Application note: Improving and simplifying the design of knock-in experiments using the TrueDesign Genome Editor

Posted: 21 June 2021 | | No comments yet

Genome editing technologies have given investigators the power to unlock a variety of new applications and experimental approaches. While gene knockouts have been achievable for several years, successful gene knock-ins have remained elusive due to the low efficiency of homology-directed repair (HDR).

Successful knock-in experiments depend on the design of the donor DNA, effectiveness of the single guide RNA (sgRNA) and Cas9 ribonucleoprotein (RNP) complex, and efficiency of delivering materials into the target cells. Here we show how our researchers have optimized these steps and demonstrate how to introduce single-nucleotide polymorphisms (SNPs) and fluorescent tags with up to 100% editing efficiency

To enable access to these optimized approaches to genome editing, we have created the Invitrogen™ TrueDesign™ Genome Editor, a free online tool for designing and ordering materials for knock-in CRISPR-Cas9-based editing. This application note describes two use cases: (1) introduction of a SNP change in the LRRK2 gene (G2019S) in induced pluripotent stem cells (iPSCs), and (2) tagging of beta-actin with GFP. For SNP changes, a recommended design for a single-stranded oligo deoxyribonucleotide (ssODN) donor is automatically designed through the online tool. For fusion proteins, primer recommendations are automatically generated for production of double-stranded donor DNA using the Invitrogen™ TrueTag™ Donor DNA Kit. Donor DNA is then cotransfected with Invitrogen™ TrueCut™ Cas9 Protein v2 and TrueGuide™ Modified Synthetic sgRNA. A detailed protocol for this transfection process is also described in this application note. Once delivered into cells, the donor DNA will be integrated into the genome of your target cells in less than a day.

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