Erratum: Corrigendum: Whole-rat conditional gene knockout via genome editing

ABSTRACT Candida albicans is the most common fungal pathogen of humans. Historically, molecular genetic analysis of this important pathogen has been hampered by the lack of stable plasmids or meiotic cell division, limited selectable markers, and inefficient methods for generating gene knockouts. The recent development of clustered regularly interspaced short palindromic repeat(s) (CRISPR)-based tools for use with C. albicans has opened the door to more efficient genome editing; however, previously reported systems have specific limitations. We report the development of an optimized CRISPR-based genome editing system for use with C. albicans. Our system is highly efficient, does not require molecular cloning, does not leave permanent markers in the genome, and supports rapid, precise genome editing in C. albicans. We also demonstrate the utility of our system for generating two independent homozygous gene knockouts in a single transformation and present a method for generating homozygous wild-type gene addbacks at the native locus. Furthermore, each step of our protocol is compatible with high-throughput strain engineering approaches, thus opening the door to the generation of a complete C. albicans gene knockout library. IMPORTANCE Candida albicans is the major fungal pathogen of humans and is the subject of intense biomedical and discovery research. Until recently, the pace of research in this field has been hampered by the lack of efficient methods for genome editing. We report the development of a highly efficient and flexible genome editing system for use with C. albicans. This system improves upon previously published C. albicans CRISPR systems and enables rapid, precise genome editing without the use of permanent markers. This new tool kit promises to expedite the pace of research on this important fungal pathogen.

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Conditional Gene Knockout

Encyclopedic Reference of Genomics and Proteomics in Molecular Medicine ◽

10.1007/3-540-29623-9_5948 ◽

2006 ◽

pp. 327-327

Keyword(s):

Gene Knockout ◽

Conditional Gene Knockout

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Prospects for Knockout of MYB60, a Transcriptional Repressor of Anthocyanin Biosynthesis, in Brassicaceae Plants by Genome Editing

KnE Life Sciences ◽

10.18502/kls.v7i1.10157 ◽

2022 ◽

Author(s):

Elena Mikhaylova ◽

Alexander Artyukhin ◽

Michael Shein ◽

Khalit Musin ◽

Anna Sukhareva ◽

...

Keyword(s):

Transcription Factors ◽

Genome Editing ◽

Agronomic Traits ◽

Anthocyanin Biosynthesis ◽

Gene Knockout ◽

Transcriptional Repressor ◽

Academic Community ◽

Plant Responses ◽

Individual Plant ◽

Brassica Napus L

The Brassicaceae plant family contains many economically important crops such as Brassica napus L., Brassica rapa L., Brassica oleracea L., Brassica juncea L., Eruca sativa Mill., Camelina sativa L. and Raphanus sativus L. Insufficient data on the genetic regulation of agronomic traits in these species complicates the editing of their genomes. In recent years, the attention of the academic community has been drawn to anthocyanin hyperaccumulation. This trait is not only beneficial for human health, but can also increase plant resistance to stress. MYB transcription factors are the main regulators of flavonoid biosynthesis in plants. Some of them are well studied in Arabidopsis thaliana. The AtMYB60 gene is a transcriptional repressor of anthocyanin biosynthesis, and it also negatively impacts plant responses to drought stress. Myb60 is one of the least studied transcription factors with similar functions in Brassicaceae. There is a high degree of homology between predicted MYB60 genes of A. thaliana and related plant species. However, functions of these homologous genes have never been studied. Gene knockout by CRISPR/Cas technology remains the easiest way to perform genome editing in order to discover the role of individual plant genes. Disruption of genes acting as negative regulators of anthocyanin biosynthesis could result in color staining of plant tissues and an increase in stress tolerance. In the present study, we investigated the AtMYB60 gene and its homologs in Brassicaceae plants and suggested universal gRNAs to knockout these genes. Keywords: CRISPR, Brassicaceae, MYB60, knockout, anthocyanin

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CRISPys: Optimal sgRNA design for editing multiple members of a gene family using the CRISPR system

10.1101/221341 ◽

2017 ◽

Cited By ~ 1

Author(s):

Gal Hyams ◽

Shiran Abadi ◽

Adi Avni ◽

Eran Halperin ◽

Eilon Shani ◽

...

Keyword(s):

Optimal Design ◽

Gene Family ◽

Genome Editing ◽

Reverse Genetics ◽

Gene Knockout ◽

Gene Families ◽

The Other ◽

List Type ◽

Suggested Approach ◽

Eukaryotic Genomes

AbstractThe discovery and development of the CRISPR-Cas9 system in the past few years has made eukaryotic genome editing, and specifically gene knockout for reverse genetics, a simpler, efficient, and effective task. The system is directed to the genomic target site by a programmed single-guide RNA (sgRNA) that base-pairs with the DNA target, subsequently leading to site-specific double-strand breaks. However, many gene families in eukaryotic genomes exhibit partially overlapping functions and, thus, the knockout of one gene might be concealed by the function of the other. In such cases, the reduced specificity of the CRISPR-Cas9 system, which may lead to the cleavage of genomic sites that are not identical to the sgRNA, can be harnessed for the simultaneous knockout of multiple homologous genes. Here, we introduce CRISPys, an algorithm for the optimal design of sgRNAs that would potentially target multiple members of a given gene family. CRISPys first clusters all the potential targets in the input sequences into a hierarchical tree structure that specifies the similarity among them. Then, sgRNAs are proposed in the internal nodes of the tree by embedding mismatches where needed, such that the cleavage efficiencies of the induced targets are maximized. We suggest several approaches for designing the optimal individual sgRNA, and an approach that provides a set of sgRNAs that also accounts for the homologous relationships among gene-family members. We further show by in-silico examination over all gene families in the Solanum lycopersicum genome that our suggested approach outperforms simpler alignment-based techniques.Graphical abstractHighlightsMany genes in eukaryotic genomes exhibit partially overlapping functions. This imposes difficulties on reverse-genetics, as the knockout of one gene might be concealed by the function of the other.We present CRISPys, a graph-based algorithm for the optimal design of CRISPR systems given a set of redundant genes.CRISPys harnesses the lack of specificity of the CRISPR-Cas9 genome editing technique, providing researchers the ability to simultaneously mutate multiple genes.We show that CRISPys outperforms existing approaches that are based on simple alignment of the input gene family.

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Efficient genome editing and gene knockout in Setaria viridis with CRISPR/Cas9 directed gene editing by the non-homologous end-joining pathway

Plant Biotechnology ◽

10.5511/plantbiotechnology.21.0407a ◽

2021 ◽

Author(s):

Marcos Fernando Basso ◽

Karoline Estefani Duarte ◽

Thais Ribeiro Santiago ◽

Wagner Rodrigo de Souza ◽

Bruno de Oliveira Garcia ◽

...

Keyword(s):

Genome Editing ◽

Gene Editing ◽

Gene Knockout ◽

Setaria Viridis ◽

End Joining ◽

Non Homologous End Joining

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Unrestrained markerless trait stacking in Nannochloropsis gaditana through combined genome editing and marker recycling technologies

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1718193115 ◽

2018 ◽

Vol 115 (30) ◽

pp. E7015-E7022 ◽

Cited By ~ 33

Author(s):

John Verruto ◽

Kristie Francis ◽

Yingjun Wang ◽

Melisa C. Low ◽

Jessica Greiner ◽

...

Keyword(s):

Genome Editing ◽

Gene Knockout ◽

Algal Species ◽

Nuclear Genome ◽

Gene Families ◽

Concept Generation ◽

Multiple Traits ◽

Nannochloropsis Gaditana ◽

Marker Recycling ◽

New Genes

Robust molecular tool kits in model and industrial microalgae are key to efficient targeted manipulation of endogenous and foreign genes in the nuclear genome for basic research and, as importantly, for the development of algal strains to produce renewable products such as biofuels. While Cas9-mediated gene knockout has been demonstrated in a small number of algal species with varying efficiency, the ability to stack traits or generate knockout mutations in two or more loci are often severely limited by selectable agent availability. This poses a critical hurdle in developing production strains, which require stacking of multiple traits, or in probing functionally redundant gene families. Here, we combine Cas9 genome editing with an inducible Cre recombinase in the industrial alga Nannochloropsis gaditana to generate a strain, NgCas9+Cre+, in which the potentially unlimited stacking of knockouts and addition of new genes is readily achievable. Cre-mediated marker recycling is first demonstrated in the removal of the selectable marker and GFP reporter transgenes associated with the Cas9/Cre construct in NgCas9+Cre+. Next, we show the proof-of-concept generation of a markerless knockout in a gene encoding an acyl-CoA oxidase (Aco1), as well as the markerless recapitulation of a 2-kb insert in the ZnCys gene 5′-UTR, which results in a doubling of wild-type lipid productivity. Finally, through an industrially oriented process, we generate mutants that exhibit up to ∼50% reduction in photosynthetic antennae size by markerless knockout of seven genes in the large light-harvesting complex gene family.

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31 PRODUCTION EFFICIENCY OF GENE KNOCKOUT PIGS USING GENOME EDITING AND SOMATIC CELL CLONING

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab31 ◽

2015 ◽

Vol 27 (1) ◽

pp. 108

Author(s):

H. Matsunari ◽

M. Watanabe ◽

K. Nakano ◽

A. Uchikura ◽

Y. Asano ◽

...

Keyword(s):

Somatic Cell ◽

Genome Editing ◽

Production Efficiency ◽

Gene Knockout ◽

Genetically Modified ◽

Zinc Finger Nucleases ◽

International Institute ◽

Induced Mutations ◽

Transcription Activator

Genome editing technologies have been used as a powerful strategy for the generation of genetically modified pigs. We previously developed genetically modified clone pigs with organogenesis-disabled phenotypes, as well as pigs exhibiting diseases with similar features to those of humans. Here, we report the production efficiency of various gene knockout cloned pigs from somatic cells that were genetically modified using zinc finger nucleases (ZFN) or transcription activator-like effector nucleases (TALEN). The ZFN- or TALEN-encoding mRNAs, which targeted 7 autosomal or X-linked genes, were introduced into porcine fetal fibroblast cells using electroporation. Clonal cell populations carrying induced mutations were selected after limiting dilution. The targeted portion of the genes was amplified using PCR, followed by sequencing and mutation analysis. Among the collected knockout cell colonies, cells showing good proliferation and morphology were selected and used for somatic cell nuclear transfer (SCNT). In vitro-matured oocytes were obtained from porcine cumulus-oocyte complexes cultured in NCSU23-based medium and were used to obtain recipient oocytes for SCNT after enucleation. SCNT was performed as reported previously (Matsunari et al. 2008). The cloned embryos were cultured for 7 days in porcine zygote medium (PZM)-5 to assess their developmental ability. Cloned embryos were transplanted into the oviduct or uterus of oestrus-synchronized recipient gilts to evaluate their competence to develop to fetuses or piglets. Cloned embryos reconstructed with 7 types of knockout cells showed equal development to blastocysts compared with those derived from the wild-type cells (54.5–83.3% v. 60.7%). Our data (Table 1) demonstrated that the reconstructed embryos derived from knockout cells could efficiently give rise to cloned offspring regardless of the type of genome editing methodology (i.e. ZFN or TALEN). Table 1.Production efficiency of gene knockout cloned pigs using genome editing This study was supported by JST, ERATO, the Nakauchi Stem Cell and Organ Regeneration Project, JST, CREST, Meiji University International Institute for Bio-Resource Research (MUIIBR), and JSPS KAKENHI Grant Number 26870630.

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