CRISPR/Cas9 for RNA Cleavage

Postdoctoral Researcher, Division of Biology Kansas State University Manhattan KS

David Meekins, Ph.D., Postdoctoral Researcher, Division of Biology, Kansas State University, Manhattan, KS USA

The CRISPR/Cas9 system is quickly becoming the most versatile method for modifying DNA targets, but researchers seeking equivalent control against RNA transcripts have been left behind.

In a new paper by O’Connell et. al, (2014) the authors describe a clever modification of the CRISPR/Cas9 system to target ssRNA. This adjustment to the current system could become the standard in RNA silencing and isolation.

To generate Cas9 cleavage of dsDNA, the target sequence must be complementary to the guide-RNA (gRNA) and must be followed by a protospacer adjacent motif (PAM). The PAM sequence activates the nuclease domain of Cas9, thereby initiating DNA cleavage via an allosteric mechanism. In the context of immunity, the PAM sequence allows Cas9 to discriminate between foreign DNA, which contain the PAM sequence and potential host genomic targets, which do not.

Cas9 with functional domains highlighted in relation to target DNA and guideRNA

Cas9 with functional domains highlighted in relation to target DNA and guide RNA

A few findings fueled the transition from Cas9 cleavage of dsDNA targets towards its application in ssRNA cleavage.

First, Cas9 can cleave ssDNA if a separate PAM-presenting oligonucleotide (PAMmer) is present to bind and activate Cas9.

Second, the HNH nuclease domain of Cas9 has been shown to cleave ssRNA.

CRISPR/Cas9 cutting RNA.  Note the critical requirement of a PAMmer sequence.

CRISPR/Cas9 cutting RNA. Note the critical requirement of a PAMmer sequence.

 

Based on these data, the authors demonstrated that a ssRNA transcript could be efficiently cleaved using Cas9-gRNA in the presence of a separate DNA PAMmer.

The authors demonstrate a range of PAMmer variations that optimize the system and generate highly specific cleavage of RNA targets without cross reactivity between different RNA transcripts or against genomic DNA. They also demonstrate a promising proof-of-principle application of the method by isolating endogenous GAPDH transcripts from HeLa cells. This indicates that the applications for this technology will far exceed the findings published in the current study.

The method presented is of prime importance to insect geneticists desiring to target, bind, or cleave target RNA without secondary effects on genomic DNA. The simplicity of the technique is particularly attractive because the complementary gRNA and PAMmer are the only components necessary in designing your tool.

There are many possible applications for a CRISPR/Cas9 system that can bind and cleave RNA specifically.

There are many possible applications for a CRISPR/Cas9 system that can bind and cleave RNA specifically.

 

There is currently some variability in the success rate of RNA silencing between different genes and insect systems. Cas9 ssRNA targeting may provide a much more consistent approach to efficiently remove specific transcripts RNA in vivo. Furthermore, the recognition of specific RNA transcripts without the need for genetically encoded tags is definitely an exciting proposition for insect genetic studies.

O’Connell MR, Oakes BL, Sternberg SH, East-Seletsky A, Kaplan M, Doudna JA (2014) Programmable RNA recognition and cleavage by CRISPR/Cas9. Nature advance online publication  10.1038/nature13769

 

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