Fragmentation of the CRISPR-Cas Type I-B signature protein Cas8b

Richter, Hagen; Rompf, Judith; Wiegel, Julia; Rau, Kristina; Randau, Lennart

doi:10.1016/j.bbagen.2017.02.026

Cited by 10 publications

(8 citation statements)

References 57 publications

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“…4e). In agreement with our findings for type I-B systems, a C-terminal portion of Cas8b containing an N-terminal methionine was previously detected; however, the authors proposed it resulted from proteolytic cleavage 35 . Type I-F1 systems lack a cas11 gene and alignment of Cas8f proteins did not detect a conserved internal translation start site, indicating internal translation initiation regions are not conserved in cas8f genes (Supplementary Information S2).…”

Section: Introductionsupporting

confidence: 93%

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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Section: Introductionsupporting

confidence: 93%

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

McBride

Schwartz

Kumar

et al. 2020

Preprint

View full text Add to dashboard Cite

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“…In fact, the subtypes I-A and I-E are the only systems that harbor a separate gene for the small subunit. In the other subtypes, the small subunit is either fused to or functionally replaced by Cas8 (Koonin et al, 2017;Richter et al, 2017). An even more minimal Cascade architecture is seen in type I-C, which lacks a Cas6 homolog (Hochstrasser et al, 2016;Nam et al, 2012), and type I-Fv (a variant of type I-F), where the large and small subunits are absent and functionally replaced by Cas5fv and Cas7fv (Gleditzsch et al, 2016;Pausch et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

The Biology of CRISPR-Cas: Backward and Forward

Hille

Richter

Wong

et al. 2018

Cell

Self Cite

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607

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In bacteria and archaea, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins constitute an adaptive immune system against phages and other foreign genetic elements. Here, we review the biology of the diverse CRISPR-Cas systems and the major progress achieved in recent years in understanding the underlying mechanisms of the three stages of CRISPR-Cas immunity: adaptation, crRNA biogenesis, and interference. The ecology and regulation of CRISPR-Cas in the context of phage infection, the roles of these systems beyond immunity, and the open questions that propel the field forward are also discussed.

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“…Further in silico analysis of the Type I–B operon identified spacer-protospacer matches and putative PAM sequences (Pyne et al., 2016). In addition, in vitro work was done wherein the Type I–B Cascade complex from C. thermocellum was reconstituted and further characterized (Richter et al., 2017). Here we report the characterization of both a native Type I–B and a Type II (GeoCas9) CRISPR/Cas genome editing system in the thermophilic, industrially relevant bacterium Clostridium thermocellum .…”

Section: Introductionmentioning

confidence: 99%

Development of both type I–B and type II CRISPR/Cas genome editing systems in the cellulolytic bacterium Clostridium thermocellum

Walker

Lanahan

Zheng

et al. 2020

Metabolic Engineering Communications

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The robust lignocellulose-solubilizing activity of C. thermocellum makes it a top candidate for consolidated bioprocessing for biofuel production. Genetic techniques for C. thermocellum have lagged behind model organisms thus limiting attempts to improve biofuel production. To improve our ability to engineer C. thermocellum, we characterized a native Type I–B and heterologous Type II Clustered Regularly-Interspaced Short Palindromic Repeat (CRISPR)/cas (CRISPR associated) systems. We repurposed the native Type I–B system for genome editing. We tested three thermophilic Cas9 variants (Type II) and found that GeoCas9, isolated from Geobacillus stearothermophilus, is active in C. thermocellum. We employed CRISPR-mediated homology directed repair to introduce a nonsense mutation into pyrF. For both editing systems, homologous recombination between the repair template and the genome appeared to be the limiting step. To overcome this limitation, we tested three novel thermophilic recombinases and demonstrated that exo/beta homologs, isolated from Acidithiobacillus caldus, are functional in C. thermocellum. For the Type I–B system an engineered strain, termed LL1586, yielded 40% genome editing efficiency at the pyrF locus and when recombineering machinery was expressed this increased to 71%. For the Type II GeoCas9 system, 12.5% genome editing efficiency was observed and when recombineering machinery was expressed, this increased to 94%. By combining the thermophilic CRISPR system (either Type I–B or Type II) with the recombinases, we developed a new tool that allows for efficient CRISPR editing. We are now poised to enable CRISPR technologies to better engineer C. thermocellum for both increased lignocellulose degradation and biofuel production.

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Fragmentation of the CRISPR-Cas Type I-B signature protein Cas8b

Cited by 10 publications

References 57 publications

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

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

The Biology of CRISPR-Cas: Backward and Forward

Development of both type I–B and type II CRISPR/Cas genome editing systems in the cellulolytic bacterium Clostridium thermocellum

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