CRISPR-Cas9 off-targeting assessment with nucleic acid duplex energy parameters
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CRISPR-Cas9 off-targeting assessment with nucleic acid duplex energy parameters. / Alkan, Ferhat; Wenzel, Anne; Anthon, Christian; Havgaard, Jakob Hull; Gorodkin, Jan.
I: Genome Biology, Bind 19, 177, 2018.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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T1 - CRISPR-Cas9 off-targeting assessment with nucleic acid duplex energy parameters
AU - Alkan, Ferhat
AU - Wenzel, Anne
AU - Anthon, Christian
AU - Havgaard, Jakob Hull
AU - Gorodkin, Jan
PY - 2018
Y1 - 2018
N2 - Background: Recent experimental efforts of CRISPR-Cas9 systems have shown that off-target binding and cleavage are a concern for the system and that this is highly dependent on the selected guide RNA (gRNA) design. Computational predictions of off-targets have been proposed as an attractive and more feasible alternative to tedious experimental efforts. However, accurate scoring of the high number of putative off-targets plays a key role for the success of computational off-targeting assessment. Results: We present an approximate binding energy model for the Cas9-gRNA-DNA complex, which systematically combines the energy parameters obtained for RNA-RNA, DNA-DNA, and RNA-DNA duplexes. Based on this model, two novel off-target assessment methods for gRNA selection in CRISPR-Cas9 applications are introduced: CRISPRoff to assign confidence scores to predicted off-targets and CRISPRspec to measure the specificity of the gRNA. We benchmark the methods against current state-of-the-art methods and show that both are in better agreement with experimental results. Furthermore, we show significant evidence supporting the inverse relationship between the on-target cleavage efficiency and specificity of the system, in which introduced binding energies are key components. Conclusions: The impact of the binding energies provides a direction for further studies of off-targeting mechanisms. The performance of CRISPRoff and CRISPRspec enables more accurate off-target evaluation for gRNA selections, prior to any CRISPR-Cas9 genome-editing application. For given gRNA sequences or all potential gRNAs in a given target region, CRISPRoff-based off-target predictions and CRISPRspec-based specificity evaluations can be carried out through our webserver at https://rth.dk/resources/crispr/.
AB - Background: Recent experimental efforts of CRISPR-Cas9 systems have shown that off-target binding and cleavage are a concern for the system and that this is highly dependent on the selected guide RNA (gRNA) design. Computational predictions of off-targets have been proposed as an attractive and more feasible alternative to tedious experimental efforts. However, accurate scoring of the high number of putative off-targets plays a key role for the success of computational off-targeting assessment. Results: We present an approximate binding energy model for the Cas9-gRNA-DNA complex, which systematically combines the energy parameters obtained for RNA-RNA, DNA-DNA, and RNA-DNA duplexes. Based on this model, two novel off-target assessment methods for gRNA selection in CRISPR-Cas9 applications are introduced: CRISPRoff to assign confidence scores to predicted off-targets and CRISPRspec to measure the specificity of the gRNA. We benchmark the methods against current state-of-the-art methods and show that both are in better agreement with experimental results. Furthermore, we show significant evidence supporting the inverse relationship between the on-target cleavage efficiency and specificity of the system, in which introduced binding energies are key components. Conclusions: The impact of the binding energies provides a direction for further studies of off-targeting mechanisms. The performance of CRISPRoff and CRISPRspec enables more accurate off-target evaluation for gRNA selections, prior to any CRISPR-Cas9 genome-editing application. For given gRNA sequences or all potential gRNAs in a given target region, CRISPRoff-based off-target predictions and CRISPRspec-based specificity evaluations can be carried out through our webserver at https://rth.dk/resources/crispr/.
KW - CRISPR-Cas9
KW - Energy models
KW - gRNA design
KW - gRNA specificity
KW - Off-target scoring
KW - Off-targets
U2 - 10.1186/s13059-018-1534-x
DO - 10.1186/s13059-018-1534-x
M3 - Journal article
C2 - 30367669
AN - SCOPUS:85055487106
VL - 19
JO - Genome Biology (Online Edition)
JF - Genome Biology (Online Edition)
SN - 1474-7596
M1 - 177
ER -
ID: 208978075