PROTOCOL 017: T7 ENDONUCLEASE GENOTYPING OF CRISPR-INDUCED GENE LESIONS

(9/2017)

 

REAGENTS

 

Genomic DNA (see PROTOCOL 016)

T7 Endonuclease I (NEB M0302)

Proteinase K (5 μg/ μl)

Primers flanking mutation

Note: product should be between 300 and 500 bp centered on predicted mutation site.

NuSieve 3:1 Agarose

1X TBE buffer

DNA loading buffer

 

BACKGROUND: This assay is based on the principle that T7 endonuclease will cut double stranded DNA at a site of mismatch. CRISPR-mediated DNA damage activates non-homologous end joining repair, which repairs double strand breaks in a non-template dependent manner. Therefore, even if CRISPR mutates both alleles in a diploid organism (e.g. zebrafish), the likelihood that repair of both alleles results in identical base incorporation is infinitesimally low. By PCR amplifying the region targeted by CRISPR, the presence of a mutation can be detected by denaturing and reannealing the PCR products followed by T7 endonuclease digestion. The assay is graphically demonstrated in the following figure: 

 

 

 

 

PROCEDURE

 

Setup PCR reaction with 2.5 μl of diluted gDNA isolated according to PROTOCOL 016.

 

Select PCR parameters suitable for primer Tm. A typical PCR protocol is shown below:

 

            95°C                2m

2          95°C                30s

55°C                30s

72°C                30s

GoTo 2            30x

72°C                10m

4°C                  Hold

 

After PCR, assemble denaturation reaction:

 

PCR reaction              10 μl

10X NEB Buffer 2        2 μl

Water                          7 μl

 

Program thermocycler as follows:

 

STEP

TEMPERATURE

RAMP RATE

TIME

Initial Denaturation

95°C

 

5 m

Annealing

95 à 85°C

85 à 25°C

-2°C/s

-0.1°C/s

 

Hold

4°C

 

 

 

Place denaturation reaction in thermocycler and proceed with denaturation/reannealing program.

 

Assemble heteroduplex digestion:

 

            Annealed PCR product from preceding step              19 μl

            T7 Endonuclease                                                        1 μl

 

Incubate reaction for 15m at 37°C

 

Add 1 μl ProtK and digest for 5m at 37°C

 

Analyze digestion products on 3% NuSieve 3:1 gel