Genome editing of cells is a quantitative process in that it is not enough simply to deliver a nuclease or a donor template, but instead they have to be delivered in sufficient quantities to achieve the desired on-target efficacy without delivering so much that it causes toxicity.[1] The relative indel formation is directly related to the amount of nuclease delivered to the cell until a plateau is reached. Similarly, the frequency of HDR is directly proportional to the amount of donor that is delivered until a plateau is reached. The primary counterforce in delivering sufficient quantities of genome editing reagents is that primary human cells have redundant and robust mechanisms to detect and inhibit the delivery of foreign nucleic acids (e.g., through the intracellular type I interferon innate immune response).[2] A secondary counterforce to efficient genome editing while maintaining cell potency is the cell’s DNA damage response (DDR) to the creation of genomic DSBs and to the donor molecules themselves (in the case of HDR).[3] Thus, for successful therapeutic editing, sufficient quantities of genome editing reagents must be delivered to cells while minimizing the inhibitory responses to the process. Specifically, the delivery of genome editing reagents as naked DNA plasmids, a process that works well in trans formed cell lines with damaged cytoplasmic DNA sensing systems, is not tolerated well by primary human cell such as HSPCs and T cells.

In addition to delivering the reagents without causing toxicity, for HDR-based editing, in which the cell needs to be in S/G₂ for HDR to occur, a manufacturing key is to develop ex vivo cell culture conditions that put the cells into cycle without compromising their long-term potency.

For meganucleases, ZFNs, and TALENs, the solution to ex vivo manufacturing is to deliver the nuclease as a highly purified messenger RNA (mRNA) with modifications to minimize detection of foreign RNA molecules, including the use pseudo-uridine, 5-methylcytosine, and HPLC purification to reduce double-stranded structures.

For CRISPR/Cas9, the current optimal delivery system is to deliver the nuclease as a ribonucleoprotein (RNP) complex. In this complex, purified Cas9 (which is easy to produce and stable) is mixed with a gRNA that is made synthetically with end-modifications to protect against degradation.[4] The gRNA is then complexed with Cas9 protein prior to delivery.

For both mRNA and RNP delivery, electroporation of cells to create pores through which the macromolecule enters is highly effective. Electroporation without contaminating DNA in the mix is surprisingly nontoxic to cells following optimization. Various electroporation devices that allow cell manufacturing of billions of cells are now available. Other methods that allow the macromolecules to enter cells may also be developed in the future. In all methods, the amount of nuclease macromolecule introduced needs to be titrated to achieve the desired ratio of efficacy to toxicity. One advantage of both mRNA and especially RNP delivery is the nuclease is present in the cell for only a short period (for Cas9 RNP for ~48 hours), which minimizes the possibility of creating breaks at off-target sites and decreases the cellular DDR. Using these nuclease delivery strategies, greater than 90% indels can be routinely achieved at the target site in primary human hematopoietic cells.

For HDR, the donor template must also be delivered in sufficient quantities. For HDR based on ssODNs, the purity of the ssODN is essential to enhance efficacy and minimize toxicity. Careful titrations of the amount of ssODN included are an important aspect of optimizing the manufacturing process. After optimization 30% to 50% HDR can be achieved in HSPCs using ssODN-based genome editing.[5]

For HDR using classic gene targeting donors and HR, recombinant adeno-associated virus serotype 6 (AAV6) has become the most effective method of delivering the donor molecule.[6-8] AAV6 has evolved to deliver its single-stranded DNA cargo to the nucleus of cells without being easily detected by cytoplasmic sensors of viral transduction.[2] The efficiency of AAV6 transduction of primary human HSPCs and T cells is enhanced by electroporation and is maximal if the AAV6 is applied to cells within 15 minutes of the electroporation.[9] As with the nuclease, ssODN, each AAV6 prep needs to be titered to achieve the desired efficacy and toxicity ratio. Using CRISPR/Cas9 RNP and AAV6, greater than 40% HDR can routinely achieved in HSPCs and up to 80% in T cells and certain loci in HSPCs. These frequencies of HDR are on allele basis, so a 40% HDR allele frequency results in between 55% to 70% HDR cell frequency depending on the ratio of monoallelic versus biallelic HDR.

Small molecules are increasingly being incorporated into the cell manufacturing process to improve efficacy and potency. These include small molecules to maintain stem cell potency,[10] inhibitors of the p53 response to minimize the DDR,[3] and small molecules that bias toward HDR rather than NHEJ for cells in situations where HDR is the desired editing event.[11,12]

References

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[1] Hendel A, et al. Quantifying on- and off-target genome editing. Trends Biotechnol. 2015;33(2):132–140.

[2] Cromer MK, et al. Global transcriptional response to CRISPR/Cas9-AAV6 based genome editing in CD34(+) Hematopoietic stem and progenitor cells. Mol Ther. 2018;26(10):2431–2442.

[3] Schiroli G, et al. Precise gene editing preserves hematopoietic stem cell function following transient p53-mediated DNA damage response. Cell Stem Cell. 2019;24(4):551–565. e8.

[4] Hendel A, et al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat Biotechnol. 2015;33(9):985–989.

[5] DeWitt MA, et al. Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells. Sci Transl Med. 2016;8(360):360ra134.

[6] Dever DP, et al. CRISPR/Cas9 beta-globin gene targeting in human haematopoietic stem cells. Nature. 2016;539(7629):384–389.

[7] Wang J, et al. Homology-driven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors. Nat Biotechnol. 2015;33(12):1256–1263.

[8] De Ravin SS, et al. Targeted gene addition in human CD34(+) hematopoietic cells for correction of X-linked chronic granulomatous disease. Nat Biotechnol. 2016;34(4):424–429.

[9] Charlesworth C, et al. Priming human repopulating hematopoietic stem and progenitor cells for Cas9/sgRNA gene targeting. Mol Ther Nucleic Acids. 2018;12:89–104. in press.

[10] Fares I, et al. Small molecule regulation of normal and leukemic stem cells. Curr Opin Hematol. 2015;22(4):309–316.

[11] Canny MD, et al. Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency. Nat Biotechnol. 2018;36(1):95–102.

[12] Certo MT, et al. Tracking genome engineering outcome at individual DNA breakpoints. Nat Methods. 2011;8(8):671–676.

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مواضيع ذات صلة

Using Engineered Nucleases to Initiate the Genome Editing Process

Base Editing

Double-stranded DNA breaks: the basis of nuclease-based genome editing

Plaque and Colony Hybridization for Clone Identification

Cloning from RNA

Plasmid Vectors

كلونة النعجة دولي Cloning Dolly the sheep

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أدوات كلونة الجين المتعاقبة

genetic engineering in industry

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