CRISPR-Cas9 has increased the accessibility of genome engineering due to its ease of use and ability to cause double-strand breaks (DSBs) at almost any locus of interest. DSBs are repaired in cells by two predominant pathways: non-homologous end joining (NHEJ) and homology-directed repair (HDR). Endogenous repair of DSBs using the NHEJ pathway typically results in functional protein disruption (knockout) whereas the HDR pathway can be used to introduce exogenous genetic content (knockin). During this webinar we will focus on the utility of a synthetic dual RNA approach to apply CRISPR-Cas9 to HDR genomic engineering applications and will provide guidelines for improving CRISPR Cas9-assisted HDR. We will also discuss the use of short single-strand DNA as a donor template for small insertions as well as plasmid DNA donor templates for large insertions. Lastly, we outline methods for characterization of HDR-generated cell lines for precise genomic engineering.
In this webinar, you will learn:
- Basics of homology-directed repair (HDR) using CRISPR-Cas9
- Selection of CRISPR RNAs (crRNAs) for HDR
- Recommendations for design of a synthetic oligo or plasmid donor template
- Application of endogenous gene tagging with GFP
- Techniques to evaluate HDR cell lines for the desired mutation
John Schiel, Ph.D.
He received his Bachelor of Science degree in Biochemistry at Colorado State University and his Ph.D. degree in Cell Biology at the University of Colorado Denver – Anschutz Medical Campus. John completed a postdoctoral fellowship at University of Massachusetts Medical School where he studied asymmetric cell division and centrosome maturation.