Aug 30, 2024
Version 2
Efficient and precise targeting of the AAVS1 safe harbour locus in hPSCs. V.2
- Dmitry Ovchinnikov1
- 1Florey Institute / UofMelbourne
Protocol Citation: Dmitry Ovchinnikov 2024. Efficient and precise targeting of the AAVS1 safe harbour locus in hPSCs.. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn6r1ml5d/v2Version created by Dmitry Ovchinnikov
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: August 25, 2024
Last Modified: August 30, 2024
Protocol Integer ID: 106712
Keywords: hPSC gene targeting, TALENs, AAVS1 allele b
Funders Acknowledgement:
ASAP MJFF
Grant ID: 000497
ARC
Grant ID: MRFF Accelerate Stem Cell program
Disclaimer
The protocol was developed while carrying out studies supported by the Australian Research Council's Special Research Initiative "Stem Cells Australia" MRFF accelerate grant and an ASAP MJFF grant 000497 (team Kirik)
Abstract
Stably genetically-modified human pluripotent stem cells (hPSCs) are increasingly being used for studies relying on the consistent expression of the transgene of interest in human stem cells and their derivatives. Most often, the robustness of the transgene expression is achieved by its introduction into one of the members of an ever-expanding set of so-called "safe harbour"loci in the human genome. Here we describe a process of an efficient generation of high-quality hPSC clones with precise homology-directed targeting of the AAVS1(PPP1R2C) locus assisted by local DNA cutting using TALEN user-customisable nucleases.
Protocol materials
ACCUTASE™ 100 mL
STEMCELL Technologies Inc.Catalog #7920
Step 2
Corning® Matrigel®CorningCatalog #354277
Step 2
Puromycin DihydrochlorideGibco - Thermo FisherCatalog #A1113803
Step 3
Preparation of reagents for successful transfection
Preparation of reagents for successful transfection
Preparation of targeting and nuclease-encoding vectors for a successful transfection
Targeting of the AAVS1/PPP1R12C locus could be aided, for instance, by the use of the 2 TALEN plasmids available from theAddgene repository (addgene.org), AAVS1 TALEN-LaddgeneCatalog #59025 AAVS1-TALEN-RaddgeneCatalog #59026 These vectors are compatible with a wide range of AAVS1-targeting plasmids, e.g. those based on AAVS1 targeting vector with puromycin selectionaddgeneCatalog #73503 Sufficient amount of the plasmid should be prepared using a midi/maxiprep commercial plasmid prep kit, preferably with an endotoxin-free purification option.
High-quality bulk or manually-passaged hPSC culture is used as a cell source for transfection. Typically, depending on survival due to passaging and electroporation which is highly hPSC line-specific, 1-2 wells of a 6-well plate with 30-60% confluent stem cell culture provides a sufficient cell number for plating into 3/6 wells after transfection.
Transfection for gene targeting
Transfection for gene targeting
1. Prepare a desired number of wells in a 6-well plate to accommodate the hPSC cell suspension after electroporation, and become "master" wells for establishing targeted clones after antibiotic selection. The wells are coated with Corning® Matrigel®CorningCatalog #354277 or similar ECM with 2x higher concentration relative to the manufacturer's instructions.
2. Prepare a suspension of hPSCs for transfection using a Neon electroporator kit (or similar device)
Equipment
new equipment
NAME
Thermo Fisher Scientific Neon™ Transfection System
BRAND
MPK5000S
SKU
This is achieved by the generation of a single-cell suspension from the existing cultures using accutase ACCUTASE™ 100 mL
STEMCELL Technologies Inc.Catalog #7920 digestion (5-7 minutes at RT), followed by a rinse/spin with culture medium.
3. According to the manufacturer's instructions, use 100μL tip to transfect 3-4x10^6 hPSCs with 3μg of AAVS1 targetting vector, and 2μg of each of the TALEN vectors (see 1.1). Use a customised protocol or this set of parameters: 1250V / 15 ms / 2 pulses. Let cells recover after the pulses in the tip for 2 mins.
4. Plate cells in 3 wells of a 6-well plate (2.1) (density could be adjusted depending on the specific cell line used), in the antibiotic-free medium (1.5mL/well) supplemented with CloneR2STEMCELL Technologies Inc.Catalog ## 100-0691 or a similar hPSC survival-promoting agent.
Selection of the correctly-targeted clones
Selection of the correctly-targeted clones
After 5 days in culture or ~95% confluency (whichever comes first), selection of the single transfected cell-derived clones could be started. For instance, for AAVS1-Px vectors (see 1.1), Puromycin DihydrochlorideGibco - Thermo FisherCatalog #A1113803 selection at 2μg/mL for 1-2 days. Single cell-derived colonies should be allowed to grow for 5-10 days after transfection (depending on the cell line/colony density/selection regiment used) before being manually passaged into individual plate wells to establish clonal lines for downstream use and characterization (genotyping).
Correctly-targeted clones could be identified by genotyping PCR from genomic DNA using a primer set, with one anchored in the AAVS1 locus outside the homology arms of the AAVS1 vector, while the reverse primer is targetted to the puromycin resistance coding region.
Primers used for genotyping of the AAVS1 targetting SA-Puro vectors:
AAVS1_PC_F
CTG CCG TCT CTC TCC TGA GT
AAVS1-SA-Puro_R3
TCG TCC GCG ACC CAC ACC TT
gDNA-based PCR: use 100-200ng of high-purity gDNA prep for a >=20uL reaction using standard PCR reaction setup (using a conventional Taq or similar polymerase)
PCR conditions used for the amplification on a conventional PCR machine:
35 cycles of:
98°C for 15 secs
68°C (decreasing 0.2 deg every cycle) for 20sec (touchdown PCR)
72°C for 30 sec,
Followed by a final extension
72°C for 5 min
Keep at 4°C
The bands can then be separated on a TAE agarose gel (0.8-1%) with a ~1.1kb band indicative of the presence of at least one targetted allele. Make sure (especially in the first runs) to include negative and positive controls for this genotyping PCR. For new AAVS1-targeting vectors Sanger sequencing is recommended.