Generation of an Akaluc knock-in human embryonic stem cell reporter line using CRISPR-Cas9 technology

Abstract Akaluc, an enzyme engineered from luciferase, provides a potential powerful tool for tracing transplanted cells in vivo because of its near-infrared emission light. To enable evaluation of its potency, we inserted the Akaluc gene at AAVS1 locus using CRISPR/Cas9 technology and generated a clonal human embryonic stem stable cell line (Named H1-AAVS1-EF1α-Akaluc-KI or AkalucHES). AkalucHES could efficiently express Akaluc and were traced easily in vivo. We verified that AkalucHES expressed the pluripotency markers and showed normal stem cell morphology. Furthermore, AkalucHES maitains normal karyotype and is able to differentiate toward three germ-layer in vivo. So the Akaluc is effective for tracing transplanted cells in vivo.