Apr 24, 2026
GBA1 HDR Donor Vector Assembly
- 1National Human Genome Research Institute
Protocol Citation: Yu Chen 2026. GBA1 HDR Donor Vector Assembly. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov11m3ovr2/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 17, 2025
Last Modified: April 24, 2026
Protocol Integer ID: 230093
Keywords: ASAPCRN, gba1 hdr donor vector, gba1
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000458
Disclaimer
This protocol describes the step to generate GBA1 HDR donor vectors.
Abstract
This protocol describes the steps to generate GBA1 HDR donor vectors.
Protocol materials
HpaI - 500 unitsNew England BiolabsCatalog #R0105S
Prepare HDR Donor Vector
The HDR donor vectors used to revert GBA1 N370S to WT were built using pDONOR-tagBFP-PSM-EGFP (Addgene, 100603) and pDONOR-tagBFP-PSM-dTOMATO (Addgene, 100604) as backbones.
Both vectors were digested with HpaI - 500 unitsNew England BiolabsCatalog #R0105S at 37 °C for 04:00:00 to generate two fragments of the vectors.
PCR purification of the two fragments of both vectors.
HiFi Assembly to Insert GBA1 Left and Right Homology Arms
Two custom gene blocks corresponding to the left 1-kb HDR arm and the right
1-kb HDR arm of GBA1 were assembled with the two fragments from pDONOR-tagBFP-PSM-EGFP, and with the two from pDONOR-tagBFP-PSM-dTOMATO using NEBuilder‱ HiFi DNA Assembly (New
England Biolabs, E5520), generating the GBA1-WT-pDONOR-tagBFP-PSM-EGFP
and GBA1-WT-pDONOR-tagBFP-PSM-dTOMATO, respectively.
Excision-only PiggyBac Transposase expression vector (PB220PA-1) was purchased from System
Biosciences.