scholarly journals Highly efficient genome editing in Xanthomonas oryzae pv.  oryzae through repurposing the endogenous type I‐C CRISPR‐Cas system

2021 ◽  
Author(s):  
Dandan Jiang ◽  
Dandan Zhang ◽  
Shengnan Li ◽  
Yueting Liang ◽  
Qianwei Zhang ◽  
...  
Keyword(s):  
Genome Editing ◽  
Xanthomonas Oryzae ◽  
Type I ◽  
Highly Efficient

scholarly journals An Efficient, Rapid, and Recyclable System for CRISPR-Mediated Genome Editing in Candida albicans

mSphere ◽  
2017 ◽  
Vol 2 (2) ◽  
Author(s):  
Namkha Nguyen ◽  
Morgan M. F. Quail ◽  
Aaron D. Hernday
Keyword(s):  
Candida Albicans ◽  
Genome Editing ◽  
Fungal Pathogen ◽  
Gene Knockout ◽  
Strain Engineering ◽  
Molecular Genetic Analysis ◽  
Content Type ◽  
Highly Efficient ◽  
Discovery Research ◽  
Gene Knockouts

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.

Highly efficient heritable genome editing in wheat using an RNA virus and bypassing tissue culture

2021 ◽  
Author(s):  
Tingdong Li ◽  
Jiacheng Hu ◽  
Yu Sun ◽  
Boshu Li ◽  
Dingliang Zhang ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Genome Editing ◽  
Rna Virus ◽  
Highly Efficient

scholarly journals Cas9 cleavage assay for pre-screening of sgRNAs using nicking triggered isothermal amplification

2016 ◽  
Vol 7 (8) ◽  
pp. 4951-4957 ◽  
Author(s):  
Kaixiang Zhang ◽  
Ruijie Deng ◽  
Yue Li ◽  
Ling Zhang ◽  
Jinghong Li
Keyword(s):  
Genome Editing ◽  
Quantitative Evaluation ◽  
Isothermal Amplification ◽  
Highly Efficient ◽  
Cleavage Efficiency ◽  
Cleavage Assay

A novel Cas9 cleavage assay was developed for quantitative evaluation of Cas9 cleavage efficiency and pre-screening of sgRNA to achieve highly specific and highly efficient CRISPR/Cas9 genome editing.

scholarly journals Highly Efficient Mouse Genome Editing by CRISPR Ribonucleoprotein Electroporation of Zygotes

2016 ◽  
Vol 291 (28) ◽  
pp. 14457-14467 ◽  
Author(s):  
Sean Chen ◽  
Benjamin Lee ◽  
Angus Yiu-Fai Lee ◽  
Andrew J. Modzelewski ◽  
Lin He
Keyword(s):  
Genome Editing ◽  
Mouse Genome ◽  
Highly Efficient

scholarly journals Using the Endogenous CRISPR-Cas System of Heliobacterium modesticaldum To Delete the Photochemical Reaction Center Core Subunit Gene

2019 ◽  
Vol 85 (23) ◽  
Author(s):  
Patricia L. Baker ◽  
Gregory S. Orf ◽  
Kimberly Kevershan ◽  
Michael E. Pyne ◽  
Taner Bicer ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Genome Editing ◽  
Reaction Center ◽  
Photochemical Reaction ◽  
Genetic Locus ◽  
Gene Replacement ◽  
Type I ◽  
Content Type ◽  
Heliobacterium Modesticaldum

ABSTRACT In Heliobacterium modesticaldum, as in many Firmicutes, deleting genes by homologous recombination using standard techniques has been extremely difficult. The cells tend to integrate the introduced plasmid into the chromosome by a single recombination event rather than perform the double recombination required to replace the targeted locus. Transformation with a vector containing only a homologous recombination template for replacement of the photochemical reaction center gene pshA produced colonies with multiple genotypes, rather than a clean gene replacement. To address this issue, we required an additional means of selection to force a clean gene replacement. In this study, we report the genetic structure of the type I-A and I-E CRISPR-Cas systems from H. modesticaldum, as well as methods to leverage the type I-A system for genome editing. In silico analysis of the CRISPR spacers revealed a potential consensus protospacer adjacent motif (PAM) required for Cas3 recognition, which was then tested using an in vivo interference assay. Introduction of a homologous recombination plasmid that carried a miniature CRISPR array targeting sequences in pshA (downstream of a naturally occurring PAM sequence) produced nonphototrophic transformants with clean replacements of the pshA gene with ∼80% efficiency. Mutants were confirmed by PCR, sequencing, optical spectroscopy, and growth characteristics. This methodology should be applicable to any genetic locus in the H. modesticaldum genome. IMPORTANCE The heliobacteria are the only phototrophic members of the largely Gram-positive phylum Firmicutes, which contains medically and industrially important members, such as Clostridium difficile and Clostridium acetobutylicum. Heliobacteria are of interest in the study of photosynthesis because their photosynthetic system is unique and the simplest known. Since their discovery in the early 1980s, work on the heliobacteria has been hindered by the lack of a genetic transformation system. The problem of introducing foreign DNA into these bacteria has been recently rectified by our group; however, issues still remained for efficient genome editing. The significance of this work is that we have characterized the endogenous type I CRISPR-Cas system in the heliobacteria and leveraged it to assist in genome editing. Using the CRISPR-Cas system allowed us to isolate transformants with precise replacement of the pshA gene encoding the main subunit of the photochemical reaction center.

scholarly journals Optimized Cas9 expression systems for highly efficient Arabidopsis genome editing facilitate isolation of complex alleles in a single generation

2019 ◽  
Vol 20 (1) ◽  
pp. 151-162 ◽  
Author(s):  
Jana Ordon ◽  
Mauro Bressan ◽  
Carola Kretschmer ◽  
Luca Dall’Osto ◽  
Sylvestre Marillonnet ◽  
...  
Keyword(s):  
Genome Editing ◽  
Arabidopsis Genome ◽  
Expression Systems ◽  
Single Generation ◽  
Highly Efficient

Highly efficient genome editing in N. gerenzanensis using an inducible CRISPR/Cas9–RecA system

2020 ◽  
Vol 42 (9) ◽  
pp. 1699-1706
Author(s):  
Xue Yue ◽  
Tianyu Xia ◽  
Shuai Wang ◽  
Huijun Dong ◽  
Yongquan Li
Keyword(s):  
Genome Editing ◽  
Highly Efficient

scholarly journals Development of both type I–B and type II CRISPR/Cas genome editing systems in the cellulolytic bacterium Clostridium thermocellum

2020 ◽  
Vol 10 ◽  
pp. e00116 ◽  
Author(s):  
Julie E. Walker ◽  
Anthony A. Lanahan ◽  
Tianyong Zheng ◽  
Camilo Toruno ◽  
Lee R. Lynd ◽  
...  
Keyword(s):  
Genome Editing ◽  
Clostridium Thermocellum ◽  
Cellulolytic Bacterium ◽  
Type I ◽  
Type Ii
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