Generation of AAVS1 integrated doxycycline-inducible CRISPR-Prime Editor human induced pluripotent stem cell line

Prime editing uses the Cas9 nickase fused to a reverse transcriptase to copy a DNA sequence into a specific locus from a ‘prime editing’ guide RNA (pegRNA), eliminating the need for double-stranded DNA breaks and donor DNA templates. To facilitate prime editing in human induced pluripotent stem cells (iPSCs), we integrated a doxycycline-inducible Prime Editor protein (PE2) into the AAVS1 genomic safe harbor locus. Prime editing of iPSCs resulted in precise insertion of three nucleotides in HEK3 locus with high efficiency, demonstrating the utility of this approach. This engineered cell line can be used to edit a single or multiple genomic loci by introducing a target-specific pegRNA for precise and effective genome editing to facilitate disease modeling and functional genetics studies.


Resource utility
This iPSC line can be used to edit any locus in the human genome using prime editing (PE) by transfecting a locus-specific prime-editing-guide-RNA and inducing the expression of the prime editor with doxycycline.This will be applicable for functional genetics studies like validating GWAS hits and disease modeling, as well as inserting tags/epitopes precisely into loci.

Resource details
The CRISPR-Cas system has revolutionized genome editing in the last decade due to its relative ease of use, lower cost and high programmability, as compared to other genetic engineering tools.There are now four classes of CRISPR-Cas tools available -nucleases, base editors, transposases and prime editors, of which, prime editors are the most versatile (Anzalone et al, 2020).Prime editors allow for precise edits of point mutations, all twelve possible base-to-base conversions, small insertions/deletions with fewer undesired mutations and with higher or similar efficiency than homology-directed repair, without double strand breaks or donor DNA.The prime editor protein (PE2) is a fusion between the S.pyogenes Cas9(H840A) nickase and an engineered Moloney Murine Leukemia Virus (MMLV) reverse transcriptase domain (Anzalone et al, 2019).This fusion protein can be directed to the desired locus by an engineered prime editing guide RNA (pegRNA) which, includes the target site in its spacer sequence and the desired edit in an extension at the 3′ end of the pegRNA.Once the target DNA is nicked, its 3′ end hybridizes to the primer binding site and using the pegRNA template, PE2 reverse transcribes the DNA with the desired edit.
We generated a human iPSC line that inducibly expresses the PE2 protein that enables gene editing at potentially any locus in the genome by transfecting a pegRNA specific for the desired target locus.(See Tables 1 and 2).This line can be used for functional genomics studies, such as validating GWAS hits and inserting epitope tags or introducing SNPs and then differentiating the line into the cell-type of interest.The prime editor was integrated at the AAVS1 'genomic safe harbor' locus within the PP1R12C gene (Mandegar et al, 2016).Transgenes integrated at this locus retain their transcriptional activity both in iPSCs, and upon differentiation into other cell types.The construct was designed such that PE2 is under transcriptional control of the Tetracycline regulatable promoter and can be activated by the addition of doxycycline when required, remaining transcriptionally inactive upon doxycycline withdrawal (Fig. 1A).Additionally, the PE2 is fused to Blue Fluorescent Protein (BFP) with a P2A peptide sequence, allowing for visualization of cells that are actively transcribing the PE2-P2A-BFP (Fig. 1E).After selecting for the integrated plasmid with the antibiotic G418, iPSC clones were selected and expanded for further characterization .The PE2-P2A-BFP-integrated cell line showed normal morphology (Fig. 1B).The correct insertion of the transgene at a single allele of the AAVS1 locus was verified by PCR amplification of the 5′ integration junction (1 kb).A different set of primers amplified across the cut site (250 bp) showed the intact WT allele (Fig. 1C).Pluripotency was verified by immunostaining for OCT3/4, SOX2, NANOG, TRA1-60 (Fig. 1D), and trilineage potential was confirmed by Scorecard at passage 35 (Fig. 1G).The cells showed normal karyotype at passage 35 (Fig. 1I).We validated the editing capability and utility of the PE2 engineered cell line by transfecting a pegRNA carrying a 3 bp (CTT) insertion targeting the HEK3 locus, followed by Sanger sequencing of the PCR-amplified DNA collected from the edited cells (Fig. 1H).

Molecular cloning
The PE2-P2A-BFP fusion was PCR amplified from pTRE3G-PE2-P2A-BFP and cloned into NotI/AflII-digested pAAVS1-NDi-CRISPRi (Addgene#73497) using the In-Fusion HD cloning kit (Takara), replacing the KRAB-dCas9-P2A-mCherry cassette.The tetracyclineinducible vector contains the reverse tetracycline-controlled transcriptional activator (rtTA) as well as the tetracycline-response element (TRE3G).The rtTA is transcribed by a strong constitutive promoter (CAG) oriented in the opposite direction of the TRE3G element, which ensures no expression of the transgene can occur without addition of doxycycline.
The vector contains left and right homology arms (HA-L/HA-R) that flank the genomiccut site in the AAVS1 locus.A splice-acceptor (SA) site and a 2A peptide sequence (T2A) downstream of the HA-L arm allows for endogenous expression of a promoterless-Neomycin gene that confers resistance to Neomycin/G418.

Immunostaining
The cells were fixed with 4% PFA for 10 min at 37 °C and washed 3 times for 5 min each with DPBS.They were then permeabilized in DPBS with 0.1% Triton for 10 min at room temperature, followed by blocking for 1 h at room temperature in DPBS/0.1% Triton X/1% BSA.Cells were incubated with primary antibodies at 4 °C overnight.The cells were then washed 3 times for 5 min each with DPBS and incubated with secondary antibody for 1 h at room temperature.After washing 3 times for 5 min each, a drop of NucBlue was added to counterstain the DNA.

Validation of the line for genome editing
The plasmid pU6-Sp-pegRNA-HEK3-CTT-ins (Addgene# 132778) expressing a pegRNA specific for a 3 bp insertion (CTT) of the HEK3 locus was electroporated into the PE2-BFP iPSCs.At 48 h post-electroporation, the cells were induced with 1.5 μg/ml Doxycycline.DNA was collected 72 h post-Doxycyclin induction and PCR-amplified, followed by Sanger sequencing to confirm target insertion.STR analysis karyotyping.