Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency

• Published in: Nature biotechnology Vol. 38; no. 1; pp. 44 - 49
• Main Authors: Nguyen, David N., Roth, Theodore L., Li, P. Jonathan, Chen, Peixin Amy, Apathy, Ryan, Mamedov, Murad R., Vo, Linda T., Tobin, Victoria R., Goodman, Daniel, Shifrut, Eric, Bluestone, Jeffrey A., Puck, Jennifer M., Szoka, Francis C., Marson, Alexander
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.01.2020; Nature Publishing Group
• Subjects: 631/1647/1511; 631/1647/1513/1967/3196; 631/61/201/2110; 631/61/350/354; Adult; Agriculture; Bioinformatics; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Biomedicine; Biotechnology; CD3 antigen; CD4 antigen; CD8 antigen; CRISPR; CRISPR-Associated Protein 9 - metabolism; Editing; Efficiency; Gene Editing; Gene sequencing; Gene targeting; Genetic engineering; Genome editing; Genomes; Genomics; Hematopoietic stem cells; Homology; Humans; Immunoregulation; Letter; Life Sciences; Lymphocytes; Lymphocytes B; Lymphocytes T; Methods; Nanoparticles; Nanoparticles - chemistry; Natural killer cells; Nuclei (cytology); Pluripotency; Polymer industry; Polymers; Polymers - chemistry; Progenitor cells; Protein Stability; Repair; Ribonucleoproteins; RNA, Guide, CRISPR-Cas Systems - metabolism; Stem cells; Toxicity; United States; California
• ISSN: 1087-0156; 1546-1696
• DOI: 10.1038/s41587-019-0325-6

Versatile and precise genome modifications are needed to create a wider range of adoptive cellular therapies 1 – 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-derived 6 hematopoietic stem progenitor cells (HSPCs). Precise genome editing is made more efficient by stabilizing Cas9 and enhancing shuttling to the nucleus.