#genetics

I've been setting up a CRISPR workflow to generate homozygous knockouts in murine embryonic stem (ES) cells, and I'm torn between two plasmid backbones that keep popping up in the literature:

• pX330 (Addgene #42230) – the classic U6‑sgRNA drive with a bare‑bones backbone and a neomycin resistance cassette.
• pSpCas9(BB)-2A‑Puro (PX459) (Addgene #62988) – same U6 promoter but adds a self‑cleaving 2A peptide linked to puromycin resistance.

Both have been reported to work, but my lab's conditions are a bit particular:

• Need strong selection to eliminate non‑transfected cells, since our ES cells are notoriously fickle.
• Prefer a vector that leaves minimal scar after knock‑in, because we plan downstream recombination with a floxed reporter.
• Concerned about off‑target activity; some papers suggest that the type of promoter driving Cas9 (EF1α vs CBh) can affect fidelity.

What have you found in practice?

• Efficiency of homozygous indel formation (any numbers are welcome)
• Toxicity or growth impacts on ES cells
• Ease of plasmid preparation (some kits recommend mini‑prep versus midi‑prep)
• Any tweaks you made – e.g., adding a GFP reporter, swapping puromycin for blasticidin, or using a modified Cas9 (high‑fidelity SpCas9‑HF1) in the same backbone.

I’m leaning towards the puromycin vector because I’ve had decent success with puromycin selection on fibroblasts, but I’m hesitant about the 2A peptide possibly affecting protein expression in ES cells.

Any detailed anecdotes, protocol tweaks, or even a simple recommendation would be greatly appreciated. Thanks for your thoughts! ??

Spent months tinkering with 23andMe V4 chip, like a bored mechanic on a cold Sunday. Every time I hit upload it throws a sample too low error, as if my DNA were a busted engine. The new model promises 650K markers, yet the report comes in a blurry, mixed line that says Possible Ancestor 34% Caucasian, 66% Undetermined, which looks like an error code in a dashboard. Key pain points: 3.6 GB dataset is the size of my 2024 Mustang’s onboard storage – who really needs that much? Ancestry Insights claims Your DNA is similar to a reference panel from 1829, the same year a 1965 Ford GT40 hit the road. The privacy policy is still vague about third‑party data sharing. Added quick note: after a couple of emails support said the issue was a bug in the new uploader. Still no fix yet.

So I was reading about cell division and I stumbled upon this thing that's been bugging me - what's the point of having so many useless genes in our DNA? Like, we've got all these genes that don't even code for proteins, they just sit there doing nothing. Is it just leftovers from our evolutionary history or is there something more to it? I mean, it's not like we're using them for anything. Anyone got any ideas on this?

I've been slogging through papers on non‑coding regions and the more I read, the more the whole "junk DNA" label feels like a lazy shortcut. Some groups cling to it, arguing that most of the genome has no known function, while others point to regulatory elements, transcriptional noise, and even evolutionary remnants that clearly do something. The crux of my frustration is that the term keeps being used as a catch‑all dismissal, yet we keep finding bits of activity that challenge that narrative. Are we just being lazy by lumping everything unknown into "junk"? Or is there merit in keeping the term to highlight how little we actually understand? I want solid arguments, not just buzzword tossing. What does the evidence say, and should we retire the phrase altogether?