CRISPR efficiency calculations for indel%?

Josephine Swiggs Malmevik @Josephine_Swiggs_Malmevik

09 September 2016 3 855 Report

Hi all,

We use the Surveyor assay and I have some questions as we get a maximum of 14% indel occurrence. We also have some questions regarding the use of the formula specified by Ran et al 2013 in Nature Protocols; where they say to use the fcut (a + b) / (a + b + c) and then the 1 - square root of (1-fcut).

If you just have time to answer one question, no worries. :)

Q1: 14% seems quite low to me, or do you get similar values? We know that our transduction of the gRNA and the Cas9 (using all-in-one lentiviral vector) is 100% due to the following puromycin selection ensuring only cells with the gRNA and Cas9 inside will survive. Has anyone obtained the reported 68% in Ran et al 2013?

(More details about our protocol: We produce lentivirus with SpCas9 and a gRNA (same vector) in 293T cells, transfer the conditioned medium for transduction of 3T3 cells, do puromycin selection and harvest 5 days after transduction of the cells. The genomic region is amplified through PCR (using Pfu) and then 360 ng is annealed using the specified annealing protocol with lowered temperature and thereafter cut using Surveyor. I think I might add a higher amount of PCR product next time for visualisation simplification

Q2: The fcut formula says to take the combined intensity of the a+b, which from the article seems to be any band in the lane that was digested including the thickest band at the top? I normally get one thick band and one faint, smaller band for my two genomic regions tested so far. (The Surveyor cuts approximately in the middle of the amplified genomic region, so tends to be onen band).

Q3: The fcut formula then says to divide this with the a+b+c, ie also including the undigested PCR product. I assume one should make sure to load the exact same amount as what is added to the Surveyor protocol? Otherwise, this of course affects the results.

Q4: Wouldn’t it be better to compare the digested products with a homodimer of a wt sample (ie one that will not be cleaved but has been digested)?

Q5: We have the capacity to do 3D, ie volume measurements, wouldn’t that be even more valuable, or will it give the same results?

Q5: What kind of indel % do you guys get using the C+G heterodimer vs G?

Thanks in advance!

Best regards,

Josephine

Ümit Pul

Hi Josephine,

I suggest to use the TIDE tool (instead of enzymatic assay) to quantify sgRNA efficiencies; it is easier to use and very reliable for the evaluation/comparison of sgRNAs.

Link to the free Webtool:

https://tide.nki.nl/

TIDE publication:

http://nar.oxfordjournals.org/content/42/22/e168

Ümit

...a new tool (ICE - Inference of CRISPR Edits) similar to TIDE is available here:

https://ice.synthego.com/#/

Reference: https://www.biorxiv.org/content/early/2018/01/20/251082

Best,

Di Xia

The calculation is correct but You can not simply compare your efficiency with Ran's paper unless you are using the same guide RNA on same cell-line. If you are worrying about your CRISPR experiment, you should use the same gRNA and cells as Ran's paper used or add a commonly used guide RNA targeting eg. AAVS1 or HPRT as your positive control.

How do I quickly find out whether an AAV plasmid is single-stranded or self-complementary?

Intracellular detection of myc using FACS?

Is there an optimal way to design a stuffer sequence for insertion into AAV vectors to ensure proper packaging?

error in running a model in Abaqus

error in modelling model in Abaqus

MD MOE RMSD

MD MOE RMSD

MGL tools

fatal error

fatal error

fatal error

fatal error

i have some problem with chtMultiRegionSimpleFoam