Deletion-based escape of CRISPR-Cas9 targeting in Lactobacillus gasseri

Microbiology
Emily StoutRodolphe Barrangou

Abstract

Lactobacillus gasseri is a human commensal which carries CRISPR-Cas, an adaptive immune system that protects the cell from invasive mobile genetic elements (MGEs). However, MGEs occasionally escape CRISPR targeting due to DNA mutations that occur in sequences involved in CRISPR interference. To better understand CRISPR escape processes, a plasmid interference assay was used to screen for mutants that escape CRISPR-Cas targeting. Plasmids containing a target sequence and a protospacer adjacent motif (PAM) were transformed for targeting by the native CRISPR-Cas system. Although the primary outcome of the assay was efficient interference, a small proportion of the transformed population overcame targeting. Mutants containing plasmids that had escaped were recovered to investigate the genetic routes of escape and their relative frequencies. Deletion of the targeting spacer in the native CRISPR array was the dominant pattern of escape, accounting for 52-70 % of the mutants from two L. gasseri strains. We repeatedly observed internal deletions in the chromosomal CRISPR array, characterized by polarized excisions from the leader end that spanned 1-15 spacers, and systematically included the leader-proximal targeting spacer. This study...Continue Reading

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Citations

Mar 25, 2019·Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences·Gayetri Ramachandran, David Bikard
Jun 9, 2020·Environmental Microbiology·Zeling XuAixin Yan
Nov 12, 2019·Frontiers in Microbiology·Yingjun Li, Nan Peng
Nov 30, 2019·BMC Genomics·Matthew A NetheryRodolphe Barrangou
Jan 28, 2020·Current Opinion in Biotechnology·Samuel M RothsteinThomas J Mansell
Apr 20, 2021·Frontiers in Microbiology·Sandra C Garrett

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