scholarly journals A transferrable and integrative type I-F Cascade for heterologous genome editing and transcription modulation

2021 ◽  
Author(s):  
Zeling Xu ◽  
Yanran Li ◽  
Huiluo Cao ◽  
Meiru Si ◽  
Guangming Zhang ◽  
...  
Keyword(s):  
Genome Editing ◽  
Sequence Information ◽  
Type I ◽  
Cascade System ◽  
Highly Active ◽  
Crispr Array ◽  
Class 1 ◽  
Transcription Modulation ◽  
One Step ◽  
Generic Genome

ABSTRACTThe Class 1 type I CRISPR-Cas systems represent the most abundant and diverse CRISPR systems in nature. However, their applications for generic genome editing have been hindered by difficulties of introducing the class-specific, multi-component effectors in heterologous hosts for functioning. Here we established a transferrable Cascade system that enables stable integration and expression of a complete and highly active I-F Cascade in the notoriously recalcitrant and diverse P. aeruginosa genomes by conjugation. The transferred Cascade displayed substantially higher DNA interference activity and greater editing capacity than the Cas9 system in diverse genetic backgrounds, including removal of the large (21-kb) integrated cassette with efficiency and simplicity. An advanced λred-I-F system enabled editing in genotypes with poor homologous recombination capacity, clinical isolates lacking sequence information, and cells containing anti-CRISPR elements Acrs. Lastly, an ‘all-in-one’ I-F Cascade-mediated CRISPRi platform was developed for transcription modulation by simultaneous introduction of the Cascade and the mini-CRISPR array expressing desired crRNA in one-step. This study provides a framework for expanding the diverse type I Cascades for widespread, heterologous genome editing and establishment of editing techniques in non-model isolates of pathogens.

Characterization and applications of Type I CRISPR-Cas systems

2020 ◽  
Vol 48 (1) ◽  
pp. 15-23 ◽  
Author(s):  
Claudio Hidalgo-Cantabrana ◽  
Rodolphe Barrangou
Keyword(s):  
Transcriptional Regulation ◽  
Genome Editing ◽  
Adaptive Immune System ◽  
Type I ◽  
Research Areas ◽  
Wide Range ◽  
Class 1 ◽  
Compact Class ◽  
Molecular Machinery ◽  
Repair Template

CRISPR-Cas constitutes the adaptive immune system of bacteria and archaea. This RNA-mediated sequence-specific recognition and targeting machinery has been used broadly for diverse applications in a wide range of organisms across the tree of life. The compact class 2 systems, that hinge on a single Cas effector nuclease have been harnessed for genome editing, transcriptional regulation, detection, imaging and other applications, in different research areas. However, most of the CRISPR-Cas systems belong to class 1, and the molecular machinery of the most widespread and diverse Type I systems afford tremendous opportunities for a broad range of applications. These highly abundant systems rely on a multi-protein effector complex, the CRISPR associated complex for antiviral defense (Cascade), which drives DNA targeting and cleavage. The complexity of these systems has somewhat hindered their widespread usage, but the pool of thousands of diverse Type I CRISPR-Cas systems opens new avenues for CRISPR-based applications in bacteria, archaea and eukaryotes. Here, we describe the features and mechanism of action of Type I CRISPR-Cas systems, illustrate how endogenous systems can be reprogrammed to target the host genome and perform genome editing and transcriptional regulation by co-delivering a minimal CRISPR array together with a repair template. Moreover, we discuss how these systems can also be used in eukaryotes. This review provides a framework for expanding the CRISPR toolbox, and repurposing the most abundant CRISPR-Cas systems for a wide range of applications.

scholarly journals Genome editing using the endogenous type I CRISPR-Cas system in Lactobacillus crispatus

2019 ◽  
Vol 116 (32) ◽  
pp. 15774-15783 ◽  
Author(s):  
Claudio Hidalgo-Cantabrana ◽  
Yong Jun Goh ◽  
Meichen Pan ◽  
Rosemary Sanozky-Dawes ◽  
Rodolphe Barrangou
Keyword(s):  
Genome Editing ◽  
Fluorescent Protein ◽  
Stop Codon ◽  
Mucosal Vaccine ◽  
Green Fluorescent Protein Gene ◽  
Type I ◽  
Lactobacillus Crispatus ◽  
Class 1 ◽  
Flexible Genome ◽  
Green Fluorescent

CRISPR-Cas systems are now widely used for genome editing and transcriptional regulation in diverse organisms. The compact and portable nature of class 2 single effector nucleases, such as Cas9 or Cas12, has facilitated directed genome modifications in plants, animals, and microbes. However, most CRISPR-Cas systems belong to the more prevalent class 1 category, which hinges on multiprotein effector complexes. In the present study, we detail how the native type I-E CRISPR-Cas system, with a 5′-AAA-3′ protospacer adjacent motif (PAM) and a 61-nucleotide guide CRISPR RNA (crRNA) can be repurposed for efficient chromosomal targeting and genome editing in Lactobacillus crispatus, an important commensal and beneficial microbe in the vaginal and intestinal tracts. Specifically, we generated diverse mutations encompassing a 643-base pair (bp) deletion (100% efficiency), a stop codon insertion (36%), and a single nucleotide substitution (19%) in the exopolysaccharide priming-glycosyl transferase (p-gtf). Additional genetic targets included a 308-bp deletion (20%) in the prophage DNA packaging Nu1 and a 730-bp insertion of the green fluorescent protein gene downstream of enolase (23%). This approach enables flexible alteration of the formerly genetically recalcitrant species L. crispatus, with potential for probiotic enhancement, biotherapeutic engineering, and mucosal vaccine delivery. These results also provide a framework for repurposing endogenous CRISPR-Cas systems for flexible genome targeting and editing, while expanding the toolbox to include one of the most abundant and diverse systems found in nature.

scholarly journals CRISPR-Cas3 induces broad and unidirectional genome editing in human cells

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Hiroyuki Morisaka ◽  
Kazuto Yoshimi ◽  
Yuya Okuzaki ◽  
Peter Gee ◽  
Yayoi Kunihiro ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Cleavage ◽  
Exon Skipping ◽  
Nuclease Activity ◽  
Human Cells ◽  
Eukaryotic Cells ◽  
Type I ◽  
Crispr System ◽  
Class 1 ◽  
Component Class

AbstractAlthough single-component Class 2 CRISPR systems, such as type II Cas9 or type V Cas12a (Cpf1), are widely used for genome editing in eukaryotic cells, the application of multi-component Class 1 CRISPR has been less developed. Here we demonstrate that type I-E CRISPR mediates distinct DNA cleavage activity in human cells. Notably, Cas3, which possesses helicase and nuclease activity, predominantly triggered several thousand base pair deletions upstream of the 5′-ARG protospacer adjacent motif (PAM), without prominent off-target activity. This Cas3-mediated directional and broad DNA degradation can be used to introduce functional gene knockouts and knock-ins. As an example of potential therapeutic applications, we show Cas3-mediated exon-skipping of the Duchenne muscular dystrophy (DMD) gene in patient-induced pluripotent stem cells (iPSCs). These findings broaden our understanding of the Class 1 CRISPR system, which may serve as a unique genome editing tool in eukaryotic cells distinct from the Class 2 CRISPR system.

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 Genome Editing in Cereals: Approaches, Applications and Challenges

2020 ◽  
Vol 21 (11) ◽  
pp. 4040 ◽  
Author(s):  
Waquar A. Ansari ◽  
Sonali U. Chandanshive ◽  
Vacha Bhatt ◽  
Altafhusain B. Nadaf ◽  
Sanskriti Vats ◽  
...  
Keyword(s):  
Genome Editing ◽  
Target Genes ◽  
Crop Improvement ◽  
Whole Genome Sequence ◽  
Cereal Crops ◽  
Sequence Information ◽  
World Population ◽  
Base Editing ◽  
Crop Varieties ◽  
Current Scenario

Over the past decades, numerous efforts were made towards the improvement of cereal crops mostly employing traditional or molecular breeding approaches. The current scenario made it possible to efficiently explore molecular understanding by targeting different genes to achieve desirable plants. To provide guaranteed food security for the rising world population particularly under vulnerable climatic condition, development of high yielding stress tolerant crops is needed. In this regard, technologies upgradation in the field of genome editing looks promising. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 is a rapidly growing genome editing technique being effectively applied in different organisms, that includes both model and crop plants. In recent times CRISPR/Cas9 is being considered as a technology which revolutionized fundamental as well as applied research in plant breeding. Genome editing using CRISPR/Cas9 system has been successfully demonstrated in many cereal crops including rice, wheat, maize, and barley. Availability of whole genome sequence information for number of crops along with the advancement in genome-editing techniques provides several possibilities to achieve desirable traits. In this review, the options available for crop improvement by implementing CRISPR/Cas9 based genome-editing techniques with special emphasis on cereal crops have been summarized. Recent advances providing opportunities to simultaneously edit many target genes were also discussed. The review also addressed recent advancements enabling precise base editing and gene expression modifications. In addition, the article also highlighted limitations such as transformation efficiency, specific promoters and most importantly the ethical and regulatory issues related to commercial release of novel crop varieties developed through genome editing.

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