Gene Silencing Through CRISPR Interference in Bacteria: Current Advances and Future Prospects

Zhang, Riyu; Xu, Wensheng; Shao, Shuai; Wang, Qiyao

doi:10.3389/fmicb.2021.635227

Cited by 44 publications

(41 citation statements)

References 48 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To discover novel players involved in competence regulation, we built a CRISPRi-based library and performed three types of screen in parallel. The interference technology offers several advantages over the classical random transposon mutagenesis (59) but the primary one is the production of conditional mutants allowing the study of essential/deleterious genes. Considering the transformation screen, the library was transiently induced, dampening the toxic-acquired phenotype due to constitutive activation of natural transformation.…”

Section: Discussionmentioning

confidence: 99%

A genome-wide CRISPRi screen reveals a StkP-mediated connection between cell-wall integrity and competence inStreptococcus salivarius

Knoops¹,

Waegemans²,

Lamontagne³

et al. 2022

Preprint

View full text Add to dashboard Cite

Competence is one of the most efficient bacterial evolutionary and adaptative strategies by synchronizing production of antibacterial compounds and integration of DNA released by dead cells. In most streptococci, this tactic is orchestrated by the ComRS system, a pheromone communication device providing a sharp time window of activation in which only part of the population is responsive. Understanding how this developmental process integrates multiple inputs to fine-tune the adequate response is a long-standing question. However, essential genes involved in the regulation of ComRS have been challenging to study. In this work, we built a conditional mutant library using CRISPR-interference and performed three complementary screens to investigate competence genetic regulation in the human commensalStreptococus salivarius. We show that initiation of competence increases upon cell-wall impairment, suggesting a connection between cell envelope stress and competence activation. Notably, we report a key role for StkP, a serine-threonine kinase known to regulate cell-wall homeostasis. We show that StkP controls competence by a mechanism that reacts to peptidoglycan fragments. Together, our data suggest a key cell-wall sensing mechanism coupling competence to cell envelope integrity.IMPORTANCESurvival of human commensal streptococci in the digestive tract requires efficient strategies which must be tightly and collectively controlled for responding to competitive pressure and drastic environmental changes. In this context, the autocrine signaling system ComRS controlling competence for natural transformation and predation in salivarius streptococci could be seen as a multi-input device integrating a variety of environmental stimuli. In this work, we revealed novel positive and negative competence modulators by using a genome-wide CRISPR- interference strategy. Notably, we highlighted an unexpected connection between bacterial envelope integrity and competence activation that involves several cell-wall sensors. Together, these results showcase how commensal streptococci can fine-tune the pheromone-based competence system by responding to multiple inputs affecting their physiological status in order to calibrate an appropriate collective behavior.

show abstract

Section: Discussionmentioning

confidence: 99%

A genome-wide CRISPRi screen reveals a StkP-mediated connection between cell-wall integrity and competence inStreptococcus salivarius

Knoops¹,

Waegemans²,

Lamontagne³

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

“…Furthermore, the CRISPR system is varied and suitable for upgrades to the genetic operating system [36,37]. The fusion of various protein motifs to Cas effector proteins has facilitated a diverse set of genetic manipulations, such as base editing, transposition, recombination, and epigenetic regulation [38][39][40][41][42][43].…”

Section: Discussionmentioning

confidence: 99%

An Optimized and Efficient CRISPR/Cas9 System for the Endophytic Fungus Pestalotiopsis fici

Huang

Yin

2021

JoF

View full text Add to dashboard Cite

Endophytic fungi are emerging as attractive producers of natural products with diverse bioactivities and novel structures. However, difficulties in the genetic manipulation of endophytic fungi limit the search of novel secondary metabolites. In this study, we improved the polyethylene glycol (PEG)-mediated protoplast transformation method by introducing the CRISPR/Cas9 system into endophytic fungus Pestalotiopsis fici. Using this approach, we performed genome editing such as site-specific gene insertion, dual-locus mutations, and long DNA fragment deletions in P. fici efficiently. The average efficiency for site-specific gene insertion and two-site gene editing was up to 48.0% and 44.4%, respectively. In addition, the genetic manipulation time with long DNA fragment (5–10 kb) deletion was greatly shortened to one week in comparison with traditional methods such as Agrobacterium tumefaciens-mediated transformation (ATMT). Taken together, the development of the CRISPR/Cas9 system in the endophytic fungus will accelerate the discovery of novel natural products and further biological study.

show abstract

“…There may be other biological significance for these incomplete CRISPR-Cas systems located on MGEs, as roles other than immunological protection have been revealed for CRISPR-Cas systems, such as signal transduction and gene regulation ( Bikard et al, 2012 ). Considering that some CRISPR locus located on MGEs can lose their endonuclease, the cascade complex would only be able to bind its target, therefore promoting steric transcriptional inhibition ( Zhang et al, 2021 ). Further experimental investigations are required to better understand the role and mechanisms of these incomplete type I-B systems located in MGEs in the biology of Clostridium novyi sensu lato .…”

Section: Discussionmentioning

confidence: 99%

Exploration of the Diversity of Clustered Regularly Interspaced Short Palindromic Repeats-Cas Systems in Clostridium novyi sensu lato

et al. 2021

View full text Add to dashboard Cite

Classified as the genospecies Clostridium novyi sensu lato and distributed into four lineages (I–IV), Clostridium botulinum (group III), Clostridium novyi, and Clostridium haemolyticum are clostridial pathogens that cause animal diseases. Clostridium novyi sensu lato contains a large mobilome consisting of plasmids and circular bacteriophages. Here, we explored clustered regularly interspaced short palindromic repeats (CRISPR) arrays and their associated proteins (Cas) to shed light on the link between evolution of CRISPR-Cas systems and the plasmid and phage composition in a study of 58 Clostridium novyi sensu lato genomes. In 55 of these genomes, types I-B (complete or partial), I-D, II-C, III-B, III-D, or V-U CRISPR-Cas systems were detected in chromosomes as well as in mobile genetic elements (MGEs). Type I-B predominated (67.2%) and was the only CRISPR type detected in the Ia, III, and IV genomic lineages. Putative type V-U CRISPR Cas14a genes were detected in two different cases: next to partial type-IB CRISPR loci on the phage encoding the botulinum neurotoxin (BoNT) in lineage Ia and in 12 lineage II genomes, as part of a putative integrative element related to a phage-inducible chromosomal island (PICI). In the putative PICI, Cas14a was associated with CRISPR arrays and restriction modification (RM) systems as part of an accessory locus. This is the first time a PICI containing such locus has been detected in C. botulinum. Mobilome composition and dynamics were also investigated based on the contents of the CRISPR arrays and the study of spacers. A large proportion of identified protospacers (20.2%) originated from Clostridium novyi sensu lato (p1_Cst, p4_BKT015925, p6_Cst, CWou-2020a, p1_BKT015925, and p2_BKT015925), confirming active exchanges within this genospecies and the key importance of specific MGEs in Clostridium novyi sensu lato.

show abstract

Gene Silencing Through CRISPR Interference in Bacteria: Current Advances and Future Prospects

Cited by 44 publications

References 48 publications

A genome-wide CRISPRi screen reveals a StkP-mediated connection between cell-wall integrity and competence inStreptococcus salivarius

A genome-wide CRISPRi screen reveals a StkP-mediated connection between cell-wall integrity and competence inStreptococcus salivarius

An Optimized and Efficient CRISPR/Cas9 System for the Endophytic Fungus Pestalotiopsis fici

Exploration of the Diversity of Clustered Regularly Interspaced Short Palindromic Repeats-Cas Systems in Clostridium novyi sensu lato

Contact Info

Product

Resources

About