How does the CRISPR guideRNA get into the Nucleus?

Hi, Alberto.

In procaryotes, no need for Cas9 to translocate into the nucleus. In eukaryotes, Cas9 protein and gRNA complex could form within the nucleus or cytoplasm after Cas9 translocates due to its NLSequence. Since Cas9 and gRNA viral vectors or plasmids or mRNA or RNA are injected or transfected or infected, Cas9 has to be made in the cytoplasm and return to the nucleus and binds to gRNA. Since the CRISPR/Cas9 systems vary as mentioned above, no one seems to be interested the sequential process at the moment.

It should also be remembered that nuclear membrane breakdown occurs at every cell cycle. There is always opportunity that both molecules bump into each other. This may explain that no study is needed for the precise space and time.

Sorry for the ref.

Qais Al-Oqaily

Thanks for the clarification.

I'm now aware why the NLS is there in the CRISPR plasmid.

Anthony Stephenson

TL;DR::: Depends on many factors.

If you use in vitro transcribed RNA and deliver into mammalian cells via electroporation or lipofection then it will likely be across many membranes. If encode it genetically and use a PolIII promoter (like a U6 cassette) and terminate with a polyU signal, it will be likely be strongly retained in the nucleus by La protein (see 10.1261/rna.065136.117 for review and other references) upon transcription and may never enter the cytosol if the cells are non-replicating -- but could transiently enter the cytosol during mitosis in many cells. Ultimately you use an NLS on your Cas9 which will drive nuclear localization through it's incredibly tight binding to gRNA. May also depend on the NLS tho.. I think large-T antigen's NLS (PKKKRKV) is dependent on importin alpha and occurs post-translationally .

I can't say that I've seen anyone actually track the location of the gRNA in mammalian cells using any of the examples I give above.

Yu Wa

Hello,

The delivery of CRISPR guide RNA (gRNA) into the nucleus, where it can effectively guide the Cas9 nuclease to the target DNA, is a critical aspect of CRISPR-Cas9 genome editing. This process can be achieved through several methods, each utilizing different mechanisms to facilitate nuclear entry. Here’s an overview of how gRNA gets into the nucleus:

Vector-Based Delivery: In many experiments, gRNA is delivered into cells using a vector, such as a plasmid DNA. Once the vector is inside the cell, it utilizes the cell's own machinery to transcribe the gRNA from the DNA template. The produced gRNA then enters the nucleus via nuclear pores.

Ribonucleoprotein (RNP) Complexes: Direct delivery of gRNA can be achieved by forming ribonucleoprotein complexes with the Cas9 protein in vitro. These complexes can be introduced into cells through methods like electroporation. Once inside the cell, they can passively diffuse through nuclear pores due to their small size.

Lipid Nanoparticles and Other Carriers: Lipid nanoparticles and other nanocarrier systems can encapsulate gRNA or RNP complexes and facilitate their entry into cells. After endocytosis, these carriers can escape from endosomes and release gRNA or RNPs, which then migrate to the nucleus.

Viral Vectors: Viral vectors like lentivirus or adeno-associated virus (AAV) can be engineered to deliver gRNA or the gRNA-Cas9 encoding sequence into cells. These viral systems have natural mechanisms for nuclear entry and can be effective in delivering gRNA to the nucleus.

Physical Methods: Methods such as microinjection or nucleofection can directly introduce gRNA or RNP complexes into the nucleus, bypassing the need for nuclear localization signals or other complex delivery mechanisms.

Nuclear Localization Signals (NLS): If the gRNA is delivered as part of a larger construct (like a plasmid or RNP complex), the presence of nuclear localization signals on Cas9 or associated molecules can enhance nuclear import.

Endogenous Transport Mechanisms: Cells have natural transport mechanisms to regulate the nuclear entry of molecules. Small molecules and RNAs can enter the nucleus through the nuclear pore complex by passive diffusion or active transport.

Optimization for Efficiency: The efficiency of gRNA delivery to the nucleus can vary based on the cell type, delivery method, and the design of the gRNA construct or delivery system. Optimization may be necessary to achieve effective genome editing.

In summary, the delivery of gRNA into the nucleus is a crucial step in CRISPR-Cas9 genome editing, and it can be achieved through various methods, each leveraging different aspects of cellular transport and molecular biology techniques. The choice of delivery method depends on the specific requirements of the experiment, including the type of cells being targeted and the desired efficiency and specificity of editing.

With this protocol list, we might find more ways to solve this problem.

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