Abstract
We developed a simple method to apply CRISPR interference by modifying an existing plasmid pCRISPathBrick containing the native S. pyogenes CRISPR assembly for Synechocystis PCC6803 and named it pCRPB1010. The technique presented here using deadCas9 is easier to implement for gene silencing in Synechocystis PCC6803 than other existing techniques as it circumvents the genome integration and segregation steps thereby significantly shortens the construction of the mutant strains. We executed CRISPR interference against well characterized photosynthetic genes to get a clear phenotype to validate the potential of pCRPB1010 and presented the work as a “proof of concept”. Targeting the non-template strand of psbO gene resulted in decreased amount of PsbO and 50% decrease in oxygen evolution rate. Targeting the template strand of psbA2 and psbA3 genes encoding the D1 subunit of photosystem II (PSII) using a single spacer against the common sequence span of the two genes, resulted in full inhibition of both genes, complete abolition of D1 protein synthesis, complete loss of oxygen evolution as well as photoautotrophic growth arrest. This is the first report of a single plasmid based, completely lesion free and episomal expression and execution of CRISPR interference in Synechocystis PCC6803.
Highlights
CRISPR-Cas9 (CRISPR associated protein 9) has revolutionized genome editing due to its simple execution and precision
With this work we report for the first time a simple method to repress multiple genes using a single plasmid containing dCas9 in Synechocystis PCC6803, and demonstrate the potential of the technique to be applied in further studies requiring gene silencing
While here we present a system consisting of the original S. pyogenes genes and the native bidirectional promoter, they used plasmid-based dCas9-dependent silencing using a single guide RNA with engineered promoters and optimized ribosome binding site
Summary
CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated protein 9) has revolutionized genome editing due to its simple execution and precision. The system was discovered in Streptococcus pyogenes and has primarily three working components: The ‘Cas9’ protein which has a single strand nuclease activity, a ‘tracrRNA’ complementary to the palindromic repeat sequences in the crRNA, which forms RNA loop for Cas binding, and ‘crRNA’, which harbors palindromic repeats separated by ‘spacer’ sequences complementary to the targeted DNA sequence [1] These three components are transcribed from a constitutive bi-directional promoter. Once such sequences are found lying next to those complementary to the crRNA, Cas will exert its nuclease activity [1] This tri-component system was further simplified to a bi-component system where the tracrRNA and crRNA were combined as a single effector RNA named gRNA and Cas was placed under inducible promoters to control its expression for genome editing [2]. This system is widely adapted for both prokaryotic and eukaryotic gene editing [3]
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