CRISPRCasIdentifier: Machine learning for accurate identification and classification of CRISPR-Cas systems

Padilha, Victor Alexandre; Alkhnbashi, Omer S.; Shah, Shiraz A.; Carvalho, André C. P. L. F. de; Backofen, Rolf

doi:10.1101/817619

Cited by 4 publications

(4 citation statements)

References 44 publications

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“…Complete and draft bacterial genomes were downloaded from NCBI. CRISPR-Cas systems were annotated using CRISPRcasIdentifier 62 and Casboundary 63 , and CRISPR arrays were extracted from genomes only containing I-E (4,991 arrays), I-F (2,632 arrays), II-A (211 arrays), and II-C (636 arrays) systems using CRISPRidentify 64 . Array orientations were then detected using CRISPRstrand 65 followed by manual curation.…”

Section: Methodsmentioning

confidence: 99%

Spacer prioritization in CRISPR–Cas9 immunity is enabled by the leader RNA

et al. 2022

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CRISPR-Cas systems store fragments of foreign DNA called spacers as immunological recordings used to combat future infections. Of the many spacers stored in a CRISPR array, the newest spacers are known to be prioritized for immune defense. However, the underlying mechanism remains unclear. Here we show that the leader region upstream of CRISPR arrays in CRISPR-Cas9 systems enhances CRISPR RNA (crRNA) processing from the newest spacer, prioritizing defense against the matching invader. Using the CRISPR-Cas9 system from Streptococcus pyogenes as a model, we found that the transcribed leader interacts with the conserved repeats bordering the newest spacer. The resulting interaction promotes tracrRNA hybridization with the second repeat, accelerating crRNA processing. Accordingly, disrupting this structure reduces the abundance of the associated crRNA and immune defense against targeted plasmids and bacteriophages. Beyond the S. pyogenes system, bioinformatics analyses revealed that leader-repeat structures appear across CRISPR-Cas9 systems. CRISPR-Cas systems thus possess an RNA-based mechanism to prioritize defense against the most recently encountered invaders.

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Section: Methodsmentioning

confidence: 99%

Spacer prioritization in CRISPR–Cas9 immunity is enabled by the leader RNA

et al. 2022

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“…In more detail, we detect the first CRISPR arrays using the ML-based CRISPRidentify (Component 4), as an array is an evidence for the existence of a CRISPR system and it allows us to determine the repeat, which is subsequently used to determine the anti-repeat part of the tracrRNA via RNA–RNA interaction prediction. Additional evidence is acquired by the identification of cas9 / cas12 genes (Component 5) using CRISPRcasIdentifier ( Padilha et al , 2020 ), and computing the distance to the formed tracrRNA candidates. Each of the listed factors is provided with the corresponding certainty score.…”

Section: Methodsmentioning

confidence: 99%

CRISPRtracrRNA: robust approach for CRISPR tracrRNA detection

et al. 2022

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Motivation The CRISPR-Cas9 system is a Type II CRISPR system that has rapidly become the most versatile and widespread tool for genome engineering. It consists of two components, the Cas9 effector protein, and a single guide RNA that combines the spacer (for identifying the target) with the tracrRNA, a trans-activating small RNA required for both crRNA maturation and interference. While there are well-established methods for screening Cas effector proteins and CRISPR arrays, the detection of tracrRNA remains the bottleneck in detecting Class 2 CRISPR systems. Results We introduce a new pipeline CRISPRtracrRNA for screening and evaluation of tracrRNA candidates in genomes. This pipeline combines evidence from different components of the Cas9-sgRNA complex. The core is a newly developed structural model via covariance models from a sequence-structure alignment of experimentally validated tracrRNAs. As additional evidence, we determine the terminator signal (required for the tracrRNA transcription) and the RNA–RNA interaction between the CRISPR array repeat and the 5′-part of the tracrRNA. Repeats are detected via an ML-based approach (CRISPRidenify). Providing further evidence, we detect the cassette containing the Cas9 (Type II CRISPR systems) and Cas12 (Type V CRISPR systems) effector protein. Our tool is the first for detecting tracrRNA for Type V systems. Availability and implementation The implementation of the CRISPRtracrRNA is available on GitHub upon requesting the access permission, (https://github.com/BackofenLab/CRISPRtracrRNA). Data generated in this study can be obtained upon request to the corresponding person: Rolf Backofen (backofen@informatik.uni-freiburg.de). Supplementary information Supplementary data are available at Bioinformatics online.

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“…Additionally, antiSMASH 6.01 was used to identify biosynthesis gene clusters (BGCs) and metabolic gene clusters (MGCs) within the genomes (Blin et al, 2019). The CRISPRCasFinder and CRISPRcasIdentifier 14 servers were used to annotate the CRISPR Cas system (Couvin et al, 2018;Padilha et al, 2020). The presence of Cas enzymes were further confirmed by blasting each sequence in the UniProt database (The UniProt Consortium, 2023).…”

Section: Genome Functional Analysismentioning

confidence: 99%

Three novel marine species of the genus Reichenbachiella exhibiting degradation of complex polysaccharides

Muhammad,

Avila,

Nedashkovskaya

et al. 2023

Front. Microbiol.

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Three novel strains designated ABR2-5T, BKB1-1T, and WSW4-B4T belonging to the genus Reichenbachiella of the phylum Bacteroidota were isolated from algae and mud samples collected in the West Sea, Korea. All three strains were enriched for genes encoding up to 216 carbohydrate-active enzymes (CAZymes), which participate in the degradation of agar, alginate, carrageenan, laminarin, and starch. The 16S rRNA sequence similarities among the three novel isolates were 94.0%–94.7%, and against all three existing species in the genus Reichenbachiella they were 93.6%–97.2%. The genome sizes of the strains ABR2-5T, BKB1-1T, and WSW4-B4T were 5.5, 4.4, and 5.0 Mb, respectively, and the GC content ranged from 41.1%–42.0%. The average nucleotide identity and the digital DNA–DNA hybridization values of each novel strain within the isolates and all existing species in the genus Reichenbachiella were in a range of 69.2%–75.5% and 17.7–18.9%, respectively, supporting the creation of three new species. The three novel strains exhibited a distinctive fatty acid profile characterized by elevated levels of iso-C15:0 (37.7%–47.4%) and C16:1 ω5c (14.4%–22.9%). Specifically, strain ABR2-5T displayed an additional higher proportion of C16:0 (13.0%). The polar lipids were phosphatidylethanolamine, unidentified lipids, aminolipids, and glycolipids. Menaquinone-7 was identified as the respiratory quinone of the isolates. A comparative genome analysis was performed using the KEGG, RAST, antiSMASH, CRISPRCasFinder, dbCAN, and dbCAN-PUL servers and CRISPRcasIdentifier software. The results revealed that the isolates harbored many key genes involved in central metabolism for the synthesis of essential amino acids and vitamins, hydrolytic enzymes, carotenoid pigments, and antimicrobial compounds. The KEGG analysis showed that the three isolates possessed a complete pathway of dissimilatory nitrate reduction to ammonium (DNRA), which is involved in the conservation of bioavailable nitrogen within the ecosystem. Moreover, all the strains possessed genes that participated in the metabolism of heavy metals, including arsenic, copper, cobalt, ferrous, and manganese. All three isolated strains contain the class 2 type II subtype C1 CRISPR-Cas system in their genomes. The distinguished phenotypic, chemotaxonomic, and genomic characteristics led us to propose that the three strains represent three novel species in the genus Reichenbachiella: R. ulvae sp. nov. (ABR2-5T = KCTC 82990T = JCM 35839T), R. agarivorans sp. nov. (BKB1-1T = KCTC 82964T = JCM 35840T), and R. carrageenanivorans sp. nov. (WSW4-B4T = KCTC 82706T = JCM 35841T).

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CRISPRCasIdentifier: Machine learning for accurate identification and classification of CRISPR-Cas systems

Cited by 4 publications

References 44 publications

Spacer prioritization in CRISPR–Cas9 immunity is enabled by the leader RNA

Spacer prioritization in CRISPR–Cas9 immunity is enabled by the leader RNA

CRISPRtracrRNA: robust approach for CRISPR tracrRNA detection

Three novel marine species of the genus Reichenbachiella exhibiting degradation of complex polysaccharides

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