This webinar covers methodologies for genome-wide screening with CRISPR/Cas9 in human pluripotent stem cells (hPSCs) and their neuronal progeny.
CRISPR/Cas9 has revolutionized the ability to precisely modify genomes and to conduct genome-wide screens in human cells. While some cell types are easily modified, hPSCs poorly tolerate Cas9 editing and are difficult to engineer.
Our speaker, Robert Ihry of the Novartis Institutes for Biomedical Research, discusses a method he has developed for efficiently engineering and screening in hPSCs using a drug-inducible Cas9.
Dr. Ihry and colleagues found that double strand breaks induced by Cas9 are toxic and kill the majority of hPSCs. They also found that this response is tp53-dependent and transient inhibition of TP53 dramatically improved the efficiency of precise transgene knock-in by over fifteenfold.
Dr. Ihry discusses how these results are directly relevant to the use of CRISPR/Cas9 in high-throughput screening. Despite Cas9-induced toxicity, high performance genome-wide screens can be performed in hPSCs by accounting for cell loss. Dr. Ihry also discusses future considerations for conducting pooled and arrayed screens in differentiated neurons.
Effective CRISPR library screens require high target specificity and even oligo representation. Charles Joseph from Twist Bioscience discusses how their massively parallel silicon-based DNA synthesis platform delivers highly accurate and uniform oligo pools for precision CRISPR library generation.