Lactobacillus gasseri CRISPR-Cas9 characterization In Vitro reveals a flexible mode of protospacer-adjacent motif recognition.

Emily M Anderson,Shawn Mcclelland,Anja Van Brabant Smith,Elena Maksimova,Rodolphe Barrangou,Žaklina Strezoska,Megan Basila,Alexandra E Briner,Shuang-Yong Xu

doi:10.1371/journal.pone.0192181

Emily M Anderson, Shawn Mcclelland + Show 7 more

Open Access

https://doi.org/10.1371/journal.pone.0192181

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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.

Highlights

The CRISPR-Cas9 (Clustered Regularly Interspaced Palindromic Repeats-CRISPR-associated protein 9) system, derived from a bacterial immune system pathway, has emerged as a dominant platform for gene editing in a host of applications, especially in mammalian cells [1,2,3]
To test for expression of Lactobacillus gasseri (Lga) Cas9, plasmids containing FLAG-tagged Lga Cas9 were transfected into HEK293T cells, and relative protein expression was determined by Western Blot
The T7EI assay (Fig 1A) indicated no gene editing on the Lga Cas9 target sites, while Streptococcus pyogenes (Spy) Cas9 displayed modest levels of activity (9%)

Summary

Introduction

The CRISPR-Cas (Clustered Regularly Interspaced Palindromic Repeats-CRISPR-associated protein 9) system, derived from a bacterial immune system pathway, has emerged as a dominant platform for gene editing in a host of applications, especially in mammalian cells [1,2,3]. The Cas nuclease can be directed to targets by loading it with two small RNAs (transactivating CRISPR-associated RNA (tracrRNA) and the target-specific CRISPR RNA (crRNA)) that enable the formation of double-stranded breaks (DSBs) at almost any desired sequence without having to re-engineer the Cas protein, a requirement of other site-specific nucleases [4, 5]. In addition to a 20-nucleotide (nt) region of complementarity between a targeting crRNA and desired target site, a short protospacer-adjacent motif (PAM) [8] is required downstream. Upon resolution of the DSB by endogenous cellular mechanisms, genes can be knocked out or their function modulated in precise way by introduction of mutations, tags, or other sequence edits [6, 7].

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