RT Journal Article SR Electronic T1 Efficient generation of endogenous fluorescent reporters by Nested CRISPR in Caenorhabditis elegans JF bioRxiv FD Cold Spring Harbor Laboratory SP 423970 DO 10.1101/423970 A1 Jeremy Vicencio A1 Carmen Martínez-Fernández A1 Xènia Serrat A1 Julián Cerón YR 2018 UL http://biorxiv.org/content/early/2018/09/26/423970.abstract AB CRISPR-based genome editing methods in model organisms are evolving at an extraordinary speed. Whereas the generation of deletion or missense mutants is quite straightforward, the production of endogenous fluorescent reporters is still inefficient. The use of plasmids with selection markers is an effective methodology, but often requires laborious and complicated cloning steps. We have established a cloning-free ribonucleoprotein-driven Nested CRISPR method that robustly produces endogenous fluorescent reporters. This methodology is based on the division of the GFP and mCherry sequences in three fragments. In the first step we use ssDNA donors (≤200 bp) to insert 5’ and 3’ fragments in the place of interest. In the second step, we use these sequences as homology regions for Homologous Directed Repair (HDR) with a dsDNA donor (PCR product, ≈700 bp) including the middle fragment, thus completing the fluorescent protein sequence. This method is advantageous because the first step with ssDNA donors is known to be very efficient, and the second step, uses universal reagents, including validated PCR products and crRNAs, to create fluorescent reporters reaching reliable editing efficiencies as high as 40%. We have also used Nested CRISPR in a non-essential gene to produce a deletion mutant in the first step and a transcriptional reporter in the second step.In the search of modifications to optimize the method, we tested synthetic sgRNAs, but we did not observe a significant increase in the efficacy compared to independently adding tracrRNA and crRNA to the injection mix. Finally, we discuss the utility of Nested CRISPR for targeted insertion of long DNA fragments in other systems and prospects of this method in the future.