Opfi: A Python package for identifying gene clusters in large genomics and metagenomics data sets

Hill, Alexis M.; Rybarski, James R.; Hu, Kuang; Finkelstein, Ilya J.; Wilke, Claus O.

doi:10.21105/joss.03678

Cited by 3 publications

(1 citation statement)

References 13 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Genomes containing CAST systems were collected from NCBI genomic databases (Pruitt et al, 2005). We searched for CRISPR-Cas systems in these genomes using Opfi, a Python library to search DNA sequencing data for putative CRISPR systems (Hill et al, 2021). First, we located all regions containing a CRISPR array that was not associated with a CAST.…”

Section: Methodsmentioning

confidence: 99%

Distinct horizontal transfer mechanisms for type I and type V CRISPR-associated transposons

Chou

Wilke

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

CRISPR-associated transposons (CASTs) have co-opted CRISPR-Cas proteins and Tn7-family transposons for RNA-guided vertical and horizontal transmission. CASTs encode minimal CRISPR arrays but lack all spacer acquisition genes. Here, we define how different CASTs target new invading mobile elements without updating their own CRISPR arrays. A bioinformatic analysis reveals that all CAST sub-families co-exist with defense-associated CRISPR-Cas systems. Using a quantitative transposition assay, we show that type I-F and I-B CASTs use CRISPR RNAs (crRNAs) from these defense systems for horizontal gene transfer. A high-resolution structure of the type I-F CAST-Cascade in complex with a type III-B crRNA reveals a sequence-independent mechanism for direct repeat recognition. Type I CASTs recognize heterologous CRISPR arrays via a short hairpin in the direct repeat of their crRNA. In contrast, type V CASTs require the Cas12k effector protein but not any crRNA for unguided transposition. This transposition causes random genomic insertions via a copy-and-paste mechanism, even with over-expression of the S15 co-factor. Conversely, a single guide RNA, in concert with S15, increases on-target integration for type V CASTs. These discoveries explain how CASTs horizontally transfer to new hosts without updating their own CRISPR arrays. More broadly, this work will guide further efforts to engineer the activity and specificity of CASTs for gene editing applications.

show abstract

Section: Methodsmentioning

confidence: 99%

Distinct horizontal transfer mechanisms for type I and type V CRISPR-associated transposons

Chou

Wilke

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

Distinct horizontal transfer mechanisms for type I and type V CRISPR-associated transposons

Hu,

Chou,

Wilke

et al. 2024

Nat Commun

View full text Add to dashboard Cite

CASTs use both CRISPR-associated proteins and Tn7-family transposons for RNA-guided vertical and horizontal transmission. CASTs encode minimal CRISPR arrays but can’t acquire new spacers. Here, we report that CASTs can co-opt defense-associated CRISPR arrays for horizontal transmission. A bioinformatic analysis shows that CASTs co-occur with defense-associated CRISPR systems, with the highest prevalence for type I-B and type V CAST sub-types. Using an E. coli quantitative transposition assay and in vitro reconstitution, we show that CASTs can use CRISPR RNAs from these defense systems. A high-resolution structure of the type I-F CAST-Cascade in complex with a type III-B CRISPR RNA reveals that Cas6 recognizes direct repeats via sequence-independent π − π interactions. In addition to using heterologous CRISPR arrays, type V CASTs can also transpose via an unguided mechanism, even when the S15 co-factor is over-expressed. Over-expressing S15 and the trans-activating CRISPR RNA or a single guide RNA reduces, but does not abrogate, off-target integration for type V CASTs. Our findings suggest that some CASTs may exploit defense-associated CRISPR arrays and that this fact must be considered when porting CASTs to heterologous bacterial hosts. More broadly, this work will guide further efforts to engineer the activity and specificity of CASTs for gene editing applications.

show abstract

Metagenomic discovery of CRISPR-associated transposons

Rybarski

Hill

et al. 2021

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

View full text Add to dashboard Cite

CRISPR-associated Tn7 transposons (CASTs) co-opt cas genes for RNA-guided transposition. CASTs are exceedingly rare in genomic databases; recent surveys have reported Tn7-like transposons that co-opt Type I-F, I-B, and V-K CRISPR effectors. Here, we expand the diversity of reported CAST systems via a bioinformatic search of metagenomic databases. We discover architectures for all known CASTs, including arrangements of the Cascade effectors, target homing modalities, and minimal V-K systems. We also describe families of CASTs that have co-opted the Type I-C and Type IV CRISPR-Cas systems. Our search for non-Tn7 CASTs identifies putative candidates that include a nuclease dead Cas12. These systems shed light on how CRISPR systems have coevolved with transposases and expand the programmable gene-editing toolkit.

show abstract

Opfi: A Python package for identifying gene clusters in large genomics and metagenomics data sets

Cited by 3 publications

References 13 publications

Distinct horizontal transfer mechanisms for type I and type V CRISPR-associated transposons

Distinct horizontal transfer mechanisms for type I and type V CRISPR-associated transposons

Distinct horizontal transfer mechanisms for type I and type V CRISPR-associated transposons

Metagenomic discovery of CRISPR-associated transposons

Contact Info

Product

Resources

About