcus pyogenes Cas9, together with a suitable guide RNA for each target sequence to be be modified, to generate a site-specific cut or nick in at least one genome target sequence, followed by the repair of the cut(s) and / or nick(s) via at least one oligonucleotide capable of hybridizing with the at least one genome target sequence, thereby highly efficiently introducing the one or more desired modification(s) into the target sequence.

Description

[0001] Sequence Listing Reference [0002] This application contains a Sequence Listing in computer readable form. This computer readable form is incorporated herein by reference. technical field [0003] The present invention provides endonucleases for guidance by employing programmable polynucleotides (eg, by Inscripta TM A method for modifying the genome of a host cell with the MAD7 enzyme or the well-known Streptococcus pyogenes Cas9) is isolated and described in conjunction with one or more single-stranded oligonucleotides as donor DNA. Background technique [0004] The so-called CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Cas9 genome editing system, originally isolated from Streptococcus pyogenes, has been widely used as a tool to modify the genomes of various microorganisms as well as higher organisms. [0005] The programmable Cas9 enzyme has two RNA-guided DNA endonuclease domains capable of targeting specific genomic sequences. This system...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90C12N15/10C12N15/113
CPCC12N15/102C12N15/113C12N2310/20C12N15/111
Inventor G.穆齐-埃里赫森N.约胡姆森
Owner NOVOZYMES AS