Genome-editing techniques, including CRISPR-Cas9 and TALENs, have leapt into the limelight through their ability to perform feats such as creating genetically engineered monkeys, ‘curing’ a human related disease in mice, and taking out huge sections of a genome.
The two systems are both based on nucleases, with the CRISPR-Cas9 system using the bacterially derived Cas-9 nuclease while TALENs are made by fusing a DNA binding domain to a nuclease. The specificity and versatility of these genome-editing techniques makes them a powerful research tool. Nature Methods named these genome-editing techniques as the “Method of the Year” in 2011, while a previous study examining the level of off-target effects found both systems to be highly specific in induced pluripotent stem cells.
Here, Wang et al. turn a critical eye to these oft-lauded techniques and examine both TALENs and the CRISPR-Cas9 system for off-target effects. They utilized Integrase Defective Lentiviral Vectors (IDLV) to find and mark double strand DNA breaks. The nuclease activity of the CRISPR-Cas9 and TALENs generate breakages, and the IDLVs could then be incorporated. The genomes were then methodically sequenced to look for incorporation of the IDLV at the intended site, and also at off-target sequences.
The researchers designed and tested four TALENs and six CRISPR-Cas9 nucleases, in addition to two CRISPR-Cas9 nucleases with only nickase activity (generating a single strand break) looking for off-target nuclease activity. The results were surprising:
- There was no off-target cleavage by the TALENs designed and tested for either gene.
- 3 of the 6 CRISPR-Cas9 nucleases had off-target effects BUT the mutant CRISPR-Cas9 nucleases with only nickase activity showed no off-target effects.
- CRISPR-Cas9 off-target effects appear to be dosage dependent, and can be minimized at lower doses.
- CRISPR-Cas9 (and nickase mutant) can tolerate single base skippage between guide RNA and target.
- IDLVs can be used to identify transient double stranded DNA breakages, such as those caused by genome editing nucleases, with an indel frequency <1%.
These results, along with those from another recent study, point to the need for careful design of CRISPR-Cas9 systems, or perhaps consideration of the mutant nickase Cas9 or TALENs as alternative genome-editing methods.
If you’re curious for more, read the full article at Nature Biotechnology, 2015.