GUIDE-seq
Genome-Wide, Unbiased Identification of DSBs Enabled by Sequencing
GUIDE-seq (Tsai et al., 2015) relies on the integration of double-stranded oligodeoxynucleotides (DSOs) into DSBs. It belongs to a family of methodsãsuch as HTGTS (Chiarle et al., 2011), LAM-HTGTS (Hu et al., 2016), and Digenome-seq (Kim et al., 2015) ãthat are aimed at detecting off-target effects of CRISPR/Cas9 and other RNA-guided nucleases (RGNs).RGN-induced DSBs are tagged by integration of blunt-ended DSOs in the genomes of living human cells. The DSO integration sites are mapped precisely in the genome at the nucleotide level with unbiased amplification and NGS.
Advantages:
- Generates global specificity landscapes for RGNs in living human cells
- Targeted sequencing reduces cost
Disadvantages:
- Can miss some targets (Kim et al., 2016)
Reagents:
Illumina Library prep and Array Kit Selector
Reviews:
Hu J., Meyers R. M., Dong J., Panchakshari R. A., Alt F. W., et al. Detecting DNA double-stranded breaks in mammalian genomes by linear amplification-mediated high-throughput genome-wide translocation sequencing. Nat Protoc. 2016;11:853-871
Lee C. M., Cradick T. J., Fine E. J. and Bao G. Nuclease Target Site Selection for Maximizing On-target Activity and Minimizing Off-target Effects in Genome Editing. Mol Ther. 2016;24:475-487
References:
Kleinstiver B. P., Tsai S. Q., Prew M. S., et al. Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells. Nat Biotechnol. 2016;
.Kleinstiver B. P., Pattanayak V., Prew M. S., et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature. 2016;529:490-495
.Yin H., Song C. Q., Dorkin J. R., et al. Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo. Nat Biotechnol. 2016;34:328-333
.Bolukbasi M. F., Gupta A., Oikemus S., et al. DNA-binding-domain fusions enhance the targeting range and precision of Cas9. Nat Methods. 2015;12:1150-1156
.Friedland A. E., Baral R., Singhal P., et al. Characterization of Staphylococcus aureus Cas9: a smaller Cas9 for all-in-one adeno-associated virus delivery and paired nickase applications. Genome Biol. 2015;16:257
Kleinstiver B. P., Prew M. S., Tsai S. Q., et al. Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition. Nat Biotechnol. 2015;33:1293-1298
Doench J. G., Fusi N., Sullender M., et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol. 2016;34:184-191
Yin H., Song C. Q., Dorkin J. R., Zhu L. J., Li Y., et al. Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo. Nat Biotechnol. 2016;34:328-333
Chari R., Mali P., Moosburner M. and Church G. M. Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach. Nat Methods. 2015;12:823-826
Kleinstiver B. P., Prew M. S., Tsai S. Q., et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature. 2015;523:481-485