![]() OffScan is not limited by a number of mismatches and/or PAM. Detecting off-target sites for large genomes will thus consume hundreds of GB of memory.Īccordingly, in this paper, we present OffScan, a universal and fast CRISPR/Cas off-target site detection tool. In addition, a trie is a space-consuming data structure, and the number of k-mers adjacent to PAM in the genome is usually huge. ![]() ![]() It is necessary to traverse the trie once for each k-mer to detect its off-target sites within several mismatches consequently, this involves a large amount of redundant computations. constructed a trie to store all candidate sgRNA ( k-mer) in K, and thus to search off-target sites by traversing the trie rather than the entire genome. The tool is partly written in OpenCL, enabling operations using an accelerator such as GPU, which can significantly speed up the searching process. It is not limited by the number of mismatches and allows various PAM sequences. CasOT can identify potential off-target sites in an acceptable period of time.Ĭas-OFFinder can search for potential off-target sites in a given genome or set of user-defined sequences. It allows mismatches in the seed and non-seed regions and provides both a single-gRNA searching mode and a paired-gRNA searching mode. ĬasOT is a Perl script that searches for potential off-target sites in any given genome, with user-specified PAM. Accordingly, we have to filter the returned off-target sites and remove those false off-targets. The off-target sites returned by alignment tools contain a number of sequences that are not adjacent to PAM. As a result, these alignment tools cannot consider the PAM sequence when searching mismatched sites. On the other hand, although alignment tools support pattern matching within several mismatches, the mismatch position in the query cannot be set. As mentioned in CRISPR-DO, for each candidate sgRNA k-mer in K, alignment tools have to scan the entire genome once to identify its off-target sites, rather than searching in K, which results in a large amount of redundant computations. However, alignment tools are not originally designed for off-target detection. Third-party alignment tools, such as BWA and Bowtie, are often used to search off-target sites. However, we must remove the sgRNA with high off-target potential from K. Then we can obtain a set K of candidates to be sgRNA. Accordingly, it is essential to identify potential off-target sites and improve sgRNA specificity.įinding target sites can generally be accomplished quite easily by scanning the whole genome for the PAM sequence, such as 5′-NGG-3′ for the CRISPR/Cas9 system. However, SpCas9 also binds 5′-NAG-3′ or 5′-NGA-3′ with low frequency. Some studies have shown that CRISPR/Cas9 non-specifically cleave DNA sites with several mismatches, generating off-target mutations with considerable frequency. 1) and the target DNA sequence can be tolerated. A few mismatches between the 5′ 20-nt sequence in sgRNA (the purple part in Fig. The off-target effect is caused by both sgRNA and Cas9. However, the off-target effect of sgRNA may lead to undesired mutations in the genome and limit the use of this technology. The performance of CRISPR/Cas is highly dependent on well-designed sgRNA. 1, sgRNA will guide Cas nuclease to the desired DNA site and create a DNA double-strand break, the repair of which leads to a variety of DNA sequence modifications. When delivered into cells, as illustrated in Fig. CRISPR/Cas is a powerful genome editing tool.
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