Peptide-enabled Ribonucleoprotein Delivery for CRISPR Engineering (PERC) in Primary Human Immune Cells and Hematopoietic Stem Cells

Overview

Journal Nat Protoc

Specialties Biology
Pathology
Science

Date 2025 Mar 3

PMID 40032999

Authors

Srishti U Sahu

Madalena Castro

Joseph J Muldoon

Kunica Asija

Stacia K Wyman

Netravathi Krishnappa

Lorena de Onate

Justin Eyquem

David N Nguyen

Ross C Wilson

Affiliations

Soon will be listed here.

Abstract

Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) is a new approach for ex vivo genome editing of primary human cells. PERC uses a single amphiphilic peptide reagent to mediate intracellular delivery of the same pre-formed CRISPR ribonucleoprotein enzymes that are broadly used in research and therapeutics, resulting in high-efficiency editing of stimulated immune cells and cultured hematopoietic stem and progenitor cells (HSPCs). PERC facilitates nuclease-mediated gene knockout, precise transgene knock-in and base editing. The protocol involves mixing the CRISPR ribonucleoprotein enzyme with peptide and then incubating with cultured cells. For efficient transgene knock-in, adeno-associated virus (AAV) homology-directed repair template (HDRT) DNA may be included. In contrast to electroporation, PERC is appealing because it needs no dedicated hardware and has less impact on cell phenotype and viability. Because of the gentle nature of PERC, delivery can be performed multiple times without substantial impact to cell health or phenotype. Editing efficiencies can surpass 90% when using either Cas9 or Cas12a in primary T cells or HSPCs. After 3 h dedicated to reagent preparation, the PERC delivery step can be completed in 1 h, with the associated cell culture steps taking 3-7 d total. Because the protocol calls for only three readily available reagents (protein, RNA and peptide) and does not require dedicated hardware for any step, PERC demands no special expertise and is exceptionally straightforward to adopt. The inherent compatibility of PERC with established cell engineering pipelines makes the protocol appealing for rapid deployment in research and clinical settings.

References

Taha E, Lee J, Hotta A . Delivery of CRISPR-Cas tools for in vivo genome editing therapy: Trends and challenges. J Control Release. 2022; 342:345-361. DOI: 10.1016/j.jconrel.2022.01.013. View

Stadtmauer E, Fraietta J, Davis M, Cohen A, Weber K, Lancaster E . CRISPR-engineered T cells in patients with refractory cancer. Science. 2020; 367(6481). PMC: 11249135. DOI: 10.1126/science.aba7365. View

Chiesa R, Georgiadis C, Syed F, Zhan H, Etuk A, Gkazi S . Base-Edited CAR7 T Cells for Relapsed T-Cell Acute Lymphoblastic Leukemia. N Engl J Med. 2023; 389(10):899-910. DOI: 10.1056/NEJMoa2300709. View

Bhokisham N, Laudermilch E, Traeger L, Bonilla T, Ruiz-Estevez M, Becker J . CRISPR-Cas System: The Current and Emerging Translational Landscape. Cells. 2023; 12(8). PMC: 10136740. DOI: 10.3390/cells12081103. View

Liu X, Li G, Liu Y, Zhou F, Huang X, Li K . Advances in CRISPR/Cas gene therapy for inborn errors of immunity. Front Immunol. 2023; 14:1111777. PMC: 10083256. DOI: 10.3389/fimmu.2023.1111777. View

Shifrut E, Carnevale J, Tobin V, Roth T, Woo J, Bui C . Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function. Cell. 2018; 175(7):1958-1971.e15. PMC: 6689405. DOI: 10.1016/j.cell.2018.10.024. View

Schmidt R, Ward C, Dajani R, Armour-Garb Z, Ota M, Allain V . Base-editing mutagenesis maps alleles to tune human T cell functions. Nature. 2023; 625(7996):805-812. PMC: 11065414. DOI: 10.1038/s41586-023-06835-6. View

Tothova Z, Krill-Burger J, Popova K, Landers C, Sievers Q, Yudovich D . Multiplex CRISPR/Cas9-Based Genome Editing in Human Hematopoietic Stem Cells Models Clonal Hematopoiesis and Myeloid Neoplasia. Cell Stem Cell. 2017; 21(4):547-555.e8. PMC: 5679060. DOI: 10.1016/j.stem.2017.07.015. View

Canver M, Smith E, Sher F, Pinello L, Sanjana N, Shalem O . BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis. Nature. 2015; 527(7577):192-7. PMC: 4644101. DOI: 10.1038/nature15521. View

10.

Ravi N, Wienert B, Wyman S, Bell H, George A, Mahalingam G . Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin. Elife. 2022; 11. PMC: 8865852. DOI: 10.7554/eLife.65421. View

11.

Cromer M, Barsan V, Jaeger E, Wang M, Hampton J, Chen F . Ultra-deep sequencing validates safety of CRISPR/Cas9 genome editing in human hematopoietic stem and progenitor cells. Nat Commun. 2022; 13(1):4724. PMC: 9372057. DOI: 10.1038/s41467-022-32233-z. View

12.

Delguidice T, Lepetit-Stoffaes J, Bordeleau L, Roberge J, Theberge V, Lauvaux C . Membrane permeabilizing amphiphilic peptide delivers recombinant transcription factor and CRISPR-Cas9/Cpf1 ribonucleoproteins in hard-to-modify cells. PLoS One. 2018; 13(4):e0195558. PMC: 5884575. DOI: 10.1371/journal.pone.0195558. View

13.

Foss D, Muldoon J, Nguyen D, Carr D, Sahu S, Hunsinger J . Peptide-mediated delivery of CRISPR enzymes for the efficient editing of primary human lymphocytes. Nat Biomed Eng. 2023; 7(5):647-660. PMC: 10129304. DOI: 10.1038/s41551-023-01032-2. View

14.

Zhang Z, Baxter A, Ren D, Qin K, Chen Z, Collins S . Efficient engineering of human and mouse primary cells using peptide-assisted genome editing. Nat Biotechnol. 2023; 42(2):305-315. PMC: 11230135. DOI: 10.1038/s41587-023-01756-1. View

15.

Rouet R, Thuma B, Roy M, Lintner N, Rubitski D, Finley J . Receptor-Mediated Delivery of CRISPR-Cas9 Endonuclease for Cell-Type-Specific Gene Editing. J Am Chem Soc. 2018; 140(21):6596-6603. PMC: 6002863. DOI: 10.1021/jacs.8b01551. View

16.

Krishnamurthy S, Wohlford-Lenane C, Kandimalla S, Sartre G, Meyerholz D, Theberge V . Engineered amphiphilic peptides enable delivery of proteins and CRISPR-associated nucleases to airway epithelia. Nat Commun. 2019; 10(1):4906. PMC: 6817825. DOI: 10.1038/s41467-019-12922-y. View

17.

Kulhankova K, Traore S, Cheng X, Benk-Fortin H, Hallee S, Harvey M . Shuttle peptide delivers base editor RNPs to rhesus monkey airway epithelial cells in vivo. Nat Commun. 2023; 14(1):8051. PMC: 10698009. DOI: 10.1038/s41467-023-43904-w. View

18.

Algayer B, OBrien A, Momose A, Murphy D, Procopio W, Tellers D . Novel pH Selective, Highly Lytic Peptides Based on a Chimeric Influenza Hemagglutinin Peptide/Cell Penetrating Peptide Motif. Molecules. 2019; 24(11). PMC: 6600388. DOI: 10.3390/molecules24112079. View

19.

Bak R, Dever D, Porteus M . CRISPR/Cas9 genome editing in human hematopoietic stem cells. Nat Protoc. 2018; 13(2):358-376. PMC: 5826598. DOI: 10.1038/nprot.2017.143. View

20.

Romero Z, Lomova A, Said S, Miggelbrink A, Kuo C, Campo-Fernandez B . Editing the Sickle Cell Disease Mutation in Human Hematopoietic Stem Cells: Comparison of Endonucleases and Homologous Donor Templates. Mol Ther. 2019; 27(8):1389-1406. PMC: 6697408. DOI: 10.1016/j.ymthe.2019.05.014. View