I have read couple of recent papers in biorxiv stating that CRISPR Cas9 gene editing does not cause significant off-target mutations invivo in mice. Is it that they could not see the off-targets for the particular guides they tested or do you normally not get off-targets for any appropriate guides that are highly ranked on specificity score?
Even if highly ranked you can still have off-targets. It depends on the target sequence, the sgRNA, and also the cas9 used (for example, you could use cas13 which was reported to have fewer off-targets and does not require a PAM sequence, but it edits RNA).
One can reduce off-targets using various sophisticated approaches (see this paper: https://www.nature.com/articles/nature17946).
Lastly, this website should help you predict off-target sites for your experiment: http://www.rgenome.net/cas-designer/
Best,
Is in-silico prediction for CRISPR off-target far from foolproof?
Do we not get off-target with >6 mismatches invivo even if there is no off-targets with
Have you seen this report:
The paper (Nature Methods) authors claims many unexpected mutation occurred while operating CRISPR on mice last year have been retracted (1). Reported today (2). The publish of the paper (1) caused CRISPR company stocks to drop.
**(1) Schaefer KA, et al. Unexpected mutations after CRISPR–Cas9 editing in vivo. Nature Methods 14, 547–548 (2017) doi:10.1038/nmeth.4293 30 May 2017
**(2) https://www.acsh.org/news/2018/03/29/crispr-science-prevails-critics-proven-target-12773 (American Council of Science and Health)
@ Shalini Reddy,
Due to the possible 'off-target' effect on other normal genes, now another field of researches using CRISPR/Cas systems to fix 'RNA' is very popular. I saw a couple of papers came out recently. The nucleases in these systems would fix on defect mRNAs (from a defect gene). So, it can avoid off-targeting on normal genes in the genome.
GENOME DAMAGE FROM CRISPR / CAS9 GENE EDITING HIGHER THAN THOUGHT
Previous research had not shown many unforeseen mutations from CRISPR/Cas9 in the DNA at the genome editing target site. To investigate this further the researchers carried out a full systematic study in both mouse and human cells and discovered that CRISPR/Cas9 frequently caused extensive mutations, but at a greater distance from the target site.
The researchers found many of the cells had large genetic rearrangements such as DNA deletions and insertions. These could lead to important genes being switched on or off, which could have major implications for CRISPR/Cas9 use in therapies. In addition, some of these changes were too far away from the target site to be seen with standard genotyping methods.
Prof Allan Bradley, corresponding author on the study from the Wellcome Sanger Institute, said: “This is the first systematic assessment of unexpected events resulting from CRISPR/Cas9 editing in therapeutically relevant cells, and we found that changes in the DNA have been seriously underestimated before now. It is important that anyone thinking of using this technology for gene therapy proceeds with caution, and looks very carefully to check for possible harmful effects.”
Michael Kosicki, the first author from the Wellcome Sanger Institute, said: “My initial experiment used CRISPR/Cas9 as a tool to study gene activity, however it became clear that something unexpected was happening. Once we realised the extent of the genetic rearrangements we studied it systematically, looking at different genes and different therapeutically relevant cell lines, and showed that the CRISPR/Cas9 effects held true.”
The work has implications for how CRISPR/Cas9 is used therapeutically and is likely to re-spark researchers’ interest in finding alternatives to the standard CRISPR/Cas9 method for gene editing.
Prof Maria Jasin, an independent researcher from Memorial Slone Kettering Cancer Centre, New York, who was not involved in the study said: “This study is the first to assess the repertoire of genomic damage arising at a CRISPR/Cas9 cleavage site. While it is not known if genomic sites in other cell lines will be affected in the same way, this study shows that further research and specific testing is needed before CRISPR/Cas9 is used clinically.”
Publication: Michael Kosicki, Kärt Tomberg and Allan Bradley (2018) Repair of CRISPR-Cas9–induced double-stranded breaks leads to large deletions and complex rearrangements. Nature Biotechnology. DOI: 10.1038/Nbt.4192