TY - JOUR
T1 - Robust genome editing via modRNA-based Cas9 or base editor in human pluripotent stem cells
AU - Haideri, Tahir
AU - Howells, Alessandro
AU - Jiang, Yuqian
AU - Yang, Jian
AU - Bao, Xiaoping
AU - Lian, Xiaojun Lance
N1 - Funding Information:
This work was supported by NIH NIBIB R21EB026035 (to X.L.L.), NIH NIAMS R01 AR072731 (to J.Y.), NSF CBET-1943696 (to X.L.L.), NSF CBET-2143064 (to X.B.), and Penn State startup funding (to X.L.L.).
Funding Information:
This work was supported by NIH NIBIB R21EB026035 (to X.L.L.), NIH NIAMS R01 AR072731 (to J.Y.), NSF CBET-1943696 (to X.L.L.), NSF CBET-2143064 (to X.B.), and Penn State startup funding (to X.L.L.). T.H. A.H. and X.L.L. designed the experiments and analyzed the results. T.H. A.H. and Y.J. performed the experiments and analyzed data. T.H. A.H. and X.L.L. wrote the manuscript. J.Y. X.B. and X.L.L. contributed to the revision of the manuscript. X.L.L. provided funding and supervised the experiments. X.L.L. T.H. A.H. and Y.J. are listed as inventors on a provisional patent application (no. 63/335,568), which was filed on April 27, 2022. The other authors declare no competing interests.
Publisher Copyright:
© 2022 The Author(s)
PY - 2022/9/19
Y1 - 2022/9/19
N2 - CRISPR systems have revolutionized biomedical research because they offer an unprecedented opportunity for genome editing. However, a bottleneck of applying CRISPR systems in human pluripotent stem cells (hPSCs) is how to deliver CRISPR effectors easily and efficiently. Here, we developed modified mRNA (modRNA)-based CRIPSR systems that utilized Cas9 and p53DD or a base editor (ABE8e) modRNA for the purposes of knocking out genes in hPSCs via simple lipid-based transfection. ABE8e modRNA was employed to disrupt the splice donor site, resulting in defective splicing of the target transcript and ultimately leading to gene knockout. Using our modRNA CRISPR systems, we achieved 73.3% ± 11.2% and 69.6 ± 3.8% knockout efficiency with Cas9 plus p53DD modRNA and ABE8e modRNA, respectively, which was significantly higher than the plasmid-based systems. In summary, we demonstrate that our non-integrating modRNA-based CRISPR methods hold great promise as more efficient and accessible techniques for genome editing of hPSCs.
AB - CRISPR systems have revolutionized biomedical research because they offer an unprecedented opportunity for genome editing. However, a bottleneck of applying CRISPR systems in human pluripotent stem cells (hPSCs) is how to deliver CRISPR effectors easily and efficiently. Here, we developed modified mRNA (modRNA)-based CRIPSR systems that utilized Cas9 and p53DD or a base editor (ABE8e) modRNA for the purposes of knocking out genes in hPSCs via simple lipid-based transfection. ABE8e modRNA was employed to disrupt the splice donor site, resulting in defective splicing of the target transcript and ultimately leading to gene knockout. Using our modRNA CRISPR systems, we achieved 73.3% ± 11.2% and 69.6 ± 3.8% knockout efficiency with Cas9 plus p53DD modRNA and ABE8e modRNA, respectively, which was significantly higher than the plasmid-based systems. In summary, we demonstrate that our non-integrating modRNA-based CRISPR methods hold great promise as more efficient and accessible techniques for genome editing of hPSCs.
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U2 - 10.1016/j.crmeth.2022.100290
DO - 10.1016/j.crmeth.2022.100290
M3 - Article
C2 - 36160051
AN - SCOPUS:85138017022
SN - 2667-2375
VL - 2
JO - Cell Reports Methods
JF - Cell Reports Methods
IS - 9
M1 - 100290
ER -