CRISPRtracrRNA: robust approach for CRISPR tracrRNA detection

Abstract

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.

CvkR is a MerR-type transcriptional repressor of class 2 type V-K CRISPR-associated transposase systems

“…Next to the CRISPR array follows a tracrRNA gene (Fig. 1 ) on the same strand 11 , 25 – 27 . Based on the previous genome-wide mapping of transcriptional start sites (TSS) 28 , we detected the tracrRNA TSS 35 nt downstream of the stop codon of the CRISPR-Cas effector gene cas12k , transcribed in the same direction (Supplementary Fig.…”

“…We searched for cas12k in close vicinity to these genes and found in 169 cases a cas12k gene or a degenerated version of it (Supplementary Table 1 ). We also searched for other CAST components around the regulators yielding 130 left end elements, 90 CRISPR arrays, 139 tRNA genes as well as 119 tracrRNA loci identified by using the CRISPRtracrRNA tool 27 . Just in 18 cases we could not find any CAST components close to the regulator.…”

Sequence modeling and design from molecular to genome scale with Evo

“…The MSA boundaries of the lobes in SpCas9 were used as boundaries for labeling the REC and NUC lobes in the sampled sequences. CRISPRtracrRNA was used to extract potential tracrRNA sequences from candidate generations and co-folded with the extracted crRNA sequence using RNAmultifold (Mitrofanov et al, 2022; Lorenz et al, 2011). Different combinations of tracrRNA and crRNA lengths were assessed as the resulting mature crRNA and tracrRNA sequences are not readily apparent from raw sequence data.…”

ARA: a flexible pipeline for automated exploration of NCBI SRA datasets

“…The Mamba implementation offers a faster interface and uses libsolv to effectively resolve the dependencies. The use of a package manager guarantees seamless installation of the required software tools along with their dependencies and has been successfully used in many recent bioinformatics projects (e.g., SCRAP [ 21 ], grenepipe [ 22 ], pyGenomeTracks [ 23 ], TransposonUltimate [ 24 ], matOptimize [ 25 ], plotsr [ 26 ], CRISPRtracrRNA [ 27 ]). The Perl modules are automatically downloaded and installed by the setup script through CPAN [ 28 ].…”