CRISPR/Cas-mediated genome editing in human pluripotent stem cells(hPSCs)offers unprecedented opportunities for developing in vitro disease modeling,drug screening and cell-based therapies.To efficiently deliver the C...CRISPR/Cas-mediated genome editing in human pluripotent stem cells(hPSCs)offers unprecedented opportunities for developing in vitro disease modeling,drug screening and cell-based therapies.To efficiently deliver the CRISPR components,here we developed two all-in-one vectors containing Cas9/gRNA and inducible Cas13d/gRNA cassettes for robust genome editing and RNA interference respectively.These vectors utilized the PiggyBac transposon system,which allows stable expression of CRISPR components in hPSCs.The Cas9 vector PB-CRISPR exhibited high efficiency(up to 99%)of inducing gene knockout in both protein-coding genes and long non-coding RNAs.The other inducible Cas13d vector achieved extremely high efficiency in RNA knockdown(98%knockdown for CD90)with optimized gRNA designs.Taken together,our PiggyBac CRISPR vectors can serve as powerful toolkits for studying gene functions in hPSCs.展开更多
基金supported by NIH R21EB026035(X.L.L.)NIH R21AI149312(C.E.C.,and X.L.L.)+1 种基金NSF CBET-1943696(X.L.L.)Penn State startup funding to X.L.L.
文摘CRISPR/Cas-mediated genome editing in human pluripotent stem cells(hPSCs)offers unprecedented opportunities for developing in vitro disease modeling,drug screening and cell-based therapies.To efficiently deliver the CRISPR components,here we developed two all-in-one vectors containing Cas9/gRNA and inducible Cas13d/gRNA cassettes for robust genome editing and RNA interference respectively.These vectors utilized the PiggyBac transposon system,which allows stable expression of CRISPR components in hPSCs.The Cas9 vector PB-CRISPR exhibited high efficiency(up to 99%)of inducing gene knockout in both protein-coding genes and long non-coding RNAs.The other inducible Cas13d vector achieved extremely high efficiency in RNA knockdown(98%knockdown for CD90)with optimized gRNA designs.Taken together,our PiggyBac CRISPR vectors can serve as powerful toolkits for studying gene functions in hPSCs.
