The CRISPR/Cas9 system sheds new lights on the biology of protozoan parasites

Grzybek, Maciej; Golonko, Aleksandra; Górska, Aleksandra; Szczepaniak, Klaudiusz; Strachecka, Aneta; Lass, Anna; Lisowski, Paweł

doi:10.1007/s00253-018-8927-3

Cited by 18 publications

(14 citation statements)

References 85 publications

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“…Because Giardia trophozoites are effectively tetraploid and lack a defined sexual cycle (Poxleitner et al , 2008), the development of molecular genetic tools in Giardia has generally lagged behind that of other parasitic protists. Although new CRISPR/Cas9 gene-editing strategies have revolutionized molecular genetics in other protists (Grzybek et al , 2018), the adaptation of CRISPR-based tools for Giardia has been hindered by Giardia 's lack of a nonhomologous end-joining (NHEJ) pathway (Morrison et al , 2007) and by the inability to target native Streptomyces pyogenes Cas9 (SpCas9) to the two nuclei. The widely used SV40 nuclear localizing signal (NLS) failed to localize full-length Cas9 (Ebneter et al , 2016), although it has been used previously in Giardia to localize exogenously expressed GFP or TetR protein to the nuclei (Elmendorf et al , 2000).…”

Section: Introductionmentioning

confidence: 99%

Robust and stable transcriptional repression inGiardiausing CRISPRi

McInally

Hagen

Nosala

et al. 2019

MBoC

113

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Giardia lamblia is a binucleate protistan parasite causing significant diarrheal disease worldwide. An inability to target Cas9 to both nuclei, combined with the lack of nonhomologous end joining and markers for positive selection, has stalled the adaptation of CRISPR/Cas9-mediated genetic tools for this widespread parasite. CRISPR interference (CRISPRi) is a modification of the CRISPR/Cas9 system that directs catalytically inactive Cas9 (dCas9) to target loci for stable transcriptional repression. Using a Giardia nuclear localization signal to target dCas9 to both nuclei, we developed efficient and stable CRISPRi-mediated transcriptional repression of exogenous and endogenous genes in Giardia. Specifically, CRISPRi knockdown of kinesin-2a and kinesin-13 causes severe flagellar length defects that mirror defects with morpholino knockdown. Knockdown of the ventral disk MBP protein also causes severe structural defects that are highly prevalent and persist in the population more than 5 d longer than defects associated with transient morpholino-based knockdown. By expressing two guide RNAs in tandem to simultaneously knock down kinesin-13 and MBP, we created a stable dual knockdown strain with both flagellar length and disk defects. The efficiency and simplicity of CRISPRi in polyploid Giardia allows rapid evaluation of knockdown phenotypes and highlights the utility of CRISPRi for emerging model systems.

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Section: Introductionmentioning

confidence: 99%

Robust and stable transcriptional repression inGiardiausing CRISPRi

McInally

Hagen

Nosala

et al. 2019

MBoC

113

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“…The requirement of only Cas9 nuclease for DNA cleavage and a single-guide RNA (sgRNA) for target specificity, has called the attention for the applicability of this system as a biotechnological tool 21,22 . The CRISPR/Cas9 tool has been applied in several prokaryotes 17,18,23 and eukaryotes 24–29 .…”

Section: Introductionmentioning

confidence: 99%

Gene silencing based on RNA-guided catalytically inactive Cas9 (dCas9): a new tool for genetic engineering in Leptospira

Fernandes

Guamán

Vasconcellos

et al. 2019

Sci Rep

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Leptospirosis is a worldwide zoonosis caused by pathogenic bacteria of the genus Leptospira, which also includes free-living saprophyte strains. Many aspects of leptospiral basic biology and virulence mechanisms remain unexplored mainly due to the lack of effective genetic tools available for these bacteria. Recently, the type II CRISPR/Cas system from Streptococcus pyogenes has been widely used as an efficient genome engineering tool in bacteria by inducing double-strand breaks (DSBs) in the desired genomic targets caused by an RNA-guided DNA endonuclease called Cas9, and the DSB repair associated machinery. In the present work, plasmids expressing heterologous S. pyogenes Cas9 in L. biflexa cells were generated, and the enzyme could be expressed with no apparent toxicity to leptospiral cells. However, L. biflexa cells were unable to repair RNA-guided Cas9-induced DSBs. Thus, we used a catalytically dead Cas9 (dCas9) to obtain gene silencing rather than disruption, in a strategy called CRISPR interference (CRISPRi). We demonstrated complete gene silencing in L. biflexa cells when both dCas9 and single-guide RNA (sgRNA) targeting the coding strand of the β-galactosidase gene were expressed simultaneously. Furthermore, when the system was applied for silencing the dnaK gene, no colonies were recovered, indicating that DnaK protein is essential in Leptospira. In addition, flagellar motor switch FliG gene silencing resulted in reduced bacterial motility. To the best of our knowledge, this is the first work applying the CRISPRi system in Leptospira and spirochetes in general, expanding the tools available for understanding leptospiral biology.

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“…More generally, many CRISPR-Cas9 editing strategies have been developed in Saccharomyces cerevisiae [69,70] as well as microbiome-associated fungi, including the opportunistic pathogen Candida albicans [71,72]. Along the same line, CRISPR tools developed for protozoan parasites like Plasmodium falciparum [73,74] could likely be adapted to commensal protozoans like Blastocystis .…”

Section: Crispr Editing Of Unicellular Eukaryotesmentioning

confidence: 99%

Editing the microbiome the CRISPR way

Ramachandran

Bikard

2019

Phil. Trans. R. Soc. B

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Our bodies are colonized by a complex ecosystem of bacteria, unicellular eukaryotes and their viruses that together play a major role in our health. Over the past few years tools derived from the prokaryotic immune system known as CRISPR-Cas have empowered researchers to modify and study organisms with unprecedented ease and efficiency. Here we discuss how various types of CRISPR-Cas systems can be used to modify the genome of gut microorganisms and bacteriophages. CRISPR-Cas systems can also be delivered to bacterial population and programmed to specifically eliminate members of the microbiome. Finally, engineered CRISPR-Cas systems can be used to control gene expression and modulate the production of metabolites and proteins. Together these tools provide exciting opportunities to investigate the complex interplay between members of the microbiome and our bodies, and present new avenues for the development of drugs that target the microbiome. This article is part of a discussion meeting issue ‘The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems’.

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The CRISPR/Cas9 system sheds new lights on the biology of protozoan parasites

Cited by 18 publications

References 85 publications

Robust and stable transcriptional repression inGiardiausing CRISPRi

Robust and stable transcriptional repression inGiardiausing CRISPRi

Gene silencing based on RNA-guided catalytically inactive Cas9 (dCas9): a new tool for genetic engineering in Leptospira

Editing the microbiome the CRISPR way

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