Date: 2018-12-01 03:36
The CRISPR-Cas9 system is routinely used for genome editing approaches in plant biotechnology. Off-targets represent undesirable additional target sites and require particular observance to avoid deleterious outcomes. There are (limited) prediction possibilities which should be considered prior to editing in vivo. After editing, results should be thoroughly examined to verify the success of the approach and occurrence of unwanted mutations.
In silico off-target prediction
It is possible to predict off-targets in silico. Several tools are available for this assay. However, a major problem is that such methods are biased, and the screening for detection of non-predicted off-targets, in vivo or in vitro, requires more efforts.
off-target prediction tools
Unbiased in vitro off-target identification
The following methods describe genome-wide in vitro assays for off-target identification.
After isolation of genomic DNA, it is digested with a nuclease, followed by whole genome sequencing. Off-targets are identified computationally (Kim et al. 2015).
Source-Code on GitHub
Genomic DNA is sheared and circularized after isolation. Residual linear DNA is degraded. The circular DNA can now be cleaved using Cas9. Cleaved ends are amplified via PCR and sequenced to detect off-targets (Tsai et al. 2017).
Source-Code on GitHub
Genomic DNA is isolated and cleaved by Cas9 ribonucleoproteins (RNPs). By fragmentation, adapter ligation, and affinity purification Cas9 cleaved fragments can be enriched. SITE-Seq libraries are created for subsequent next generation sequencing on the Illumina platform (Cameron et al. 2017).
Tsai, S. Q., Nguyen, N. T., Malagon-Lopez, J., Topkar, V. V., Aryee, M. J., & Joung, J. K. (2017). CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR–Cas9 nuclease off-targets. Nature methods, 14(6): 607-614.
Cameron, P. et al. (2017). Mapping the genomic landscape of CRISPR–Cas9 cleavage. Nature methods, 14(6): 600-606.
Kim, D., Bae, S., Park, J., Kim, E., Kim, S., Yu, H. R., Hwang, J., Kim, J. & Kim, J. S. (2015). Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nature methods, 12(3): 237-243.