Reprogramming the endogenous type III-A CRISPR-Cas system for genome editing, RNA interference and CRISPRi screening in Mycobacterium tuberculosis

AbstractMycobacterium tuberculosis (M.tb) causes the current leading infectious disease. Examination of the functional genomics of M.tb and development of drugs and vaccines are hampered by the complicated and time-consuming genetic manipulation techniques for M.tb. Here, we reprogrammed M.tb endogenous type III-A CRISPR-Cas10 system for simple and efficient gene editing, RNA interference and screening via simple delivery of a plasmid harboring a mini-CRISPR array, thereby avoiding the introduction of exogenous proteins and minimizing proteotoxicity. We demonstrated that M.tb genes were efficiently and specifically knocked-in/out by this system, which was confirmed by whole-genome sequencing. This system was further employed for single and simultaneous multiple-gene RNA interference. Moreover, we successfully applied this system for genome-wide CRISPR interference screening to identify the in-vitro and intracellular growth-regulating genes. This system can be extensively used to explore the functional genomics of M.tb and facilitate the development of new anti-Mycobacterial drugs and vaccines.SummaryTuberculosis caused by Mycobacterium tuberculosis (M.tb) is the current leading infectious disease affecting more than ten million people annually. To dissect the functional genomics and understand its virulence, persistence, and antibiotics resistance, a powerful genome editing tool and high-throughput screening methods are desperately wanted. Our study developed an efficient and a robust tool for genome editing and RNA interference in M.tb using its endogenous CRISPR cas10 system. Moreover, the system has been successfully applied for genome-wide CRISPR interference screening. This tool could be employed to explore the functional genomics of M.tb and facilitate the development of anti-M.tb drugs and vaccines.

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Genome-Wide RNA Interference: Functional Genomics in the Postgenomics Era

Cold Spring Harbor Protocols ◽

10.1101/pdb.top097550 ◽

2017 ◽

Vol 2017 (9) ◽

pp. pdb.top097550 ◽

Cited By ~ 1

Author(s):

Katerina Politi ◽

Narendra Wajapeyee

Keyword(s):

Rna Interference ◽

Functional Genomics ◽

Genome Wide

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Reprogramming Mycobacterium tuberculosis CRISPR System for Gene Editing and Genome-wide RNA Interference Screening

Genomics Proteomics & Bioinformatics ◽

10.1016/j.gpb.2021.01.008 ◽

2021 ◽

Author(s):

Khaista Rahman ◽

Muhammad Jamal ◽

Xi Chen ◽

Wei Zhou ◽

Bin Yang ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Rna Interference ◽

Gene Editing ◽

Crispr System ◽

Genome Wide

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The Use of Functional Genomics in Conjunction with Metabolomics forMycobacterium tuberculosisResearch

Disease Markers ◽

10.1155/2014/124218 ◽

2014 ◽

Vol 2014 ◽

pp. 1-12 ◽

Cited By ~ 13

Author(s):

Conrad C. Swanepoel ◽

Du Toit Loots

Keyword(s):

Infectious Disease ◽

Drug Resistance ◽

Mycobacterium Tuberculosis ◽

Systems Biology ◽

Functional Genomics ◽

The Past ◽

Holistic Understanding ◽

Transcriptomics Data ◽

Genomic Studies ◽

Complex Genome

Tuberculosis (TB), caused byMycobacterium tuberculosis, is a fatal infectious disease, resulting in 1.4 million deaths globally per annum. Over the past three decades, genomic studies have been conducted in an attempt to elucidate the functionality of the genome of the pathogen. However, many aspects of this complex genome remain largely unexplored, as approaches like genomics, proteomics, and transcriptomics have failed to characterize them successfully. In turn, metabolomics, which is relatively new to the “omics” revolution, has shown great potential for investigating biological systems or their modifications. Furthermore, when these data are interpreted in combination with previously acquired genomics, proteomics and transcriptomics data, using what is termed a systems biology approach, a more holistic understanding of these systems can be achieved. In this review we discuss how metabolomics has contributed so far to characterizing TB, with emphasis on the resulting improved elucidation ofM. tuberculosisin terms of (1) metabolism, (2) growth and replication, (3) pathogenicity, and (4) drug resistance, from the perspective of systems biology.

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Computational approach for siRNA Based RNA Interference of ATP synthase subunit alpha coding gene of different strains of Mycobacterium tuberculosis

International Journal of Pharmaceutical Research ◽

10.31838/ijpr/2019.11.02.008 ◽

2019 ◽

Vol 11 (02) ◽

Keyword(s):

Mycobacterium Tuberculosis ◽

Rna Interference ◽

Atp Synthase ◽

Computational Approach ◽

Different Strains

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Functional Genomics in Xenopus laevis: Towards Transgene-Driven RNA Interference and Cell-Specific Transgene Expression

Current Genomics ◽

10.2174/1389202033490132 ◽

2003 ◽

Vol 4 (8) ◽

pp. 699-711 ◽

Cited By ~ 6

Author(s):

Ron Dirks ◽

Gerrit Bouw ◽

Rick Huizen ◽

Eric Jansen ◽

Gerard Martens

Keyword(s):

Rna Interference ◽

Xenopus Laevis ◽

Functional Genomics ◽

Transgene Expression

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Editing GWAS: experimental approaches to dissect and exploit disease-associated genetic variation

Genome Medicine ◽

10.1186/s13073-021-00857-3 ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Shuquan Rao ◽

Yao Yao ◽

Daniel E. Bauer

Keyword(s):

Genome Editing ◽

Genetic Variants ◽

Association Studies ◽

Genome Wide Association ◽

Genome Wide Association Studies ◽

Functional Studies ◽

Functional Genetics ◽

Genome Wide ◽

Causal Variants ◽

Experimental Approaches

AbstractGenome-wide association studies (GWAS) have uncovered thousands of genetic variants that influence risk for human diseases and traits. Yet understanding the mechanisms by which these genetic variants, mainly noncoding, have an impact on associated diseases and traits remains a significant hurdle. In this review, we discuss emerging experimental approaches that are being applied for functional studies of causal variants and translational advances from GWAS findings to disease prevention and treatment. We highlight the use of genome editing technologies in GWAS functional studies to modify genomic sequences, with proof-of-principle examples. We discuss the challenges in interrogating causal variants, points for consideration in experimental design and interpretation of GWAS locus mechanisms, and the potential for novel therapeutic opportunities. With the accumulation of knowledge of functional genetics, therapeutic genome editing based on GWAS discoveries will become increasingly feasible.

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Recent advances in CRISPR/Cas9 and applications for wheat functional genomics and breeding

aBIOTECH ◽

10.1007/s42994-021-00042-5 ◽

2021 ◽

Author(s):

Jun Li ◽

Yan Li ◽

Ligeng Ma

Keyword(s):

Functional Genomics ◽

Genome Editing ◽

Functional Annotation ◽

Genome Engineering ◽

Agronomic Traits ◽

Wheat Breeding ◽

Breeding Programs ◽

Base Editing ◽

The World ◽

World Food Security

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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Learning a genome-wide score of human–mouse conservation at the functional genomics level

Nature Communications ◽

10.1038/s41467-021-22653-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Soo Bin Kwon ◽

Jason Ernst

Keyword(s):

Mouse Model ◽

Functional Genomics ◽

Functional Genomic ◽

Transcriptomic Data ◽

Model Studies ◽

Genome Wide ◽

A Genome ◽

Important Challenge ◽

Genomic Regions ◽

Human And Mouse

AbstractIdentifying genomic regions with functional genomic properties that are conserved between human and mouse is an important challenge in the context of mouse model studies. To address this, we develop a method to learn a score of evidence of conservation at the functional genomics level by integrating information from a compendium of epigenomic, transcription factor binding, and transcriptomic data from human and mouse. The method, Learning Evidence of Conservation from Integrated Functional genomic annotations (LECIF), trains neural networks to generate this score for the human and mouse genomes. The resulting LECIF score highlights human and mouse regions with shared functional genomic properties and captures correspondence of biologically similar human and mouse annotations. Analysis with independent datasets shows the score also highlights loci associated with similar phenotypes in both species. LECIF will be a resource for mouse model studies by identifying loci whose functional genomic properties are likely conserved.

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