| Products/Services Used | Details | Operation |
|---|---|---|
| Gene Fragments | Assembly compatible overhangs was ordered from Genscript and cloned in the digested plasmid. The resulting plasmid was then digested with XhoI and IlluminaPBS-Filler-mU6 gene fragment (Genscript) that was PCR amplified using the following primers: 5′-GATCCACTTTGGCGCCGGCCTCGAGCAG-3′ and 5′-CTTTCAAGACCTAGGGCCCCCCTCGAGCCCGGGCATGCTCTTCAACCTCAATAACTGGAGTTATATGGACCATTGTTCTAGCGCTGATCCGACG-3′. Gibson assembly overhangs were added as before, and the arrays were synthesized by Genscript as gene fragments.A gene fragment without the saCas9 tracrRNA was synthesized by Genscript and cloned in the plasmid cut position by Gibson Assembly. A gene fragment without the spCas9 tracrRNA was synthesized by Genscript and cloned in the plasmid cut position by Gibson Assembly (Supplementary Data 3). | Get A Quote |
Mapping genetic interactions (GIs) is crucial for understanding genetic network complexity. In this study, we investigate the utility of Cas13d, a CRISPR system targeting RNA, for GI mapping and compare it to Cas9 and Cas12a, two DNA nucleases commonly used for GI mapping. We find that Cas13d induces faster target gene perturbation and generates more uniform cell populations with double perturbations than Cas9 or Cas12a. We then encounter Cas13d gRNA-gRNA interference when concatenating gRNAs targeting different genes into one gRNA array, which we overcome by a dual promoter gRNA expression strategy. Moreover, by concatenating three gRNAs targeting the same gene into one array, we are able to maximize the Cas13... More
