Characterization and Repurposing of Type I and Type II CRISPR–Cas Systems in Bacteria

Hidalgo-Cantabrana, Claudio; Goh, Yong Jun; Barrangou, Rodolphe

doi:10.1016/j.jmb.2018.09.013

Cited by 42 publications

(25 citation statements)

References 80 publications

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“…However, the current toolbox is limited to only a few Cas9, Cas12, and Cas13 effector proteins, predominantly optimized for use in eukaryotes. With thousands of native CRISPR-Cas systems widely occurring in bacteria and archaea, we have the opportunity to repurpose endogenous systems in their native host for genome editing, provided we can characterize their guide RNAs and targeting PAM sequences (15). Harnessing the endogenous machinery enables efficient genome editing simply by delivering a CRISPR array, together with desired repair templates.…”

Section: Discussionmentioning

confidence: 99%

“…In type II systems, the tracrRNA has a complementary region to the CRISPR repeat sequence of the crRNA, allowing creation of the duplex crRNA/tracrRNA. Therefore, the repeat sequence of type II-A was used to identify the tracrRNA in the CRISPR locus as previously described (15), and the interaction between crRNA and tracrRNA was then predicted using the NUPACK web server and depicted manually.…”

Section: Discussionmentioning

confidence: 99%

“…published studies detailing the activity of CRISPR-Cas systems in their native hosts and lack of fundamental understanding regarding type I CRISPR arrays, accompanying Cas proteins, and corresponding guide CRISPR RNAs (crRNAs) and targeting PAMs necessary for molecular tool development (15). To date, only a handful of type I CRISPR-Cas systems have been characterized, most notably the model type I-E CRISPR-Cas system from Escherichia coli, which was actually the first observed CRISPR locus over 3 decades ago (16) and has been used more recently to demonstrate the dependency of CRISPR immunity on crRNA targeting (17,18).…”

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confidence: 99%

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

Hidalgo-Cantabrana

Goh

Pan

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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131

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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

Hidalgo-Cantabrana

Goh

Pan

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

131

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“…There has been significant recent interest in the biotechnological utilization of type I systems in both bacteria and eukaryotes. Internal translation in these type I systems needs to be considered when using CRISPR-Cas for applications in organisms with differing translational machinery, such as eukaryotes ( 40, 41 ). In these cases, cas11 might need to be encoded from a separate gene.…”

Section: Discussionmentioning

confidence: 99%

Diverse CRISPR-Cas complexes require independent translation of small and large subunits from a single gene

McBride

Schwartz

Kumar

et al. 2020

Preprint

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CRISPR-Cas adaptive immune systems provide bacteria and archaea with defense16 against their viruses and other mobile genetic elements 1 . CRISPR-Cas immunity 17 involves the formation of ribonucleoprotein complexes that specifically bind and 18 degrade foreign nucleic acids 2,3 . Despite advances in the biotechnological exploitation 19 of select systems, multiple CRISPR-Cas types remain uncharacterized 4 . Here, we 20 investigated a type I-D system from Synechocystis and revealed the Cascade complex, 21 which is required for interference, forms a hybrid ribonucleoprotein complex that is 22 structurally and genetically related to both type I and III systems 5-8 . Surprisingly, the 23 type I-D complex contained multiple functionally-important small subunit proteins 24 encoded from an internal in-frame translation initiation site within the large subunit 25 gene, cas10d. Structural analysis revealed that these small subunits bound the 26 complex similarly to Cas11 small subunits in other type I and III systems, where they 27 are encoded as a separate gene. We show that internal translation of small subunits 28 from within large subunit genes is conserved across diverse type I-D, I-B and I-C 29 systems, which account for ~23% of all sequenced CRISPR-Cas systems 4 . Indeed, we 30 demonstrate that small subunits are expressed from within the cas8c large subunit 31 gene in the Desulfovibrio vulgaris type I-C system. Our work reveals an unexpected 32 aspect of CRISPR-Cas evolution and expansion of the coding potential from within 1 single cas genes. 2 3

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“…It was also reported that overexpressing a catalytically dead Cas9 (dCas9) in Escherichia coli resulted in abnormal morphology and retarded growth, indicating that the cytotoxicity of Cas9 is not solely caused by DNA cleavage but possibly transient non-specific DNA binding across the genome (Cho et al, 2018). Therefore, using endogenous CRISPR-Cas systems of the host for genome engineering could be an effective way to overcome the restriction (Hidalgo-Cantabrana et al, 2019a). In comparison with the imported class 2 systems, all the protein components of endogenous type I systems are present in the cells, excluding any heterologous nuclease.…”

Section: Introductionmentioning

confidence: 99%

Endogenous Type I CRISPR-Cas: From Foreign DNA Defense to Prokaryotic Engineering

Zheng

Wang

et al. 2020

Front. Bioeng. Biotechnol.

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Establishment of production platforms through prokaryotic engineering in microbial organisms would be one of the most efficient means for chemicals, protein, and biofuels production. Despite the fact that CRISPR (clustered regularly interspaced short palindromic repeats)-based technologies have readily emerged as powerful and versatile tools for genetic manipulations, their applications are generally limited in prokaryotes, possibly owing to the large size and severe cytotoxicity of the heterogeneous Cas (CRISPR-associated) effector. Nevertheless, the rich natural occurrence of CRISPR-Cas systems in many bacteria and most archaea holds great potential for endogenous CRISPR-based prokaryotic engineering. The endogenous CRISPR-Cas systems, with type I systems that constitute the most abundant and diverse group, would be repurposed as genetic manipulation tools once they are identified and characterized as functional in their native hosts. This article reviews the major progress made in understanding the mechanisms of invading DNA immunity by type I CRISPR-Cas and summarizes the practical applications of endogenous type I CRISPR-based toolkits for prokaryotic engineering.

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Characterization and Repurposing of Type I and Type II CRISPR–Cas Systems in Bacteria

Cited by 42 publications

References 80 publications

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

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

Diverse CRISPR-Cas complexes require independent translation of small and large subunits from a single gene

Endogenous Type I CRISPR-Cas: From Foreign DNA Defense to Prokaryotic Engineering

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