STRAIGHT-IN: A platform for high-throughput targeting of large DNA payloads into human pluripotent stem cells

Abstract

Inserting large DNA payloads (>10 kb) into specific genomic sites of mammalian cells remains challenging. Applications ranging from synthetic biology to evaluating the pathogenicity of disease-associated variants for precision medicine initiatives would greatly benefit from tools that facilitate this process. Here, we merge the strengths of different classes of site-specific recombinases and combine these with CRISPR/Cas9-mediated homologous recombination to develop a strategy for stringent site-specific replacement of genomic fragments at least 50 kb in size in human induced pluripotent stem cells (hiPSCs). We demonstrate the versatility of STRAIGHT-IN (Serine and Tyrosine Recombinase Assisted Integration of Genes for High-Throughput INvestigation) by: (i) inserting various combinations of fluorescent reporters into hiPSCs to assess excitation-contraction coupling cascade in derivative cardiomyocytes, and; (ii) simultaneously targeting multiple variants associated with inherited cardiac arrhythmic disorder into a pool of hiPSCs. STRAIGHT-IN offers a precise approach to generate genetically-matched panels of hiPSC lines efficiently and cost-effectively.

Programmable RNA-Guided Large DNA Transgenesis by CRISPR/Cas9 and Site-Specific Integrase Bxb1

“…In sum, a key obstacle in genetic engineering remains the lack of sophisticated and economical techniques for precise integration of kb-sized DNA in human cells and animals. To overcome these limitations, a new toolkit—an RNA-guided Cas9-Bxb1 toolbox ( Anzalone et al, 2021 ; Grandela et al, 2021 ; Ioannidi et al, 2021 ; Low et al, 2021 )—has recently been developed that expands the breadth of precision transgenesis, genetic engineering, cell-based treatments, and synthetic biology.…”

“…Modified ES cells can be taken to germline transmission. (C) The Straight-IN (adapted from Grandela et al, 2021 ) tool uses a combination of Bxb1 (or phiC31)- and cre-mediated recombination to insert a safe harbor locus in human iPSCs for large DNA transgenesis. Similarly to the ICE approach, Straight-IN uses a heterologous loxP and loxP* site-floxed landing pad cassette containing an attP site, an inactive antibiotic resistance gene (*BsdR), and a fluorescent reporter gene driven by a constitutive promoter such as pgk1.…”

“… Grandela et al (2021) recently presented STRAIGHT-IN (Serine and Tyrosine Recombinase Assisted Integration of Genes for High-Throughput INvestigation), a novel approach for integrating large DNA payloads into hiPSCs that combines the benefits of both serine (Bxb1) and tyrosine recombinases (cre or Flp) ( Figure 5C ). The authors first generated hiPSCs with a landing pad cassette containing attachment sites ( attP ) for Bxb1 at the safe harbor locus AAVS1 using CRISPR/Cas9-mediated HDR.…”

“…Further, the validation strategy is designed to enable early validation of construct integration. Grandela et al (2021) recently presented STRAIGHT-IN (Serine and Tyrosine Recombinase Assisted Integration of Genes for High-Throughput INvestigation), a novel approach for integrating large DNA payloads into hiPSCs that combines the benefits of both serine (Bxb1) and tyrosine recombinases (cre or Flp) (Figure 5C). The authors first generated hiPSCs with a landing pad cassette containing attachment sites (attP) for Bxb1 BOX 1 | Site-specific serine recombinases Site-specific recombinases are enzymes that cut and rejoin DNA strands at precise genomic locations.…”