A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.


–end && reference


— later I’ll explain a little bit.

Maximum expected accuracy structural neighbors of an RNA secondary structure

Since RNA molecules regulate genes and control alternative splicing by allostery, it is important to develop algorithms to predict RNA conformational switches. Some tools, such as paRNAss, RNAshapes and RNAbor, can be used to predict potential conformational switches; nevertheless, no existent tool can detect general (i.e., not family specific) entire riboswitches (both aptamer and expression platform) with accuracy. Thus, the development of additional algorithms to detect conformational switches seems important, especially since the difference in free energy between the two metastable secondary structures may be as large as 15-20 kcal/mol. It has recently emerged that RNA secondary structure can be more accurately predicted by computing the maximum expected accuracy (MEA) structure, rather than the minimum free energy (MFE) structure.


Source code for RNAborMEA can be downloaded from http://sourceforge.net/projects/rnabormea/ or http://bioinformatics.bc.edu/clotelab/RNAborMEA/

“RISC” of Bacteria and Archaea

Restriction-modification systems, abortive-phage phenotypes, toxin-antitoxins and other innate defense systems, in the past, have been shown in familiar chapters in typical microbiology textbook, while now what if I say in prokaryotes world “RISC” can serve a role for new kind of antiviral defense, in addition the “RNAi” can even be engineered and designed to lead to target gene silencing, would you believe me?

You must have ever heard CRISPR/Cas (CRISPR Associated proteins) System if you have ever read this Science paper [1]. Exactly as the title said, CRISPR, Clustered Regularly Inter-spaced Short Palindromic Repeat, serves as the leading role to provide the “memory” as an adaptive immunity, akin to a blacklist of unwanted visitors, like plasmids or viruses genome.

CRISPR/Cas has different types based on Cas family. Three modules of Cas proteins are Cmr, Cst, Csa. It is an old story in bacteria world as it had been firstly identified in E.coli in 1987. Most have been reported to head for invading DNA, while here what I introduce to you now is an unique and intriguing discovery that in achaeon Pyrococcus furiosus which thrives best under extremely high temperatures, CRISPR/Cmr (one subtype of CRISPR/Cas) targets invading RNA, rather than DNA, thus what I called “RNAi” can makes sense.

In general, the context of CRISPR RNA (crRNA) is typically a sandwich, repeat-sequence-repeat. The internal sequence is termed guide sequence which is complementary to invading RNA only for CRISPR/Cmr, and it is identical to invading DNA for most other cases of CRISPR/Cas.

–How CRISPR/Cmr works?


As recalled, the internal sequence of crRNA is complementary to invading RNA as the “seed” region, and more importantly only Cmr complex can contribute to RNA cleavage.

The 8 nt 5′-end tag, among the short repeat sequence in crRNA, will lead crRNA to bind to invading RNA. It is suggested that the 8 nt 5′-end tag plays a discrimination function to classify self-RNA and non-self RNA.

Once crRNA and invading RNA get paired, hydrolysis of the target RNA takes place at a fixed distance, 14nt, from the 3′-end of the small guide RNA. Thus the invading RNA will be degraded and its expression will be turned OFF. In this way Pyrococcus furiosus help themselves against foreign viruses invading with RNA gnome.

Thus I cannot help raising an analogy between CRISPR/Cas system and noted RISC (RNA-induced siliencing complex) in eukaryotes [2]. They all have the progess: processing to be matured, base-pair induced target cleavage.

–Can CRISPR/Cmr engineered?

Yes, we can. The magic is the 8nt 5′-end tag, whose sequence is AUUGAAAG. Scientists had hacked the “immune” system to suppress target gene expression [3], here with the example beta-lactamase (bla) mRNA. The internal sequence, or guide sequence had been designed complementary to 5′-end bla mRNA sequence with the required 5′-end tag. Good result is the gene get silenced which shows promise to another novel silencing systems in bacterium and archaea.


CRISPR/Cmr with RNA as its target is just one subtype of CRISPR/Cas system. Other types target DNA. 

If we took a deep look at the general features of all the systems, and compare them with eukaryotic RNAi side-by-side,[4] there are still lots of questions remained unsolved, and mechanisms left mysterious.

How the invading sequence get integrated into CRISPR loci?

CRISPR/Cas system is adaptive immune system, not innate. Bacteria and archaea are not born with it, and they need immune stimulation at first to gain a short of sequence from invading virus or plasmids. And it is the short foreign sequence that gets integrated into CRISPR loci between two short repeat sequences and enables crRNA to bind with RNA/DNA.

But what is mechanisms of the acquisition? Unknown[4].

How to discriminate self or invading?

For CRISPR/Cas system that target DNA, the 5′-end tag (in short repeat region) is critical for distinguishing self from non-self. If the 5′-end tag mismatches the invading DNA, the invaders must die. If the tag precisely matches foreign DNA, it is considered as the host CRISPR locus itself and does not “attack”. What about  CRISPR/Cmr? 

To discern the function of 5′-end tag in CRISPR/Cmr targeting RNA. Three disturbance experiments are conducted. When the 5′-end tag is totally deleted, substituted by other types of sequences (one is precisely complementary to itself, two are with just first one or two bases complementary to itself), these three new tags are no more original 5′-end tag leading to the silencing effect disappear, just as expected. Thus it can be concluded that 5′-end tag is sufficient and critical for RNA silencing. 

But an interesting experiment leaves the tag’s function more confusing[3]. If the target transcript sequence is complementary to the tag, even though the target is known to be CRISPR hosts sequence, the RNA cleavage is not prevented. Just like shown in the right half figure, the target sequence cannot escape from being killed even it is complementary to the 5′-end tag. Thus 5′-end seems not to be the key commander in discrimination, or there are other molecules hold the key? At least, another unknown issue. 

What can Synbio do?

A DNA silencing systems in bacteria, and novel RNA silencing system in archaea!!! It leaves up to you.


–end &&reference

  1. Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., Romero, D. A., et al. (2007). CRISPR provides acquired resistance against viruses in prokaryotes. Science (New York, N.Y.), 315(5819), 1709-12. doi:10.1126/science.1138140
  2. van der Oost, J., & Brouns, S. J. J. (2009). RNAi: prokaryotes get in on the act. Cell, 139(5), 863-5. doi:10.1016/j.cell.2009.11.018
  3. Hale, C. R., Majumdar, S., Elmore, J., Pfister, N., Compton, M., Olson, S., Resch, A. M., et al. (2012). Essential Features and Rational Design of CRISPR RNAs that Function with the Cas RAMP Module Complex to Cleave RNAs. Molecular Cell, 1-11. Elsevier Inc. doi:10.1016/j.molcel.2011.10.023
  4. Wiedenheft, B., Sternberg, S. H., & Doudna, J. a. (2012). RNA-guided genetic silencing systems in bacteria and archaea. Nature, 482(7385), 331-338. doi:10.1038/nature10886

Copyright: The attached figures belong to publications with reference number, respectively.

microRNAs quantitative assay with Splinted Ligation

Do you still use Northern Blot to quantitate microRNAs expression? Here I recommend Splinted Ligation Assay[1,2], though this method is an old story (published in 2007).


Detection of miRNAs using splinted ligation. Schematic depiction of the assay process. As described in the text, the assay involves: (1) Labeling of the ligation oligonucleotide; (2) concurrent annealing of the ligation oligonucleotide and miRNA to a bridge oligonucleotide; (3) linking of the ligation oligonucleotide to the miRNA by DNA ligase; (4) removal of labeled phosphate from unligated oligonucleotide; and (5) fractionation on a denaturing gel.

–end && reference

[1] Maroney, P. a, Chamnongpol, S., Souret, F., & Nilsen, T. W. (2007). A rapid, quantitative assay for direct detection of microRNAs and other small RNAs using splinted ligation. RNA (New York, N.Y.), 13(6), 930-6. doi:10.1261/rna.518107

[2] Maroney, P. A., Chamnongpol, S., Souret, F., & Nilsen, T. W. (2008). Direct detection of small RNAs using splinted ligation. Nat. Protocols, 3(2), 279-287. Nature Publishing Group. Retrieved from http://dx.doi.org/10.1038/nprot.2007.530

Computer Program Enrich Understandings to RNA motifs

DNA carries and passes the genetic information with nucleotides sequences, but in some virus RNA does the job instead of DNA.  In addition, the 3D structure of RNA, RNA motif, shows more various roles in cellular functions. It means that only with sequence information we cannot fully understand the RNA roles and functions.

Detecting and exploring RNA motif are prone to lie in modeling, engineering, and this is partly why we need computational biology.

RNA structural motifs are the building blocks of the complex RNA architecture. Identification of non-coding RNA structural motifs is a critical step towards understanding of their structures and functionalities. In this article, we present a clustering approach for de novo RNA structural motif identification. We applied our approach on a data set containing 5S, 16S and 23S rRNAs and rediscovered many known motifs including GNRA tetraloop, kink-turn, C-loop, sarcin–ricin, reverse kink-turn, hook-turn, E-loop and tandem-sheared motifs, with higher accuracy than the state-of-the-art clustering method. We also identified a number of potential novel instances of GNRA tetraloop, kink-turn, sarcin–ricin and tandem-sheared motifs. More importantly, several novel structural motif families have been revealed by our clustering analysis. We identified a highly asymmetric bulge loop motif that resembles the rope sling. We also found an internal loop motif that can significantly increase the twist of the helix. Finally, we discovered a subfamily of hexaloop motif, which has significantly different geometry comparing to the currently known hexaloop motif. Our discoveries presented in this article have largely increased current knowledge of RNA structural motifs.

Researchers at University of Central Florida [1] used a complex computer program to analyze RNA motifs — the subunits that make up RNA (ribonucleic acid).

–end &&reference

[1] C. Zhong, S. Zhang. Clustering RNA structural motifs in ribosomal RNAs using secondary structural alignment.Nucleic Acids Research, 2011; 40 (3): 1307 DOI:10.1093/nar/gkr804