[DIYbio] Re: How does/would sperm-based germline modification work?

For genome engineering, if you want to modify a native gene (editing, knock-out, insertion of a tag) and CRISPR is the best one right now. If you want to add one or more genes, there are several possibilities (replicable episome, retrovirus, transposons, plasmid integration, artificial chromosomes) but I think it is better to avoid anything that integrate in the genome due to the possible deleterious effects (even more for germ lines modification). For replicative episomes/artificial chromosomes, one problem might be the possibility of cross-over with other chromosomes which could be problematic (not sure if it is known). 

On Saturday, 28 March 2015 11:58:55 UTC, Mega [Andreas Stuermer] wrote:

http://www.pnas.org/content/103/47/17672.full

Here is a protocol how to do it. Basically, sperm has a layer that protects it from foreign DNA (alledgedly) so you purify it, mix it with DNA and do artificial/instrumental insemination of a female specimen. This episome is said to be totally stable, and is replicated during mitosis (almost as stable as a chromosome). It has an element that attaches to the chromosomes during mitosis and is co-replicated. It does not integrate so no chance of getting cancer from random integration and hetereochromatin unwinding/activation of downstream genes.
There also is an improved version of the plasmid with a native human promoter (gets less silenced).