Researchers at Stanford University using TriLink's Cas9 mRNA report that chemical modification of CRISPR RNA guide strands can enhance CRISPR-Cas9-mediated genome editing efficiency in human primary cells. They also identify mRNA as an optimal delivery vehicle for Cas9 when compared to plasmid.
|Representative image of modified sgRNA. Stars indicate placement of modifications.
The CRISPR-Cas9 system has revolutionized the field of genome editing due to its efficiency, ease of use, and cost effectiveness, however, gene editing in primary human cells has remained problematic. The findings presented here are a major breakthrough for the CRISPR-Cas9 field, particularly for therapeutic applications.
The CRISPR guide strands (short for clustered, regularly interspaced, short palindromic repeats) are short pieces of RNA that guide the CRISPR associated protein (Cas9) to a particular genomic site for cleavage and either homologous recombination or non-homologous end joining (NHEJ) occurs. In nature, there are two guide RNAs, termed CRISPR RNA (crRNA) and trans-activating crRNA. These are often combined to yield a ~ 100 nucleotide chimeric single guide RNA (sgRNA).
Published in Nature Biotechnology, Dr. Matthew Porteus’ lab sought to improve editing efficiency in primary human cells. They tested three variations of chemically modified sgRNA. The variations included: 1 )2'-O-methyl , 2) 2'-O-methyl 3' phosphorothiate (MS) or 3) 2'-O-methyl 3' thioPACE (MSP). These modifications were incorporated on the terminal three nucleotides of both the 5' and 3' ends and were selected for their increased stability and decreased immunostimulatory properties.
The team then chose three target genes: 1) interleukin 2 receptor, gamma (IL2RG) 2) hemoglobin, beta (HBB) and 3) C-C chemokine receptor type 5 (CCR5). These genes were chosen because they are known to have relatively high gene editing frequencies in cell lines. Dr. Porteus comments, “We have been actively studying these genes because of their therapeutic importance for years and had identified highly active sgRNAs that worked in cell lines but had been stymied on trying to get high frequencies of editing in primary blood cells using the CRISPR system.”
|"With these results we are now poised to begin exploring the therapeutic promise in editing primary blood cells using the CRISPR/Cas9 system..."
- Dr. Matthew Porteus
They first tested modified sgRNAs co-transfected with Cas9 plasmid in human cell line K562 and looked at indel frequency, which is indicative of non-homologous end joining (NHEJ). sgRNA modified with MS or MSP produced indel frequencies 20 times greater than unmodified or M-modified sgRNAs. MS- or MSP-sgRNAs were also significantly better when measuring homologous recombination. Importantly, they found that the modified sgRNAs generally retained specificity; however they did find varying levels of off-target activity. Further experiments suggest that the increased editing efficiency was due to increased sgRNA stability.
The authors also reported similar findings in stimulated primary T cells. They transfected Cas9 mRNA or Cas9 protein with sgRNAs targeting IL2RG or HBB and found that the MS- or MSP-modified sgRNA performed far better than the unmodified or M-modified sgRNA. They found that the modification frequencies were similar in CD4+, CD8+, and the total T cell population. In contrast to stimulated T cells, unstimulated T cells were more difficult to edit. Cas9 mRNA co-transfected with modified sgRNA produced indel frequencies of 6.6%-22.2% which varied based on the donor source of T-cells.
Additionally, they also reported that Cas9 mRNA with 5-methylcytidine and pseudouridine significantly improved gene editing efficiency when compared to plasmid and Cas9 protein complexed to sgRNA in stimulated T cells. Notably, Cas9 protein complexed with sgRNA had a better on-target: off-target ratio than sgRNA co-delivered with Cas9 plasmid or Cas9 mRNA.
A report indicated that the use of two guide strands with different (but closely aligned) target sequences improved indel frequency. Here, the authors similarly co-transfected two sgRNAs targeting CCR5 with Cas9 mRNA. As with the previous study, the authors also found that two MS- or MSP-modified sgRNAs performed better than a single modified sgRNA in both human primary T cells (~10-20% increase) and CD34+ hematopoietic stem/progenitor cells (HSPCs; ~100% increase). Dr. Porteus notes,"With these results we are now poised to begin exploring the therapeutic promise in editing primary blood cells using the CRISPR/Cas9 system that has been until now been based on more hype than data."
Taken together, this research provides key insights into increasing gene editing efficiency in primary human cells with CRISPR-Cas9 system. It is feasible to infer that these modified sgRNAs may also increase editing efficacy with other cell types. This would be extremely valuable for both basic research and therapeutic applications.
In addition to increased efficacy through modification, the authors also note that the use of chemically synthesized guide strands provides several other advantages over expressed or in vitro transcribed sgRNAs, such as robust and scalable manufacturing, greater flexibility with design and the ability to do site specific modifications. They also note that a completely RNA-based CRISPR system is known to display decreased toxicity. As with all potential therapeutics, researchers should understand the implications of intellectual property for any given system.
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