| Title |
DNA interference is controlled by R-loop length in a type I-F1 CRISPR-Cas system / |
| Authors |
Tuminauskaitė, Donata ; Norkūnaitė, Danguolė ; Fiodorovaitė, Marija ; Tumas, Šarūnas ; Songailienė, Inga ; Tamulaitienė, Giedrė ; Šinkūnas, Tomas |
| DOI |
10.1186/s12915-020-00799-z |
| Full Text |
|
| Is Part of |
BMC Biology.. London : BMC. 2020, vol.18, art. no. 65, p. [1-16].. eISSN 1741-7007 |
| Keywords [eng] |
CRISPR protection ; CRISPR-Cas immunity ; Cas2/3 ; Cas3 nuclease/helicase ; Cascade ; Csy ; DNA degradation ; DNA interference ; R-loop ; type I-F |
| Abstract [eng] |
Background: CRISPR-Cas systems, which provide adaptive immunity against foreign nucleic acids in prokaryotes, can serve as useful molecular tools for multiple applications in genome engineering. Diverse CRISPR-Cas systems originating from distinct prokaryotes function through a common mechanism involving the assembly of small crRNA molecules and Cas proteins into a ribonucleoprotein (RNP) effector complex, and formation of an R-loop structure upon binding to the target DNA. Extensive research on the I-E subtype established the prototypical mechanism of DNA interference in type I systems, where the coordinated action of a ribonucleoprotein Cascade complex and Cas3 protein destroys foreign DNA. However, diverse protein composition between type I subtypes suggests differences in the mechanism of DNA interference that could be exploited for novel practical applications that call for further exploration of these systems. Results: Here we examined the mechanism of DNA interference provided by the type I-F1 system from Aggregatibacter actinomycetemcomitans D7S-1 (Aa). We show that functional Aa-Cascade complexes can be assembled not only with WT spacer of 32 nt but also with shorter or longer (14-176 nt) spacers. All complexes guided by the spacer bind to the target DNA sequence (protospacer) forming an R-loop when a C or CT protospacer adjacent motif (PAM) is present immediately upstream the protospacer (at -1 or -2,-1 position, respectively). The range of spacer and protospacer complementarity predetermine the length of the R-loop; however, only R-loops of WT length or longer trigger the nuclease/helicase Cas2/3, which initiates ATP-dependent unidirectional degradation at the PAM-distal end of the WT R-loop. Meanwhile, truncation of the WT R-loop at the PAM-distal end abolishes Cas2/3 cleavage. Conclusions: We provide a comprehensive characterisation of the DNA interference mechanism in the type I-F1 CRISPR-Cas system, which is different from the type I-E in a few aspects. First, DNA cleavage initiation, which usually happens at the PAM-proximal end in type I-E, is shifted to the PAM-distal end of WT R-loop in the type I-F1. Second, the R-loop length controls on/off switch of DNA interference in the type I-F1, while cleavage initiation is less restricted in the type I-E. These results indicate that DNA interference in type I-F1 systems is governed through a checkpoint provided by the Cascade complex, which verifies the appropriate length for the R-loop. |
| Published |
London : BMC |
| Type |
Journal article |
| Language |
English |
| Publication date |
2020 |
| CC license |
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