Exploiting off‐targeting in guide‐<scp>RNA</scp>s for <scp>CRISPR</scp> systems for simultaneous editing of multiple genes

Ferreira, Raphael; Gatto, Francesco; Nielsen, Jens

doi:10.1002/1873-3468.12835

Cited by 11 publications

(9 citation statements)

References 32 publications

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“…Gene deletions were performed in the strain IMX581 using CRISPR/Cas9 as described previously ( 34 ). The diagnostic primers and the repair fragments (listed in Dataset S1 ) were designed using the Yeastriction webtool ( yeastriction.tnw.tudelft.nl/ ), with the exception of the PLB1 and PLB2 deletions, which were carried out with a single gRNA promiscuously targeting both PLB 1 and PLB2 , as described previously ( 35 ).…”

Section: Methodsmentioning

confidence: 99%

Redirection of lipid flux toward phospholipids in yeast increases fatty acid turnover and secretion

Ferreira

Teixeira

Siewers

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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SignificanceReplacement of nonrenewable petrochemicals and liquid fuels requires sustainable production of oleochemicals. Free fatty acids (FFAs) are versatile molecules that can be produced by microbial fermentation and are used as precursors for production of these oleochemicals. In the past few years, we have seen major advancements in improving the yeast Saccharomyces cerevisiae for FFA production. Despite these successes, lipid metabolism is highly complex, and the pathways and metabolites involved in the formation of FFAs in yeast remain incompletely understood. In this work, we make important advancements in understanding the dynamics of FFA formation in the cell and explore the role of phospholipids in this process.

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Section: Methodsmentioning

confidence: 99%

Redirection of lipid flux toward phospholipids in yeast increases fatty acid turnover and secretion

Ferreira

Teixeira

Siewers

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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“…A commercial service performed the sequencing, analysed the editing of the target, integrated the characteristics of the target and compiled the sequencing results ( Table 1). The higher the GC content in the target, the harder to be off target [18][19][20][21]. Further analysis of mutant plants from the T3 generation showed that CRISPR/Cas9 technology produced the following mutation types: homozygous mutations, heterozygous mutations and double allele mutations.…”

Section: Sequence Analysis Of the Target Mutation Of Positive Plantsmentioning

confidence: 99%

Construction and Analysis of GmFAD2-1A and GmFAD2-2A Soybean Fatty Acid Desaturase Mutants Based on CRISPR/Cas9 Technology

Lü

Wang

et al. 2020

IJMS

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The soybean fatty acid desaturase family is composed of seven genes, but the function of each gene has not been reported. Bioinformatics was used to analyse the structure of genes in this family, as well as the correlation between Δ12-fatty acid desaturase II (FAD2) expression and oleic acid content on different days after flowering of soybean. In the present study, CRISPR/Cas9 technology was used to construct single and double mutant knockout vectors of functional genes in the FAD2 family. Analysis of the molecular biology and expression patterns of genes in the FAD2 family, namely, GmFAD2-1A (Glyma.10G278000) and GmFAD2-2A (Glyma.19G147300), showed that they had little homology with other soybean FAD2 genes, and that their function was slightly changed. Sequencing of the target showed that the editing efficiency of the GmFAD2-1A and GmFAD2-2A genes was 95% and 55.56%, respectively, and that the double mutant editing efficiency was 66.67%. The mutations were divided into two main types, as follows: base deletion and insertion. A near-infrared grain analyser determined the following results: In the T2 generation, the oleic acid content increased from 17.10% to 73.50%; the linoleic acid content decreased from 62.91% to 12.23%; the protein content increased from 37.69% to 41.16%; in the T3 generation, the oleic acid content increased from 19.15% to 72.02%; the linoleic acid content decreased from 56.58% to 17.27%. In addition, the protein content increased from 37.52% to 40.58% compared to that of the JN38 control variety.

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“…This phenomenon is known to be accentuated in regions with sequences similar to the original sequence, e.g. paralogs genes or retrotransposons regions [24]. This feature, so far, severely hinders the technology to enter into advanced clinical phases.…”

Section: Conclusion and Discussionmentioning

confidence: 99%

Advancing biotechnology with CRISPR/Cas9: recent applications and patent landscape

Ferreira

David

Nielsen

2018

Journal of Industrial Microbiology and Biotechnology

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Clustered regularly interspaced short palindromic repeats (CRISPR) is poised to become one of the key scientific discoveries of the twenty-first century. Originating from prokaryotic and archaeal immune systems to counter phage invasions, CRISPRbased applications have been tailored for manipulating a broad range of living organisms. From the different elucidated types of CRISPR mechanisms, the type II system adapted from Streptococcus pyogenes has been the most exploited as a tool for genome engineering and gene regulation. In this review, we describe the different applications of CRISPR/Cas9 technology in the industrial biotechnology field. Next, we detail the current status of the patent landscape, highlighting its exploitation through different companies, and conclude with future perspectives of this technology.

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Exploiting off‐targeting in guide‐RNAs for CRISPR systems for simultaneous editing of multiple genes

Cited by 11 publications

References 32 publications

Redirection of lipid flux toward phospholipids in yeast increases fatty acid turnover and secretion

Redirection of lipid flux toward phospholipids in yeast increases fatty acid turnover and secretion

Construction and Analysis of GmFAD2-1A and GmFAD2-2A Soybean Fatty Acid Desaturase Mutants Based on CRISPR/Cas9 Technology

Advancing biotechnology with CRISPR/Cas9: recent applications and patent landscape

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