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Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases.

Bae S, Park J, Kim JS - Bioinformatics (2014)

Bottom Line: CRISPR/Cas-derived RNA-guided engineered nucleases (RGENs) enable genome editing in cultured cells, animals and plants, but are limited by off-target mutations.Unlike other algorithms currently available for identification of RGEN off-target sites, Cas-OFFinder is not limited by the number of mismatches and allows variations in protospacer-adjacent motif sequences recognized by Cas9, the essential protein component in RGENs.Cas-OFFinder is available as a command-line program or accessible via our website.

View Article: PubMed Central - PubMed

Affiliation: National Creative Research Initiatives Center for Genome Engineering, Department of Chemistry and Department of Physics and Astronomy, Seoul National University, 599 Gwanak-ro, Seoul 151-742, South Korea.

ABSTRACT

Summary: The Type II clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system is an adaptive immune response in prokaryotes, protecting host cells against invading phages or plasmids by cleaving these foreign DNA species in a targeted manner. CRISPR/Cas-derived RNA-guided engineered nucleases (RGENs) enable genome editing in cultured cells, animals and plants, but are limited by off-target mutations. Here, we present a novel algorithm termed Cas-OFFinder that searches for potential off-target sites in a given genome or user-defined sequences. Unlike other algorithms currently available for identification of RGEN off-target sites, Cas-OFFinder is not limited by the number of mismatches and allows variations in protospacer-adjacent motif sequences recognized by Cas9, the essential protein component in RGENs. Cas-OFFinder is available as a command-line program or accessible via our website.

Availability and implementation: Cas-OFFinder free access at http://www.rgenome.net/cas-offinder.

Contact: baesau@snu.ac.kr or jskim01@snu.ac.kr.

Show MeSH
(A) The scheme of Cas-OFFinder. (B) The workflow of Cas-OFFinder. (C) Running time per target site as a function of the number of input target sites via CPU (black squares) and GPU (red circles)
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btu048-F1: (A) The scheme of Cas-OFFinder. (B) The workflow of Cas-OFFinder. (C) Running time per target site as a function of the number of input target sites via CPU (black squares) and GPU (red circles)

Mentions: Versions of Cas9 derived from three different species have been exploited to edit genes in human cells. These Cas9 proteins recognize different PAM sequences. Cas9 originated from Streptococcus pyogenes (SpCas9) recognizes 5′-NGG-3′ PAM sequences and, to a lesser extent, 5′-NAG-3′. Cas9 from Streptococcus thermophilus (StCas9) (Cong et al., 2013) and that from Neisseria meningitidis (NmCas9) (Hou et al., 2013) recognizes 5′-NNAGAAW-3′ (W = A or T) and 5′-NNNNGMTT-3′ (M = A or C), respectively. The degeneracy in PAM recognition by Cas9 must be accounted for when searching for potential off-target sites. In the case of SpCas9, Cas-OFFinder first compiles all the 23-bp DNA sequences composed of 20-bp sequences corresponding to the sgRNA sequence of interest and the 5′-NRG-3′ PAM sequences (Fig. 1A). Cas-OFFinder then compares all the compiled sequences with the query sequence and counts the number of mismatched bases in the 20-bp sgRNA sequence.Fig. 1.


Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases.

Bae S, Park J, Kim JS - Bioinformatics (2014)

(A) The scheme of Cas-OFFinder. (B) The workflow of Cas-OFFinder. (C) Running time per target site as a function of the number of input target sites via CPU (black squares) and GPU (red circles)
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4016707&req=5

btu048-F1: (A) The scheme of Cas-OFFinder. (B) The workflow of Cas-OFFinder. (C) Running time per target site as a function of the number of input target sites via CPU (black squares) and GPU (red circles)
Mentions: Versions of Cas9 derived from three different species have been exploited to edit genes in human cells. These Cas9 proteins recognize different PAM sequences. Cas9 originated from Streptococcus pyogenes (SpCas9) recognizes 5′-NGG-3′ PAM sequences and, to a lesser extent, 5′-NAG-3′. Cas9 from Streptococcus thermophilus (StCas9) (Cong et al., 2013) and that from Neisseria meningitidis (NmCas9) (Hou et al., 2013) recognizes 5′-NNAGAAW-3′ (W = A or T) and 5′-NNNNGMTT-3′ (M = A or C), respectively. The degeneracy in PAM recognition by Cas9 must be accounted for when searching for potential off-target sites. In the case of SpCas9, Cas-OFFinder first compiles all the 23-bp DNA sequences composed of 20-bp sequences corresponding to the sgRNA sequence of interest and the 5′-NRG-3′ PAM sequences (Fig. 1A). Cas-OFFinder then compares all the compiled sequences with the query sequence and counts the number of mismatched bases in the 20-bp sgRNA sequence.Fig. 1.

Bottom Line: CRISPR/Cas-derived RNA-guided engineered nucleases (RGENs) enable genome editing in cultured cells, animals and plants, but are limited by off-target mutations.Unlike other algorithms currently available for identification of RGEN off-target sites, Cas-OFFinder is not limited by the number of mismatches and allows variations in protospacer-adjacent motif sequences recognized by Cas9, the essential protein component in RGENs.Cas-OFFinder is available as a command-line program or accessible via our website.

View Article: PubMed Central - PubMed

Affiliation: National Creative Research Initiatives Center for Genome Engineering, Department of Chemistry and Department of Physics and Astronomy, Seoul National University, 599 Gwanak-ro, Seoul 151-742, South Korea.

ABSTRACT

Summary: The Type II clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system is an adaptive immune response in prokaryotes, protecting host cells against invading phages or plasmids by cleaving these foreign DNA species in a targeted manner. CRISPR/Cas-derived RNA-guided engineered nucleases (RGENs) enable genome editing in cultured cells, animals and plants, but are limited by off-target mutations. Here, we present a novel algorithm termed Cas-OFFinder that searches for potential off-target sites in a given genome or user-defined sequences. Unlike other algorithms currently available for identification of RGEN off-target sites, Cas-OFFinder is not limited by the number of mismatches and allows variations in protospacer-adjacent motif sequences recognized by Cas9, the essential protein component in RGENs. Cas-OFFinder is available as a command-line program or accessible via our website.

Availability and implementation: Cas-OFFinder free access at http://www.rgenome.net/cas-offinder.

Contact: baesau@snu.ac.kr or jskim01@snu.ac.kr.

Show MeSH