Generation of Gene Knockout and Gene Replacement with Complete Removal of Full-length Endogenous Transcript Using CRISPR-Trap

Jonas Mechtersheimer; Stefan Reber; Marc-David Ruepp

doi:10.21769/BioProtoc.3052

Generation of Gene Knockout and Gene Replacement with Complete Removal of Full-length Endogenous Transcript Using CRISPR-Trap

Bio Protoc. 2018 Oct 20;8(20):e3052. doi: 10.21769/BioProtoc.3052.

Authors

Jonas Mechtersheimer¹, Stefan Reber¹, Marc-David Ruepp¹

Affiliation

¹ United Kingdom Dementia Research Institute Centre at King's College London, Institute of Psychiatry, Psychology and Neuroscience, King's College London, Maurice Wohl Clinical Neuroscience Institute, London, UK.

Abstract

This protocol describes the application of the CRISPR-Trap from designing of the gene targeting strategy to validation of successfully edited clones that was validated on various human cell lines, among them human induced pluripotent stem cells (hiPSCs). The advantage of CRISPR-Trap over conventional approaches is the complete removal of any endogenous full-length transcript from the target gene. CRISPR-Trap is applicable for any target gene with no or little coding sequence in its first exon. Several human cell lines and different genes have so far been edited successfully with CRISPR-Trap.

Keywords: CRISPR; CRISPR-Trap; Gene editing; Gene knockout; Gene replacement; hiPSCs.