Programmable gene insertion in human cells with a laboratory-evolved CRISPR-associated transposase
Authors/Creators
Description
Abstract
Programmable gene integration in human cells has the potential to enable mutation-agnostic treatments for loss-of-function genetic diseases and facilitate many applications in the life sciences. CRISPR-associated transposases (CASTs) catalyze RNA-guided DNA integration but thus far demonstrate minimal activity in human cells. Using phage-assisted continuous evolution (PACE), we identified CAST variants with ≥200-fold average improved integration activity. The evolved CAST system (evoCAST) achieves ~10-30% integration efficiencies of kilobase-size DNA cargoes in human cells across 14 tested genomic target sites, including safe harbor loci, sites used for immunotherapy, and genes implicated in loss-of-function diseases, with undetected indels and low levels of off-target integration. Collectively, our findings establish a platform for the laboratory evolution of CASTs and advance a versatile system for programmable gene integration in living systems.
Table of contents
Description of files uploaded
Off-target_ATAC_code.zip: Compressed folder of python/bash scripts used to calculate ATAC scores of off-target evoCAST integration events, and perform bootstrap analysis (fig. S25D)
HTS-IntegrationEfficiencyScript.zip: Compressed folder of script used to analyze integration efficiencies for CAST using HTS data, adapted from Lampe, et al. NBT 2023. Script also calculates distribution of T-RL insertion sites.
240111_1921_xSL0283.ncf: Raw flow-cytommetry data of TnsB-VP64 reporter assay (fig. S18, A and B). Example gating shown in Lampe, et al. NBT 2023.
240111_1921_xSL0283_Heat Map1.csv: CSV file of tdTomato MFI for TnsB-VP64 reporter assay (fig. S18, A and B). Example gating shown in Lampe, et al. NBT 2023.
220713_QCascade-structure-AF2.cxs: Chimera session of AlphaFold2-predicted PseQCascade (fig. S21A, C, and D).
PseTnsAB-STC.pse: PyMOL session of AlphaFold3-predicted PseTnsAB strand-transfer complex (Fig. 3E, figs. S16 and S18C).
PseTnsC7-DNA.pse: PyMOL session of AlphaFold3-predicted PseTnsC heptamer and target DNA (fig. S8).
PseTnsC7-TnsBhook.pse: PyMOL session of AlphaFold3-predicted PseTnsC heptamer in complex witih a PseTnsB C-terminal hook (Fig. 3F).
All ".ddpcrone" and "WellAnnotations.xlsx" files: Raw ddPCR files (.ddpcrone) for Fig. 5A and 5I, representative of ddPCR data throughout study. Well identities and efficiency calculations are in the ".xlsx" file. Figures S5 and S6 discuss ddPCR quantitation in depth.
ExampleGating-figS25H.ai and figS25H-flowquantitation.xlsx: Example flow cytometry gating (.ai) and % mCherry+ values (.xlsx) for off-target integration reporter assay (fig. S25H).
FigS18D_Western.jpg: Uncropped western blot image for fig. S18D.
UDiTaS analysis scripts are in the linked GitHub repository (https://github.com/sternberglab/Witte_Lampe_Eitzinger_et_al_2024)
Files
240111_1921_xSL0283_Heat Map1.csv
Files
(291.2 MB)
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Additional details
Software
- Repository URL
- https://github.com/sternberglab/Witte_Lampe_Eitzinger_et_al_2024
- Programming language
- Python
- Development Status
- Active