CRISPR KO vs CRISPR KI experiment using CRISPR-Cas9

References/Resources: https://www.patreon.com/the_Crux

All videos on Genetic Engineering: https://www.youtube.com/playlist?list=PL0Ymnd-zt4Ij9qp5ziU0TkyF42LUcgHQf

How to design a CRISPR experiment? How to perform surveyor assay to check gRNA targeting efficiency? How to perform a CRISPR knock-out experiment? How to perform a CRISPR knock-in experiment? What is the difference between the steps to perform a crispr deletion and crispr insertion experiment? What are the considerations for donor DNA design for CRISPR Knock-IN experiment? How to screen CRISPR edited cells? How to obtain clonal edited cell lines from a CRISPR experiment? This video is a technical and conceptual discussion around the practical design of a CRISPR experiment.

I discuss design details of a CRISPR Cas9 experimental protocol workflow, with a focus around the donor DNA design and validation of the Cas9-editing in cells. I discuss gRNA verification using surveyor assay or T7E1 assay. After verification, I highlight considerations around CRISPR KO and CRISPR KI experiments, especially when designing donor DNA templates for knock-in (insertion) experiments. Finally, I discuss how to obtain CRISPR edited cells lines, screening, and validating heterozygous or homozygous edited cells from a CRISPR experiment.

00:00 Types of CRISPR experiments
01:09 Delivery of sgRNA and Cas9
01:54 gRNA Design rules
02:25 Surveyor and T7E1 Assay
06:04 CRISPR KO experiment
07:13 CRISPR KI experiment
07:53 CRISPR KI Donor DNA and HDR
10:09 CRISPR KO with HDR
10:53 Outcomes of a CRISPR experiment
12:42 Selection and Screening markers
14:06 Clone expansion
14:41 Distinguish Hets vs Homs
16:25 Validate using SNPs

CRISPR-Cas9 genome editing Mechanism: https://youtu.be/7ESZTE6rjLI
gRNA Design for CRISPR-Cas9 using CRISPOR/CHOPCHOP: https://youtu.be/r4puDgc9rBs

References/Resources: https://www.patreon.com/the_Crux

*Must watch playlists*
All videos on Genetic Engineering: https://www.youtube.com/playlist?list=PL0Ymnd-zt4Ij9qp5ziU0TkyF42LUcgHQf
All videos on DNA Replication: https://youtube.com/playlist?list=PL0Ymnd-zt4IgLefDaeqicJbCBnbLqnDJ7
All videos on Transcription: https://www.youtube.com/playlist?list=PL0Ymnd-zt4Ij2VcAOHNUweftSElVsc-MX
All videos on Translation: https://www.youtube.com/playlist?list=PL0Ymnd-zt4IjAAAMlPLDIXbs692Yp14xa

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