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If you could write software that could distinguish off-target CRISPR mutations from sequencing data, you'd have quite a valuable tool. Shouldn't be too difficu
by gravelc 9y ago
If you could write software that could distinguish off-target CRISPR mutations from sequencing data, you'd have quite a valuable tool. Shouldn't be too difficult if indistinguishablity is a declared myth.
- amelius 9y agoThat's pretty easy, because exact matching is easy. Just perform sequencing on the new genome, and map the sequences back to the original genome (with edits). If you can't find an exact match, you know something is wrong. You can also look at the statistics: if some sequences are more prevalent than they should be, you know something is wrong.
- folli 9y agoAnd how would you differentiate from classical breeding over X decades?
- gravelc 9y agoSequencing has errors - some in the PCR stage so not corrected by greater read depth for short reads (long reads are much more error prone). Genomes are dynamic - they change. You are looking at the progeny or regenerated plant, not the original plant. Exact matching will not get you far.
- amelius 9y agoYeah, but still it's a pretty easy problem compared to de-novo sequencing, since you have a good reference to map to.
- Obi_Juan_Kenobi 9y agoOkay, no. Stop a second. To use an analogy that HN might understand, doing that would be like using those giant javascript frameworks to create a distributed, dynamic website to serve a couple lines of static text. The answer is blotting. You do Southerns and Northerns and you'll see exactly whether or not you have off-targets. This is super basic stuff, and very reliable.
- folli 9y agoIn your analogy, Southern and Northern would be like using Fortran on a punched card, while high-throughput sequencing is using a modern programming language.