When working with CRISPR approaches all kind of mutations can occur through NHEJ. Most frequently I and others see small (1-5nt) deletions, less frequently insertions, sometimes lager deletions.
The biggest deltion I have found recently on a mouse ES cell projest is 350 nt. In a recent report from Alan Bradley () a much bi...
Crimson Pixie
over 4 years ago
Designing a 8kb replacement using HDR
I am new to CRISPR and have been doing deletions so far using NHEJ. We have a project that needs 5 SNPs in a span of 8 kb to be altered. So we are considering if we can replace the 8kb fragment using a donor DNA in a cell line. Has anyone done something like? Any suggestions are welcome.
Brown Siren
almost 5 years ago
Plasmids undergoing NHEJ?
I am trying to screen for gRNA's targeting a plasmid that is being co-transfected into HEK 293T cells. However, when I sequence around the cut site for TIDE, I am not seeing any detectable indel formations.
Has anyone done this before? Can plasmids undergo NHEJ, or is it that the plasmids undergo degradation after cutting?...
Gold Selkie
over 5 years ago
Small Molecules to enhance HDR
I did a literature search tailored to find ways to increase HDR in Human IPS - ES cells. I was interested in trying drugs to enhance Homology directed repair.
Has anyone has tried any of the following drugs- constructs in their lab
or know of any which I have left off the list.
I will be introducing a SNP into an IPSC ...
Azure Dragon
over 6 years ago
THP-1 CRISPR protocol
Using the protocol outlined below, I am unable to detect integration of our donor construct by PCR or GFP expression in THP-1 cells. Does anyone have a protocol or a reference to the use of recombinant Cas9 protein in THP-1 cells using nucleofection, or a suggestion to improve our current protocol? THP-1 cells are recalcitr...
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