Genome Editing Efficiency Analysis

Genomic DNA was extracted using the PureLink Genomic DNA Mini Kit (Invitrogen). Specific genomic loci were amplified using Velocity DNA Polymerase (Bioline). Off-target loci represent the top predicted off-target sites in the CRISPR Design Tool (crispr.mit.edu)^{68 (link)}. PCR products were gel-extracted (NucleoSpin Gel and PCR Clean-up kit, Clontech) and sent for Sanger sequencing. Sequencing results could then be uploaded to the Synthego ICE Analysis tool (v3) allowing for inference of the percent indels in the sample. For deep sequencing, the gel-extracted products were pooled and prepared for sequencing via paired-end 2 × 150 bp iSeq (Illumina, San Diego, CA) sequencing in-house.

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Stevens C.S., Carmichael J., Watkinson R., Kowdle S., Reis R.A., Hamane K., Jang J., Park A., Pernet O., Khamaikawin W., Hong P., Thibault P., Gowlikar A., An D.S, & Lee B. (2024). A temperature-sensitive and interferon-silent Sendai virus vector for CRISPR-Cas9 delivery and gene editing in primary human cells. bioRxiv.

Publication Preprint 2024

PureLink Genomic DNA Mini Kit Velocity DNA Polymerase NucleoSpin Gel and PCR Clean-up kit

Corresponding Organization : Icahn School of Medicine at Mount Sinai

Other organizations : Black AIDS Institute, University of California, Los Angeles

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Variable analysis

independent variables

Specific genomic loci amplified using Velocity DNA Polymerase (Bioline)

dependent variables

Percent indels in the sample

control variables

Genomic DNA extracted using the PureLink Genomic DNA Mini Kit (Invitrogen)
Off-target loci represent the top predicted off-target sites in the CRISPR Design Tool (crispr.mit.edu)
PCR products gel-extracted (NucleoSpin Gel and PCR Clean-up kit, Clontech) and sent for Sanger sequencing
Sequencing results uploaded to the Synthego ICE Analysis tool (v3) for inference of percent indels
Gel-extracted products pooled and prepared for paired-end 2 × 150 bp iSeq (Illumina, San Diego, CA) sequencing in-house

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