Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) in primary human immune cells and hematopoietic stem cells
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Abstract
Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) is a new approach for ex vivo genome editing of primary human cells. PERC uses a single amphiphilic peptide reagent to mediate intracellular delivery of the same pre-formed CRISPR ribonucleoprotein enzymes that are broadly used in research and therapeutics, resulting in high-efficiency editing of stimulated immune cells and cultured hematopoietic stem and progenitor cells (HSPCs). PERC facilitates nuclease-mediated gene knockout, precise transgene knock-in, and base editing. PERC involves mixing the CRISPR ribonucleoprotein enzyme with peptide and then incubating the formulation with cultured cells. For efficient transgene knock-in, adeno-associated virus (AAV) bearing homology-directed repair template DNA may be included. In contrast to electroporation, PERC is appealing as it requires no dedicated hardware and has less impact on cell phenotype and viability. Due to the gentle nature of PERC, delivery can be performed multiple times without substantial impact to cell health or phenotype. Here we report methods for improved PERC-mediated editing of T cells as well as novel methods for PERC-mediated editing of HSPCs, including knockout and precise knock-in. Editing efficiencies can surpass 90% using either Cas9 or Cas12a in primary T cells or HSPCs. Because PERC calls for only three readily available reagents - protein, RNA, and peptide - and does not require dedicated hardware for any step, PERC demands no special expertise and is exceptionally straightforward to adopt. The inherent compatibility of PERC with established cell engineering pipelines makes this approach appealing for rapid deployment in research and clinical settings.
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ABSTRACT
Very exciting technology, great work to the authors!
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PERC is gentle on cells, permitting sequential editing of multiple loci. As previously reported, this is one way to minimize chromosomal translocations1
This is a really exciting implication that I hadn't considered before!
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Fig. 2 and Fig. 3
I found this figures a little difficult to fully understand. Here are my small notes about what would improve the presentation, again, please take it or leave it:
Colors: It would be helpful to have a legend that explains that different colors uses (light blue, dark blue, white, etc) . It took me a while to see the triangle vs. circle for washed and unwashed, but im not sure how the colors connect.
Some stats would be helpful here! It can be difficult to asses the differences just by eye. It seems that sometimes washed vs. unwashed are different in terms of edited cell yield (like in the HSPCs) but then are the same for other metrics like % editing and % NHEJ? It would be useful to in the figure have some comparisons and indications of if the differences are statistically significant.
This might just be a biorxiv …
Fig. 2 and Fig. 3
I found this figures a little difficult to fully understand. Here are my small notes about what would improve the presentation, again, please take it or leave it:
Colors: It would be helpful to have a legend that explains that different colors uses (light blue, dark blue, white, etc) . It took me a while to see the triangle vs. circle for washed and unwashed, but im not sure how the colors connect.
Some stats would be helpful here! It can be difficult to asses the differences just by eye. It seems that sometimes washed vs. unwashed are different in terms of edited cell yield (like in the HSPCs) but then are the same for other metrics like % editing and % NHEJ? It would be useful to in the figure have some comparisons and indications of if the differences are statistically significant.
This might just be a biorxiv figure display issue, but in 2c, 2f, 3f, 4c, and 4f the white NT bars are missing some of their outlines.
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INTRODUCTION
This is a really helpful introduction to the technology. I appreciate the level of detail provided here, its clear the authors are being very thoughtful about enabling others to use this approach. Overall I found the description of the technology to clear and rigorous. I left some comments about details that I would wonder as a non-expert user if i were trying to get something similar off the ground, please take it or leave it.
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The peptide-only condition can be used to test a given cell type’s sensitivity to the peptide, although we note that peptide-mediated toxicity can be exacerbated by the absence of RNP cargo.
How do you measure cell sensitivity? Is this overall viability, or are there other important metrics to consider?
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Assessing editing efficiency
I really appreciate your step-by-step breakdown of how to evaluate editing success! It could be useful to also explain how to evaluate off-target editing. Do you have a routine approach for this?
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For synthesis, we recommend ≥95% purity as assessed by HPLC, which is also used for purification. It has been suggested that an acid exchange step (using HCl or acetate to displace trifluoroacetic acid)
So helpful! thanks for including this detail.
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Two PERC peptides are commercially available: INF7TAT-A5K (A5K)15 and INF7TAT-P55 (P55).
Where are they commercially available from?
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T cells and HSPCs are relatively fragile and generally resistant to transfection
I can't tell if you are including LNPs under the transfection umbrella or not. I naively would assume yes but am not an expert. Below you talk about LNPs being a viable option for T cell / HSPC delivery, but this sentence up top is suggests otherwise. If you are referring to a different type of transfection reagents here being non-ideal or T cell/HSPC delivery , can you specify ?
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Spacing PERC delivery steps by ≥ 2 d allows each RNP to be metabolized by the cell26,
Can you clarify if the cells are dividing during this time frame? Or if this is due to the overall stability of the RNP in the cell itself.
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NF7TAT peptide had served as the basis for a prior screen for lytic activity in red blood cells as a proxy for endosomal escape20. Our screen of INF7TAT variants in T cells15 identified A5K as well as three additional activating INF7TAT substitutions (G1K, G20L and Y22N) that we have now incorporated in a single peptide: INF7TAT-P55 (henceforth P55)
Could you briefly explain a little bit more about your peptide reagent? How do the mutations impact activity?
Also, why aren't the peptides lytic in this context? Do the mutations reduce this activity?
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