Hello everyone,
I came across a paper (DOI: https://doi.org/10.1016/j.cell.2024.12.025) where the authors constructed an endogenous fusion protein using CRISPR knock-in. Instead of inserting the fusion tag (e.g., GFP) at the C-terminal end of the last exon, they removed the entire coding sequence (from the first exon to the last exon) while keeping only the UTR regions. They then inserted an intronless CDS of the target gene along with the fusion protein sequence in the homology-directed repair (HDR) template.
I have two main questions:
I would appreciate any insights from those who have experience with CRISPR-mediated knock-in strategies. Thank you!
Replacing the entire coding sequence (CDS) of a gene with an intronless version to create endogenous fusion proteins is not a common CRISPR knock-in strategy. This approach can disrupt natural gene expression patterns and regulatory mechanisms, as introns play crucial roles in transcriptional regulation, mRNA stability, and alternative splicing.
Instead, more precise methods have been developed to tag endogenous proteins while preserving gene integrity. One such method is the Targeted Knock-In with Two (TKIT) guides approach, which utilizes CRISPR/Cas9 to insert tags into specific genomic locations without altering the coding sequence. This strategy involves designing two guide RNAs that target non-coding regions flanking the coding sequence, allowing for the insertion of tags such as fluorescent proteins. TKIT has been successfully used to label endogenous synaptic proteins in mouse primary cultured neurons, achieving efficiencies up to 42%.
Jaba Tkemaladze Thank you for your detailed response! I really appreciate your insights, and I now have a much clearer understanding of the approach.