TY - JOUR
T1 - Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency
AU - Nguyen, David N.
AU - Roth, Theodore L.
AU - Li, P. Jonathan
AU - Chen, Peixin Amy
AU - Apathy, Ryan
AU - Mamedov, Murad R.
AU - Vo, Linda T.
AU - Tobin, Victoria R.
AU - Goodman, Daniel
AU - Shifrut, Eric
AU - Bluestone, Jeffrey A.
AU - Puck, Jennifer M.
AU - Szoka, Francis C.
AU - Marson, Alexander
N1 - Publisher Copyright:
© 2019, The Author(s), under exclusive licence to Springer Nature America, Inc.
PY - 2020/1/1
Y1 - 2020/1/1
N2 - Versatile and precise genome modifications are needed to create a wider range of adoptive cellular therapies1–5. Here we report two improvements that increase the efficiency of CRISPR–Cas9-based genome editing in clinically relevant primary cell types. Truncated Cas9 target sequences (tCTSs) added at the ends of the homology-directed repair (HDR) template interact with Cas9 ribonucleoproteins (RNPs) to shuttle the template to the nucleus, enhancing HDR efficiency approximately two- to fourfold. Furthermore, stabilizing Cas9 RNPs into nanoparticles with polyglutamic acid further improves editing efficiency by approximately twofold, reduces toxicity, and enables lyophilized storage without loss of activity. Combining the two improvements increases gene targeting efficiency even at reduced HDR template doses, yielding approximately two to six times as many viable edited cells across multiple genomic loci in diverse cell types, such as bulk (CD3+) T cells, CD8+ T cells, CD4+ T cells, regulatory T cells (Tregs), γδ T cells, B cells, natural killer cells, and primary and induced pluripotent stem cell-derived6 hematopoietic stem progenitor cells (HSPCs).
AB - Versatile and precise genome modifications are needed to create a wider range of adoptive cellular therapies1–5. Here we report two improvements that increase the efficiency of CRISPR–Cas9-based genome editing in clinically relevant primary cell types. Truncated Cas9 target sequences (tCTSs) added at the ends of the homology-directed repair (HDR) template interact with Cas9 ribonucleoproteins (RNPs) to shuttle the template to the nucleus, enhancing HDR efficiency approximately two- to fourfold. Furthermore, stabilizing Cas9 RNPs into nanoparticles with polyglutamic acid further improves editing efficiency by approximately twofold, reduces toxicity, and enables lyophilized storage without loss of activity. Combining the two improvements increases gene targeting efficiency even at reduced HDR template doses, yielding approximately two to six times as many viable edited cells across multiple genomic loci in diverse cell types, such as bulk (CD3+) T cells, CD8+ T cells, CD4+ T cells, regulatory T cells (Tregs), γδ T cells, B cells, natural killer cells, and primary and induced pluripotent stem cell-derived6 hematopoietic stem progenitor cells (HSPCs).
UR - http://www.scopus.com/inward/record.url?scp=85076435884&partnerID=8YFLogxK
U2 - 10.1038/s41587-019-0325-6
DO - 10.1038/s41587-019-0325-6
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C2 - 31819258
AN - SCOPUS:85076435884
SN - 1087-0156
VL - 38
SP - 44
EP - 49
JO - Nature Biotechnology
JF - Nature Biotechnology
IS - 1
ER -