TY - JOUR
T1 - Optimized Guide RNA Selection Improves Streptococcus pyogenes Cas9 Gene Editing of Human Hematopoietic Stem and Progenitor Cells
AU - Verhagen, Han J. M. P.
AU - Kuijk, Carlijn
AU - Rutgers, Laurens
AU - Kokke, Anne M.
AU - van der Meulen, Santhe A.
AU - van Mierlo, Gerard
AU - Voermans, Carlijn
AU - van den Akker, Emile
N1 - Funding Information: This work was supported by ZONMW game changer (116004203). Publisher Copyright: © 2022 Mary Ann Liebert, Inc., publishers.
PY - 2022/10/1
Y1 - 2022/10/1
N2 - Ribonucleoproteins (RNPs) are frequently applied for therapeutic gene editing as well as fundamental research because the method is fast, viral free, and shows fewest off target effects. We evaluated various parameters to genetically engineer human hematopoietic stem and progenitor cells (HSPCs) using Streptococcus pyogenes Cas9 (spCas9) RNPs, and achieve gene editing efficiencies up to 80%. We find that guide RNA (gRNA) design is critical to achieve high gene editing efficiencies. However, finding effective gRNAs for HSPCs can be challenging, while the contribution of numerous in silico models is unclear. By screening more than 120 gRNAs, our data demonstrate that in silico gRNA prediction models are ineffective. In this study, we established a time- and cost-efficient in vitro transcribed gRNA screening model in K562 cells that predicts effective gRNAs for HSPCs. RNP based screening thus outperforms in silico modeling and we report that gene editing is equally efficient in distinct CD34+ HSPC subpopulations. Furthermore, no effects on cell proliferation, differentiation, or in vitro hematopoietic lineage commitment were observed. Finally, no upregulation of p21 expression was found, suggesting unperturbed HSPC homeostasis.
AB - Ribonucleoproteins (RNPs) are frequently applied for therapeutic gene editing as well as fundamental research because the method is fast, viral free, and shows fewest off target effects. We evaluated various parameters to genetically engineer human hematopoietic stem and progenitor cells (HSPCs) using Streptococcus pyogenes Cas9 (spCas9) RNPs, and achieve gene editing efficiencies up to 80%. We find that guide RNA (gRNA) design is critical to achieve high gene editing efficiencies. However, finding effective gRNAs for HSPCs can be challenging, while the contribution of numerous in silico models is unclear. By screening more than 120 gRNAs, our data demonstrate that in silico gRNA prediction models are ineffective. In this study, we established a time- and cost-efficient in vitro transcribed gRNA screening model in K562 cells that predicts effective gRNAs for HSPCs. RNP based screening thus outperforms in silico modeling and we report that gene editing is equally efficient in distinct CD34+ HSPC subpopulations. Furthermore, no effects on cell proliferation, differentiation, or in vitro hematopoietic lineage commitment were observed. Finally, no upregulation of p21 expression was found, suggesting unperturbed HSPC homeostasis.
UR - http://www.scopus.com/inward/record.url?scp=85142507003&partnerID=8YFLogxK
U2 - https://doi.org/10.1089/crispr.2021.0112
DO - https://doi.org/10.1089/crispr.2021.0112
M3 - Article
C2 - 36169633
SN - 2573-1599
VL - 5
SP - 702
EP - 716
JO - CRISPR Journal
JF - CRISPR Journal
IS - 5
ER -