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Case Study // JUN 30 2014

Genome editing mediated by efficient transfection of dimeric CRISPR/Fok1 nuclease encoding plasmids

Lower off-target effects through the use of dimeric RNA-guided Fok1 nucleases.

"HEK293 cells were transfected with 750 ng of pSQT1601 nuclease plasmid, 250 ng of gRNA expression plasmid and 10 ng of tdTomato expression plasmid, using TransIT®-LT1 (Mirus Bio) according to the manufacturer’s instructions and analyzed for NHEJ-mediated mutagenesis 3d after transfection."

- Tsai, et al.1

Background:

The ability to quickly and easily alter the genome ushers forth an era in which relevant disease models can be created, pathways can be better analyzed and therapeutic possibilities become reality. A variety of genome editing methodologies are available including Zinc Finger Nucleases (ZFNs) and Transcription Activation Effector-like Nucleases (TALENs), but now the use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) has come to the forefront as a much more effective strategy. Transfection, or nucleic acid delivery to the cell, is a necessary requirement of genome editing techniques.

Transfection was first harnessed in 1973 with the development of calcium phosphate precipitation as a means to deliver nucleic acid to cells. Since that time there have been multiple approaches taken to enhance DNA delivery through virus delivery, mechanical methods (e.g. electroporation), chemical delivery or the use of new polymers. Mirus Bio has been at the forefront in driving this technology forward. Our very first reagent, TransIT®-LT1, was introduced in 1996 and still serves as a standard by which other reagents are measured. This product is known for maintaining a low toxicity profile, while providing robust DNA delivery in various cell types.

In the present case study, we highlight the use of TransIT®-LT1 to transfect HEK293 cells with a modification of the CRISPR system that allows for increased specificity, high efficiency, and a less prohibitive guide RNA design.

Dimeric CRISPR RNA-guided Fok1 nucleases for highly specific genome editing.
1Tsai SQ, Wyvekens N, Khayter C, Foden JA, Thapar V, Reyon D, Goodwin MJ, and Joung JK. Nat Biotech, 2014 April 25, Epub ahead of print doi:10.1038/nbt.2908, PMID 24770325

What is genome editing?

Genome editing refers to a technique whereby DNA is inserted, replaced or deleted from a genome by way of wild type or chimeric nucleases. These nucleases are directed to specific sequences within the genome and mediate changes through homologous recombination (HR) or non-homologous end-joining (NHEJ). The three most common methods for genome editing make use of ZFNs, TALENs and CRISPR. Publications in early 2013 established CRISPR as a viable tool for genome editing and work quickly progressed to broad application in mammalian systems. CRISPR has some distinct advantages over ZFN and TALEN approaches such as being simple, cheaper, more efficient, and the ability to be multiplexed through addition of multiple guide RNAs.

Does a requirement for dimerization increase CRISPR specificity?

One of the concerns with the CRISPR technique as a whole is whether off-target effects are increased. The sequence specificity for CRISPR is less than that of ZFNs or TALENs. In early iterations of the CRISPR system, WT recombinant Cas9 caused double strand DNA cleavage to facilitate HR or NHEJ mechanisms. As a result, off-target effects also resulted in a degree of genome alteration. An improvement over this early form of CRISPR was the introduction of a mutation in Cas9 that specifies a single-strand DNA nick rather than a double stranded break. A single nick as the result of an off-target effect is repaired by the cell’s endogenous base-excision repair pathway whereas two nicks in close proximity proceed down the HR or NHEJ pathways and create insertions or deletions (INDELs). As an additional improvement, Tsai, et al.sup>1 hypothesized that introducing a dual specificity to the system would further decrease off-target effects, especially if the dual specificity was dictated by a dimer formation requirement for enzymatic activity.

To achieve this aim Tsai, et al.sup>1 borrowed a page from the ZFN and TALEN approach and rather than relying on Cas9 to to drive strand cleavage, they used Fok1 nuclease. A chimeric protein comprising the Cas9 guide RNA binding domain coupled with the Fok1 dimerization and nucleic acid cleavage domain was created. Since Fok1 nuclease requires dimerization, cleavage should only occur when both guide RNAs (both halves of the cleavage site) are brought into close proximity such that the two halves of the Fok1 nuclease are able to associate.

In addition, Tsai, et al.sup>1 developed a system for generating guide RNAs that is less prohibitive than former mechanisms. Multiple guide RNAs are created from a single mU6 promoter and then cleaved into individual RNA elements by a Csy4 protease. This allows multiple guide RNAs to be made from a single promoter on a single plasmid and removes some sequence specificity limitations imparted by mU6 (requires a G to be present on the 5’ end of the guide RNA). This approach makes it much easier to multiplex guide RNAs against various target genomic loci.

In this publication, Tsai, et al.sup>1 was able to show that TransIT®-LT1 transfection of HeLa cells with constructs expressing RNA-guided Fok1 nucleases brought about mutations in 11 of 12 target sites. The mutation frequency varied from 2.9% to 20.7% overall and exhibited results similar to what was achieved using electroporation in U2OS cells.

The Cas9-Fok1 dimer approach can induce mutations with high efficiency and with nearly undetectable off-target effects, providing a better tool for conducting genome editing.

For more information, please visit our CRISPR/Cas Genome Editing page.

Other Mirus Bio reagents useful in genome editing:

  • TransIT-X2® - an animal origin free reagent capable of delivering DNA and/or siRNA with high efficiency in a broad spectrum of cell types.
  • TransIT®-2020 - an animal origin free reagent specifically developed to deliver DNA with high efficiency in difficult-to-transfect cell types.

Free samples of Mirus Bio transfection reagents are available upon request.

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