Simple and efficient delivery of CRISPR genome editing systems in primary cells remains a major challenge. Here, we describe an engineered Peptide-Assisted Genome Editing (PAGE) CRISPR-Cas system for rapid and robust editing of primary cells. PAGE couples a cell-penetrating Cas protein with a cell-penetrating endosomal escape peptide in a 30-minute incubation that yields up to ~98% editing efficiency in primary human and mouse T cells. PAGE provides a broadly generalizable platform for next generation genome engineering in primary cells. CITATION INFORMATION: Zhang Zhen, Baxter Amy E, Ren Diqiu, Qin Kunhua, Chen Zeyu, Collins Sierra M., Huang Hua, Komar Chad A., Bailer Peter F., Parker Jared B., Blobel Gerd A., Kohli Rahul M., Wherry E. John*, Berger Shelley,*, and Shi Junwei*. Peptide-assisted genome editing permits efficient CRISPR engineering of primary T cells. Overall design: The purpose of this experiment is to compare the gene expression changes following opCas12a-CPP RNP delivery by PAGE or electroporation on CAR19 T cells. 0.5 ~ 2 million CAR19 T cells were collected at 6 hours and 3 days post PAGE and electroporation.. Cells were washed once with PBS and resuspend in 0.5 ml of TRIzol (Thermo 15596018) and stored at -80°C. RNA was extracted using RNA Clean & Concentrator-5 (ZYMO R1013) following manufacture?s instruction. mRNA was isolated from 500 ng of RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB E7490L) to prepare libraries with NEBNext Ultra II Directional RNA Library Prep Kit (NEB E7760L) following manufacture?s instruction. RNA libraries were pooled and sequenced with NextSeq 500/550 High Output Kit (75 cycles) v2.5 kit (illumina 20024906) on NextSeq 550 platform (illumina) using paired-end sequencing (42:6:0:42).