Target preference of Type III-A CRISPR-Cas complexes at the transcription bubble

Jennifer A Doudna,Eva Nogales,Abhishek J Aditham,Tina Y Liu,Jun-Jie Liu

doi:10.1038/s41467-019-10780-2

Abstract

Type III-A CRISPR-Cas systems are prokaryotic RNA-guided adaptive immune systems that use a protein-RNA complex, Csm, for transcription-dependent immunity against foreign DNA. Csm can cleave RNA and single-stranded DNA (ssDNA), but whether it targets one or both nucleic acids during transcription elongation is unknown. Here, we show that binding of a Thermus thermophilus (T. thermophilus) Csm (TthCsm) to a nascent transcript in a transcription elongation complex (TEC) promotes tethering but not direct contact of TthCsm with RNA polymerase (RNAP). Biochemical experiments show that both TthCsm and Staphylococcus epidermidis (S. epidermidis) Csm (SepCsm) cleave RNA transcripts, but not ssDNA, at the transcription bubble. Taken together, these results suggest that Type III systems primarily target transcripts, instead of unwound ssDNA in TECs, for immunity against double-stranded DNA (dsDNA) phages and plasmids. This reveals similarities between Csm and eukaryotic RNA interference, which also uses RNA-guided RNA targeting to silence actively transcribed genes.

Highlights

Type III-A CRISPR-Cas systems are prokaryotic RNA-guided adaptive immune systems that use a protein-RNA complex, Csm, for transcription-dependent immunity against foreign DNA
To test whether TthCsm interacts with RNA and DNA at an RNA polymerase (RNAP)-bound transcription bubble, we first tested whether nascent RNA recruits TthCsm to a transcription bubble in vitro
We observed that TthCsm subunits co-eluted with RNAP only when the RNA contained a sequence that was complementary to the CRISPR RNA (crRNA) (Fig. 1c, d, “target RNA”), but not when the target was replaced by an unrelated sequence (Fig. 1c, d, “non-target RNA”)

Summary

Introduction

Type III-A CRISPR-Cas systems are prokaryotic RNA-guided adaptive immune systems that use a protein-RNA complex, Csm, for transcription-dependent immunity against foreign DNA. Biochemical experiments show that both TthCsm and Staphylococcus epidermidis (S. epidermidis) Csm (SepCsm) cleave RNA transcripts, but not ssDNA, at the transcription bubble Taken together, these results suggest that Type III systems primarily target transcripts, instead of unwound ssDNA in TECs, for immunity against double-stranded DNA (dsDNA) phages and plasmids. The lack of a physical interaction between Csm and RNAP helps to explain the prevalence of Type III CRISPRCas systems across different species of bacteria and archaea, as there would be no requirement for adaptation to specific RNAPs in each new host These findings reveal similarities between Type III CRISPR-Cas systems and the eukaryotic RNA interference pathway, which uses an RNA-guided complex, RISC (RNAinduced silencing complex), to cleave RNA transcripts for gene silencing[15]

Methods

Results

Conclusion