scholarly journals Efficient Genome Editing in Setaria italica Using CRISPR/Cas9 and Base Editors

2022 ◽  
Vol 12 ◽  
Author(s):  
Zhen Liang ◽  
Yuqing Wu ◽  
Lingling Ma ◽  
Yingjie Guo ◽  
Yidong Ran

The genome editing toolbox based on CRISPR/Cas9 has brought revolutionary changes to agricultural and plant scientific research. With the development of stable genetic transformation protocols, a highly efficient genome editing system for foxtail millet (Setaria italica) is required. In the present study, we use the CRISPR/Cas9 single- and multi-gene knockout system to target the SiFMBP, SiDof4, SiBADH2, SiGBSS1, and SiIPK1 genes in the foxtail millet protoplasts to screen out highly efficient targeted sgRNAs. Then, we recovered homozygous mutant plants with most of the targeted genes through an Agrobacterium-mediated genetic transformation of foxtail millet. The mutagenesis frequency in the T0 generation was as high as 100%, and it was passed stably on to the next generation. After screening these targeted edited events, we did not detect off-target mutations at potential sites. Based on this system, we have achieved base editing successfully using two base editors (CBE and ABE) to target the SiALS and SiACC genes of foxtail millet. By utilizing CBE to target the SiALS gene, we created a homozygous herbicide-tolerant mutant plant. The current system could enhance the analysis of functional genomics and genetic improvement of foxtail millet.

mSphere ◽  
2017 ◽  
Vol 2 (2) ◽  
Author(s):  
Namkha Nguyen ◽  
Morgan M. F. Quail ◽  
Aaron D. Hernday

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.


Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. SCI-22-SCI-22
Author(s):  
Feng Zhang

Precision genome editing, which can be used to alter specific DNA sequences, is a powerful tool for understanding the molecular circuitry underlying cellular processes. Over the past several years, we and others have harnessed microbial CRISPR-Cas systems for use as platforms for a range of genome manipulations, including single and multiplex gene knockout, gene activation, and large-scale screening applications. Recently, we discovered and characterized several novel CRISPR systems that target RNA, including the CRISPR-Cas13 family. We developed a toolbox for RNA modulation based on Cas13, including methods for highly specific RNA knockdown, transcript imaging, and precision base editing. During our initial characterization of Cas13, we observed that Cas13 also exhibits so-called non-specific "collateral" RNase activity in vitro, which we capitalized on to create SHERLOCK, a highly sensitive and specific CRISPR diagnostic platform. We are continuing to refine and extend CRISPR-based technologies as well as explore microbial diversity to find new enzymes and systems that can be adapted for use as molecular biology tools and novel therapeutics. Disclosures Zhang: Arbor Biotechnologies: Consultancy, Equity Ownership; Sherlock Biosciences: Consultancy, Equity Ownership; Pairwise Plants: Consultancy, Equity Ownership; Beam Therapeutics: Consultancy, Equity Ownership; Editas Medicine: Consultancy, Equity Ownership.


2015 ◽  
Vol 30 (5) ◽  
pp. 389-395 ◽  
Author(s):  
Sandra Korge ◽  
Astrid Grudziecki ◽  
Achim Kramer

2018 ◽  
Vol 84 (23) ◽  
Author(s):  
Yu Wang ◽  
Shanshan Wang ◽  
Weizhong Chen ◽  
Liqiang Song ◽  
Yifei Zhang ◽  
...  

ABSTRACTKlebsiella pneumoniaeis a promising industrial microorganism as well as a major human pathogen. The recent emergence of carbapenem-resistantK. pneumoniaehas posed a serious threat to public health worldwide, emphasizing a dire need for novel therapeutic means against drug-resistantK. pneumoniae. Despite the critical importance of genetics in bioengineering, physiology studies, and therapeutic-means development, genome editing, in particular, the highly desirable scarless genetic manipulation inK. pneumoniae, is often time-consuming and laborious. Here, we report a two-plasmid system, pCasKP-pSGKP, used for precise and iterative genome editing inK. pneumoniae. By harnessing the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 genome cleavage system and the lambda Red recombination system, pCasKP-pSGKP enabled highly efficient genome editing inK. pneumoniaeusing a short repair template. Moreover, we developed a cytidine base-editing system, pBECKP, for precise C→T conversion in both the chromosomal and plasmid-borne genes by engineering the fusion of the cytidine deaminase APOBEC1 and a Cas9 nickase. By using both the pCasKP-pSGKP and the pBECKP tools, theblaKPC-2gene was confirmed to be the major factor that contributed to the carbapenem resistance of a hypermucoviscous carbapenem-resistantK. pneumoniaestrain. The development of the two editing tools will significantly facilitate the genetic engineering ofK. pneumoniae.IMPORTANCEGenetics is a key means to study bacterial physiology. However, the highly desirable scarless genetic manipulation is often time-consuming and laborious for the major human pathogenK. pneumoniae. We developed a CRISPR-Cas9-mediated genome-editing method and a cytidine base-editing system, enabling rapid, highly efficient, and iterative genome editing in both industrial and clinically isolatedK. pneumoniaestrains. We applied both tools in dissecting the drug resistance mechanism of a hypermucoviscous carbapenem-resistantK. pneumoniaestrain, elucidating that theblaKPC-2gene was the major factor that contributed to the carbapenem resistance of the hypermucoviscous carbapenem-resistantK. pneumoniaestrain. Utilization of the two tools will dramatically accelerate a wide variety of investigations in diverseK. pneumoniaestrains and relevantEnterobacteriaceaespecies, such as gene characterization, drug discovery, and metabolic engineering.


2013 ◽  
Vol 38 (5) ◽  
pp. 800-807
Author(s):  
Hui ZHI ◽  
Zhen-Gang NIU ◽  
Guan-Qing JIA ◽  
Yang CHAI ◽  
Wei LI ◽  
...  

1940 ◽  
Vol 32 (6) ◽  
pp. 426-438 ◽  
Author(s):  
H. W. Li ◽  
J. C. Meng ◽  
C. H. Li

aBIOTECH ◽  
2021 ◽  
Author(s):  
Jun Li ◽  
Yan Li ◽  
Ligeng Ma

AbstractCommon wheat (Triticum aestivum L.) is one of the three major food crops in the world; thus, wheat breeding programs are important for world food security. Characterizing the genes that control important agronomic traits and finding new ways to alter them are necessary to improve wheat breeding. Functional genomics and breeding in polyploid wheat has been greatly accelerated by the advent of several powerful tools, especially CRISPR/Cas9 genome editing technology, which allows multiplex genome engineering. Here, we describe the development of CRISPR/Cas9, which has revolutionized the field of genome editing. In addition, we emphasize technological breakthroughs (e.g., base editing and prime editing) based on CRISPR/Cas9. We also summarize recent applications and advances in the functional annotation and breeding of wheat, and we introduce the production of CRISPR-edited DNA-free wheat. Combined with other achievements, CRISPR and CRISPR-based genome editing will speed progress in wheat biology and promote sustainable agriculture.


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