JUNCTION PCR

For KI: Can be applied to determine successful integration of your exogenous DNA sequence.

To check for targeted (= at the intended genomic location) integration make sure one primer (P1/P4) is binding outside of the homology arm and the other primer (P2/P3) is located in your DNA sequence you want to insert. By this you eliminate any false positive that could occur due to random integration (off-target) of your donor DNA into the genome.

For larger KI (>1kb) it is advisable to check both - 5' and 3' junctions to confirm complete integration 

X

For KO: Can be applied to determine successful deletion (> 100bp) of your DNA sequence of interest 

To check for successful excision design primers that will allow you to distinguish wt and tg band on an agarose gel

 100 bp - 3 kb deletion           > 4 kb (wt amplification tricky)

T7 / SURVEYOR ASSAY

Allows for the detection of indels

Following amplification over the cut site, PCR products (~ 500bp) are denatured and re-annealed. Possible heteroduplex formation - hybridization of wt and targeted strand - leads to the formation of DNA bulges in the presence of indels which are cleaved by T7 / surveyor endonuclease. Cleavage products can be analyzed on agarose gel. 

TIDE (Tracking of Indels by Decomposition) 

Allows for the analysis of raw sequencing results 

Following amplification over the cut site, PCR products (~ 700bp) are subjected to Sanger sequencing (include wt control for reference). Algorithm allows for the identification of actual indel sizes and editing frequencies in a pool of cells. 

RESTRICTION ANALYSIS

Most useful to identify correct insertion of point mutations

 

Loss or gain (preferred) of unique restriction site possible. PCR amplify over the region of interest (include wt control), purify, and digest PCR products (500-700bp) with unique restriction enzyme. Note, it is advisable NOT to have the cut site in the centre of your PCR product to enable cleavage products to be separated on agarose gel.

CLONING (e.g. TOPO CLONING)

To verify sequence integrity of individual alleles

Following amplification over the cut site, PCR products (~ 500bp) are cloned into vectors and transformed into E.Coli. Pick 12 colonies for Sanger sequencing and check for indel sizes by aligning to wt sequence

Possible outcomes:

12 x wt

 

    wt

X

6 x wt

6 x -2bp deletion 

 

heterozygous

(out-of frame deletion)

6 x wt

6 x -3bp deletion 

 

heterozygous 

(in-frame deletion)

X

6 x +4bp insertion

6 x -2bp deletion 

 

homozygous KO

(out-of frame deletion)

SANGER SEQUENCING

Always check sequence integrity. Note, allow at least 30bp distance between primer and region of interest to account for poor sequence quality 

All above methods are useful for initial screening purposes to identify potential positive clones/pups. Nevertheless, we always recommend to seq. verify both alleles!