The lab meeting involved discussing the T7E1 assay method for detecting off-target effects of CRISPR/Cas9 genome editing. They aimed to determine the sensitivity, specificity, and ability to detect off-targets in different cell types. The T7E1 assay involves PCR amplification of potential off-target sites followed by cleavage with the T7E1 endonuclease which recognizes mismatches, to indicate editing at off-target locations. They also discussed how enhancing homologous recombination during gene therapy could promote use of the preferred HR repair pathway over non-homologous end joining.

1. Lab Meeting
1 Oct 2021
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2. Methods detecting off-target -- T7E1 assay
Determinations
- Sensitivity
- How many off-target it can detect
- Specificity
- Whether it can work with variety of cell
type
- Purpose
- gRNA optimization (good design gRNA
prevents off-target)
- Looking in whole genome of edited cells
Off-target Effects in CRISPR/Cas9-mediated Genome Engineering, 2015
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3. WT
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Methods detecting off-target -- T7E1 assay
gRNA+Cas9 (no donor)
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Extract genomic DNA
PCR covering
region of gene
editing
T7E – endonuclease
recognizing
heteroduplex
(mismatch detection)
https://horizondiscovery.com/en/resources/featured-articles/considerations-for-t7-endonuclease-i-t7ei-mismatch-assays
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4. Volume = intensity of band
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5. Enhanced HR during gene therapy meaning?
• Promote cell to enter S-G2 phase
• Increase cell division
• Synchronize cells
• During S-G2 phase
• HR>NHEJ at normal state
• HR>>>>NHEJ at man-made manipulation
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