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Updated: Feb 15, 2026

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing
Published on: January 20, 2018
Lactobacillus gasseri CRISPR-Cas9 characterization In Vitro reveals a flexible mode of protospacer-adjacent motif
Emily M Anderson1, Shawn McClelland1, Elena Maksimova1
1Dharmacon, a Horizon Discovery Group Company, Lafayette, CO, United States of America.
Abstract:
While the CRISPR-Cas9 system from S. pyogenes is a powerful genome engineering tool, additional programmed nucleases would enable added flexibility in targeting space and multiplexing. Here, we characterized a CRISPR-Cas9 system from L. gasseri and found that it has modest activity in a cell-free lysate assay but no activity in mammalian cells even when altering promoter, position of tag sequences and NLS, and length of crRNA:tracrRNA. In the lysate assay we tested over 400 sequential crRNA target sequences and found that the Lga Cas9 PAM is NNGA/NDRA, different than NTAA predicted from the native bacterial host. In addition, we found multiple instances of consecutive crRNA target sites, indicating flexibility in either PAM sequence or distance from the crRNA target site. This work highlights the need for characterization of new CRISPR systems and highlights the non-triviality of porting them into eukaryotes as gene editing tools.
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