EENdb - Utilities  >  EEN efficiency detection methods
Summary of EEN efficiency detection methods
These efficiency detection methods can also be applied to off-target sites to evaluate the specificity of EENs.
Note: New methods may be developed in the future.
ClassificationMethodDescriptionComments
Based on cleavage of EENs in vitro cleavage testing - Simply digest the DNA substrate containing the target site by EENs in vitro and examine/compare the length of products after digestion.
- The efficiency of EENs can be evaluated by comparing the digested products and the undigested substrate.
Based on reporter gene reporter gene disruption assay - If the target gene is a reporter gene (eGFP, resistance genes, etc.) itself, the efficiency of EENs can be evaluated by measuring the activity of the reporter gene.
- The accurate efficiency cannot be evaluated if positive selection is applied.
Applicable to NHEJ and HR.
reporter gene correction assay - A frame-shift mutated reporter gene inserted by the EENs target site after the start codon is constructed, which may be corrected by NHEJ indels.
- This is a positive selection process and cannot be used to evaluate the efficiency of EENs.
Applicable to NHEJ.
reporter gene addition / replacing assay - Use HR addition or HR replacing to introduce a functional reporter gene into the EENs target site.
- This is a positive selection process and cannot be used to evaluate the efficiency of EENs.
Applicable to HR.
SSA [single strand annealing] assay - Need to construct a reporter system consisting of two inactive homologous arms of a reporter gene which are separated by the EEN site.
- DSB-induced homologous recombination may restore a functional reporter gene, whose activity represents the efficiency of EENs.
- SSA assay is often performed in bacteria or yeasts, but can also be applied in in vitro cultured cells.
- This method can be used to evaluate the efficiency of EENs simultaneously.
Applicable to HR.
Based on PCR direct PCR - Direct detection of the target effect by PCR amplification of the modified sequences induced by EENs.
- Intended big change of the target sequences is required.
- This is a positive selection process and cannot be used to evaluate the efficiency of EENs.
Applicable to NHEJ-ODC, NHEJ-DisDel, and most types of HR.
HRMA
[high resolution melt analysis]
- Mix the PCR products spanning the EENs target site from EENs-treated (and wild-type) samples.
- The curve of fluorescence data (presents double-strand DNAs) to temperature in renaturing and then denaturing of the mixed PCR products is different to that of wild-type product only, if mismatches exist.
- Hard to evaluate the efficiency of EENs directly.
Applicable to NHEJ.
Based on PCR & sequencing direct sequencing - Just directly sequence the PCR products spanning the EENs target site. Applicable to NHEJ and HR.
high-throughput sequencing - Sequence the PCR products spanning the EENs target site via high-throughput sequencing (deep sequencing) instead of the traditional direct sequencing method.
- Similar to the direct sequencing method, the efficiency of EENs can be evaluated by comparison of the numbers of WT sequences and mutated sequences.
Applicable to NHEJ and HR.
Based on PCR & cleavage of other endonucleases mismatch-detection nuclease assay - Mix the PCR products spanning the EENs target site from EENs-treated (and wild-type) samples.
- A mismatch loop may be generated during renaturing of double-strands DNA after denaturing the mixed PCR products if one of the two strands carries an indels and the other not, which can be cleaved by mismatch-detection nucleases (CEL-1, T7E1, etc.).
- The efficiency of EENs can be evaluated by comparing the digested and undigested products.
Applicable to NHEJ.
restriction-enzyme-resistance assay (after PCR) - The EENs target site is designed to contain a restriction enzyme (RE) site in the spacer and the RE site may be destroyed by small indels induced by EENs.
- The PCR products containing these types of mutated target site are resistant to the RE digestion while the WT sequences will be digested.
- Not precise RFLP because the mutations caused by indel are uncontrollable.
- The efficiency of EENs can be approximately evaluated by comparing the digested and undigested (mutated) PCR products.
Applicable to NHEJ.

TALE-NT supports to find RE sites in the spacers of TALEN target sites.
restriction-enzyme-resistance assay (before PCR) - Similar to the above method, except that the steps of RE-digestion and PCR amplification are swapped.
- The genomic DNA preparation is first digested by the RE so that the unchanged WT sequences around the EENs target site are digested and cannot be PCR amplified. After the RE digestion, the genomic DNA containing target site mutations (with RE site mutated) remains intact and can be detected by PCR amplification.
- This is a positive selection process and cannot be used to evaluate the efficiency of EENs.
Applicable to NHEJ.

TALE-NT supports to find RE sites in the spacers of TALEN target sites.
PCR-RFLP
[restriction fragment length polymorphism]
- Use HR replacing to mutate an old RE site or to introduce a new RE site in the EENs target site.
- The PCR products containing the changed RE site will get different fragments when digested by the RE.
- The efficiency of EENs can be approximately evaluated by comparing the digested and undigested PCR products.
Applicable to HR.

TALE-NT supports to find RE sites in the spacers of TALEN target sites.
Based on cleavage of other nuclease (without PCR) Southern blotting - If the RE sites inside or nearby the EENs target site is changed, a Southern blot can discover and evoluate the efficiency of EENs.
- Similar to the methods based on PCR & cleavage of other nuclease, except for no PCR amplification step is taken.
Applicable to HR.



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