Development of cloning-free, PCR-based sgRNA synthesis using CRISPR/ Cas9 for rapid Generation of Mice Models
DOI:
https://doi.org/10.48165/jlas.2026.9.2.2Keywords:
CRISPR/Cas9, Single guide RNA (sgRNA), PCR-based synthesis, Genome editing,, In-vitro Transcription (IVT)Abstract
The mouse models are vital for studying human biology, diseases, and therapeutic interventions. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology has revolutionized the creation of genetically modified animal models by enabling precise genome modifications across various species. The design and synthesis of guide RNAs (gRNAs) are essential steps in this system, but traditional methods require cloning crRNA and tracrRNA sequences into Cas expression vectors, a slow, labor-intensive process that often creates a bottleneck for large-scale applications. To address these challenges, we developed a rapid, PCR-based, cloning-free method for the synthesis of dsDNA transcription template. Our method creates in vitro transcription (IVT) templates with the T7 promoter, guide RNA, and tra crRNA scaffold using an overhang-extension PCR protocol. Single-stranded oligonucleotides are used to construct the tracrRNA sequence (80 bp), and overhang primers add the 5’- T7 promoter-crRNA-3’ sequence to the double-stranded ~80 bp tracrRNA scaffold. This strategy eliminates the need for cloning, reducing gRNA synthesis time from weeks to about 6 hours. The activity of the synthesized gRNAs was confirmed through in vitro cleavage assays and the successful creation of mouse models. This optimized process offers a scalable, affordable way to accelerate CRISPR-based genome modification. Our PCR-based approach makes gRNA synthesis more accessible by drastically reducing time and labor requirements, offering considerable advantages across genetics, developmental biology, disease modeling, and transgenic core facilities.
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