Non-viral targeted gene insertion method for cell engineering

Abstract

In one aspect, a method of treating an NK cell to induce a gene edit. The method may include contacting an NK cell with a transfection composition including poly-L-glutamic acid (y- PGA), a single-stranded oligo deoxynucleotide (ssODN) enhancer, or a combination thereof; contacting the NK cell with an editing composition including: a DNA-PK inhibitor, and an HD AC inhibitor; electroporating the NK cell; and contacting the NK cell with a recovery composition including DNase I. In another aspect, a method of preparing an engineered NK cell comprising a fluorescent reporter at an endogenous locus. In another aspect, a method of treating an NK cell to introduce an exogenous nucleic acid.

Description

[0001] NON- VIRAL TARGETED GENE INSERTION METHOD FOR CELL ENGINEERING

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 665,765, filed on lune 28, 2024, the disclosure of which is incorporated by reference herein in its entirety.

[0004] SEQUENCE LISTING

[0005] This application contains a Sequence Listing electronically submitted via EFS-Web to the United States Patent and Trademark Office as an XML file entitled “0110.000751W001.xml” having a size of 251,914 bytes and created on May 29, 2025. The information contained in the Sequence Listing is incorporated by reference herein.

[0006] GOVERNMENT FUNDING

[0007] This invention was made with government support under CA283892, CA065493, and CAI 11412 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0008] SUMMARY

[0009] In one aspect, the present disclosure relates to a method of treating an NK cell to induce a gene edit. Generally, the method includes contacting the NK cell with a transfection composition, contacting the NK cell with an editing composition, contacting the NK cell with a repair template, electroporating the NK cell, and contacting the NK cell with a recovery composition. The transfection composition generally can include poly-L-glutamic acid (y-PGA), a single-stranded oligo deoxynucleotide (ssODN) enhancer, or a combination thereof. The editing composition can generally include a DNA-PK inhibitor and an HD AC inhibitor. The recovery composition can generally include DNase I. In one or more embodiments, the NK cell includes a feeder-expanded NK cell and / or the NK cell has been previously cryopreserved In one or more embodiments, the method further includes contacting the cell with editing reagents., such as Cas9 and a guide RNA, such as a single guide RNA (sgRNA).

[0010] In one or more embodiments, the recovery composition further includes N-acetyl- cysteine, L-glutathione, or a combination thereof. In one or more embodiments, the recovery composition further includes emricasan, trans-ISRIB, or a combination thereof. In one or more embodiments, the DNA-PK inhibitor includes M3814 or AZD-7648. In one or more embodiments, the HD AC inhibitor includes Trichostatin A. In one or more embodiments, the repair template includes plasmid DNA, single-stranded DNA, or double-stranded DNA. In one or more embodiments, the repair template includes a tCTS. In one or more embodiments, at least 100 NK cells are treated. In one or more embodiments, at least 50% of the treated NK cells include the gene edit following treatment.

[0011] In another aspect, the present disclosure relates to a method of increasing the efficiency of gene editing in an NK cell including contacting the NK cell with poly-L-glutamic acid (PGA) and gene editing reagents.

[0012] In another aspect, the present disclosure relates to an edited NK cell prepared using the method of any preceding embodiment. In one or more embodiments, the NK cell is derived from a human NK cell. In one or more embodiments, the NK cell includes an inserted DNA sequence at the human CISH locus or the human CD96 locus. In one or more embodiments, the NK cell includes an inserted DNA sequence at one or more of the TOMM70, CTCF, ACTG1, MAP4, EEFIGJ, YWHAQ, HNRNPA1, ENO J, or GAPDH \oci.

[0013] In another aspect, the present disclosure relates to an editing composition for use in gene editing an NK cell including Trichostatin A and a DNA-PK inhibitor including AZD-7648, M3814, or a combination thereof. In one or more embodiments, a method of using the editing composition of includes contacting an NK cell with gene editing reagents and the editing composition.

[0014] In another aspect, the present disclosure relates to a recovery composition for use in gene editing an NK cell including DNasel; an apoptosis inhibitor including emricasan, and / or trans- ISRIB inhibitor; and an antioxidant including N-acetylcysteine, L-glutathione, or a combination thereof. In one or more embodiments, a method of using the recovery composition includes contacting an NK cell with gene editing reagents and the recovery composition. In another aspect, the present disclosure relates to a cell therapy reagent including an edited NK cell produced using the methods described herein.

[0015] In one or more embodiments, the edited NK cell includes a chimeric antigen receptor (CAR)-NK cell, optionally wherein the CAR targets CD 19, CD22, or both CD 19 and CD22.

[0016] In another aspect, the present disclosure relates to a method of preparing an engineered NK cell including a fluorescent reporter at an endogenous locus, the method including: contacting an NK cell with a transfection composition including: poly-L-glutamic acid (y-PGA), a single-stranded oligo deoxynucleotide (ssODN) enhancer, or a combination thereof; contacting the NK cell with an editing composition including: a DNA-PK inhibitor, and an HDAC inhibitor; contacting the NK cell with a repair template including a nucleic acid sequence encoding a fluorescent reporter; electroporating the NK cell; and contacting the NK cell with a recovery composition including DNase I. In one or more embodiments, the endogenous locus includes the TOMM70, CTCF, ACTG1, MAP4, EEF1G, YWHAQ, HNRNPA1, EN01, or GAPDH locus, particularly the PFKB4 locus. In one or more embodiments, the repair template includes a nucleic acid sequence having at least 80% identity to a region of the endogenous locus.

[0017] In another aspect, the present disclosure relates to a method of treating an NK cell to introduce an exogenous nucleic acid, the method including: contacting the NK cell with a transfection composition including: poly-L-glutamic acid (y-PGA), a single-stranded oligo deoxynucleotide (ssODN) enhancer, or a combination thereof; contacting the NK cell with an editing composition including: a DNA-PK inhibitor, and an HDAC inhibitor; contacting the NK cell with an exogenous nucleic acid; electroporating the NK cell; and contacting the NK cell with a recovery composition including DNase I. In one or more embodiments, the exogenous nucleic acid includes DNA, such as a DNA plasmid.

[0018] The above summary is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

[0019] BRIEF DESCRIPTION OF THE FIGURES FIG. 1 . Editing strategy and validation in human primary NK cells. (A) A schematic illustration of the knock-in strategy. Cas9 RNP preassembled with CD96 targeting sgRNA makes a double-strand break in the CD96 locus. A template for knock-in encodes mNeonGreen and associated 3’UTR, BGH poly A is flanked by homology arm. (B) A schematic of the knock- in strategy. The isolated human primary NK cells were expanded with irradiated mIL21-41BBL K562 in BO medium supplemented with 50 lU / mL IL-2 until electroporation on Day 5. A medium change was performed on Day 6 followed by additional two days recovery before flow cytometry analysis. (C) Successful knock-in results using the method described in Figure 1A and B. The Cas9 RNP only efficiently eliminated the expression of CD96. The HDR template only had a minimum random integration event. The combination of Cas9 RNP and HDR template only generated the successful knock-in and robust mNeonGreen expression.

[0020] FIG. 2. (A) Incorporation of Poly-L-glutamic acid sodium salt (PGA) or short single- strand DNA (ssODN) enhancer enhances the knock-in efficiency. (B) Statistical analysis of knock-in results across six independent donors. The incorporation of PGA or ssODN enhancers improved the knock-in efficiency from two-fold to four-fold, which preserved the knock-out efficiency.

[0021] FIG. 3 DNA-PK inhibitors enhance the knock-in efficiency in a dose-dependent manner. (A) M3814 improved the knock-in efficiency up to 2.5-fold and became plateau at 1 μM. (B) AZD-7648 increases the knock-in efficiency up to 2.5-fold and became plateau at 0.5 μM. Both DNA-PK inhibitors dampened the cell recovery, but the relative mNeonGreen+ knock-in cell number increased.

[0022] FIG. 4. Representative flow cytometry figures of the knock-in efficiency improvement with or without the incorporation of 1 μM M3814 or AZD-7648.

[0023] FIG. 5. Effects of Trichostatin A. (A) Trichostatin A can enhances the knock-in efficiency in a dose-dependent manner. Trichostatin A reached the best balance between cell recovery and knock-in efficiency at 0.05 μM. (B) Representative flow cytometry figure of knock-in efficiency with or without 0.05 μM Trichostatin A. The incorporation of Trichostatin A increased the knock-in efficiency around 50%.

[0024] FIG. 6. Effects of M3814, AZD-7648, and Trichostatin A. (A) The mix-and-match test of M3814, AZD-7648, and Trichostatin A. The combination of Trichostatin A with either M3814 or AZD-7648 had the optimal knock-in efficiency improvement. The knock-in efficiency improved three-fold and the relative mNeonGreen+ cell numbers increased 1 .7-fold at average, when using the combination of Trichostatin A and M3814. (B) Representative figure showing that the combination Trichostatin A and M3814 increased the knock-in efficiency from 12.6% to 50.8%.

[0025] FIG. 7 (A) Emricasan enhanced the cell recovery in a dose-dependent manner. (B) Trans- ISRIB inhibitor also enhanced the cell recovery in a dose-dependent manner. Both small molecules maintained the knock-in efficiency and were none-low toxic to cells. Emricasan and trans-ISRIB inhibitor improved the cell recovery 30% at 0.25 and 0.5 μM.

[0026] FIG. 8. L-glutathione and N-acetyl-cysteine improved the cell recovery in a dose- dependent manner. At 7.5 μM, L-glutathione improved the cell recovery by 2.5-fold and knock- in efficiency by 30%. The relative mNeonGreen+ cell number increased three-fold at this concentration. At 2.5 μM, N-acetyl-cysteine improved the cell recovery by two-fold and the relative mNeonGreen+ cell number increased two-fold.

[0027] FIG. 9. The mix-and-match test of Emricasan, trans-ISRIB, and L-glutathione. Each small molecule only and combination groups enhanced the cell recovery. Among the three small molecule-only groups, the L-glutathione provided the highest cell recovery around two-fold. The combination of Emricasan, trans-ISRIB, and L-glutathione increased the robustness of cell recovery improvement around 3.7-fold.

[0028] FIG. 10. Incorporation of DNase I after electroporation enhanced the cell recovery by 50% without sacrificing the knock-in efficiency.

[0029] FIG. 11. The knock-in efficiency using different amount of dsDNA, ssDNA, and plasmid HDR knock-in templates. The dsDNA provided the optimal knock-in efficiency and cell recovery. ssDNA had a lower cellular toxicity, but the efficiency was lower than dsDNA. The plasmid provided the highest knock-in efficiency but had a strong cellular toxicity. The optimal knock-in efficiency and cell recovery was the condition using 2 μg dsDNA HDR knock-in temple.

[0030] FIG. 12. Knock-in efficiency and cell recovery when using different modified HDR knock-in template. tCTS modified ssDNA template provided the highest knock-in efficiency by 1.7-fold and relative mNeonGreen+ cell number by two-fold, compared to non-modified dsDNA. In the plasmid group, the nanoplasmid outperformed the pUC19, pUCmu, and minicircle plasmid in knock-in efficiency and cell recovery. FIG. 13. Combinatorial analysis of each optimized strategy. (A) The combination of PGA, DNase I, HDR cocktail (1 μM M3814 and 0.05 μM Trichostatin A), Recovery cocktail (2.5 μM Emricasan, 0.5 μM trans-ISRIB, and 7.5 mM L-glutathione) and Optimized HDRT (tCTS modified ssDNA) improved the knock-in efficiency by 7-fold and relative mNeonGreen+ cell number by 8-fold. The average absolute mNeonGreen+ cell number was around 6xl05cells from electroporation input of IxlO6cells. (B) Representative flow cytometry figures before / after optimization. The knock-in efficiency increased from 5.82% to 51.4%.

[0031] FIG. 14. CAR19 knock in strategy and results. (A) A schematic representing the c-myc epitope-tagged anti-CD19 chimeric antigen receptor (CAR19) knock-in strategy. The original mNeonGreen payload was swapped by codon-optimized CAR19. (B) Representative CAR19 knock-in flow cytometry data from two independent donors. The CAR19 knock-in efficiency (c- myc tag +) for donors 1 and 2 were 17.2% and 23.1%, respectively.

[0032] FIG. 15. CISH anti-CD22 / 19 CAR with IL- 15 knock-in. A synthetic positive feedback loop using a gene switch robustly enhanced NK cell function. (A) An anti-CD22 / 19 CAR plus sIL15 knock-in template was designed to target the CISH locus. (B) A schematic representation of a synthetic positive feedback loop. The CISH promoter primed by IL-2 in culture medium trigger the expression of anti-CD22 / 19 CAR and sIL15. Both anti-CD22 / 19 CAR and sIL15 can further positively contribute to CISH promoter activity and lead to a positive feedback loop for stable transgene expression. Meanwhile, the original CISH gene was terminated by a stop codon encoded in the knock-in template. (C) A representative figure of anti-CD22 / 19 CAR labeling. The anti-CD22 / 19 CAR expressed by the CISH promoter can be validated on flow cytometry. Anti-CD22 / 19 CAR can capture the soluble recombinant CD19 and CD22 proteins. CISH O exhibited better NK cell effector functions than the control. The anti-CD22 / 19 CAR expressed by the CISH promoter further enhanced the functionality.

[0033] FIG. 16. PFKFB4 IL-12 knock-in as proof of concept of hypoxia-inducible gene expression. (A) A schematic illustration of the transgene knock-in design. The knock-in template included IL-12 and truncated NGFR (tNGFR) for flow cytometry validation. (B) A schematic representation of one potential mechanism of hypoxia-restricted transgene expression. The PFKFB4 promoter is controlled by HIF-la, which is degraded by the E3 ubiquitin ligase system when oxygen is present. When environmental oxygen levels are low, such as in the tumor microenvironment, HIF-la is stabilized and starts to boost the transcription of the PFKFB4 gene. The transgene tethered to the PFKFB4 gene can be expressed upon hypoxia. (C) A representative flow cytometry figure. tNGFR reporter expression was triggered by low oxygen culture conditions.

[0034] FIG. 17. Endogenous protein tag design and efficiency. (A) Schematic representations of endogenous protein tag design. To tag endogenous protein, a linker is incorporated between endogenous protein and fluorescent transgene. (B) A statistical view of knock-in results from six independent donors. (C) Representative flow cytometry plot for tagged loci.

[0035] FIG. 18. Characterization of the recovery and HDR cocktails on electroporated cells. (A) The effect of HDR enhancer (HDR), Recovery cocktail (Recovery), and a combination of both (optimized) is independent of pulse conditions. Each dot represents one individual pulse program of Lonza 4D nuclefector. Data points were normalized to the average of live cell counts and %mNeonGreen+. Plasmid DNA can be toxic to NK cells and prevents meaningful cell recovery after electroporation. (B) Plasmid donors outperformed dsHDRT in knock-in efficiency (solid black line represents the average of 1 ug dsHDRT control). (C) The addition of a recovery cocktail rescued 100% of cells back, compared to the initial entry (solid black line).

[0036] FIG. 19. Comparison of freshly isolated and cryopreserved NK cells as starting materials. Two common cry opreservation reagents - FBS + 10% DMOS and CryoStor CS10 are equally functional for non-viral knock-in. Cryopreserved NK cells tended to have lower knock-in efficiency, but higher cell recovery post-electroporation.

[0037] FIG. 20. The effect of culture medium and supplement on knock-in efficiency and cell recovery. Each tested medium was able to support NK cell expansion. Each tested medium was able to conduct knock-in in NK cells. Each tested supplement was able to allow knock-in in NK cells. Although the knock-in efficiency is comparable across different culture medium conditions, the cell recovery varies. Immune SR and CellVive SS (a serum substitute) worked in the platform, demonstrating that the optimized platform can support serum-free non-viral genome editing in NK cells.

[0038] FIG. 21. A non-viral knock-in platform can perform serum -free manufacturing of CAR- NK cells, and the results were comparable between serum and serum-free conditions.

[0039] FIG. 22. Tri-locus knock-in. (A) A schematic representation of NCR3-mNeonGreen, TOMM70-mKate2, and CTCF-mTagBFP knock-in strategy in NK cells. (B) A triple positive population can be observed across six different donors. (C) Representative gating strategies of flow cytometry data.

[0040] FIG. 23. The titratable transgene expression system design and results. (A) Schematic representations of two targeting strategies are used for development. N-terminus targeting strategy directly separates mNeonGreen and endogenous protein with P2A. C-terminus targeting strategy often targets a DNA region away from the stop codon. The additional rescued sequence is introduced into the template to ensure the integrity of the endogenous protein and separate the mNeonGreen by P2A. Because mNeonGreen is separated from endogenous protein, the expression level is independent of the protein turnover rate of endogenous protein. (B) The OpenCell database provides reference mRNA abundance of selected loci in human cells. The knock-in result shows that all selected loci can knock-in. (C) A representative figure of the knock-in result. (D) A representative histogram plot of positive population and mean fluorescent intensity (MFI) comparison.

[0041] FIG. 24. Feasibility of the non-viral engineering platforms of the present disclosure for use in therapeutically relevant NK cell engineering. GAPDH was selected as a locus for engineering. (A) An illustration of the GAPDH targeting strategy. A set of various-sized transgene constructs were designed for knock-in. (B) The results of different editing, including 1071 bp of mbIL15 (membrane-bound), 2361 bp of anti-CD19 CAR (CAR19) with sIL15 (soluble), 3129 bp of anti-CD22 / 19 CAR (CAR22 / 19), and 4238 bp of a complex payload consisting of anti-Mesothelin CAR (CARmeso), anti-PD-1 ScFv (aPDl ScFv), and anti-B7-H3 tri-specific killer engager (B7H3 TriKE). The results were confirmed in two independent donors.

[0042] FIG. 25. sgRNA screening results of GISH targeting. (A) A schematic illustration of GISH mRNA transcripts. In Intron 1, there are two predicted transcription start sites. If targeting exon 1 for knock-out, the protein can potentially be rescued by these two alternative transcripts. Thus, sgRNAs were designed targeting exon 2 or 3. (B) The results of sgRNA screening. Exon 2 sgRNA 5 has the highest %KO. This demonstrates that one sgRNA can provide more than 90% Indel and %KO.

[0043] FIG. 26. The CISH-sIL15 positive feedback loop is able to perform the self-enrichment process. (A) Different amounts of IL-2 were added to the culture medium. Edited NK cells could proliferate without the addition of extra IL -2. (B) The percentage of CAR+ cells increased more than two-fold in cytokine cytokine-free expansion condition. The percentage of CAR+ cells decreased along with the increased IL-2 concentration, demonstrating that the bystander cells receive IL-2 signaling to proliferate and take up the population. (C) Quantification of the expansion fold of edited CAR+ and bystander NK cells and double-confirm our hypothesis. (D) A schematic illustration of a hypothesized model of self-enrichment. Autocrine secretion of sIL15 allowed CISH-CAR NK cells to proliferate without additional IL-2 supplement. The lack of IL-2 suppressed bystander NK cell expansion, leading to an enriched CAR+ population.

[0044] FIG. 27. Screening different sgRNAs targeting exon 1 for the N-terminus and exon 14 for the C-terminus. Exon 14 sgRNA 1 showed the highest indel installation.

[0045] FIG. 28. PFKFB4 CAR.B7H3 as an additional example of hypoxia-inducible gene expression, illustration of the transgene knock-in design. (A) A schematic representation of the knock-in template including a B7H3 CAR and sIL15. (B) A schematic representation of one potential mechanism of hypoxia-restricted CAR.B7H3 expression. (C) A representative flow cytometry figure. CAR.B7H3 expression was triggered by low oxygen culture conditions.

[0046] DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0047] Cell therapy is a powerful tool with the potential to improve efficacy and decrease side- effects of treating disease such as cancer. Many cell therapy clinical trials use T cells as a platform for therapeutic design. However, NK cells are an alternative cell candidate showing a potent cancer-killing and cytokine production capacity to orchestrate the immune response. Unlike T cells, NK cells are safe for allogenic transfer, have a minimal risk of cytokine release syndrome, and hold a potential for off-the-shelf product development. While NK cells are a strong candidate for cancer immunotherapy, there is a need for effective non-viral gene editing tools to prepare reagents such as CAR NK cells.

[0048] Conventional virus-based gene editing methods often rely on random integration of synthetic promoter-driven genes. Random integration has a risk of oncogene activation or tumor- suppressor gene inhibition, which may lead to tumorigenesis. In addition, synthetic promoters are prone to silencing over time and often yield non-uniform gene expression. Further, clinical- grade virus manufacturing is cumbersome and cost-prohibitive. In contrast, the cost-effective, non-viral, targeted gene insertion method presented herein may maximize the potential of engineered NK cell therapy. Previous publications have shown that while many existing non-viral targeted gene insertion methods are efficient in T cells, the same methods are significantly less effective in NK cells. For example, one method reported efficiency up to 60% in T cells and only 3-5% non-viral gene insertion efficiency in NK cells (see, e.g., Nguyen et al. 2020 Nature Biotechnology, 38:44- 49).

[0049] In one aspect, the present disclosure relates to optimized methods of gene editing in NK cell. As is described herein, the inventors tested four different avenues of gene editing including stabilization of DNA nuclease, DNA repair pathway manipulation, DNA template design, and cell recovery optimization. Surprisingly, stabilization of DNA nuclease is improved gene transfer efficiency the most. Also presented herein is composition for enhancing gene knock-in in NK cells. In one or more embodiments, this cocktail increases the efficiency of gene knock-in by up to 50%. Also presented herein is an optimized template design for gene knock-in in NK cells. In one or more embodiments, NK cells treated with the methods and compositions described herein show knock-in efficiency of up to 63%.

[0050] Some methods described herein include electroporation. Notably, cells treated with some cocktail treatments and strong electroporation pulses during gene editing demonstrate worse recovery. To address this newly identified issue, the inventors developed a cell recovery cocktail to rescue the viability after engineering. In one or more embodiments, the cell recovery cocktail improves cell viability to at most 80% viable cells.

[0051] Compositions for gene editing

[0052] In one aspect, the present disclosure relates to compositions for use during gene editing. The compositions described herein may be used during any stage of gene editing. The compositions describe herein are generally characterized by the step of a gene editing method during which they are used. In one or more embodiments, the present disclosure relates to a transfection composition that is generally present during transfection of an NK cell. In one or more embodiments, the present disclosure relates to an editing composition that is generally present with editing reagents in an NK cell. IN one or more embodiments, the present disclosure relates to a recovery composition that is present during and / or following transfection or transduction of an NK cell. However, it should be understood that the components of each composition may be additionally useful at multiple timepoints during gene editing an NK cell. In one or more embodiments, the present disclosure relates to a transfection composition. Typically, the transfection composition is applied to an NK cell while editing reagents are introduced to the cell. Any suitable method of introducing editing reagents to an NK cell may be compatible with the transfection composition. In one or more embodiments, the transfection composition is used before, during, or after transfection, transduction, or transformation of the NK cell. In one or more embodiments, transfection includes electroporation. In one or more embodiments, transformation includes chemical transformation. Typically, the transfection composition is used in combination with a non-viral gene transfer method.

[0053] In one or more embodiments, the transfection composition stabilizes the gene editing reagents before or during transfection or transduction. For example, the transfection composition may prevent degradation of the gene editing reagents, such as a guide nucleic acid or a nuclease, such as Cas9.

[0054] In one or more embodiments, a transfection composition includes poly-L-y-glutamic acid (y-PGA). In one or more embodiments, a transfection composition includes a single-stranded oligodeoxynucleotide (ssODN). The ssODN typically encodes the desired edit. Methods of providing ssODNs in gene editing compositions are known to the art.

[0055] In one or more embodiments, the transfection composition includes cell medium. In one or more embodiments, the transfection composition includes DMEM (high glucose, ThermoFisher Scientific), RPMI-1640 (ThermoFisher Scientific), IMDM (ThermoFisher Scientific), NK Xpander (ThermoFisher Scientific), or NK MACS (Miltenyi Biotec). In one or more embodiments, the transfection composition includes human AB serum, fetal bovine serum (FB)S, human platelet lysate, Immune SR (such as that provided by ThermoFisher Scientific), or CellVive Serum Substitute (BioLegend).

[0056] In another aspect, the present disclosure relates to a method of using a transfection composition. In one or more embodiments, a method includes contacting an NK cell with a transfection composition and transfecting the NK cell to introduce editing reagents. In one or more embodiments, a method of using a transfection composition results in a higher percentage of transduced NK cells as compared to a method that does not include contacting the NK cells with a transfection composition. In one or more embodiments, the present disclosure relates to an editing composition. Typically, an editing composition is intended for use with NK cells while the NK cells have been contacted with editing reagents.

[0057] In one or more embodiments, an editing composition increases the rate of editing in NK cells.

[0058] In one or more embodiments, the editing composition includes an HD AC inhibitor. In one or more of these embodiments, the HD AC inhibitor includes Trichostatin A, dacinostat, CUDC-101, AR-42, M344, NKL 22, splitomicin, sodium butyrate, curcumin, scriptaid, 4- phenylbutyric acid, GSK3117391, BML-210, sulforaphane, raddeanin A, sinapinic acid, or a combination thereof.

[0059] In one or more embodiments, the editing composition includes a DNA-PK inhibitor. In one or more embodiments, the DNA-PK inhibitor includes AZD-7648, M3814, LY294002, Wortmannin, NU7441, PI- 103, T0070907, Torin 2, NU7026, PIK-75 HC1, KU-0060648, CC- 115, Samotolisib, PP121, SF2523, YU238259, LTURM34, VX-984, BAY-8400, AMA-37, XRD-0394, NU5455, or a combination thereof. In one or more of these embodiments, the DNA- PK inhibitor includes AZD-7648, M3814, or a combination thereof.

[0060] In another aspect, the present disclosure relates to a method of using an editing composition. In one or more embodiments, a method includes contacting an NK cell with an editing composition and gene editing reagents. In one or more embodiments, a method of using an editing composition results in a higher percentage of edited NK cells as compared to a method that does not include contacting the NK cells with an editing composition.

[0061] In one or more embodiments, a composition is a recovery composition. Typically, a recovery composition is intended for use with NK cells after the NK cells have been contacted with editing reagents. For example, a recovery composition may be applied to NK cells following transfection with a polynucleotide. Additionally or alternatively, NK cells may be contacted with the recovery composition before the cells are contacted with editing reagents so that the recovery composition is present simultaneously with the editing reagents.

[0062] In one or more embodiments, a recovery composition increases NK cell recovery following contacting the cell with editing reagents. For example, electroporation often decreases NK cell fitness. A significant portion of NK cells often die following electroporation, resulting in production of fewer edited NK cells. However, electroporation may be desirable because of the high percentage of cells that receive electroporated reagents. To increase the efficiency of NK cell editing using electroporation, some recovery compositions described herein decrease NK cell mortality following electroporation.

[0063] In one or more embodiments, a recovery composition increases NK cell viability following contact with editing reagents. NK cells treated with a recovery composition may exhibit increased cell viability relative to NK cells contacted with editing reagents in the absence of the recovery composition.

[0064] In one or more embodiments, a recovery composition includes DNase, benzonase, or pulmozyme. In one or more embodiments, DNase includes DNasel, turbo DNase, DNase I-XT, DENARASE, or a combination thereof.

[0065] In one or more embodiments, a recovery composition includes an antioxidant. In one or more embodiments, the antioxidant includes N-acetylcysteine, L-glutathione, necrostatin-1, EVP4593, GSK2982772, GSK481, LY364947, OD36, PK68, RIPK1-IN-7, necrostatin-34, tuxobertinib, GSK3145095, or a combination thereof. In one or more of these embodiments, the antioxidant includes N-acetylcysteine, L-glutathione, or a combination thereof.

[0066] In one or more embodiments, the recovery composition includes an apoptosis inhibitor. In one or more of these embodiments, the apoptosis inhibitor includes emricasan, VX-765, Ac- DEVD-CHO, Q-VD-Oph, Z-VAD-FMK, Z-IETD-FMK, Z-VAD(OH)-FMK, Z-DEVD-FMK, or a combination thereof. In one or more embodiments, the recovery composition includes a trans- ISRIB inhibitor, a PERK inhibitor, or a combination thereof. In one or more of these embodiments, GSK2606414, GSK2656157, salubrinal, Sal003, azoramide, BTdCPU, CCT020312, AMG PERK 44, MK-28. In one or more embodiments, the recovery composition includes emricasan, a trans-ISRIB inhibitor, or a combination thereof.

[0067] In another aspect, the present disclosure relates to a method of using a recovery composition. In one or more embodiments, a method includes contacting an NK cell with a recovery composition and gene editing reagents. In one or more embodiments, a method of using a recovery composition results in a higher percentage of viable NK cells than a comparable method that does not include a recovery composition.

[0068] Methods of gene editing In one aspect, the present disclosure relates to methods of gene editing. Generally, a method includes providing an NK cell and transfecting the NK cell with gene editing reagents.

[0069] In one or more embodiments, a method includes providing an NK cell. In one or more embodiments, a purified source of NK cells may be purchased. In one or more embodiments, NK cells may be isolated from peripheral blood mononuclear cells (PBMCs). The NK cell may be from a mammal, such as a mouse, a non-human primate, or a human.

[0070] In one or more embodiments, the NK cells are feeder-expanded NK cells. Often, non- feeder expanded NK cells are used for gene editing methods. However, feeder-expanded NK cells may be available in larger numbers, increasing the number of edited NK cells that are produced. For example, feeder cells having certain desirable membrane markers may yield NK cells that exhibit advantageous characteristics. In one or more embodiments, NK cells are expanded using K562 feeder cells. The K562 feeder cells may have membrane bound IL-21 (mbIL21) and 4-1BBL. In one or more embodiments, the K562 feeder cells increase NK cell telomere length. In one or more embodiments, the K562 feeder cells enhance NK cell proliferation, subsequently increasing the frequency of the cell cycle. Cells exhibiting a more frequent cell cycle may exhibit increased rates of gene knock-in.

[0071] In one or more embodiments, the method includes contacting the NK cell with a transfection composition before and / or while transfecting the NK cell. The transfection composition may include any of the transfection compositions described herein. In one or more embodiments, the transfection composition includes v-PGA and a repair template, such as an ssODN. Typically, transfecting the NK cell includes non-virally transferring gene editing reagents to the NK cell. In one or more embodiments, transfecting the NK cell comprises electroporating the NK cell. The electroporation cycle used may be, for example, DK-125, DN- 100, DP-100, EH-100, EH-111, EH-115, EH-140, EN-138, EN-151, EO-115, EO-148, EO-151, ER- 100, FA- 100, FB-151, or FI-115 on the Lonza 4D Nucleofector, or a similar electroporation cycle on another machine. In one or more embodiments, the transfection composition increases the rate of NK cell transfection.

[0072] In one or more embodiments, the method further comprises contacting the NK cell with an editing composition. Typically, the NK cell is contacted with the editing composition following transfection of the editing reagents. The editing composition may include any editing compositions described herein. In one or more embodiments, the editing composition includes a DNA-PK inhibitor and an HD AC inhibitor. In one or more embodiments, the DNA-PK inhibitor includes M3814, AZD-7648, or a combination thereof. In one or more embodiments, the HDAC inhibitor includes Trichostatin A. The editing composition may increase the rate of editing the NK cell.

[0073] In one or more embodiments, the gene editing reagents include a nucleotide-guided nuclease, such as Cas9. The gene editing reagents may include any CRISPR-associated (Cas) protein, such as LbCasl2A, AsCasl2a, AsCasl2a Ultra, CasX, AsCasl2fl, NanoCas, S. aureus Cas9, S. pyogenes Cas9, or Cpf 1. In one or more embodiments, the gene editing reagents include a guide nucleic acid, such as a CRISPR guide RNA (crRNA) or a single guide RNA(sgRNA). In one or more embodiments, the gene editing reagents include a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), a non-nucleotide guided nuclease, or a combination thereof.

[0074] In one or more embodiments, the method includes inserting a gene at a known locus within the genome of the NK cell. In other words, the method includes targeted gene editing in an NK cell. The gene may be inserted at a known locus using homology arms that are complementary to either side of the locus.

[0075] In one or more embodiments, the method includes contacting the cells with a repair template. The repair template may be a homology-directed repair (HDR) template. In one or more embodiments, the repair template may include a truncated Cas9 targeting sequence (tCTS). In one or more embodiments, the HDR template may be single-stranded DNA(ssDNA) or double-stranded DNA (dsDNA), such as plasmid DNA. As is described herein, plasmid DNA may advantageously adapt to high-scale cell engineering, such as production of CAR cells for therapeutic purposes. However, plasmid DNA is typically thought to be more cytotoxic than some other forms of DNA. Thus, linear dsDNA or ssDNA may be used as an HDR template in some embodiments.

[0076] The repair template may include any of the nucleic acid sequences described herein. In one or more embodiments, the repair template includes a nucleic acid sequence encoding a reporter, such as a fluorescent protein. In one or more embodiments, the repair template includes a nucleic acid sequence encoding a CAR. In one or more embodiments, the repair template includes a nucleic acid sequence encoding an anti-CD19 CAR, an anti-CD22 CAR, or both an anti-CD19 CAR and an anti-CD22 CAR. In one or more embodiments, the repair template includes a nucleic acid sequence encoding an anti-PSMA CAR. In one or more embodiments, the repair template includes a nucleic acid sequence encoding an anti-B7H3 CAR. Other suitable CAR targets for use with the cells and methods of the present disclosure include CD33, CLEC12A, mesothelin, PSMA, CD70, PDL1, CSPG4, EGFR, CD 133, and HER2.

[0077] In one or more embodiments, the repair template includes a nucleic acid sequence encoding an scFv, such as an anti-PSMAl scFv. In one or more embodiments, the repair template includes a nucleic acid sequence encoding IL-15, IL-12, IL-18, IL-21, or a combination thereof. In one or more embodiments, the repair template includes a nucleic acid sequence encoding a trispecific killer engager (TriKE). In one or more embodiments, the repair template includes a nucleic acid sequence encoding a bispecific killer engager (BiKE). In one or more embodiments, the repair template includes a nucleic acid sequence encoding a bispecific T cell engager (BiTE).

[0078] In one or more embodiments, the method includes contacting the NK cell with an exogenous nucleic acid. As is described herein, contacting cells, particularly NK cells, with exogenous DNA such as plasmid DNA may result in NK cell death. The compositions and methods described herein advantageously reduce NK cell death following contacting NK cells with exogenous DNA. As it is used herein, an “exogenous” nucleic acid is a nucleic acid not natively present in a cell. An exogenous nucleic acid may include a sequence natively present in a cell, but presented in a different context, typically in a form other than genomic DNA. An exogenous nucleic acid may be a DNA plasmid.

[0079] In one or more embodiments, the method includes incubating the NK cell following editing. In one or more embodiments, the method includes expanding the edited NK cells.

[0080] In one or more embodiments, the method further includes contacting the NK cell with a recovery composition. The NK cell is typically contacted with the recovery composition following transfection of the NK cell. The recovery composition may increase the percentage of viable NK cells following editing.

[0081] The recovery composition may include any recovery composition described herein. In one or more embodiments, the recovery composition includes DNasel. In one or more embodiments, the recovery composition includes an antioxidant, such as N-actetyl cysteine, L- glutathione, or a combination thereof. In one or more embodiments, the recovery composition includes Emricasan, trans-ISRIB, or a combination thereof. To quantify the rates of transfection, editing, and viability of NK cells, it may be advantageous to include more than one NK cell in a method described herein. In one or more embodiments, at least 10, at least 100, at least 1,000, or at least 5,000 NK cells are treated.

[0082] In one or more embodiments, a method yields at least 50% transfection efficiency of NK cells. In one or more embodiments, a method including a transfection composition results in a higher transfection efficiency as compared to a similar method not including a recovery composition.

[0083] In one or more embodiments, a method yields at least 50% editing efficiency of NK cells. In one or more embodiments, a method including an editing composition results in a higher editing efficiency as compared to a similar method not including an editing composition.

[0084] In one or more embodiments, a method yields at least 50% viability of NK cells following editing. In one or more embodiments, a method including a recovery composition results in a higher cell viability following editing as compared to a similar method not including a recovery composition.

[0085] In one or more embodiments, a method includes expanding the NK cells following gene editing.

[0086] In another aspect, the present disclosure relates to a cell therapy product including one or more edited NK cells described herein. For example, an NK cell may be edited to incorporate a chimeric antigen receptor (CAR) construct, producing CAR-NK cells. In one or more embodiments, a CAR may target a tumor-associated antigen, such as CD 19.

[0087] Engineered cells

[0088] In another aspect, the present disclosure relates to engineered NK cells. An engineered NK cell of the present disclosure may be prepared using the methods described herein. Typically, the engineered NK cell includes a genetic insertion, such as a gene knock-in. In one or more embodiments, the engineered cell is a human cell.

[0089] The location of the gene knock-in may be chose to provide a particular pattern of gene expression. In one or more embodiments, an engineered cell includes a genetic insertion in or around the PFKFB4 gene. While described in the context of the human PFKFB4 gene, other analogous genes in other organisms are contemplated. In one or more embodiments, the engineered cell may include a genetic insertion at a locus whose expression is environment- dependent. For example, the PFKFB4 gene is expressed during hypoxia. Thus, the engineered cell may include a genetic insertion at a locus whose expression is dependent upon hypoxic conditions.

[0090] The location of a gene knock-in may also be chose for a particular level of gene expression. In one or more embodies, the engineered cell includes a genetic insertion at the locus of the TOMM70, CTCF, ACTG1, MAP4, EEF1G, YWHAQ, HNRNPA1, ENO J, or GAPDH gene.

[0091] The engineered cell may include any useful gene knock-in. In one or more embodiments, the engineered cell includes a reporter, such as a nucleic acid sequence encoding a fluorescent protein. In one or more embodiments, the engineered cell includes a therapeutic construct, such as nucleic acid sequence encoding a CAR. In one or more embodiments, the engineered cell includes a nucleic acid sequence encoding an anti-CD19 CAR, an anti-CD22 CAR, or both an anti-CD19 CAR and an anti-CD22 CAR. In one or more embodiments, the engineered cell includes a nucleic acid sequence encoding an anti-PSMA CAR. In one or more embodiments, the engineered cell includes a nucleic acid sequence encoding an anti-B7H3 CAR. Other suitable CAR targets for use with the cells and methods of the present disclosure include CD33, CLEC12A, mesothelin, PSMA, CD70, PDL1, CSPG4, EGFR, CD 133, and HER2.

[0092] In one or more embodiments, the engineered cell includes a nucleic acid sequence encoding an scFv, such as an anti-PSMAl scFv. In one or more embodiments, an engineered cell includes a nucleic acid sequence encoding IL-15, IL-12, IL-18, IL-21, or a combination thereof. In one or more embodiments, the engineered cell includes a nucleic acid sequence encoding a trispecific killer engager (TriKE). In one or more embodiments, the engineered cell includes a nucleic acid sequence encoding a bispecific killer engager (BiKE). In one or more embodiments, the engineered cell includes a nucleic acid sequence encoding a bispecific T cell engager (BiTE).

[0093] In the preceding description and following claims, the term “and / or” means one or all of the listed elements or a combination of any two or more of the listed elements; unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one; and the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0094] As used herein, “have,” “has,” “having,” “include,” “includes,” “including,” “comprise,” “comprises,” “comprising” or the like are used in their open-ended inclusive sense, and generally mean “include, but not limited to,” “includes, but not limited to,” or “including, but not limited to.” Further, wherever embodiments are described herein with the language “have,” “has,” “having,” “include,” “includes,” “including,” “comprise,” “comprises,” “comprising” and the like, otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of’ are also provided. The term “consisting of’ means including, and limited to, that which follows the phrase “consisting of.” That is, “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. The term “consisting essentially of’ indicates that any elements listed after the phrase are included, and that other elements than those listed may be included provided that those elements do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements.

[0095] As used herein, the word “exemplary” means to serve as an illustrative example and should not be construed as preferred or advantageous over other embodiments.

[0096] As used herein, the terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.

[0097] In the preceding description, particular embodiments may be described in isolation for clarity. Reference throughout this specification to “one embodiment,” “an embodiment,” “certain embodiments,” “one or more embodiments,” or “some embodiments,” etc., means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments. Thus, features described in the context of one embodiment may be combined with features described in the context of a different embodiment except where the features are necessarily mutually exclusive.

[0098] In several places throughout the above description, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

[0099] For any method disclosed herein that includes discrete steps, the steps may be performed in any feasible order. And, as appropriate, any combination of two or more steps may be performed simultaneously.

[0100] EXEMPLARY EMBODIMENTS

[0101] Embodiment l is a method of treating an NK cell to induce a gene edit, the method including: contacting the NK cell with a transfection composition including: poly-L-glutamic acid (y-PGA), a single-stranded oligo deoxynucleotide (ssODN) enhancer, or a combination thereof; contacting the NK cell with an editing composition including: a DNA-PK inhibitor, and an HD AC inhibitor; contacting the NK cell with a repair template; electroporating the NK cell; and contacting the NK cell with a recovery composition including DNase I.

[0102] Embodiment 2 is the method of embodiment 1, wherein the NK cell includes a feeder- expanded NK cell.

[0103] Embodiment 3 is the method of embodiment 1, wherein the NK cell has been previously cryopreserved.

[0104] Embodiment 4 is the method of embodiment 1, further including contacting the cell with editing reagents.

[0105] Embodiment 5 is the method of embodiment 4, wherein the editing reagents include Cas9 and a guide RNA, such as a single guide RNA (sgRNA).

[0106] Embodiment 6 is the method of any preceding embodiment, wherein the recovery composition further includes N-acetyl-cysteine, L-glutathione, or a combination thereof.

[0107] Embodiment 7 is the method of any preceding embodiment, wherein the recovery composition further includes emricasan, trans-ISRIB, or a combination thereof. Embodiment 8 is the method of any preceding embodiment, wherein the DNA-PK inhibitor includes M3814 or AZD-7648.

[0108] Embodiment 9 is the method of any preceding embodiment, wherein the HDAC inhibitor includes Trichostatin A.

[0109] Embodiment 10 is the method of any preceding embodiment, wherein the repair template includes plasmid DNA, single- stranded DNA, or double-stranded DNA.

[0110] Embodiment 11 is the method of embodiment 10, wherein the repair template includes a tCTS.

[0111] Embodiment 12 is the method of any preceding embodiment, wherein at least 100 NK cells are treated.

[0112] Embodiment 13 is the method of embodiment 12, wherein at least 50% of the treated NK cells include the gene edit following treatment.

[0113] Embodiment 14 is a method of increasing the efficiency of gene editing in an NK cell including contacting the NK cell with poly-L-glutamic acid (PGA) and gene editing reagents.

[0114] Embodiment 15 is an edited NK cell prepared using the method of any preceding embodiment.

[0115] Embodiment 16 is the edited NK cell of embodiment 15, wherein the NK cell is derived from a human NK cell.

[0116] Embodiment 17 is the edited NK cell of embodiment 16, wherein the NK cell includes an inserted DNA sequence at the human CISH locus or the human CD96 locus.

[0117] Embodiment 18 is the edited NK cell of embodiment 17, wherein the NK cell includes an inserted DNA sequence at one or more of the TOMM70, CTCF, ACTG1, MAP4, EEF1G, YWHAQ, HNRNPA1, ENO I, or GAPDHloci.

[0118] Embodiment 19 is an editing composition for use in gene editing an NK cell including Trichostatin A and a DNA-PK inhibitor including AZD-7648, M3814, or a combination thereof.

[0119] Embodiment 20 is a method of using the editing composition of embodiment 19 including contacting an NK cell with gene editing reagents and the editing composition.

[0120] Embodiment 21 is a recovery composition for use in gene editing an NK cell including DNasel; an apoptosis inhibitor including emricasan, and / or trans-ISRIB inhibitor; and an antioxidant including N-acetylcysteine, L-glutathione, or a combination thereof. Embodiment 22 is a method of using the recovery composition of embodiment 21 including contacting an NK cell with gene editing reagents and the recovery composition.

[0121] Embodiment 23 is a cell therapy reagent including an edited NK cell produced using the method of any preceding embodiment.

[0122] Embodiment 24 is the cell therapy reagent of any preceding embodiment, wherein the edited NK cell includes a chimeric antigen receptor (CAR)-NK cell.

[0123] Embodiment 25 is the cell therapy reagent of any preceding embodiment, wherein the CAR targets CD 19, CD22, or both CD 19 and CD22.

[0124] Embodiment 26 is a method of preparing an engineered NK cell including a fluorescent reporter at an endogenous locus, the method including: contacting an NK cell with a transfection composition including: poly-L-glutamic acid (γ-PGA), a single-stranded oligo deoxynucleotide (ssODN) enhancer, or a combination thereof; contacting the NK cell with an editing composition including: a DNA-PK inhibitor, and an HD AC inhibitor; contacting the NK cell with a repair template including a nucleic acid sequence encoding a fluorescent reporter; electroporating the NK cell; and contacting the NK cell with a recovery composition including DNase I.

[0125] Embodiment 27 is the method of any preceding embodiment, wherein the endogenous locus includes the TOMM70, CTCF, ACTG1, MAP4, EEF1G, YWHAQ, HNRNPA1, EN01, or GAPDH locus.

[0126] Embodiment 28 is the method of any preceding embodiment, wherein the endogenous locus includes a hypoxia-sensitive locus, such as the PFKB4 locus.

[0127] Embodiment 29 is the method of any preceding embodiment, wherein the repair template includes a nucleic acid sequence having at least 80% identity to a region of the endogenous locus.

[0128] Embodiment 30 is a method of treating an NK cell to introduce an exogenous nucleic acid, the method including: contacting the NK cell with a transfection composition including: poly-L-glutamic acid (y-PGA), a single-stranded oligo deoxynucleotide (ssODN) enhancer, or a combination thereof; contacting the NK cell with an editing composition including: a DNA-PK inhibitor, and an HD AC inhibitor; contacting the NK cell with an exogenous nucleic acid; electroporating the NK cell; and contacting the NK cell with a recovery composition including DNase I.

[0129] Embodiment 31 is the method of any preceding embodiment, wherein the exogenous nucleic acid includes DNA.

[0130] Embodiment 32 is the method of any preceding embodiment, wherein the exogenous nucleic acid includes a DNA plasmid.

[0131] EXAMPLES

[0132] The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

[0133] Example 1- NK cell editing enhancement by small molecules

[0134] NK cells were isolated and treated with editing reagents to knock in mNeonGreen at the CD96 locus. Different small molecule reagents were tested to determine whether they increased editing rates.

[0135] Isolation, culture, and expansion of human primary NK cells

[0136] Human PBMCs were isolated from healthy donor fresh blood using Ficoll (GE Healthcare) density centrifugation. Human primary NK cells were isolated from PBMC using Human NK Cell Enrichment Kit (STEMCELL Technologies) according to manufacturer instructions. Isolated NK cells were then expanded by 100 Gy-irradiated mbIL21-41BBL K562 feeder cells at 1 :2 NK Feeder ratio in BO medium (DMEM / F-12 (2: 1) supplemented with 10% human AB serum, 20 μM 2-mercaptoethanol, 50 μM ethanolamine, 20 μg / mL Ascorbic acid, 5 ng / mL Sodium Selenite, and 100 U / mL Penicillin / Streptomycin) with 50 lU / mL recombinant human IL-2. The medium doubling and fresh IL-2 supplement was performed on Day 3. Plasmid cloning

[0137] All plasmid cloning was conducted using isothermal reaction by NEBuilder HiFi DNA Assembly (New England Biolabs). All gene fragments were chemically synthesized (Twist Biosciences). The mNeonGreen protein sequence was derived from Shaner et al, 2013 (PMID: 23524392). The CAR19 protein sequence was derived from Zah et al, 2016 (PMID: 27059623). All protein sequence was codon-optimized by GENSMART Codon Optimization Tool (GenScript). The assembled plasmids were transformed into NEB Stable competent cells (New England Biolabs). All plasmids were sequence-verified by nanopore-based sequencing (Azenta Life Science). The pCD96-mNeonGreen contains P2A-mNeonGreen gene linked with 3'-UTR, BGH poly A, and flanked by 1000 bp homology arms on both 3' and 5' end. The pCD96-CAR19 contains P2A-CAR19 gene linked with 3'-UTR, BGH poly A, and flanked by 1000 bp homology arms on both 3' and 5' end.

[0138] DNase I, small molecule , and poly-cationic polymer preparation

[0139] The M3814, AZD-7648 and Trichostain A were purchased from Selleckchem and resuspend in DMSO at 50 mM, 20 mM, 100 mM. The L-glutathione was purchased from Merck and resuspended in PBS at 150 mM. The Emricasan and trans-ISRIB were purchased from Cayman Chemical and resuspended in DMSO at 10 mM and 5 mM. The Poly-L-glutamic acid sodium salt (PGA, molecular weightl 5,000-50,000) was purchased from Merck and resuspended in nuclease-free water at 100 mg / mL. DNase I was purchased from Roche and resuspended in water at 10 mg / mL. The ssODN enhancer was purchased from Integrated DNA Technologies and resuspended in TE buffer at 100 μM. All small molecules were stored at -20 °C until electroporation, except for PGA and DNase I, which were stored -80 °C.

[0140] Electroporation

[0141] All electroporation was conducted by the following protocol unless stated otherwise. For Cas9 RNP assembly, 1 μL of 120 μM sgRNA was pre-mixed with 1 μL of 100 mg / mL PGA. One and half μL of 40 μM Cas9 was then slowly mixed into sgRNA:PGA mixture. The Cas9 RNP mixture was then incubated at 37 °C for 15 minutes. After incubation, the HDR template was added into the RNP mixture and incubated at room temperature for at least five minutes. For electroporation, 1 e6 expanded NK cells were centrifuges at 120x for five minutes. The cell pellet was then resuspended in 19 μL P3 Nucleofection buffer (Lonza) and mixed with the RNP / template. The cell mixture was then transfer into a 16-well nucleofection strip (Lonza) and the nucleofection was conducted using EH-115 program. Immediately after nucleofection, 80 μL of pre- warmed BO medium was added and the cells were allowed to recover at 37 °C for 20 minutes. After 20 minutes, the cell suspension was transfer into a 12-well recovery plate pre- filled with BO medium supplemented with 50 lU / mL IL-2, 1 μM M3814, 0.05 μM Trichostatin A, 2.5 μM Emricasan, 0.5 μM trans-ISRIB, 7.5 mM L-glutathione, and 0.01 mg / mL DNase I. The cells were then incubated at 37 °C for 18 to 24 hours. Next day, the cells were centrifuged at 400xg for five minutes and medium was refreshed by B0 medium supplemented with 50 lU / mL IL-2. The cells were then cultured for another 48 hours before flow cytometry analysis.

[0142] Flow cytometry

[0143] For flow cytometry analysis, NK cells were harvested by centrifuge at 400x for five minutes and transferred into a U-bottom 96-well plate followed by PBS wash. The washed NK cells were then stained by Zombie Violet live / dead reagent (BioLegend) as per manufacture’s instruction. After staining, flow buffer (PBS supplemented with 0.5% human serum and 0.05 mM EDTA) was added into the staining to stop the reaction. The cells were then pelleted by centrifuge at 400xg for three minutes and resuspended in 150 μL to 300 μL of flow cytometry buffer. One to ten μL of COUNTBRIGHT Absolute Counting Beads (ThermoFisher Scientific) was added into each tube for cell recovery monitor. relative cell recovery (fold) = (live cell count of control / bead count of control) / (live cell count of sample / bead count of sample)

[0144] Absolute cell recovery (cell numbers) = (Live cell count x % mNeonGreen+) x (1x 104 / beads count).

[0145] NK cells were treated with the HDR template and Cas9 RNP separately to confirm that both were required for successful editing. Only NK cells treated with both the HDR template and Cas9 RNP exhibited mNeonGreen expression as measured by flow cytometry (FIG. 1). NK cells treated with PGA or the ssODN enhancer were compared to NK cells treated without any enhancer. Both PGA and the ssODN enhancer increased the percent of cells expressing mNeonGreen and the percent of cells exhibiting CD96 knockout (FIG. 2). Next, the effects of M3814 and AZD-7648 were tested. M3814 and AZD-7648 are both DNA-PK inhibitors. Both were found to increase knock-in efficiency in a dose-dependent manner (FIG. 3, 4). Both M3814 and AZD-7648 decreased cell recovery, but the overall rate of editing still increased (FIG. 3, 4). Trichostatin A was tested for its effect on knock in efficiency and cell recovery. Trichostatin A was found to increase knock-in efficiency in a dose-dependent manner but also to decrease cell recovery (FIG. 5). Finally, combinations of M3814, AZD-7648, and Trichostatin A were tested. The combination of Trichostatin A and M3814 was found to increase knock-in efficiency from 12.6% to 50.8% (FIG. 6).

[0146] Next, emricasan and trans-ISRIB inhibitor were tested for their effect on cell recovery following knock-in. Emricasan and trans-ISRIB inhibitor both enhanced cell recovery in a dose- dependent manner (FIG. 7). L-glutathione and N-acetyl-L-cysteine were also tested for their effect on cell recovery following knock-in. Both L-glutathione and N-acetyl-cysteine improved the cell recovery in a dose-dependent manner (FIG. 8). The synergistic effects of emricasan, trans-ISRIB, and L-glutathione following knock-in were tested, and the combination of emricasan, trans-ISRIB, and L-glutathione increased the robustness of cell recovery improvement around 3.7-fold (FIG. 9).

[0147] Next, DNase I was added to cells following electroporation to determine whether it would improve editing efficiency and / or cell recovery. Adding DNasel after electroporation enhanced the cell recovery by 50% without sacrificing the knock-in efficiency (FIG. 10).

[0148] From this Experiment, the combination of Emricasan, trans-ISRIB, and L-glutathione or addition of DNase I was found to optimally improve cell recovery. In addition, the combination of Trichostatin A and M3814 was found to increase knock-in efficiency. In sum, small molecule and DNasel enhancers of NK cell editing were identified.

[0149] Example 2- NK cell editing enhancement using different repair template formats

[0150] NK cells were isolated and treated with editing reagents to knock in mNeonGreen at the CD96 locus. Different donor formats were tested to determine whether they increased editing rates. NK cells and plasmids were prepared as described in Example 1. Double-strand HDR template synthesis

[0151] Double-strand HDR templates (dsHDRT) were synthesized by PCR-amplification using KAPA HiFi HOTSTART PCR kit (Roche). The purity is confirmed by 1% TAE agarose gel analysis. The PCR products were purified by QIAquick PCR Purification Kit (Qiagen) and eluted in nuclease-free distilled water. The purified dsHDRT was then concentrated by ethanol precipitation and formulated in TE buffer (10 mM Tris, 0.1 mM EDTA, pH 7.5) at 1 μg / μL to 2 μg / μL. The dsHDRT was stored at -20 °C until electroporation.

[0152] Single-strand HDR template synthesis

[0153] The single-strand HDR templates (ssHDRT) were synthesized using Guide-it Long ssDNA Production System v2 (Takara) according to manufacturer’s instructions. First, the dsDNA precursors were prepared by PCR-amplification with a phosphorylated reverse primer. The PCR products were purified using NucleoSpin Gel & PCR Clean-up Mini kit (Macherey- Nagel). Second, 1 μg of PCR products were used for strandase A / B digestion. The digested products were confirmed by 1% TAE agarose gel analysis and purified by NucleoSpin Gel & PCR Clean-up Mini kit. Third, the single-strand DNA was then concentrated by ethanol precipitation and formulated in TE buffer at 1 μg / μL to 2 μg / μL. For tCTS hybridization, the single-strand DNA was formulated in duplex buffer (30 mM HEPES, pH 7.5, 100 mM potassium acetate). The ssHDRT was stored at -20 °C until electroporation.

[0154] Single-strand tCTS HDR template hybridization

[0155] The annealing oligos were resuspended in duplex buffer at 100 μM. The purified single- strand DNA (ssDNA) containing tCTS modification sites were mixed with annealing oligos at 1:5 ssDNA: oligo ratio. The mixture was then heated up to 95 °C for three minutes, followed by cooling at 0.1 °C / sec until room temperature in PCR machine. The tCTS ssHDRT was stored at - 20 °C until electroporation.

[0156] Minicircle plasmid HDR template production

[0157] The parental minicircle plasmid was subcloned from pTubb3-MC (Addgene: 87112) by swapping the original insertion with the CD96-mNeonGreen template containing 300 bp homology arm amplified from pCD96-mNeon Green. The parental plasmid was transformed into ZYCY10P3S2T strain and sequence-verified by nanopore-based sequencing. The transformed ZYCY10P3S2T was then stored as glycerol stock. Upon production, the transformed ZYCY10P3S2T was inoculated into 100 mL of Terrific-Broth (ThermoFisher Scientific) and incubated at 30 °C for 18 hours. After 18 hours, the volume original culture was doubled by 100 mL TB supplemented with 0.02 M NaOH and 0.02% L-arabinose. The final concentration of NaOH and L-arabinose were 0.01 M and 0.01%. The minicircle digestion was then induced at 32 °C for 5 hours. After induction, the minicircle plasmid was extracted and purified by ZymoPURE II Plasmid Maxiprep Kit (Zymo Research) as per manufacturer’s instructions. The final product was concentrated by ethanol precipitation and formulated in in TE buffer at 2 μg / μL. The mini circle plasmid was stored at -20 °C until electroporation. pUC19, pUCmu, and nanoplasmid vector-based HDR template production

[0158] For generation of pUC19 and pUCmu vector-based HDR template, the CD96- mNeonGreen containing 300 bp homology arm was subcloned into empty pUC19 and pUCmu as described above. For plasmid production, the glycerol stock of identified bacterial clone was inoculated into 100 mL of 2X YT medium and incubate at 37 °C for 18 hours. After 18 hours, the plasmids were extracted and purified by ZymoPURE II Plasmid Maxiprep Kit (Zymo Research) as per manufacturer’s instructions. The final product was concentrated by ethanol precipitation and formulated in in TE buffer at 2 μg / μL. The plasmids were stored at -20 °C until electroporation. The nanoplasmid was subcloned and produced by Aldevron. The final product of nanoplasmid was formulated in TE buffer at 2 μg / μL and stored at -20 °C until electroporation.

[0159] When compared to unmodified ssDNA and plasmids HDR knock-in templates, unmodified dsDNA provided optimal cell recovery and knock-in efficiency (FIG. 11). However, an ssDNA template modified with truncated Cas9 Targeting Sequence (tCTS) provided the highest knock-in efficiency when compared to other HDR knock-in templates (FIG. 12). The tCTS modified ssDNA HDR knock-in template was tested in combination with the small molecule effectors identified in Example 1, which showed that the combination of PGA, DNase I, HDR cocktail Emricasan, trans-ISRIB, and L-glutathione recovery cocktail, and tCTS ssDNA HDR knock-in template improved the knock-in efficiency by 7-fold and relative mNeonGreen+ cell number by 8-fold relative to baseline conditions (FIG. 13).

[0160] Example 3- NK cell editing using recovery and HDR cocktails and different electroporation conditions

[0161] NK cells were isolated and treated with editing reagents to knock in mNeonGreen at the CD96 locus using the conditions identified in Example 2. Different electroporation conditions and supplements were tested.

[0162] Medium and supplement comparison

[0163] NK cell expansion was conducted using the aforementioned method with different media and supplement conditions. To test the suitability of different media for NK cell expansion, DMEM (high glucose, ThermoFisher Scientific), RPMI-1640 (ThermoFisher Scientific), IMDM (ThermoFisher Scientific), NK Xpander (ThermoFisher Scientific), and NK MACS (Miltenyi Biotec) were supplemented with 10% human AB serum (Access Biologies) and compared in NK cell culture. To test the suitability of different supplements for NK cell expansion, 10% human AB serum, 10% FBS, 5% human platelet lysate (EliteCell Biomedical), 5% Immune SR (ThermoFisher Scientific), and 5% CellVive Serum Substitute (BioLegend) were mixed with IMDM and tested in culture.

[0164] Cry opreservation of NK cells

[0165] NK cells were purified by using the aforementioned method. Immediately after enrichment, NK cells were centrifuged at 750 xg for 5 min and resuspended in pre-cooled FBS + 10% DMSO or CryStor CS10 (StemCell Technologies) at the cell density of 5x 106cells / mL. NK cell suspension was aliquoted into 2 mL cryogenic vials and allowed to cool down to -80 °C overnight in a CoolCell container (Corning). The next day, cryopreserved NK cell vials were transferred to a liquid nitrogen tank for long-term storage.

[0166] Electroporation pulse screening

[0167] Electroporation was conducted by the aforementioned method with different pulse programs. The following pulse programs on the Lonza 4D Nucleofector were used for screening, DK-125, DN-100, DP-100, EH-100, EH-11 1, EH-115, EH-140, EN-138, EN-151, EO-115, EO- 148, EO-151, ER-100, FA-100, FB-151, and FI-115.

[0168] The effect of HDR enhancer (HDR), Recovery cocktail (Recovery), or a combination of both (optimized) was independent of pulse conditions (FIG. 18 A, 18C). This indicates that the HDR and Recovery cocktails could be used to improve editing and viability for a variety of electroporation conditions, not just those tested. Further, electroporated cells exhibited increased viability and editing efficiency when treated with dsDNA HDR templates than with plasmid HDR templates (FIG. 18B).

[0169] Regarding cry opreservation techniques, cells preserved in either FBS + 10% DMSO or in the CryoStar CS10 exhibited improved viability following electroporation and after expansion relative to freshly isolated cells (FIG. 19). When different media were screened, rates of knock- in were comparable, and the highest level of viability was observed in cells treated with DMEM or IMDM, optionally with NK MACS (FIG. 20). Notably, cells were able to survive in serum- free conditions to rates comparable to serum-containing conditions (CellVive SS).

[0170] Example 4- CAR NK cell editing and positive feedback loop

[0171] First, NK cells were edited to incorporate an anti-CD19 CAR construct at the CD96 exon 2 locus. The incorporation strategy was similar to that described in Example 1. Both donor templates resulted in robust incorporation. In addition, recovery cocktails were tested to mitigate plasmid toxicity. Plasmids are sometimes preferable for manufacturing of CAR-NK cells, so identifying improved conditions for plasmid-based editing was desirable.

[0172] CAR19 Validation

[0173] For CAR19 validation, the c-myc tag staining was preformed after live / dead staining. The cell pellet was resuspended in 100 μL of 1 :50 diluted ALEXA FLUOR 647 anti-c-myc tag, clone 9B11 (Cell Signaling Technology) and incubated at 4 °C in the dark for 30 minutes. After staining, the cells were washed twice by flow cytometry buffer before analysis. All the analysis was performed using FlowJo v10.10. The cell recovery data calculation was performed using the equations described in Example 1.

[0174] Recombinant protein staining CAR-engineered NK cells were stained with different recombinant proteins. All recombinant proteins, including CD19, CD22, B7H3, CD33, CLEC12A, mesothelin, and PSMA were derived from ACRO Biosystems. All staining procedures were conducted as per the manufacturer’s suggestions.

[0175] First, an anti-CD19 CAR was inserted at the CD96 exon 2 locus. Insertion of up to 23.2% was observed (FIG. 14). Next, an anti CD22 / 19 CAR was inserted at the CISH exon 2 locus. The CAR insertion construct was designed so that expression of the CAR construct would initiate a positive feedback loop and result in more CAR expression (FIG. 15). Both the anti-CD19 and the anti-CD22 CARs were detected in edited cells. CD107a, IFNy, and TNFa were analyzed to determine the ability of the CAR to activate the edited cells when incubated with tumor targets. CD107a was used to evaluate NK cell cytolytic degranulation, while IFNy and TNFa were used to evaluate the inflammatory potential of the activated NK cells.

[0176] To test whether improved cell recovery could increase the percentage of CAR+ cells, cells were treated with either a dsDNA HDR template (dsHDRT) or a plasmid HDR template (pUCmu) and then treated with either 10% human antibody serum (hAB) or 5% CellVive SR (FIG. 21). Cells treated with dsHDRT and 5% CellVive SR exhibited the highest levels of viability, overall CAR+ cells, and viable, CAR+ cells (FIG. 21).

[0177] Example 5- Endogenous fluorescent tagging of proteins in NK cells

[0178] The improved HDR conditions identified in earlier Examples were used to create a variety of reporter cell lines harboring fluorescent proteins at endogenous loci. For example, mNeonGreen was inserted at the NCR3 exon 4 locus, mKate2 at the TOMM70 exon 4 locus, and mTagBFP2 at the CTCF locus (FIG. 17). Cells were treated to introduce one, two, or all three of these reporters (FIG. 22). Successful insertion was observed in all combinations, indicating feasibility of single, dual, and triple reporter systems (FIG. 22).

[0179] Traditional virus-mediated transgene expression level is often highly heterogeneous in the transduced population and uncontrollable. To identify loci that would support different levels of gene expression, mNeonGreen was inserted into nine housekeeping genes (TOMM70, CTC , ACTG1, MAP4, EEF1G, YWHAQ, HNRNPA1, EN01, and GAPDH) (FIG. 17, 23). mNeonGreen levels were measured in each edited cell population. Between these nine loci, a variety of expression levels were observed, indicating their potential usefulness as reporters. These loci are robust reporters, titratable to the user’s desired expression level.

[0180] Example 6- Robust expression of therapeutically relevant constructs under control of GAPDH promoter

[0181] Constructs for inserts of 1.071kilobases (kb), 2.361 kb, 3.129 kb, and 4.248 kb at the GAPDH locus were designed (FIG. 24A). AsCasl2a Ultra was used to insert each construct into donor NK cells. Each construct was successfully inserted at the GAPDH locus, showing that knock-in of constructs up to about 4.3 kb is feasible using the methods described herein (FIG. 24B). Notably, this size exceeds the limit of AAV-mediated gene knock-in.

[0182] Example 7- Gene conversion using CISH promoter

[0183] A one-step knock-in strategy designed to simultaneously insert a CAR construct and knock down the CISH protein was designed (FIG. 25). This one-step knock-in strategy can minimize the manufacturing cumbersomeness and improve the stability of transgene expression. Five sgRNAs were screened for creation of either an indel or successful knock-in of the CD 19 / 22 CAR at exon 2 of CISH (FIG. 25B). A similar strategy was designed for exon 3.

[0184] Genomic DNA extraction and ICE analysis

[0185] NK cells pulsed with Cas9 RNP were rested in BO medium for 72 hours after electroporation. After resting, lx 105NK cells were washed once with PBS, resuspended in 30 μL of QuickExtraction solution (LGC Biosearch Technologies), and incubated at 65 °C for 15 minutes, followed by heat-inactivation at 98 °C for 5 minutes. Extracted genomic DNA was stored at -20 °C until the PCR reaction. The targeted region was amplified by Platinum SuperFi II PCR Master Mixes (ThermoFisher Scientific) and purified by QIAquick PCR Purification Kit (Qiagen). The purified DNA was sequenced by Sanger Sequencing (Azenta) and analyzed by ICE software (Synthego).

[0186] High rates of editing were detected in cells treated with any of the sgRNAs targeting exon 2 (FIG. 25B). Appreciable rates of editing were observed in cells treated with the sgRNA targeting exon 3 (FIG. 25B). Cells treated with IL-2 showed a dose-dependent proliferation inhibition (FIG. 26). This was thought to reflect a positive feedback loop whereby the edited cell population self-selected for successfully edited CAR NK cells, represented schematically in FIG. 26D.

[0187] Example 8- Hypoxia-inducible gene expression under control of PFKB4 promoter

[0188] Delivering transgene directly into tumor microenvironment is a preferred strategy versus systematic release of biologies. An efficient strategy was designed to target transgenes into exon 1 or exon 14 of PFKFB4 which is a hypoxia inducible gene (FIG. 16). The expression of a transgene inserted into C-terminus of the PFKFB4 locus is restricted by oxygen level. This restricted expression pattern could allow for precision cytokine release or CAR expression within a hypoxic tumor microenvironment.

[0189] Hypoxia induction of PFKFB4 locus knock-in transgene expression

[0190] Followed by 72-hour recovery post-electroporation, PFKFB4 edited NK cells were allowed to rest in a 20% Oxygen incubator for 16-24 hours. After resting, NK cells were incubated in a 1%, 0.3 PSI AVATAR chamber (Xcellbio) for 24 hours before flow cytometry analysis.

[0191] High rates of indels were observed in cells treated with sgRNA targeting either PFKB4 exon 1 or exon 14 (FIG. 27). Higher levels of mNeonGreen were detected when cells were grown in a hypoxic environment, indicating that expression of the transgenes was restricted to hypoxia (FIG. 16). As an additional example of hypoxia-induced gene expression, a construct to knock in a B7H3 CAR and sIL15 at the PFKFB4 exon 14 locus was prepared (FIG. 28A). NK cells were treated as described above to knock in the B7H3 CAR / sIL15 construct. Higher levels of the B7H3 CAR (labeled CAR.B7H3) were observed when cells were grown in hypoxic conditions (FIG. 28C). This demonstrated that the hypoxia-induced gene expression strategy was suitable for expression of a therapeutic construct.

[0192] The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

[0193] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0194] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0195] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[0196] SEQUENCE LISTING FREE TEXT

[0197] SEQ ID NO: 1 - CD96-dsHDRT-F

[0198] CTCCTCATTT ATATTGTCTC ATGCATCTC

[0199] SEQ ID NO: 2 - CD96-dsHDRT-R

[0200] ATTTGTTGAA TGAGAGAGAG CTGAATG

[0201] SEQ ID NO: 3 - CD96-Biotin-F

[0202] / 5Biosg / CTCCTCATTT ATATTGTCTC ATGCATCTC SEQ ID NO: 4 - CD96-Biotin-R

[0203] / 5Biosg / ATTTGTTGAA TGAGAGAGAG CTGAATG

[0204] SEQ ID NO: 5 - CD96-AmC6-F

[0205] / 5AmMC6 / CTCCTCATTT ATATTGTCTC ATGCATCTC

[0206] SEQ ID NO: 6 - CD96-AmC6-R

[0207] / 5AmMC6 / ATTTGTTGAA TGAGAGAGAG CTGAATG

[0208] SEQ ID NO: 7 - CD96-PS-F

[0209] C*T*C*CTCATTTATATTGTCTC ATGCATCTC

[0210] SEQ ID NO: 8 - CD96-PS-R

[0211] A*T*T*TGTTGAATGAGAGAGAG CTGAATG

[0212] SEQ ID NO: 9 - CD96-Biotin-PS-F

[0213] / 5Biosg / C*T*C*CTCATTT ATATTGTCTC ATGCATCTC

[0214] SEQ ID NO: 10 - CD96-Biotin-PS-R

[0215] / 5Biosg / A*T*T*TGTTGAA TGAGAGAGAG CTGAATG

[0216] SEQ ID NO: 11 - CD96-AmC6-PS-F

[0217] / 5AmMC6 / C*T*C*CTCATTT ATATTGTCTC ATGCATCTC

[0218] SEQ ID NO: 12 - CD96-AmC6-PS-R

[0219] / 5AmMC6 / A*T*T*TGTTGAA TGAGAGAGAG CTGAATG

[0220] SEQ ID NO: 13 - CD96-tCTS-dsHDRT-F

[0221] TGGCGGGACT AGTGGCTAGA CAGATGCAAT GGTCCAAGGC TCCTCATTTA TATTGTCTCA TGCATCTC

[0222] SEQ ID NO: 14 - CD96-tCTS-dsHDRT-R

[0223] TGGCGGGACT AGTGGCTAGA CAGATGCAAT GGTCCAAGGA TTTGTTGAAT GAGAGAGAGC TGAATG SEQ ID NO: 15 - CD96-end-closed-dsHDRT-F

[0224] GGCGGGACTA GTGGCTATCA GCACACAATT GCCCATTATA CGCGCGTATA

[0225] ATGGACTATT GTGTGCTGAT ACTCCTCATT TATATTGTCT CATGCATCTC

[0226] SEQ ID NO: 16 - CD96-end-closed dsHDRT-R

[0227] GGCGGGACTA GTGGCTATCA GCACACAATA GTCCATTATA CGCGCGTATA

[0228] ATGGGCAATT GTGTGCTGAT AATTTGTTGA ATGAGAGAGA GCTGAATG

[0229] SEQ ID NO: 17 - CD96-ssHDRT-F

[0230] CTCCTCATTT ATATTGTCTC ATGCATCTC

[0231] SEQ ID NO: 18 - CD96-ssHDRT-R

[0232] / 5Phos / ATTTGTTGAA TGAGAGAGAG CTGAATG

[0233] SEQ ID NO: 19 - CD96-tCTS-ssHDRT-F

[0234] TAGACAGATG CAATGGTCCA AGGCTCCTCA TTTATATTGT CTCATGCATC TC

[0235] SEQ ID NO: 20 - CD96-tCTS-ssHDRT-R

[0236] / 5Phos / TAGACAGATG CAATGGTCCA AGGATTTGTT GAATGAGAGA GAGCTGAATG

[0237] SEQ ID NO: 21 - CD96-tCTS-ssHRDT annealing oligo 1

[0238] GAGACAATAT AAATGAGGAG CCTTGGACCA TTGCATCTGT CTA

[0239] SEQ ID NO: 22 - CD96-tCTS-ssHRDT annealing oligo 2

[0240] TAGACAGATG CAATGGTCCA AGGATTTGTT GAATGAGAGA GAG

[0241] SEQ ID NO: 23 - pCD96-mNeonGreen (pUC19)

[0242] CCTGTGAGTCACTTGTGACTTTCACAGAAACTCCTGAGAATGGGTCAAAATGGACTCTGCACTT AAGGAATATGTCTTGTTCAGTCAGTGGAAGGTACGAGTGTATGCTTGTTCTGTATCCAGAGGGC AT T CAGAC TAAAAT C TACAACC T T C T CAT T CAGACACACGGTAAGCATAAC T GGTAGAGGGAT G TGCTTTCATTCAGCTCTCTCTCATTCAACAAATGGTACCGAGCTCGAAGGCGTAATCATGGTCA TAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCA TAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACT GCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGG AGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCG TTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGG

[0243] GGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCC

[0244]  TAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACC CACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAG TGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGT AGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCT CGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCC CATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCC GCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAA GATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACC GAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTG CTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCA GTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTC TGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGT T GAAT AC T C AT AC T C T T C C T T T T T CAAT AT T AT T GAAGC AT T T AT C AG G G T T AT T G T C T C AT GA GCGGATACATACGCGTCGCGAGGCCATATGGCTCCTCATTTATATTGTCTCATGCATCTCTATC TGAGTGAGACACAAGCTC TACT C TAT TCCTAAAGCAGCCAGGGAGAAATTTCCTCAGTTGCTCC CCTCACCTTACTGAAGTGACTAGGGTTTTTAATTAAGCGTCATTCTATGTGAATTATTTAAACT CATGGCTCATGTTCTTTCTTTTCAGGAGTTTGGGAAAAAACAGTCAACACAGAAGAAAATGTTT ATGCTACACTTGGCTCTGATGTCAACCTGACCTGCCAAAC

[0245] SEQ ID NO: 25 - pCD96-mNeonGreen (Nanoplasmid)

[0246] CTCGCGAATGCATCTAGGGGACGGCCGCTAGCCCGCCTAATGAGCGGGCTTTTTTTTGGCTTGT T G T C C C AAC C G T T AAC C T T AAAG C T T T AAAAG C C T T T AT T T C T T T T T T T T C T T AT AAA C T T AAAAC C T T AGAGGC T AT T T AAG T T GC T GAT T TAT AT T AAT T T TAT T GT T CAAACAT GAGAG C T T G T AC G T GAAAC T GAGAG C T T AG T AC G T T AG C C T GAGAGC T T AG T AC G T T AG C C AT GAG GGTTTAGTTCGTTAAACATGAGAGCTTAGTACGTTAAACATGAGAGCTTAGTACGTACTATCAA CAGGTTGAACTGCTGATCCACGTTGTGGTAGAATTGGTAAAGAGAGTCGTGTAAAATATCGAGT TCGCACATCTTGTTGTCTGATTATTGATTTTTGGCGAAACCATTTGATCATATGACAAGATGTG TATCTACCTTAACTTAATGATTTTGATAAAAATCATTAGGAGCTAGCATTGGGTCATCGGATCC CGGGCCCGTCGACCCAGGCTCCTCATTTATATTGTCTCATGCATCTCTATCTGAGTGAGACACA AGCTCTACTCTATTCCTAAAGCAGCCAGGGAGAAATTTCCTCAGTTGCTCCCCTCACCTTACTG AAG T GAC TAGG G T T T T TAAT T AG C G T CAT T C TAT G T GAAT TAT T T AAAC T CAT GG C T CAT G T T CTTTCTTTTCAGGAGTTTGGGAAAAAACAGTCAACACAGAAGAAAATGTTTATGCTACACTTGG CTCTGATGTCAACCTGACCTGCCAAACACAGACAGTAGGCTTCTTCGTGCAGATGCAATGGTCT GGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGAC CTATGGTGAGCAAAGGCGAGGAAGATAACATGGCTTCTCTGCCCGCTACACACGAGCTGCATAT CTTCGGCAGCATCAACGGCGTGGATTTCGACATGGTCGGCCAGGGCACAGGCAACCCTAATGAT GGATATGAGGAACTGAATCTGAAGTCTACAAAGGGCGACCTGCAGTTCAGCCCCTGGATCCTGG TTCCTCACATCGGCTACGGCTTCCACCAGTACCTGCCTTACCCAGACGGAATGAGCCCTTTCCA GGCCGCTATGGTGGACGGCTCTGGGTACCAGGTGCACCGGACCATGCAATTTGAGGACGGCGCC TCTCTGACCGTGAACTACCGGTACACCTACGAGGGCAGCCACATTAAGGGAGAAGCCCAGGTGA AGGGAACCGGATTTCCAGCCGACGGTCCTGTGATGACCAACAGCCTGACAGCCGCCGATTGGTG CAGAAGCAAAAAGACCTACCCCAACGACAAGACCATCATCAGCACATTTAAGTGGTCCTACACC ACAGGCAACGGCAAAAGATACAGGTCCACCGCCAGAACCACCTATACATTCGCCAAGCCTATGG CCGCCAATTACCTGAAAAACCAGCCTATGTACGTGTTCAGAAAGACGGAACTGAAGCACAGCAA GACCGAGCTCAACTTCAAGGAATGGCAGAAGGCCTTCACCGACGTGATGGGCATGGACGAGCTG TACAAGTGATAATAGGCGGCCGCGTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTC GACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTG GAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTA GGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAA TAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCCAAGGTCACCAATAAGATAGACCTGATT GCTGTCTATCATCCCCAATACGGCTTCTACTGTGCCTATGGGAGACCCTGTGAGTCACTTGTGA CTTTCACAGAAACTCCTGAGAATGGGTCAAAATGGACTCTGCACTTAAGGAATATGTCTTGTTC AGTCAGTGGAAGGTACGAGTGTATGCTTGTTCTGTATCCAGAGGGCATTCAGACTAAAATCTAC AACCTTCTCATTCAGACACACGGTAAGCATAACTGGTAGAGGGATGTGCTTTCATTCAGCTCTC T C T C AT T C AAC AAAT G G T AC

[0247] SEQ ID NO: 26 - pCD96-mNeonGreen (Minicircle, parental)

[0248] CGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGC CAACGCTATGTCCTGATAGCGGTCCGCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAG CGGCCATTTTCCACCATGATATTCGGCAAGCAGGCATCGCCATGGGTCACGACGAGATCCTCGC CGTCGGGCATGCTCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTC GTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGT TTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAG CCATGATGGATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTC GCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACG CCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCTTGCAGTTCATTCAGGGCACCGGACA GGTCGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGA GCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAA CCTGCGTGCAATCCATCTTGTTCAATCATGCGAAACGATCCTCATCCTGTCTCTTGATCAGAGC TTGATCCCCTGCGCCATCAGATCCTTGGCGGCAAGAAAGCCATCCAGTTTACTTTGCAGGGCTT CCCAACCTTACCAGAGGGCGCCCCAGCTGGCAATTCCGGTTCGCTTGCTGTCCATAAAACCGCC CAGTCTAGCTATCGCCATGTAAGCCCACTGCAAGCTACCTGCTTTCTCTTTGCGCTTGCGTTTT CCCTTGTCCAGATAGCCCAGTAGCTGACATTCATCCGGGGTCAGCACCGTTTCTGCGGACTGGC TTTCTACGCGCTCGAGGGGGGGCCAAACGGTCTCCAGCTTGGCTGTTTTGGCGGATGAGAGAAG ATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTG GCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGC CGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAA GGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGT AGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAG GACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTT TGCGTTTCTACAAACTCTTTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAC C AAAAT C C C T T AAC G T GAG T T T T C G T T C C AC T GAG C G T C AGAC C C C G T AGAAAAGAT C AAAG GA TCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTAC CAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAG CAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAAC TCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCG ATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGG CTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATAC CTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGG TAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCT TTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGG GGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGC CTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTT TGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAA GCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATAT GGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCG CTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGG CTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCA GAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCAGATCAATTCGCGCGCGAAGGCGAAG CGGCATGCATAATGTGCCTGTCAAATGGACGAAGCAGGGATTCTGCAAACCCTATGCTACTCCG TCAAGCCGTCAATTGTCTGATTCGTTACCAATTATGACAACTTGACGGCTACATCATTCACTTT TTCTTCACAACCGGCACGGAACTCGCTCGGGCTGGCCCCGGTGCATTTTTTAAATACCCGCGAG AAATAGAGTTGATCGTCAAAACCAACATTGCGACCGACGGTGGCGATAGGCATCCGGGTGGTGC TCAAAAGCAGCTTCGCCTGGCTGATACGTTGGTCCTCGCGCCAGCTTAAGACGCTAATCCCTAA CTGCTGGCGGAAAAGATGTGACAGACGCGACGGCGACAAGCAAACATGCTGTGCGACGCTGGCG ATTCTTCTCTCATCCGCCAAAACAGCCAAGCTGGAGACCGTTTGACATTACCCTGTTATCCCTA GAT AC AT TACCCTGTTATCC C AGAT GAC AT AC CCTGTTATCCC T AGAT GAC AT TACCCTGTTAT C C C AGAT GAC AT TAG CCTGTTATCCC T AGAT AC AT TACCCTGTTATCC CAGAT GAC AT AC C C T G T T AT C C C T AGAT GACAT TACCCTGTTATCC CAGAT GAC AT T AC C C T G T TAT C C C T AGAT AC AT T ACCCTGTTATCC CAGAT GAC AT AC CCTGTTATCCC T AGAT GACAT TACCCTGTTATCC CAGAT G AC AT T AC CCTGTTATCCC T AGAT AC AT TACCCTGTTATCC CAGAT GAC AT AC CCTGTTATCCCT AGAT GACAT TACCCTGTTATCC CAGAT GACAT T AC CCTGTTATCCC T AGAT AC AT TACCCTGTT AT C C CAGAT GAC AT AC CCTGTTATCCC T AGAT GACAT TACCCTGTTATCC CAGAT GACAT T AC C CTGTTATCCC T AGAT AC AT TACCCTGTTATCC CAGAT GAC AT AC CCTGTTATCCC T AGAT GAC A T T ACCCTGTTATCC CAGAT GACAT TAG CCTGTTATCCC T AGAT AC AT TACCCTGTTATCC C AGA T GAC AT AC CCTGTTATCCC T AGAT GACAT TACCCTGTTATCC CAGAT GACAT TACCCTGTTATC C C T AGAT AC AT TACCCTGTTATCC CAGAT GACAT AC CCTGTTATCCC T AGAT GACAT T AC C C T G TTATCCCAGATAAACTCAATGATGATGATGATGATGGTCGAGACTCAGCGGCCGCGCCCCAACT GGGGTAACCTTTGGGCTCCCCGGGCGCGGTAATCAGCATCATGATGTGGTACCACATCATGATG C T GAT T AT AAGAAT G C G G C C GC C AC AC TCTAGTGGATCTC GAG T T AAT AAT T C AGAAGAAC T C G TCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATAC

[0249] SEQ ID NO: 27 - pCD96-mNeonGreen (Minicircle)

[0250] GTGCCAGGGCGTGCCCTTGAGTTCTCTCAGTTGGGGACTATAAGCTGCGAGCAACTTCACTTGG GTATGCCGGCGGTAGCGCTCTCCTCATTTATATTGTCTCATGCATCTCTATCTGAGTGAGACAC AAGCTCTACTCTATTCCTAAAGCAGCCAGGGAGAAATTTCCTCAGTTGCTCCCCTCACCTTACT GAAGTGACTAGGGTTTTTAATTAAGCGTCATTCTATGTGAATTATTTAAACTCATGGCTCATGT TCTTTCTTTT C AG GAG T T T G GGAAAAAAC AG T C AAC AC AGAAGAAAAT GTTTATGC TAG AC T T G GCTCTGATGTCAACCTGACCTGCCAAACACAGACAGTAGGCTTCTTCGTGCAGATGCAATGGTC TGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGA CCTATGGTGAGCAAAGGCGAGGAAGATAACATGGCTTCTCTGCCCGCTACACACGAGCTGCATA TCTTCGGCAGCATCAACGGCGTGGATTTCGACATGGTCGGCCAGGGCACAGGCAACCCTAATGA TGGATATGAGGAACTGAATCTGAAGTCTACAAAGGGCGACCTGCAGTTCAGCCCCTGGATCCTG GTTCCTCACATCGGCTACGGCTTCCACCAGTACCTGCCTTACCCAGACGGAATGAGCCCTTTCC AGGCCGCTATGGTGGACGGCTCTGGGTACCAGGTGCACCGGACCATGCAATTTGAGGACGGCGC CTCTCTGACCGTGAACTACCGGTACACCTACGAGGGCAGCCACATTAAGGGAGAAGCCCAGGTG AAGGGAACCGGATTTCCAGCCGACGGTCCTGTGATGACCAACAGCCTGACAGCCGCCGATTGGT G C AGAAG C AAAAAGAC C T AC C C C AAC GAC AAGAC CAT CAT C AG C AC AT T T AAG T GG T C C T AC AC CACAGGCAACGGCAAAAGATACAGGTCCACCGCCAGAACCACCTATACATTCGCCAAGCCTATG GCCGCCAATTACCTGAAAAACCAGCCTATGTACGTGTTCAGAAAGACGGAACTGAAGCACAGCA AGACCGAGCTCAACTTCAAGGAATGGCAGAAGGCCTTCACCGACGTGATGGGCATGGACGAGCT GTACAAGTGATAATAGGCGGCCGCGTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCT CGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCT GGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGT AGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACA ATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCCAAGGTCACCAATAAGATAGACCTGAT TGCTGTCTATCATCCCCAATACGGCTTCTACTGTGCCTATGGGAGACCCTGTGAGTCACTTGTG ACTTTCACAGAAACTCCTGAGAATGGGTCAAAATGGACTCTGCACTTAAGGAATATGTCTTGTT CAGTCAGTGGAAGGTACGAGTGTATGCTTGTTCTGTATCCAGAGGGCATTCAGACTAAAATCTA CAACCTTCTCATTCAGACACACGGTAAGCATAACTGGTAGAGGGATGTGCTTTCATTCAGCTCT CTCTCATTCAACAAATGGGCCCG

[0251] SEQ ID NO: 28 - pCD96-CAR19

[0252] CCAAGGTCACCAATAAGATAGACCTGATTGCTGTCTATCATCCCCAATACGGCTTCTACTGTGC CTATGGGAGACCCTGTGAGTCACTTGTGACTTTCACAGAAACTCCTGAGAATGGGTCAAAATGG ACTCTGCACTTAAGGAATATGTCTTGTTCAGTCAGTGGAAGGTACGAGTGTATGCTTGTTCTGT AT CCAGAGGGCAT T CAGAC TAAAAT C TACAACCT T C T CAT T CAGACACACGGTAAGCATAAC T G G T AGAG GGATGTGCTTTCATT GAG CTCTCTCTCATT C AAC AAAT G TAG AT T T T AAGAAAAGC AA GGTTAGGCTTGGTGCTCAGCATACAAAGGTTGAGCAGAAAGAGCTACTGTCTGATAAGAGAGAC AGGAAGTGACCACAGCATTTAAGTAAATAAATGTAAACTTGCAACAGTGATAAGTGTCGATAAT AAAT AC T G T AAC AG T C T T G T GAAG GAGC AC AT C AC AGAG G C C AT T GAAAT T G T C C T T T AGAAAT TAACACCATAGCTTAAGTCTAAAGAATGAGTAGACACTACCACAATTTAGGAGGTGGGGCTGAG GCAGAGTGGGATTGTGAGTAATCCcagagagagaaaagagcttgcatgaagctctttggagtgt ttgaagaaatgggaaaatgccagtgggacagcaatacagcaattaaaggataaggcaggtaagg accagatcatgttggacctggtcagcttcccttttaaaaggtttctattttgatctcagaacca taagaagacatggaagtatttccaacaaaaggatggcatgacatttataggctgactgtatttg tgttgtttaaaaaaaaaaagtcat ctgactttcatgtgaaaatgaggtgaagatggacaagaa ggagaaggtagagacaactcaggagaccacctcagtagcccagaaaagagagagtggtagttca ggtagatatagaaggaaaatggcaaattttgttcattgggCCCATGGCCAAGCTTGCATGGTTT TTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAA ACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGT TCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCT CATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGC ACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCC GGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTAT GTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTAT TTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGG CAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAA AAAGGAT C T C AAGAAGAT CCTTTGATCTTTTCTACTAC C AAT G C T T AAT C AG T GAG G C AC C T AT CTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACG ATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGG CTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAAC TTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTT AATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTA TGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAA AAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCA CTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTG TGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTG CCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGA AAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAAC CCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAA AACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATA CTCTTCCTTTTT CAATAT TAT T GAAGCAT T TAT CAGGGT TAT T GT C T CAT GAGCGGATACATAC GCGTCGCGAGGCCATATGGttttgagatggagtctcactctgttgccaggctggagtgcagtgg catgatttcggctcactgtaatctccgcctcccaggttcaagcgattctcctgcttcagcctcc caagtagctgggactacaggcgtgcaccaccacgcccagctaatttttgtatttttagtagaga cggggttttgccgtgttggccaggatggtctggttctcttgacctcatgatctgcccacctcag cctcccaaagtgct ggattacaggcgtgagccactgcgccaggccTGGAGgtgtgactttgag catactgtttcatctctctgtggcccagtttcttcctcataaaaaggggttaacaacatcacct atggcgtgtgagggggattaaacaagataagatatgaaaggcccttgtaaacatgtatttttca aagaaattgctatacacatatCATTTATTTTTGCAGTTTCAGAAAGCATCACGTGTGTATTATA TTCAGCTGTCGTTTTATAACCCAAAAAGCCAATTATATTAAACGTAAGAAGAATCGTCATACTA AGTTTGCTGGTGTGCTTCAATTTTCAAATAGGCTTAATTTTATTCACAGGTAGTCTGAAACCTT C AAT TATATTTTGGT C AGAC C AAC AC AT AGAT AAAT AT T T AGAAG T C AG TAATTACCTCTAGCT TCTCTACGTAAATGCCTCCTCATTTATATTGTCTCATGCATCTCTATCTGAGTGAGACACAAGC TCTACTCTATTCCTAAAGCAGCCAGGGAGAAATTTCCTCAGTTGCTCCCCTCACCTTACTGAAG TGACTAGGGTTTTTAATTAAGCGTCATTCTATGTGAATTATTTAAACTCATGGCTCATGTTCTT T C T T T T CAGGAGT T TGGGAAAAAACAGT CAACACAGAAGAAAATGT T TAT GC TACAC T T GGC T C TGATGTCAACCTGACCTGCCAAACACAGACAGTAGGCTTCTTCGTGCAGATGCAATGGTCTGGC TCTGGCGCCACTAACTTTAGCCTGCTGAAGCAGGCTGGTGATGTGGAGGAGAATCCTGGGCCCA TGGCTCTGCCCGTGACCGCACTGCTGCTCCCACTTGCTCTGCTGCTACACGCGGCACGCCCTGA AC AGAAG CTGATTTCT GAAGAAGAC C T G GAG AT C C AGAT GAG C C AGAC CAC AT C GAG C C T G T C A GCTTCCTTGGGGGACAGGGTGACAATCTCTTGCCGAGCGTCACAGGACATCTCCAAGTACCTCA ACTGGTACCAACAGAAGCCCGATGGGACCGTGAAGCTGTTAATCTACCATACTTCTCGCCTGCA CTCGGGTGTCCCTTCTCGATTTAGTGGTTCTGGCTCCGGTACCGACTACTCCTTGACCATCTCC AATCTGGAGCAGGAGGACATCGCCACCTACTTCTGTCAACAGGGCAACACGCTACCTTACACCT TCGGCGGCGGCACTAAACTTGAGATCACCGGCTCCACTAGCGGGTCCGGCAAGCCCGGATCTGG TGAGGGCTCTACCAAGGGTGAAGTGAAGCTGCAGGAGAGCGGGCCCGGCCTGGTGGCTCCCTCC CAGAGCCTGTCCGTTACCTGCACCGTGTCCGGCGTTTCCTTGCCCGACTATGGCGTCAGTTGGA TCCGTCAGCCGCCGCGCAAAGGCCTGGAGTGGCTGGGGGTGATTTGGGGCAGCGAGACCACCTA TTACAACAGCGCGCTCAAGTCTAGACTGACCATCATAAAGGACAACTCCAAAAGTCAGGTATTC CTGAAAATGAACTCCCTCCAGACAGATGACACGGCCATC TAG TACT GT GCCAAGCAC TAT TACT ACGGTGGAAGCTACGCCATGGATTATTGGGGCCAGGGTACCTCGGTCACCGTGAGCTCTATTGA GGTGATGTACCCTCCACCCTACCTGGACAACGAGAAGTCCAACGGCACTATCATTCACGTCAAG GGAAAGCACCTGTGCCCCTCCCCGCTGTTCCCGGGACCCTCGAAGCCATTCTGGGTCCTGGTGG TGGTAGGCGGCGTCCTCGCTTGCTACTCTTTATTGGTGACGGTGGCGTTCATCATCTTCTGGGT GCGTAGTAAGCGCAGCCGGCTTCTCCATAGCGATTACATGAACATGACCCCCCGTAGGCCCGGC CCTACCCGCAAGCATTACCAGCCGTACGCACCCCCACGGGACTTCGCCGCGTACCGGAGCCGTG TCAAATTTTCACGCTCGGCCGACGCCCCTGCTTATCAGCAGGGTCAGAATCAGCTGTACAACGA GTTGAACCTGGGCCGCCGGGAGGAGTACGATGTGCTGGACAAACGCCGCGGGCGCGACCCGGAG

[0253] ATGGGCGGGAAGCCGCGCCGCAAGAACCCACAGGAGGGCCTGTACAACGAGCTGCAGAAGGACA AGATGGCGGAGGCCTACTCCGAGATCGGAATGAAGGGGGAGCGCCGTCGCGGCAAGGGCCACGA

[0254] TGGCCTTTATCAGGGTCTTTCCACTGCCACGAAAGACACCTACGACGCCCTCCACATGCAAGCC

[0255] CTGCCTCCTAGGTGATAATAGGCGGCCGCGTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATC

[0256] AGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTG

[0257] ACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTC

[0258] TGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGA

[0259] AGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG

[0260] SEQ ID NO: 29 - CD96 sgRNA

[0261] CGTGCAGATG CAATGGTCCA

[0262] SEQ ID NO:30 - GZMB sgRNA

[0263] TCCATAGGAGACAATGCCCT

[0264] SEQ ID NO: 31 - TOMM70 sgRNA

[0265] C C AC C AAC AT T AT AAAAC AG

[0266] SEQ ID NO:32 - NCR3 sgRNA

[0267] T GACC T GGAAAGGT CCAAGA

[0268] SEQ ID NO:33 MAP4 sgRNA

[0269] TTAATGCATCTGCAAGACTG

[0270] SEQ ID NO:34 - CTCF sgRNA

[0271] TCAGCATGATGGACCGGTGA

[0272] SEQ ID NO:35 - ACTG1 sgRNA

[0273] GTACGCCAACACGGTGCTGT

[0274] SEQ ID NO:36 - EEF1G sgRNA

[0275] AC TAG GAG T CAT AC AC AT G G

[0276] SEQ ID NO:37 - YWHAQ sgRNA

[0277] AAGAAGGATGACACCCTGTA

[0278] SEQ ID NO:38 - HNRNPA1 sgRNA

[0279] TTGTACTTTTCAGGTGGCTA

[0280] SEQ ID NOG 9 - ENO1 sgRNA

[0281] CCCTTGGCCAAGTAAGCTGT

[0282] SEQ ID NO: 40 - GAPDH crRNA

[0283] AT C T T C TAGGTAT GACAACGA

[0284] SEQ ID NO:41 - CISH exon 2 sgRNA 1

[0285] TCCACAGCCAGCAAAGGACG SEQ ID NO: 42 - CISH exon 2 sgRNA 2

[0286] TACCCTCTGCCACCTCCTCG

[0287] SEQ ID NO: 43 - CISH exon 2 sgRNA 3

[0288] AGAGAGTGAGCCAAAGGTGC

[0289] SEQ ID NO: 44 - CISH exon 2 sgRNA 4

[0290] GCCAAAGGTGCTGGACCCAG

[0291] SEQ ID NO:45 - CISH exon 2 sgRNA 5

[0292] C AGAC T GAG C AGAT T C C C GA

[0293] SEQ ID NO: 46 - CISH exon 3 sgRNA 1

[0294] CTGTCAGTGAAAACCACTCG

[0295] SEQ ID NO: 47 - CISH exon 3 sgRNA 2

[0296] ACTCAATGCGTACATTGGTG

[0297] SEQ ID NO:48- CISH exon 3 sgRNA 3

[0298] TGGACTCCAACTGCTTGTCC

[0299] SEQ ID NO:49 - CISH exon 3 sgRNA 4

[0300] AAGGCTGACCACATCCGGAA

[0301] SEQ ID NO: 50 - PFKFB4 exon 1 sgRNA 1

[0302] GTTCTGTGTCAATTCCCGTG

[0303] SEQ ID NO: 51 - PFKFB4 exon 1 sgRNA 2

[0304] GGATGCCATACAGCAATGGG

[0305] SEQ ID NO: 52 - PFKFB4 exon 1 sgRNA 3

[0306] CTGCACGCTTGCCAGCGCGG

[0307] SEQ ID NO: 53 - PFKFB4 exon 1 sgRNA 4

[0308] GGTTCTGTGTCAATTCCCGT

[0309] SEQ ID NO: 54 - PFKFB4 exon 1 sgRNA 5

[0310] ATCTGGATGCCATACAGCAA

[0311] SEQ ID NO: 55 - PFKFB4 exon 1 sgRNA 6

[0312] GCGCTGGCAAGCGTGCAGAG

[0313] SEQ ID NO:56 - PFKFB4 exon 13 sgRNA 1

[0314] G GAG AT C T C AAGAC C T C C AG

[0315] SEQ ID NO: 57- PFKFB4 exon 13 sgRNA 2

[0316] ACCGTGACAAGGGCTTCCTC SEQ ID NO: 58 - PFKFB4 exon 13 sgRNA 3

[0317] GTGAGCAGGCACCGTGACAA

[0318] SEQ ID NO:59 - GZMB left homology arm

[0319] AACAGCCAACGGTCCCACATACCTCCGATCTCAGGATCTGGGGGACATGACGGAGGCTGGCCCC TGGGATGAGGTGAAGCAGTAACAATGTCCAGGGCCAGAGCTTGGCAGCTGGGGGCCACCAGCGG CCTGCCCTGCCCTCTGGTCTCCCACATGTAGGCTGTGCAAGTTGGCCTTTTCTAAAAGGGGGCT TGAGATGGAAGAGAGGGCAGGACCCGGAGGAGCATCAGCTCAGTCCTTCCACTCTCTATTCACA GGGGGACTCTGGAGGCCCTCTTGTGTGTAACAAGGTGGCCCAGG

[0320] SEQ ID NO: 60 - GZMB right homology arm

[0321] GCATTGTCTCCTATGGACGAAACAATGGCATGCCTCCACGAGCCTGCACCAAAGTCTCAAGCTT TGTACACTGGATAAAGAAAACCATGAAACGCTACTAACTACAGGAAGCAAACTAAGCCCCCGCT GTAATGAAACACCTTCTCTGGAGCCAAGTCCAGATTTACACTGGGAGAGGTGCCAGCAACTGAA TAAATACCTCTTAGCTGAGTGGAAAAGCTGGTTTCTTGTTTATTCATTGACCCTCATTCTCAGG CACCACATCTGCGCTATGCAGGCCAATGACACAATTTTGCTGTT

[0322] SEQ ID NO:61 - GZMB-mNeonGreen KI template plasmid

[0323] AAT C AT G G T C AT AG C T G T T T C C T G T G T GAAAT T G T T AT C C G C T C AC AT T C C AC ACAAC AT AC G AGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCG TTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCC AACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCT GCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCC ACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACC GTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAA TCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCT GGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTC TCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGT CGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCC GGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTG GTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAA CTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGA AAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTT GCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGG GTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGG AT C T T C AC C T AGAT C C T T T T AAAT T AAAAAT GAAG T T T T AAAT C AAT C T AAAG TAT AT AT GAG T AAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATT TCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCA TCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAA TAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCA GTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTT GTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCG GTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTT CGGTCCTCCGATCGTTGTCAGAAGTAAGTTTGCCGCAGTGTTATCACTCATGGTTATGGCAGCA CTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAA CCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGA AAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACT GAT CT T CAGCAT C T T T TAC T T T CACCAGCGT T T C T GGGT GAGCAAAAACAGGAAGGCAAAAT GO CGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATAT TATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAA ATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCAT TATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTC GGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAG CGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTG GCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCG CACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTT GGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGC AAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGT CAGGTCGACCCAGGGGTCAGACTTCATGTAGCATCACTTACAACCTTCATTGAATATTGTGAAG AAAC T TAG T GG T CAGAC T T CAT G TAC CAT CAC T T AC AC C T T CAT T GAAT AT T G T GAAG T GC T T G T T GAAT C T GAGAC T T AAAAAT TTTGTTCTTT T AGAG GAAAC AT G GAGAAAG C C AT T GAC AT G T TCAACAAAGCTATTAACCTGGCCAAATCGGAAATGGAGATGGCCCATCTGTATTCACTTTGCGA TGCCGCCCATGCCCAGACAGAAGTTGCAAAGAAATACGGATTAAAACCACCAACATTAGGAAGT GGGGGCACCAGCGGAATGGTGTCCAAAGGGGAGGAAGACAACATGGCCTCTCTTCCGGCCACTC ATGAACTCCACATCTTCGGTAGCATTAACGGCGTTGATTTCGACATGGTCGGCCAGGGCACTGG CAACCCTAACGACGGCTACGAGGAGCTCAACCTGAAGTCCACCAAGGGGGACCTGCAGTTTTCA CCGTGGATCCTGGTGCCTCATATTGGATACGGCTTTCACCAGTACCTGCCCTATCCCGACGGTA TGTCTCCCTTCCAGGCCGCCATGGTGGATGGCTCCGGCTACCAGGTCCACCGTACGATGCAGTT CGAGGACGGGGCTTCCCTAACCGTCAACTACAGGTACACCTATGAGGGTAGCCACATCAAGGGA GAGGCTCAGGTAAAGGGTACCGGCTTCCCGGCGGACGGCCCTGTGATGACCAACAGCCTGACCG CAGCGGACTGGTGCCGGTCCAAGAAGACTTACCCCAACGACAAAACCATCATCTCTACGTTCAA ATGGTCTTACACAACTGGTAATGGCAAGCGTTACCGCTCGACCGCTCGCACCACCTACACCTTC GCCAAGCCCATGGCTGCCAACTACTTGAAAAACCAGCCAATGTACGTGTTCCGCAAGACAGAGC TGAAGCACTCGAAGACCGAGCTGAACTTCAAGGAGTGGCAAAAGGCGTTCACCGATGTGATGGG CATGGATGAGCTGTACAAGTAAAACAGGGGGAAAGCAGACTGACCCTCTTTTTAAAAGTTTACC CCCTCTTCAACTGAACCCTAAAGACACTGTCATGAACTGTGTTGAATGGTGGAAATCAGTATTT CTGTTTGTGGTGTTGTTATTTGTTACATCTGTTTCATGTCTAGGTGTTGTGGGTGTGGCTGTTG AAGGAAGTTTGCAGTCTTGCAGCTTTTATTCCCTGTGCAACAAAAGATTAGAACATGTTAAAGG GAT T T T T AAAT AAAG T T GCAAAGAG T ACAAAT GAT AAT T GGC CAT GCAAAT AAAAAC T GAT T T G GTACCGAGCTCGAAGGCGT

[0324] SEQ ID NO:69 - TOMM70-mKate2 KI template plasmid

[0325] AAT C AT G G T C AT AG C T G T T T C C T G T G T GAAAT T G T T AT C C G C T C AC AAT T C C AC AC AAC AT AC G AGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCG TTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCC AACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCT GCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCC ACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACC GTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAA TCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCT GGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTC TCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGT CGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCC GGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTG GTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAA CTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGA AAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTT GCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGG GTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGG AT C T T C AC C T AGAT C C T T T T AAT T AAAAT GAAG T T T T AAT C AT C T AAG T T T AT GAG T AAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATT TCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCA TCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAA TAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCA GTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTT GTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCG GTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTT CGGTCCTCCGATCGTTGTCAGAAGTAAGTTTGCCGCAGTGTTATCACTCATGGTTATGGCAGCA CTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAA CCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGA TAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGA AAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACT GAT CT T CAGCAT C T T T TAC T T T CACCAGCGT T T C T GGGT GAGCAAAAACAGGAAGGCAAAAT GC CGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATAT TATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAA ATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCAT TATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTC GGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAG CGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTG GCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCG CACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTT GGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGC AAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGT CAGGTCGACCCAGGGGTCAGACTTCATGTAGCATCACTTACAACCTTCATTGAATATTGTGAAG AAAC T TAG T GG T CAGAC T T CAT G TAC CAT CAC T T ACAAC C T T CAT T GAAT AT T G T G AG T GC T T G T T GAAT C T GAGAC T T AAAAAT TTTGTTCTTT T AGAG GAAAC AT G GAGAAAG C C AT T GAC AT G T TCAACAAAGCTATTAACCTGGCCAAATCGGAAATGGAGATGGCCCATCTGTATTCACTTTGCGA TGCCGCCCATGCCCAGACAGAAGTTGCAAAGAAATACGGATTAAAACCACCAACATTAGGAAGT GGGGGCACCAGCGGAATGGTGTCCGAGCTGATCAAGGAGAACATGCACATGAAACTGTACATGG AGGGCACCGTCAACAACCACCACTTTAAGTGCACTTCAGAGGGCGAGGGCAAGCCCTATGAGGG CACTCAGACTATGCGTATCAAGGCCGTGGAGGGTGGACCCCTGCCCTTCGCCTTCGACATCCTG GCCACGAGCTTTATGTATGGCTCCAAGACCTTCATTAACCATACCCAGGGCATCCCTGACTTTT TCAAGCAGAGCTTCCCGGAGGGCTTCACCTGGGAGCGGGTCACCACATACGAGGACGGCGGGGT GCTCACCGCCACCCAGGACACCAGTCTTCAAGATGGGTGCCTGATCTACAACGTGAAGATACGT GGTGTCAATTTCCCCTCTAATGGGCCAGTGATGCAGAAGAAGACCCTTGGATGGGAGGCTTCCA CCGAGACTCTATACCCAGCAGATGGGGGATTGGAAGGTCGAGCGGACATGGCCCTCAAACTGGT GGGCGGCGGCCACTTGATTTGTAACCTGAAGACGACCTACAGGTCGAAGAAGCCTGCCAAGAAC CTGAAAATGCCTGGCGTGTACTACGTAGACCGCCGCCTGGAGCGCATCAAAGAAGCGGACAAGG AGACCTACGTCGAACAGCATGAGGTGGCTGTTGCTCGCTACTGTGATCTCCCGTCTAAGCTGGG CCACCGCTAAAACAGGGGGAAAGCAGACTGACCCTCTTTTTAAAAGTTTACCCCCTCTTCAACT GAACCCTAAAGACACTGTCATGAACTGTGTTGAATGGTGGAAATCAGTATTTCTGTTTGTGGTG TTGTTATTTGTTACATCTGTTTCATGTCTAGGTGTTGTGGGTGTGGCTGTTGAAGGAAGTTTGC AG T C T T G C AGC T T T TAT TCCCTGTG C AAC AAAAGAT T AGAAC AT G T T AAAG G GAT T T T T AAAT A AAGTTGCAAAGAGTACAAATGATAATTGGCCATGCAAATAAAAACTGATTTGGTACCGAGCTCG AAGGCGT

[0326] SEQ ID NO: 70 - TOMM70 dsHDRT F primer

[0327] G G T C AGAC T T C AT G TAG CAT GAG T T AC

[0328] SEQ ID NO:71 - TOMM70 dsHDRT R primer

[0329] AAAT C AG TTTTTATTTGCATGGC C AAT TATCATTTGTAC

[0330] SEQ ID NO:72 - NCR3 left homology arm

[0331] ACAGAGACCAGGAAGAGGAGAGCCTCGGGACTGCAACACTGAGCAGCTCCTGTCCTCTCTCTGA CCAGGCCACTGTCACATGGGAACACACTGCCACTCCTCAGATGGGCCCCGAGGAGTGATTCCAG AGCCCAGATGTCCCTAGTCCTCTTCAAAAGACCCCAATAAATCTGCCCCACCACTAACTCCTCA TGAGTCTCAAGTGTTTTCTTCTCCATTCTCCAGATGCCAAATCTACTCTCTCCGGATTCCCCCA ACTCTGAACTTTCCCTTCCACCAGGTCTGACCTGGAAAGGTCCA

[0332] SEQ ID NO:73 - NCR3 right homology arm

[0333] AGAAGGCAGCTGCCGGCTGTGGTCCCAGCGCCCCTCCCACCACCATGTGGGAGCTCAGCACATC TGCTTCCCCCAGTCCCAGGAGGCTGAGCCTGATTGTCCTGAGAAATGGGAAGGATCAGATATGA CTCCTCCTTGGCAACTGCCCTTTCCTGCCAGGCCCACACATACCCTCTTCTGGCTGTTAGGGGA GCTTGGGTCCCTGAACACTGTCATTCACCCAATAAATTACTATTTGACCCCAGAGTGGGTGGAA GGGTGAGCCATGTG

[0334] SEQ ID NO: 74 - NCR3 -mNeonGreen KI template plasmid

[0335] AAT C AT G G T C AT AG C T G T T T C C T G T G T GAAAT T G T T AT C C G C T C AC AAT T C C AC AC AAC AT AC G AGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCG TTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCC AACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCT GCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCC ACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACC GTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAA TCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCT GGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTC TCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGT CGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCC GGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTG GTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAA CTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGA AAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTT GCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGG GTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGG AT C T T C AC C T AGAT C C T T T T AAAT T AAAAAT GAAG T T T T AAAT C AAT C T AAAG TAT AT AT GAG T AAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATT TCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCA TCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAA TAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCA GTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTT GTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCG GTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTT CGGTCCTCCGATCGTTGTCAGAAGTAAGTTTGCCGCAGTGTTATCACTCATGGTTATGGCAGCA CTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAA CCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGA TAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGA AAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACT GAT CT T CAGCAT C T T T TAC T T T CACCAGCGT T T C T GGGT GAGCAAAAACAGGAAGGCAAAAT GO CGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATAT TATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAA ATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCAT TATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTC GGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAG CGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTG GCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCG CACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTT GGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGC AAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGT CAGGTCGACCCAGGACAGAGACCAGGAAGAGGAGAGCCTCGGGACTGCAACACTGAGCAGCTCC TGTCCTCTCTCTGACCAGGCCACTGTCACATGGGAACACACTGCCACTCCTCAGATGGGCCCCG AGGAGTGATTCCAGAGCCCAGATGTCCCTAGTCCTCTTCAAAAGACCCCAATAAATCTGCCCCA CCACTAACTCCTCATGAGTCTCAAGTGTTTTCTTCTCCATTCTCCAGATGCCAAATCTACTCTC TCCGGATTCCCCCAACTCTGAACTTTCCCTTCCACCAGGTCTGACCTGGAAAGGTCCACGTCGC CAGCTGCCTGCCGTGGTCCCTGCTCCCCTGCCCCCACCGTGCGGCAGCTCCGCGCACTTGCTCC CCCCGGTGCCCGGCGGTGGAAGTGGGGGCACCAGCGGAATGGTGTCCAAAGGGGAGGAAGACAA CATGGCCTCTCTTCCGGCCACTCATGAACTCCACATCTTCGGTAGCATTAACGGCGTTGATTTC GACATGGTCGGCCAGGGCACTGGCAACCCTAACGACGGCTACGAGGAGCTCAACCTGAAGTCCA CCAAGGGGGACCTGCAGTTTTCACCGTGGATCCTGGTGCCTCATATTGGATACGGCTTTCACCA GTACCTGCCCTATCCCGACGGTATGTCTCCCTTCCAGGCCGCCATGGTGGATGGCTCCGGCTAC CAGGTCCACCGTACGATGCAGTTCGAGGACGGGGCTTCCCTAACCGTCAACTACAGGTACACCT ATGAGGGTAGCCACATCAAGGGAGAGGCTCAGGTAAAGGGTACCGGCTTCCCGGCGGACGGCCC TGTGATGACCAACAGCCTGACCGCAGCGGACTGGTGCCGGTCCAAGAAGACTTACCCCAACGAC AAAACCATCATCTCTACGTTCAAATGGTCTTACACAACTGGTAATGGCAAGCGTTACCGCTCGA CCGCTCGCACCACCTACACCTTCGCCAAGCCCATGGCTGCCAACTACTTGAAAAACCAGCCAAT GTACGTGTTCCGCAAGACAGAGCTGAAGCACTCGAAGACCGAGCTGAACTTCAAGGAGTGGCAA AAGGCGTTCACCGATGTGATGGGCATGGATGAGCTGTACAAGTGATAGTAAAGAAGGCAGCTGC CGGCTGTGGTCCCAGCGCCCCTCCCACCACCATGTGGGAGCTCAGCACATCTGCTTCCCCCAGT CCCAGGAGGCTGAGCCTGATTGTCCTGAGAAATGGGAAGGATCAGATATGACTCCTCCTTGGCA ACTGCCCTTTCCTGCCAGGCCCACACATACCCTCTTCTGGCTGTTAGGGGAGCTTGGGTCCCTG AACACTGTCATTCACCCAATAAATTACTATTTGACCCCAGAGTGGGTGGAAGGGTGAGCCATGT GGGTACCGAGCTCGAAGGCGT

[0336] SEQ ID NO:75 - NCR3 dsHDRT F primer

[0337] ACAGAGACCAGGAAGAGGAGAGCCTC

[0338] TAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCA GTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTT GTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCG GTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTT CGGTCCTCCGATCGTTGTCAGAAGTAAGTTTGCCGCAGTGTTATCACTCATGGTTATGGCAGCA CTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAA CCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGA TAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGA AAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACT GAT CT T CAGCAT C T T T TAG T T T CACCAGCGT T T C T GGGT GAGCAAAAACAGGAAGGCAAAAT GC CGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATAT TATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAA ATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCAT TATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTC GGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAG CGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTG GCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCG CACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTT GGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGC AAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGT CAGGTCGACCCAGGATAGAAAGGACACAATTGCAATTGCTTTGTCCTGCCACCACCACCTCCCT CCACCCCCAGAAATGTGAGAAAAGAGTAACTAGCGGTGAGTTACCTTGGGTCAGACTGTCCATT TCTTAGCTTTATTTTTCTCATCTTTCAAATGAGGGAGCCTCTTCTAGCTGTAAAATCTTAGATT TTGATTCTTTTGTTTGTTTCAAACAATTGACAAGAGTTTCTAGAAATGTTTTTCCTTTTGCAGT GCTCGTTACATGAAAGATCTTTTTCCTCTTCACCAGGCAGTTGCAGTGGTGCAGAATGGTGTCC AAAGGGGAGGAAGACAACATGGCCTCTCTTCCGGCCACTCATGAACTCCACATCTTCGGTAGCA TTAACGGCGTTGATTTCGACATGGTCGGCCAGGGCACTGGCAACCCTAACGACGGCTACGAGGA GCTCAACCTGAAGTCCACCAAGGGGGACCTGCAGTTTTCACCGTGGATCCTGGTGCCTCATATT GGATACGGCTTTCACCAGTACCTGCCCTATCCCGACGGTATGTCTCCCTTCCAGGCCGCCATGG TGGATGGCTCCGGCTACCAGGTCCACCGTACGATGCAGTTCGAGGACGGGGCTTCCCTAACCGT CAACTACAGGTACACCTATGAGGGTAGCCACATCAAGGGAGAGGCTCAGGTAAAGGGTACCGGC TTCCCGGCGGACGGCCCTGTGATGACCAACAGCCTGACCGCAGCGGACTGGTGCCGGTCCAAGA AGACTTACCCCAACGACAAAACCATCATCTCTACGTTCAAATGGTCTTACACAACTGGTAATGG CAAGCGTTACCGCTCGACCGCTCGCACCACCTACACCTTCGCCAAGCCCATGGCTGCCAACTAC TTGAAAAACCAGCCAATGTACGTGTTCCGCAAGACAGAGCTGAAGCACTCGAAGACCGAGCTGA ACTTCAAGGAGTGGCAAAAGGCGTTCACCGATGTGATGGGCATGGATGAGCTGTACAAGGGCAC TGGTGCAGGCTCCGGGGCTGATCTCAGTCTTGCAGATGCATTAACAGAACCATCTCCAGACATT GAGGGAGAGATAAAGCGGGACTTCATTGCCACACTAGAGGCAGAGGCCTTTGATGATGTTGTGG GAGAAAC T G T T G GAAAAAC AGAC T AT AT T C C T C T C C T GGAT G T T GAT GAGAAAAC C G G GAAC T C AGAG T C AAAGAAGAAAC C G T GC T C AGAAAC T AG C C AGAT T GAAGG T AAG T AT T T AAAC C AGAC T GCTTATATATGTTCAGAAAGCAGGTTAGGGATTATGCCTTTGGTATAATCTTGGAAACTGGGTA CCGAGCTCGAAGGCGT

[0339] SEQ ID NO: 82 - MAP4 dsHDRT F primer

[0340] ATAGAAAGGACACAAT T GCAAT T GC

[0341] SEQ ID NO: 83 - MAP4 dsHDRT R primer C AG T T T C C AAGAT T AT AC C AAAG G C

[0342] SEQ ID NO: 84 - CTCF left homology arm

[0343] GCTATCTAATAAGGCTGTCCTGCATTGCTGACATCCCGTTCGCTGTCAGTCTAAAAGACCCTTG TGATTCTTGGGGCTTTAATGGACCATTTGTTCTGTCTGTGCTCTTCTTTGCCAGCAACAGCTAT GATT C AG G T T GAAGAC C AGAAT AC AG G T G C AAT T GAGAAC AT T AT AG T T GAAG T AAAAAAAGAG CCAGATGCTGAGCCCGCAGAGGGAGAGGAAGAGGAGGCCCAGCCAGCTGCCACAGATGCCCCCA ACGGAGACCTCACGCCCGAGATGATCCTCAGCATGATGGACCGG

[0344] SEQ ID NO:85 - CTCF right homology arm

[0345] TGATGGCGGAGCCTTGTGCGTCGCCAGGACTTCTCTGGGCTGTGTTTAAACGGCCCGCATCTTA ATTTTTCTCCCTTCTTTCTTTTTTTGGCTTTGGGAAAAGCATCATTTTACCAAACATACCGAGA AC GAAAAC T T C AAG GAT GAT G T T AGAAAAAAAT G T GAT T T AAC T AGAAC TTGCTGTCTGATGTT AGCAAATCATGGAATGTTCTGAGTCCCTGAGGGTTTACTGTGAAGTGCTGAGGACAGTGTTGAC AACTAACTCGTTTTCCTAGATGGAAACGGAGACATTGACCCCTC

[0346] SEQ ID NO:86 - CTCF-mNeonGreen KI template plasmid

[0347] CGCGTCGCGAGGCCATATGGGCTATCTAATAAGGCTGTCCTGCATTGCTGACATCCCGTTCGCT GTCAGTCTAAAAGACCCTTGTGATTCTTGGGGCTTTAATGGACCATTTGTTCTGTCTGTGCTCT T C T T T GC CAGCAACAGC T AT CAT T CAGG T T GAAGAC CAGAAT ACAGG T GCAAT T GAGAACAT TA TAGTTGAAGTAAAAAAAGAGCCAGATGCTGAGCCCGCAGAGGGAGAGGAAGAGGAGGCCCAGCC AGCTGCCACAGATGCCCCCAACGGAGACCTCACGCCCGAGATGATCCTCAGCATGATGGACCGG GGAAGTGGGGGCACCAGCGGAATGGTGTCCAAAGGGGAGGAAGACAACATGGCCTCTCTTCCGG CCACTCATGAACTCCACATCTTCGGTAGCATTAACGGCGTTGATTTCGACATGGTCGGCCAGGG CACTGGCAACCCTAACGACGGCTACGAGGAGCTCAACCTGAAGTCCACCAAGGGGGACCTGCAG TTTTCACCGTGGATCCTGGTGCCTCATATTGGATACGGCTTTCACCAGTACCTGCCCTATCCCG ACGGTATGTCTCCCTTCCAGGCCGCCATGGTGGATGGCTCCGGCTACCAGGTCCACCGTACGAT GCAGTTCGAGGACGGGGCTTCCCTAACCGTCAACTACAGGTACACCTATGAGGGTAGCCACATC AAGGGAGAGGCTCAGGTAAAGGGTACCGGCTTCCCGGCGGACGGCCCTGTGATGACCAACAGCC TGACCGCAGCGGACTGGTGCCGGTCCAAGAAGACTTACCCCAACGACAAAACCATCATCTCTAC GTTCAAATGGTCTTACACAACTGGTAATGGCAAGCGTTACCGCTCGACCGCTCGCACCACCTAC ACCTTCGCCAAGCCCATGGCTGCCAACTACTTGAAAAACCAGCCAATGTACGTGTTCCGCAAGA CAGAGCTGAAGCACTCGAAGACCGAGCTGAACTTCAAGGAGTGGCAAAAGGCGTTCACCGATGT GATGGGCATGGATGAGCTGTACAAGTGATGGCGGAGCCTTGTGCGTCGCCAGGACTTCTCTGGG CTGTGTTTAAACGGCCCGCATCTTAATTTTTCTCCCTTCTTTCTTTTTTTGGCTTTGGGAAAAG CAT CAT T T TAG C AAAC AT AC C GAGAAC GAAAAC T T C AAG GAT GAT G T T AGAAAAAAAT G T GAT T TAACTAGAACTTGCTGTCTGATGTTAGCAAATCATGGAATGTTCTGAGTCCCTGAGGGTTTACT G T GAAG T G C T GAG GAC AG T G T T GAC AAC T AAC TCGTTTTCC T AGAT G GAAAC G GAGACAT T GAC CCCTCCCCATGGCCAAGCTTGCATGGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAA AAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCC CCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCT TTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTA GGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTA TCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCA CTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCC TAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTC GGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTG TTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTAC TACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTG CCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGC AATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGA AGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCC GGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGG CATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGG CGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTG TCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTAC TGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAA TAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATA GCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTT ACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTT ACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAA GGGCGACACGGAAATGT T GAATAC T CAT AC TCTTCCTTTTT CAATAT TAT T GAAGCAT T TAT CA GGGTTATTGTCTCATGAGCGGATACATA

[0348] SEQ ID NO: 87 - CTCF-mTagBFP KI template plasmid

[0349] CGCGTCGCGAGGCCATATGGGCTATCTAATAAGGCTGTCCTGCATTGCTGACATCCCGTTCGCT GTCAGTCTAAAAGACCCTTGTGATTCTTGGGGCTTTAATGGACCATTTGTTCTGTCTGTGCTCT T C T T T GC CAGCAACAGC T AT CAT T CAGG T T GAAGAC CAGAAT ACAGG T GCAAT T GAGAACAT TA TAGTTGAAGTAAAAAAAGAGCCAGATGCTGAGCCCGCAGAGGGAGAGGAAGAGGAGGCCCAGCC AGCTGCCACAGATGCCCCCAACGGAGACCTCACGCCCGAGATGATCCTCAGCATGATGGACCGG GGAAGTGGGGGCACCAGCGGAATGGTGTCTAAAGGGGAGGAGCTGATTAAGGAGAACATGCATA TGAAACTTTACATGGAGGGTACAGTTGACAACCACCACTTTAAGTGCACTTCGGAGGGCGAGGG TAAACCTTACGAGGGCACCCAGACTATGCGAATCAAGGTGGTGGAGGGCGGACCCCTGCCCTTC GCTTTCGACATCCTAGCCACCTCCTTCCTGTACGGCTCCAAGACCTTCATTAATCACACGCAAG GCATCCCCGACTTTTTTAAGCAGAGCTTCCCGGAGGGCTTCACCTGGGAGCGGGTCACCACATA TGAGGATGGCGGCGTCTTGACTGCAACCCAGGACACCTCGCTGCAGGATGGGTGCCTGATCTAC AACGTGAAGATCCGTGGTGTCAACTTCACCAGCAACGGCCCTGTGATGCAGAAGAAAACGCTTG GATGGGAAGCGTTCACCGAGACTCTCTACCCCGCTGACGGCGGTTTGGAAGGGCGCAACGACAT GGCGCTGAAGCTGGTGGGCGGAAGTCACCTCATCGCCAATGCGAAGACCACCTACAGGTCCAAG AAGCCGGCCAAGAACCTGAAAATGCCTGGCGTGTACTACGTGGATTACCGCCTGGAGCGCATCA AGGAGGCCAACAACGAGACCTATGTAGAACAGCATGAGGTGGCTGTCGCCCGCTACTGTGACCT GCCATCTAAGCTGGGCCACAAGCTCAACTGATGGCGGAGCCTTGTGCGTCGCCAGGACTTCTCT GGGCTGTGTTTAAACGGCCCGCATCTTAATTTTTCTCCCTTCTTTCTTTTTTTGGCTTTGGGAA AAG CAT CAT T T TAG CAAAC AT AC C GAGAAC GAAAAC T T C AAG GAT GAT G T T AGAAAAAAAT G T G ATTTAACTAGAACTTGCTGTCTGATGTTAGCAAATCATGGAATGTTCTGAGTCCCTGAGGGTTT ACTGTGAAGTGCTGAGGACAGTGTTGACAACTAACTCGTTTTCCTAGATGGAAACGGAGACATT GACCCCTCCCCATGGCCAAGCTTGCATGGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCA CAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTT CCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCG CCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGT GTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCC TTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAG CCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTG GCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACC TTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTT TTGTTTGCAAGCAGCAGATTACGCGCAGAA AAAAGGATCTCAAGAAGATCCTTTGATCTTTTC TACTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAG TTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGC TGCAATG TACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCC GGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTT GCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTAC AGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCA AGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCG TTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCT TACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGA GAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCAC ATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGAT CTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCT TTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAA TAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTA T C AGG G TTATTGTCTCAT GAGC G GAT AC AT A

[0350] SEQ ID NO:88 - CTCF dsHDRT F primer

[0351] GCTATCTAATAAGGCTGTCCTGCATTG

[0352] SEQ ID NO: 89 - CTCF dsHDRT R primer

[0353] GAGGGGTCAATGTCTCCGTTTCCATC

[0354] SEQ ID NO: 90 - ACTG1 left homology arm

[0355] ATGGCCACCGCCGCATCCTCCTCTTCTCTGGAGAAGAGCTACGAGCTGCCCGATGGCCAGGTCA TCACCATTGGCAATGAGCGGTTCCGGTGTCCGGAGGCGCTGTTCCAGCCTTCCTTCCTGGGTAG GTGTTGTGAGCTAAAGGTTTCTACTCTTACATCCTCGGTGACACAGCATCACTAAGGGAGGGCT CTGTCCCCTAGGTATGGAATCTTGCGGCATCCACGAGACCACCTTCAACTCCATCATGAAGTGT GACGTGGACATCCGCAAAGACCTGTACGCCAACACGGTGCTGTC

[0356] SEQ ID NO:91 - ACTG1 right homology arm

[0357] GGGCGGCACCACCATGTACCCGGGCATTGCCGACAGGATGCAGAAGGAGATCACCGCCCTGGCG CCCAGCACCATGAAGATCAAGGTGAGTCGAGGGGTTGGTGGCCCTCTGCCTGGCTCGGGAGAGC TGACTGGGGGGCGCTCTGTGAGCTGAAGCCGTGCCTGGCTGTCTTTGCAGATCATCGCACCCCC AGAGCGCAAGTACTCGGTGTGGATCGGTGGCTCCATCCTGGCCTCACTGTCCACCTTCCAGCAG ATGTGGATTAGCAAGCAGGAGTACGACGAGTCGGGCCCCTCCAT

[0358] SEQ ID NO:92 - ACTG1 coding region restore amino acid sequence

[0359] GGTTMYPGIADRMQKEI TALAPSTMKIKI IAPPERKYSVWIGGS ILASLSTFQQMWI SKQEYDE SGPS IVHRKCF

[0360] SEQ ID NO:93 - ACTG1 coding region restore DNA sequence

[0361] GGAGGCACCACTATGTATCCTGGCATCGCGGACCGCATGCAGAAGGAGATCACCGCACTGGCCC CGTCGACCATGAAAATCAAGATCATCGCCCCCCCAGAACGTAAGTACTCCGTGTGGATCGGTGG CTCCATCCTGGCTTCTCTCAGCACGTTCCAGCAGATGTGGATTTCCAAGCAGGAGTACGATGAG AGCGGGCCCTCTATTGTCCACCGCAAGTGCTTT SEQ ID NO:94 - ACTGl-mNeonGreen KI template plasmid

[0362] CGCGTCGCGAGGCCATATGGATGGCCACCGCCGCATCCTCCTCTTCTCTGGAGAAGAGCTACGA GCTGCCCGATGGCCAGGTCATCACCATTGGCAATGAGCGGTTCCGGTGTCCGGAGGCGCTGTTC CAGCCTTCCTTCCTGGGTAGGTGTTGTGAGCTAAAGGTTTCTACTCTTACATCCTCGGTGACAC AGCATCACTAAGGGAGGGCTCTGTCCCCTAGGTATGGAATCTTGCGGCATCCACGAGACCACCT TCAACTCCATCATGAAGTGTGACGTGGACATCCGCAAAGACCTGTACGCCAACACGGTGCTGTC CGGAGGCACCACTATGTATCCTGGCATCGCGGACCGCATGCAGAAGGAGATCACCGCACTGGCC CCGTCGACCATGAAAATCAAGATCATCGCCCCCCCAGAACGTAAGTACTCCGTGTGGATCGGTG GCTCCATCCTGGCTTCTCTCAGCACGTTCCAGCAGATGTGGATTTCCAAGCAGGAGTACGATGA GAGCGGGCCCTCTATTGTCCACCGCAAGTGCTTTGGCTCTGGCGCCACTAACTTTAGCCTGCTG AAGCAGGCTGGTGATGTGGAGGAGAATCCTGGGCCCGGAAGTGGGGGCACCAGCGGAATGGTGT CCAAAGGGGAGGAAGACAACATGGCCTCTCTTCCGGCCACTCATGAACTCCACATCTTCGGTAG CATTAACGGCGTTGATTTCGACATGGTCGGCCAGGGCACTGGCAACCCTAACGACGGCTACGAG GAGCTCAACCTGAAGTCCACCAAGGGGGACCTGCAGTTTTCACCGTGGATCCTGGTGCCTCATA TTGGATACGGCTTTCACCAGTACCTGCCCTATCCCGACGGTATGTCTCCCTTCCAGGCCGCCAT GGTGGATGGCTCCGGCTACCAGGTCCACCGTACGATGCAGTTCGAGGACGGGGCTTCCCTAACC GTCAACTACAGGTACACCTATGAGGGTAGCCACATCAAGGGAGAGGCTCAGGTAAAGGGTACCG GCTTCCCGGCGGACGGCCCTGTGATGACCAACAGCCTGACCGCAGCGGACTGGTGCCGGTCCAA GAAGAC T T AC C C C AAC GAC AAAAC CATCATCTCTACGTT C AAT G G T C T T AC AC AAC T G G T AAT GGCAAGCGTTACCGCTCGACCGCTCGCACCACCTACACCTTCGCCAAGCCCATGGCTGCCAACT ACTTGAAAAACCAGCCAATGTACGTGTTCCGCAAGACAGAGCTGAAGCACTCGAAGACCGAGCT GAACTTCAAGGAGTGGCAAAAGGCGTTCACCGATGTGATGGGCATGGATGAGCTGTACAAGTGA TAGTAAGGGCGGCACCACCATGTACCCGGGCATTGCCGACAGGATGCAGAAGGAGATCACCGCC CTGGCGCCCAGCACCATGAAGATCAAGGTGAGTCGAGGGGTTGGTGGCCCTCTGCCTGGCTCGG GAGAGCTGACTGGGGGGCGCTCTGTGAGCTGAAGCCGTGCCTGGCTGTCTTTGCAGATCATCGC ACCCCCAGAGCGCAAGTACTCGGTGTGGATCGGTGGCTCCATCCTGGCCTCACTGTCCACCTTC CAGCAGATGTGGATTAGCAAGCAGGAGTACGACGAGTCGGGCCCCTCCATCCCATGGCCAAGCT TGCATGGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAG AGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGC GCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGT GGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTG GGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTG AGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAG AGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGA AGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCT CTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTAC GC GCAGAAAAAAAGGAT C T CAAGAAGAT C C T T T GAT C T T T T C T AC T AC CAAT GC T T AAT CAG T G AGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTA GATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCA CGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTG GTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAG TTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCG TCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCA TGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGC AGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGA TGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGA GTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCT CATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGT TCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTG GGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTG AAT AC T C T AC T C T T C C T T T T T CAAT AT T AT T GAAG C AT T T T C AG G G T T AT T G T C T C T GAG C GGATACATA

[0363] SEQ ID NO: 95 - EEF1G left homology arm

[0364] GCAACCTTCCTCCTTATTGATTGACCCCTGTGCTTTCTTCCTCTTAGTACCTTTGTGTTGGATG AATTTAAGCGCAAGTACTCCAATGAGGACACACTCTCTGTGGCACTGCCATATTTCTGGGAGCA CTTTGATAAGGACGGCTGGTCCCTGTGGTACTCAGAGTATCGCTTCCCTGAAGAACTCACTCAG ACCTTCATGAGCTGCAATCTCATCACTGGTAAGAGAGTGGGTCTGAGAAGGAAGGAGAACAAGG TGTAGCAAGTGCTGTGATTCCAAAGGCTGAATGTTCTCCCTTGCCGCTTCAGGAATGTTCCAGC GACTGGACAAGCTGAGGAAGAATGCCTTCGCCAGTGTCATCCTTTTTGGAACCAACAATAGCAG CTCCATTTCTGGAGTCTGGGTCTTCCGAGGCCAGGAGCTTGCCTTTCCGGTGAGGAAGGTGGAG GAGAGGAGTCTCTTTAGGGTGGGGCAGGGATATAAGAAGCACTGTGTTTTATTTCTGAGTTGAT GGATGTTTAGGAGGGTTCAGGAATGCTTATAGGAAGGAGCAGTTCATGTTCACAGGAGACCTGG GGTCTGATGACATGTGTTTGAGCTGGAAGTAACACAGGTGTGGGGTGGTGTCTCATAACTTGAC CACATGCTGGCTTTTTATCCCCAGCTGAGTCCAGATTGGCAGGTGGACTACGAGTCATAC

[0365] SEQ ID NO: 96 - EEF1G right homology arm

[0366] ACATGGCGGAAACTGGATCCTGGCAGCGAGGAGACCCAGACGCTGGTTCGAGAGTACTTTTCCT GGGAGGGGGCCTTCCAGCATGTGGGCAAAGCCTTCAATCAGGGCAAGATCTTCAAGTGAACATC TCTTGCCATCACCTAGCTGCCTGCACCTGCCCTTCAGGGAGATGGGGGTCATTAAAGGAAACTG AACATTGAACCCTTTCCTGTCCTGCCTCCTTTGTGAGTGGTGGCGTCTTCAAGGGGAGTAGATA TGTTGAAGCAGGGTACTTTCTGTGTTCGCAGTACCTCGATATGCAGGAGAGAAGACTTCAGAAG

[0367] AACAGAAATAGAGGCCTGTGTTCACTTGTGATTTTAATTACCCTGCCTCCTACGGTGATGACTG TCTTCTAGCTGGGAAAGAGCGGAAGCAGTTGGGGCGGGTAGTGACATGGGGCTTTGTGACTACC TAATTCTTAGGGATCAGATTCCAGGCCTTTCCCAGTGAAGGGAGAGAATAGCTGCTAATAACAA GAG C T AAC G T G T AT AAT T C AC AGAG C GC AAAG GAAC T T TAG T TATTTCCTG T AAC TGTCCTGT GAGGTAGGTAGCTGACAATATGCAAGGGTTG

[0368] SEQ ID NO:97 - EEF1G coding region restore amino acid sequence

[0369] TWRKLDPGSEETQTLVREYFSWEGAFQHVGKAFNQGKI FK

[0370] SEQ ID NO: 98 - EEF1G coding region restore DNA sequence

[0371] ACCTGGCGTAAGCTCGACCCCGGCTCCGAGGAGACCCAGACTCTGGTGCGCGAGTACTTTAGCT GGGAAGGTGCGTTCCAGCACGTCGGGAAGGCCTTCAACCAGGGCAAGATCTTCAAA

[0372] SEQ ID NO:99 - EEFIG-mNeonGreen KI template plasmid

[0373] CGCGTCGCGAGGCCATATGGGCAACCTTCCTCCTTATTGATTGACCCCTGTGCTTTCTTCCTCT TAGTACCTTTGTGTTGGATGAATTTAAGCGCAAGTACTCCAATGAGGACACACTCTCTGTGGCA CTGCCATATTTCTGGGAGCACTTTGATAAGGACGGCTGGTCCCTGTGGTACTCAGAGTATCGCT TCCCTGAAGAACTCACTCAGACCTTCATGAGCTGCAATCTCATCACTGGTAAGAGAGTGGGTCT GAGAAGGAAGGAGAACAAGGTGTAGCAAGTGCTGTGATTCCAAAGGCTGAATGTTCTCCCTTGC CGCTTCAGGAATGTTCCAGCGACTGGACAAGCTGAGGAAGAATGCCTTCGCCAGTGTCATCCTT TTTGGAACCAACAATAGCAGCTCCATTTCTGGAGTCTGGGTCTTCCGAGGCCAGGAGCTTGCCT CCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAG TGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATC CAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTT TCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAAT GTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCAT GAGCGGATACATA

[0374] SEQ ID NO: 100 - YWHAQ left homology arm

[0375] GCTCCAGTGGTATATGTATGTATGTATGTATGTCATTTTGTAAGTGCATAGTGAAAGGTCTGCA AGGATGTACTTTGGGAAGGTGATGGTCTTTGGGGTTTTGAGAGAGAACTTCAGTCTTAGTCAAG ATCTCCATTGTTT GAG GAAT TTACTACTCTTGTAGTTAATTTT GAT AT AAC AAT AC T AC T T AG T G T AAT T G C C T AAT C T AGAAAT AG T AAC T T AAT AT GAT T C T T C T T T T T C T AG C T T T G GAC AT C AG ACAGT GCAGGAGAAGAAT GT GAT GCGGCAGAAGGGGC TGAAAAC

[0376] SEQ ID NO: 101 - YWHAQ right homology arm

[0377] TAAATCTATACAGGGTGTCATCCTTCTTTCCTTCAAGAAACCTTTTTACACATCTCCATTCCTT AT T C C AC TTGGATTTCCTATAG C AAAGAAAC CCATTCATGTGTATG GAAT C AAC TGTTTATAGT CTTTTCACACTGCAGCTTTGGGAAAACTTCATTCCTTGATTTGTGTTTGTCTTGGCCTTCCTGG T G T GC AG T AC T G C T G T AGAAAAG T AT T AAT AG C T T C AT T T C AT AT AAACAT AAG T AAC T C C C AA AC AC T T AT G T AGAG GAC T AAAAAT GTATCTGGTATT T AAG T AAT

[0378] SEQ ID NO: 102 - YWHAQ -mNeonGreen KI template plasmid

[0379] CGCGTCGCGAGGCCATATGGGCTCCAGTGGTATATGTATGTATGTATGTATGTCATTTTGTAAG TGCATAGTGAAAGGTCTGCAAGGATGTACTTTGGGAAGGTGATGGTCTTTGGGGTTTTGAGAGA GAACTTCAGTCTTAGTCAAGATCTCCATTGTTTGAGGAATTTACTACTCTTGTAGTTAATTTTG AT AT AAC AAT AC T AC T T AG T G T AAT T GC C T AAT C T AGAAAT AG T AAC T T AAT AT GATTCTTCTT TTTCTAGCTTTGGACATCAGACAGTGCAGGAGAAGAATGTGATGCGGCAGAAGGGGCTGAAAAC GGCTCTGGCGCCACTAACTTTAGCCTGCTGAAGCAGGCTGGTGATGTGGAGGAGAATCCTGGGC CCATGGTGTCCAAAGGGGAGGAAGACAACATGGCCTCTCTTCCGGCCACTCATGAACTCCACAT CTTCGGTAGCATTAACGGCGTTGATTTCGACATGGTCGGCCAGGGCACTGGCAACCCTAACGAC GGCTACGAGGAGCTCAACCTGAAGTCCACCAAGGGGGACCTGCAGTTTTCACCGTGGATCCTGG TGCCTCATATTGGATACGGCTTTCACCAGTACCTGCCCTATCCCGACGGTATGTCTCCCTTCCA GGCCGCCATGGTGGATGGCTCCGGCTACCAGGTCCACCGTACGATGCAGTTCGAGGACGGGGCT TCCCTAACCGTCAACTACAGGTACACCTATGAGGGTAGCCACATCAAGGGAGAGGCTCAGGTAA AGGGTACCGGCTTCCCGGCGGACGGCCCTGTGATGACCAACAGCCTGACCGCAGCGGACTGGTG CCGGTCCAAGAAGACTTACCCCAACGACAAAACCATCATCTCTACGTTCAAATGGTCTTACACA ACTGGTAATGGCAAGCGTTACCGCTCGACCGCTCGCACCACCTACACCTTCGCCAAGCCCATGG CTGCCAACTACTTGAAAAACCAGCCAATGTACGTGTTCCGCAAGACAGAGCTGAAGCACTCGAA GACCGAGCTGAACTTCAAGGAGTGGCAAAAGGCGTTCACCGATGTGATGGGCATGGATGAGCTG T ACAAG T GAT AG T AAT AAAT C T AT AC AG G G T G T C AT C C T T C T T T C C T T CAAGAAAC C T T T T T AC AC AT C T C C AT T C C T T AT T C C AC T T G GAT T T C C T AT G C AAAGAAAC C C AT T C T G T G T AT GGAA TCAACTGTTTATAGTCTTTTCACACTGCAGCTTTGGGAAAACTTCATTCCTTGATTTGTGTTTG TCTTGGCCTTCCTGGTGTGCAGTACTGCTGTAGAAAAGTATTAATAGCTTCATTTCATATAAAC AT AAG T AAC T C C C AAAC AC T T AT G T AGAG GAC T AAAAAT GTATCTGGTATT T AAG T AAT C C C AT GGCCAAGCTTGCATGGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGC TCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCT CCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTC

[0380]  TGTAACAGTCTGATCGTGACGCTGAATAAATGTCTTTTTTTTAATGTGCTGTGTAAAGTTAGTC TACTCTTAAGCCATCTTGGTAAATTTCCCCAACAGTGTGAAGTTAGAATTCCTTCAGGGT

[0381] SEQ ID NO: 105 - HNRNPA1 coding region restore amino acid sequence YGGSSSSSSYGSGRRF

[0382] SEQ ID NO: 106 - HNRNPA1 coding region restore DNA sequence

[0383] TACGGTGGGAGTAGCTCGTCCTCTAGCTACGGCTCCGGCCGCCGCTTC

[0384] SEQ ID NO: 107 - HNRNPA1 -mNeonGreen KI template plasmid

[0385] CGCGTCGCGAGGCCATATGGAGGGTATGCTTGTGCCACTCTGAAAATCTCTTTATTTTATGTCA TGGTGAGTTAGGCCAGTTTTCTTTGTATTACTGGATTATTCAACTGAATGCCTTTCCCAGAGAA TGAAATGCAAAGATTGGAGTCACCATAGTTTGGGAGAAAGGAAGGCTGATAACTCAACCTTATT T TAT T C T GACT GC TAAACAGAAT T GGAAAC TAACAT CAT CC T CAGGTAACAGAT AAGGCCC T C TTTCCCATTCATAGGAAGCAATTTTGGAGGTGGTGGAAGCTACAATGATTTTGGGAATTACAAC AATCAGTCTTCAAATTTTGGACCCATGAAGGGAGGAAATTTTGGAGGCAGAAGCTCTGGCCCCT ATGGCGGTGGAGGCCAATACTTTGCAAAACCACGAAACCAAGGTATGGTATCTATGTAATTTTG GATAATGTCAAAAGAGTGTCTGTAGCTACTGCTGGGAAGAAAGCCCTTTAACTGCTATGTCTGG G C AGC AAAAC GTTTATAGTT T AGAAC C T T C AGAAAG TGATAATTTGAT CAC AAAT T AGAAAAAT CATGGGACCTCTTTACCACCTCCCTTGTAGTAGGGCCATTTTTAAATGGCCAGACACTTGAATT T AAC TTTTATTATCC C AAT T GAAAAC AT T C T G T T GG C AC T T T GAAAC T T T AAAGAAAAAT TGTACTTTTCAGGTGGATACGGTGGGAGTAGCTCGTCCTCTAGCTACGGCTCCGGCCGCCGCTT CGGCTCTGGCGCCACTAACTTTAGCCTGCTGAAGCAGGCTGGTGATGTGGAGGAGAATCCTGGG CCCATGGTGTCCAAAGGGGAGGAAGACAACATGGCCTCTCTTCCGGCCACTCATGAACTCCACA TCTTCGGTAGCATTAACGGCGTTGATTTCGACATGGTCGGCCAGGGCACTGGCAACCCTAACGA CGGCTACGAGGAGCTCAACCTGAAGTCCACCAAGGGGGACCTGCAGTTTTCACCGTGGATCCTG GTGCCTCATATTGGATACGGCTTTCACCAGTACCTGCCCTATCCCGACGGTATGTCTCCCTTCC AGGCCGCCATGGTGGATGGCTCCGGCTACCAGGTCCACCGTACGATGCAGTTCGAGGACGGGGC TTCCCTAACCGTCAACTACAGGTACACCTATGAGGGTAGCCACATCAAGGGAGAGGCTCAGGTA AAGGGTACCGGCTTCCCGGCGGACGGCCCTGTGATGACCAACAGCCTGACCGCAGCGGACTGGT G C C GG T C C AAGAAGAC T T AC C C C AAC GAC AAAAC CATCATCTCTACGTT C AAAT GG T C T TAG AC AACTGGTAATGGCAAGCGTTACCGCTCGACCGCTCGCACCACCTACACCTTCGCCAAGCCCATG GCTGCCAACTACTTGAAAAACCAGCCAATGTACGTGTTCCGCAAGACAGAGCTGAAGCACTCGA AGACCGAGCTGAACTTCAAGGAGTGGCAAAAGGCGTTCACCGATGTGATGGGCATGGATGAGCT GTACAAGTGATAATAGCTATGGCGGTTCCAGCAGCAGCAGTAGCTATGGCAGTGGCAGAAGATT TTAATTAGGTAAGTAAGCACCTTTTTGTGTGTTGACATAATTTTTTAAATTGCTGATGAACCCA ATAACCCTAATGTAGCTGAGCAGTGCAACATAGTTAACATTATAATTGCAGTAATTGTGGATAT AAAGT TAATAT T CAGAT CAGCAAAAT T T GT GGGAAACAAAC T T GATAT TGGAT T GTAGCC T T GA G T C T T AAT AT G T T T AGAT T AAC AAC TTTATTCCATATTGTT C AC AG GAAAC AAAG C T T AGC AG GAGAGGAGAGCCAGAGAAGT GACAGGGAAGC TACAGGT TACAACAGAT T T GT GAAC T CAGCCAA GCACAGTGGTGGCAGGGCCTAGCTGCTACAAAGAAGACATGTTTTAGACAAATACTCATGTGTA TGGGCAAAAAACTCGAGGACTGTATTTGTGACTAATTGTATAACAGGTTATTTTAGTTTCTGTT CTGTGGAAAGTGTAAAGCATTCCAACAAAGGGTTTTAATGTAGATTTTTTTTTTTGCACCCCAT GCTGTTGATTGCTAAATGTAACAGTCTGATCGTGACGCTGAATAAATGTCTTTTTTTTAATGTG CTGTGTAAAGTTAGTCTACTCTTAAGCCATCTTGGTAAATTTCCCCAACAGTGTGAAGTTAGAA TTCCTTCAGGGTCCCATGGCCAAGCTTGCATGGTTTTTCCATAGGCTCCGCCCCCCTGACGAGC ATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGC GTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTG TCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTT CGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTG CGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCA

[0386] GCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGT GGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGT TACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGT TTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCT

[0387] TTTCTACTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCC ATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCA GTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCC AGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAAT

[0388] TGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTG CTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACG ATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCG ATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATT

[0389] CTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATT CTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCG CCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAA GGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGC AT C T T T TAG T T T CACCAGCGT T T C T GGGT GAGCAAAAACAGGAAGGCAAAAT GCCGCAAAAAAG GGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCA TTTATCAGGGTTATTGTCTCATGAGCGGATACATA

[0390] SEQ ID NO: 108 - ENO1 left homology arm

[0391] AACATCAAGATCCAAACAGCTCACACATTTTGTCTTTTAATAAGGTCACTCTCCTTTTCTCCCC ATGCCATTTATTTGTTGTCCCTT C AAT AT T T T T T AAAAAAC AAAAG CAT T AGAAT TTTTATATT CCAAGAAATGGCAATGCTGACATTTTACTTTGCTCCTCTGCAACACTGACGGGGAGAGGAGAGG AAGAGAAGGGGCGGTGGGTGGAGGGCACCTAGCATGGTGAGTTGGGATGAGGAGAACTAAATAC TTTTCTATATCTGGCTTCTCAGAGAAGCACAAGTTTAGAGGGTTTAAAGAAGGTGGACAAACTG GAGAGTTCTGTGTTCTCAACTTCCCAGGAGTGGGGCTGTGTCTTTGACCACATCTAGGATGGGA AAACTTAAAACTTGAGGTCTGACTTTTCTTTTTTCCTCCCCATCTCTTTACCTTTCTCCTTCCC AAGAATTGAAGAGGAGCTGGGCAGCAAGGCTAAGTTTGCCGGCAGGAACTTCAGAAACCCCTTG GCCAAG

[0392] SEQ ID NO: 109 - ENO1 right homology arm

[0393] TAAGCTGTGGGCAGGCAAGCCCTTCGGTCACCTGTTGGCTACACAGACCCCTCCCCTCGTGTCA

[0394] GCTCAGGCAGCTCGAGGCCCCCGACCAACACTTGCAGGGGTCCCTGCTAGTTAGCGCCCCACCG

[0395] CCGTGGAGTTCGTACCGCTTCCTTAGAACTTCTACAGAAGCCAAGCTCCCTGGAGCCCTGTTGG

[0396] CAGCTCTAGCTTTGCAGTCGTGTAATTGGCCCAAGTCATTGTTTTTCTCGCCTCACTTTCCACC

[0397] AAGTGTCTAGAGTCATGTGAGCCTCGTGTCATCTCCGGGGTGGCCACAGGCTAGATCCCCGGTG

[0398] GT T TTGTGCTCAAAATAAAAAGCCTCAGTGACCCATGAGAATACTCCGTGTGCCTGTGTATGTC

[0399] TGGAACAATCTGGGTCTGTCCTTAGTGTTCAGGGGCCCCTGGCGAAAGGGTGTCGGCCTTTGAG

[0400] AACCAAGGTTTGGTGTCGTAACAGAAAAGGATGAGGATCCATTGGGGTCATTTTCCTGGGTCCT

[0401] TGGGAAGGGAACCAGAGGCAAAGGCAAGAGAAGAGACATACACGGTCCCTCCAGGATAGTGGGG

[0402] TCAAAACAGAGTCTAAGCTGGAAGGCAGAGCGAGACTTTGTTTGGACACCTTGTTTGGAAGAAA

[0403] CTTCAAATGCAGGCCTATTTGTAGATAATCTTAAGGATGGTAGCTACTAGGTTACACAGC  CGCCTCCATCCAGTCTATTAATTGTTGCCGGGTkAGCTAGAGTAAGTAGTTCGCCAGTTAATAGT TTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTT CATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGC GGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATG GTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTG GTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGC GTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGT TCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTC GTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGG AAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTC C T T T T T CAATAT TAT T GAAGCAT T TATCAGGGT TAT T GT C T CAT GAG CG GAT AC AT A

[0404] SEQ ID NO: 111 - GAPDH left homology arm

[0405] CTGAGGCTCCCACCTTTCTCATCCAAGACTGGCTCCTCCCTGCCGGGGCTGCGTGCAACCCTGG GGTTGGGGGTTCTGGGGACTGGCTTTCCCATAATTTCCTTTCAAGGTGGGGAGGGAGGTAGAGG GGTGATGTGGGGAGTACGCTGCAGGGCCTCACTCCTTTTGCAGACCACAGTCCATGCCATCACT GCCACCCAGAAGACTGTGGATGGCCCCTCCGGGAAACTGTGGCGTGATGGCCGCGGGGCTCTCC AGAACATCATCCCTGCCTCTACTGGCGCTGCCAAGGCTGTGGGCAAGGTCATCCCTGAGCTGAA CGGGAAGCTCACTGGCATGGCCTTCCGTGTCCCCACTGCCAACGTGTCAGTGGTGGACCTGACC TGCCGTCTAGAAAAACCTGCCAAATATGATGACATCAAGAAGGTGGTGAAGCAGGCGTCGGAGG GCCCCCTCAAGGGCATCCTGGGCTACACTGAGCACCAGGTGGTCTCCTCTGACTTCAACAGCGA CACCCACTCCTCCACCTTTGACGCTGGGGCTGGCATTGCCCTCAACGACCACTTTGTCAAGCTC ATTTCCTGGTATGTGGCTGGGGCCAGAGACTGGCTCTTAAAAAGTGCAGGGTCTGGCGCCCTCT GGTGGCTGGCTCAGAAAAAGGGCCCTGACAACTCTTT

[0406] SEQ ID NO: 112 - GAPDH right homology arm

[0407] ATTTGGCTACAGCAACAGGGTGGTGGACCTCATGGCCCACATGGCCTCCAAGGAGTAAGACCCC TGGACCACCAGCCCCAGCAAGAGCACAAGAGGAAGAGAGAGACCCTCACTGCTGGGGAGTCCCT GCCACACTCAGTCCCCCACCACACTGAATCTCCCCTCCTCACAGTTGCCATGTAGACCCCTTGA AGAGGGGAGGGGCCTAGGGAGCCGCACCTTGTCATGTACCATCAATAAAGTACCCTGTGCTCAA CCAGTTACTTGTCCTGTCTTATTCTAGGGTCTGGGGCAGAGGGGAGGGAAGCTGGGCTTGTGTC AAGGTGAGACATTCTTGCTGGGGAGGGACCTGGTATGTTCTCCTCAGACTGAGGGTAGGGCCTC CAAACAGCCTTGCTTGCTTCGAGAACCATTTGCTTCCCGCTCAGACGTCTTGAGTGCTACAGGA AGCTGGCACCACTACTTCAGAGAACAAGGCCTTTTCCTCTCCTCGCTCCAGTCCTAGGCTATCT GCTGTTGGCCAAACATGGAAGAAGCTATTCTGTGGGCAGCCCCAGGGAGGCTGACAGGTGGAGG AAGTCAGGGCTCGCACTGGGCTCTGACGCTGACTGGTTAGTGGAGCTCAGCCTGGAGCTGAGCT GCAGCGGGCAATTCCAGCTTGGCCTCCGCAGCTGTGAGGTCTTGAGCACGTGCTCTATTG

[0408] SEQ ID NO: 113 - GAPDH coding region restore amino acid sequence

[0409] FGYSNRWDLMAHMASKE

[0410] SEQ ID NO: 114 - GAPDH coding region restore DNA sequence

[0411] TTCGGATATAGCAATAGAGTGGTCGATCTGATGGCTCATATGGCTAGCAAAGAG

[0412] SEQ ID NO: 115 - GAPDH-mNeonGreen KI template plasmid

[0413] CGCGTCGCGAGGCCATATGGCCTGCCAAATATGATGACATCAAGAAGGTGGTGAAGCAGGCGTC GGAGGGCCCCCTCAAGGGCATCCTGGGCTACACTGAGCACCAGGTGGTCTCCTCTGACTTCAAC AGCGACACCCACTCCTCCACCTTTGACGCTGGGGCTGGCATTGCCCTCAACGACCACTTTGTCA AGCTCATTTCCTGGTATGTGGCTGGGGCCAGAGACTGGCTCTTAAAAAGTGCAGGGTCTGGCGC CCTCTGGTGGCTGGCTCAGAAAAAGGGCCCTGACAACTCTTTACATCTTCTAGGTATGACAACG AGTTCGGATATAGCAATAGAGTGGTCGATCTGATGGCTCATATGGCTAGCAAAGAGGGAAGCGG AGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTATGGTT TCGAAAGGAGAGGAAGACAACATGGCTTCCTTGCCTGCCACGCATGAGCTGCACATCTTCGGAA GCATTAATGGGGTCGATTTCGACATGGTGGGCCAGGGCACCGGCAATCCAAACGACGGCTATGA GGAGCTGAACCTGAAGTCCACCAAGGGTGATCTGCAGTTTTCACCGTGGATCCTGGTGCCTCAT ATTGGCTACGGCTTTCACCAGTACCTGCCCTATCCCGACGGAATGTCTCCCTTCCAGGCCGCCA TGGTGGATGGCTCCGGTTACCAGGTCCACCGCACCATGCAGTTCGAGGACGGTGCTAGTCTGAC CGTCAACTACAGGTACACCTACGAGGGCAGCCACATCAAGGGGGAGGCTCAGGTGAAGGGTACT GGCTTCCCGGCGGACGGGCCGGTGATGACCAACTCCCTAACTGCTGCGGACTGGTGCCGGTCCA AGAAGAC T T AC C C C AAC GAG AAGAC CAT CATCTCTACTTT T AAAT G G T C T T AC AC C AC AG GC AA CGGCAAGCGTTACCGCTCGACCGCACGTACAACCTACACCTTCGCCAAGCCTATGGCCGCCAAC TACCTCAAGAACCAGCCAATGTACGTGTTCCGCAAAACGGAGCTTAAGCACAGCAAAACCGAGC TCAACTTCAAGGAGTGGCAAAAGGCGTTCACCGACGTGATGGGTATGGATGAACTGTACAAGTG ATAGTAAGCGGCCGCGTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGC CTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGC CACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCAT TCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGC ATGCTGGGGATGCGGTGGGCTCTATGGATTTGGCTACAGCAACAGGGTGGTGGACCTCATGGCC CACATGGCCTCCAAGGAGTAAGACCCCTGGACCACCAGCCCCAGCAAGAGCACAAGAGGAAGAG AGAGACCCTCACTGCTGGGGAGTCCCTGCCACACTCAGTCCCCCACCACACTGAATCTCCCCTC CTCACAGTTGCCATGTAGACCCCTTGAAGAGGGGAGGGGCCTAGGGAGCCGCACCTTGTCATGT ACCATCAATAAAGTACCCTGTGCTCAACCAGTTACTTGTCCTGTCTTATTCTAGGGTCTGGGGC AGAGGGGAGGGAAGCTGGGCTTGTGTCAAGCCCATGGCCAAGCTTGCATGGTTTTTCCATAGGC TCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGG ACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTG CCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCAC GCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCC CGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACAC GACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTG CTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTG CGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACC ACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTC AAGAAGATCCTTTGATCTTTTCTACTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATC TGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGG GCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTT ATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCC TCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGC GCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATT CAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTT AGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTA TGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGA GTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCA ATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTT

[0414] CGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGC ACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGG CAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTT T T CAATAT TAT T GAAGCAT T TAT CAGGGT TAT T GT C T CAT GAGCGGATACATA

[0415] SEQ ID NO: 116 - GAPDH-mbIL15 KI template plasmid

[0416] CGCGTCGCGAGGCCATATGGCTGAGGCTCCCACCTTTCTCATCCAAGACTGGCTCCTCCCTGCC

[0417] GGGGCTGCGTGCAACCCTGGGGTTGGGGGTTCTGGGGACTGGCTTTCCCATAATTTCCTTTCAA

[0418] GGTGGGGAGGGAGGTAGAGGGGTGATGTGGGGAGTACGCTGCAGGGCCTCACTCCTTTTGCAGA

[0419] CCACAGTCCATGCCATCACTGCCACCCAGAAGACTGTGGATGGCCCCTCCGGGAAACTGTGGCG

[0420] TGATGGCCGCGGGGCTCTCCAGAACATCATCCCTGCCTCTACTGGCGCTGCCAAGGCTGTGGGC

[0421] AAGGTCATCCCTGAGCTGAACGGGAAGCTCACTGGCATGGCCTTCCGTGTCCCCACTGCCAACG

[0422] TGTCAGTGGTGGACCTGACCTGCCGTCTAGAAAAACCTGCCAAATATGATGACATCAAGAAGGT

[0423] GGTGAAGCAGGCGTCGGAGGGCCCCCTCAAGGGCATCCTGGGCTACACTGAGCACCAGGTGGTC

[0424] TCCTCTGACTTCAACAGCGACACCCACTCCTCCACCTTTGACGCTGGGGCTGGCATTGCCCTCA

[0425] ACGACCACTTTGTCAAGCTCATTTCCTGGTATGTGGCTGGGGCCAGAGACTGGCTCTTAAAAAG

[0426] TGCAGGGTCTGGCGCCCTCTGGTGGCTGGCTCAGAAAAAGGGCCCTGACAACTCTTTACATCTT

[0427] CTAGGTATGACAACGAGTTCGGATATAGCAATAGAGTGGTCGATCTGATGGCTCATATGGCTAG

[0428] CAAAGAGGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAAC

[0429] CCTGGACCTATGGCCCTCCCTGTCACCGCCCTGCTCCTACCCCTGGCCTTGCTGCTGCACGCCG

[0430] CGCGCCCCAACTGGGTCAATGTGATTTCCGACCTGAAGAAGATTGAGGACCTCATCCAGTCTAT

[0431] GCATATTGACGCTACCCTGTACACCGAGAGCGATGTGCACCCCTCTTGTAAGGTGACCGCCATG

[0432] AAGTGCTTCCTGCTGGAACTGCAGGTCATCTCGCTTGAGTCTGGTGATGCTAGCATCCATGACA

[0433] CCGTGGAGAACCTGATCATCCTGGCCAACAACTCCTTGAGCAGTAATGGCAACGTGACTGAGTC

[0434] CGGCTGCAAAGAATGCGAGGAGCTGGAGGAGAAAAACATCAAGGAGTTCTTGCAGTCCTTCGTC

[0435] CACATCGTACAGATGTTTATTAACACGAGTGCGTTGAGCAACTCCATCATGTATTTCTCGCACT

[0436] TTGTGCCAGTGTTCCTGCCCGCCAAGCCTACCACAACTCCGGCGCCGCGTCCCCCGACTCCCGC

[0437] ACCGACCATCGCATCACAGCCACTGTCTCTGCGCCCAGAGGCCTGTCGGCCTGCGGCTGGAGGC

[0438] GCGGTGCACACGCGCGGCCTGGACTTCGCTTGTGACATCTACATATGGGCTCCTCTAGCTGGTA

[0439] CCTGCGGGGTTCTCCTGCTTTCCCTGGTGATCACCTGATAGTAAGCGGCCGCGTCGAGTCTAGA

[0440] GGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTT

[0441] GCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAA

[0442] TGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAG

[0443] GACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG

[0444] ATTTGGCTACAGCAACAGGGTGGTGGACCTCATGGCCCACATGGCCTCCAAGGAGTAAGACCCC

[0445] TGGACCACCAGCCCCAGCAAGAGCACAAGAGGAAGAGAGAGACCCTCACTGCTGGGGAGTCCCT

[0446] GCCACACTCAGTCCCCCACCACACTGAATCTCCCCTCCTCACAGTTGCCATGTAGACCCCTTGA

[0447] AGAGGGGAGGGGCCTAGGGAGCCGCACCTTGTCATGTACCATCAATAAAGTACCCTGTGCTCAA

[0448] CCAGTTACTTGTCCTGTCTTATTCTAGGGTCTGGGGCAGAGGGGAGGGAAGCTGGGCTTGTGTC

[0449] AAGGTGAGACATTCTTGCTGGGGAGGGACCTGGTATGTTCTCCTCAGACTGAGGGTAGGGCCTC

[0450] CAAACAGCCTTGCTTGCTTCGAGAACCATTTGCTTCCCGCTCAGACGTCTTGAGTGCTACAGGA

[0451] AGCTGGCACCACTACTTCAGAGAACAAGGCCTTTTCCTCTCCTCGCTCCAGTCCTAGGCTATCT

[0452] GCTGTTGGCCAAACATGGAAGAAGCTATTCTGTGGGCAGCCCCAGGGAGGCTGACAGGTGGAGG

[0453] AAGTCAGGGCTCGCACTGGGCTCTGACGCTGACTGGTTAGTGGAGCTCAGCCTGGAGCTGAGCT

[0454] GCAGCGGGCAATTCCAGCTTGGCCTCCGCAGCTGTGAGGTCTTGAGCACGTGCTCTATTGCCCA

[0455] TGGCCAAGCTTGCATGGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACG

[0456] CTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGC TCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTT CGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCG CTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAAC TATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACA GGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGG CTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGA GTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGC AGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACTACCAATGC TTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCC CCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACC GCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAG CGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTA GAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGT GTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACA TGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTA AGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCC ATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATG CGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTT TAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTT GAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACC AGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACAC G GAAAT G T T GAAT AC T C AT AC T C T T C C T T T T T C AAT AT T AT T GAAG C AT T T AT C AG G G T T AT T G TCTCATGAGCGGATACATA

[0457] SEQ ID NO: 117 - GAPDH-CAR19-sIL15 KI template plasmid

[0458] CGCGTCGCGAGGCCATATGGCTGAGGCTCCCACCTTTCTCATCCAAGACTGGCTCCTCCCTGCC GGGGCTGCGTGCAACCCTGGGGTTGGGGGTTCTGGGGACTGGCTTTCCCATAATTTCCTTTCAA GGTGGGGAGGGAGGTAGAGGGGTGATGTGGGGAGTACGCTGCAGGGCCTCACTCCTTTTGCAGA CCACAGTCCATGCCATCACTGCCACCCAGAAGACTGTGGATGGCCCCTCCGGGAAACTGTGGCG TGATGGCCGCGGGGCTCTCCAGAACATCATCCCTGCCTCTACTGGCGCTGCCAAGGCTGTGGGC AAGGTCATCCCTGAGCTGAACGGGAAGCTCACTGGCATGGCCTTCCGTGTCCCCACTGCCAACG TGTCAGTGGTGGACCTGACCTGCCGTCTAGAAAAACCTGCCAAATATGATGACATCAAGAAGGT GGTGAAGCAGGCGTCGGAGGGCCCCCTCAAGGGCATCCTGGGCTACACTGAGCACCAGGTGGTC TCCTCTGACTTCAACAGCGACACCCACTCCTCCACCTTTGACGCTGGGGCTGGCATTGCCCTCA ACGACCACTTTGTCAAGCTCATTTCCTGGTATGTGGCTGGGGCCAGAGACTGGCTCTTAAAAAG TGCAGGGTCTGGCGCCCTCTGGTGGCTGGCTCAGAAAAAGGGCCCTGACAACTCTTTACATCTT CTAGGTATGACAACGAGTTCGGATATAGCAATAGAGTGGTCGATCTGATGGCTCATATGGCTAG CAAAGAGGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAAC CCTGGACCTATGGCGCTTCCCGTGACCGCCCTGTTGCTGCCCCTGGCTCTCCTCTTGCACGCCG CTCGGCCTGACTACAAGGATGACGATGATAAGGACATCCAGATGACCCAGACGACCTCGTCCCT GTCTGCGTCCCTGGGGGACCGCGTCACCATCTCATGTCGTGCTTCTCAGGACATCTCTAAGTAC CTAAACTGGTACCAGCAGAAGCCCGACGGCACCGTCAAGCTGCTTATCTACCATACCTCCCGCC TGCACTCTGGCGTCCCATCGCGCTTCTCGGGTAGCGGATCTGGCACCGACTACAGCCTGACCAT CAGCAACCTAGAACAGGAGGACATCGCCACTTACTTCTGTCAACAGGGTAACACGCTACCCTAT ACCTTCGGCGGCGGCACTAAGCTGGAGATTACCGGTTCGACCAGCGGCTCCGGGAAGCCCGGTT CCGGAGAGGGCTCCACTAAAGGCGAGGTGAAACTCCAGGAGAGCGGCCCCGGCTTAGTGGCTCC TTCCCAGAGCCTGTCCGTGACCTGCACTGTAAGCGGCGTGTCGCTGCCCGACTACGGTGTCAGT TGGATCCGCCAGCCACCCCGCAAGGGGCTGGAGTGGCTCGGTGTGATCTGGGGCTCCGAGACCA C T T AT T AC AAC TCCGCTCT T AAAAG T AG G T T GAG AAT CAT C AAGGAC AAC T C AAAG T C T C AAG T C T T C C T C AAAAT GAAT AG T T T G C AGAC T GAT GAT AC CGCGATCTATTACTGTGC C AAG C AC TAG TACTACGGCGGTAGCTACGCAATGGATTATTGGGGCCAGGGAACATCCGTTACCGTGTCCTCTA TTGAGGTGATGTACCCCCCCCCGTACCTGGACAACGAGAAATCCAATGGGACCATCATCCACGT TAAGGGCAAGCACCTGTGCCCATCCCCGCTCTTCCCTGGGCCCTCTAAGCCCTTCTGGGTCCTA GTGGTGGTCGGTGGAGTGCTGGCGTGCTACTCTCTGCTGGTGACAGTGGCGTTCATAATCTTCT GGGTGCGCTCTAAGCGCAGCCGCCTCCTCCACAGTGACTACATGAACATGACTCCGCGACGCCC CGGCCCGACGCGCAAGCACTACCAGCCGTACGCCCCGCCTAGGGACTTTGCAGCGTATAGGTCG CGCGTAAAATTTTCCCGCTCGGCGGACGCTCCCGCCTACCAGCAGGGACAGAACCAGCTCTACA ACGAGTTAAACCTGGGCCGCCGCGAGGAGTACGACGTGCTGGATAAGCGTAGAGGCCGCGATCC AGAGATGGGCGGCAAGCCGCGCCGCAAAAACCCTCAGGAGGGCCTGTACAACGAGCTGCAGAAG GACAAAATGGCCGAGGCCTACAGCGAGATCGGTATGAAGGGCGAGCGGCGTCGGGGCAAGGGAC ATGACGGCCTGTACCAGGGCCTTTCCACCGCAACCAAGGATACCTACGACGCACTGCACATGCA GGCCCTGCCGCCGCGGCGAAGAAAGCGCGGATCCGGTGAGGGGCGTGGCAGTCTGCTGACCTGC GGGGATGTGGAGGAGAATCCAGGACCTATGTATCGTATGCAGCTGCTGTCTTGTATTGCTCTGT CGTTGGCCCTTGTTACAAACAGCAATTGGGTCAATGTAATCAGCGACCTGAAGAAGATAGAAGA CCTGATCCAGTCCATGCATATTGACGCCACCCTGTACACGGAGAGCGATGTGCACCCCTCTTGT AAGGTGACTGCCATGAAGTGCTTTCTGCTGGAGCTCCAGGTCATTTCGCTGGAGTCAGGAGACG CTTCGATCCATGACACCGTGGAGAACCTGATTATCCTGGCCAACAACTCCCTGTCCTCCAACGG CAATGTGACCGAGAGCGGGTGCAAAGAGTGCGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTC CTGCAGTCCTTCGTCCACATCGTGCAGATGTTTATTAACACTTCATGATAGTAAGCGGCCGCGT CGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCA TCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTT CCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGG GGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTG GGCTCTATGGATTTGGCTACAGCAACAGGGTGGTGGACCTCATGGCCCACATGGCCTCCAAGGA G T AAGAC C C C T G G C C AC C AGC C C C AGC AAGAG C C AAGAG GAAG GAGAG C C C T C C T GC T G GGGAGTCCCTGCCACACTCAGTCCCCCACCACACTGAATCTCCCCTCCTCACAGTTGCCATGTA GACCCCTTGAAGAGGGGAGGGGCCTAGGGAGCCGCACCTTGTCATGTACCATCAATAAAGTACC CTGTGCTCAACCAGTTACTTGTCCTGTCTTATTCTAGGGTCTGGGGCAGAGGGGAGGGAAGCTG GGCTTGTGTCAAGGTGAGACATTCTTGCTGGGGAGGGACCTGGTATGTTCTCCTCAGACTGAGG GTAGGGCCTCCAAACAGCCTTGCTTGCTTCGAGAACCATTTGCTTCCCGCTCAGACGTCTTGAG TGCTACAGGAAGCTGGCACCACTACTTCAGAGAACAAGGCCTTTTCCTCTCCTCGCTCCAGTCC TAGGCTATCTGCTGTTGGCCAAACATGGAAGAAGCTATTCTGTGGGCAGCCCCAGGGAGGCTGA CAGGTGGAGGAAGTCAGGGCTCGCACTGGGCTCTGACGCTGACTGGTTAGTGGAGCTCAGCCTG GAGCTGAGCTGCAGCGGGCAATTCCAGCTTGGCCTCCGCAGCTGTGAGGTCTTGAGCACGTGCT CTATTGCCCATGGCCAAGCTTGCATGGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACA AAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCC CCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCC TTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGT AGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTT ATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCC ACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGC CTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTT CGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTT GTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTA CTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTT GCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTG

[0459] CAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGG

[0460] AAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGC

[0461] CGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAG

[0462] GCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAG

[0463] GCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTT

[0464] GTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTA

[0465] CTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGA

[0466] ATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACAT

[0467] AGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCT

[0468] TACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTT

[0469] TACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATA

[0470] AGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATC AGGGTTATTGTCTCATGAGCGGATACATA

[0471] SEQ ID NO: 118 - GAPDH-CAR22.19-sIL15 KI template plasmid

[0472] CGCGTCGCGAGGCCATATGGCTGAGGCTCCCACCTTTCTCATCCAAGACTGGCTCCTCCCTGCC

[0473] GGGGCTGCGTGCAACCCTGGGGTTGGGGGTTCTGGGGACTGGCTTTCCCATAATTTCCTTTCAA

[0474] GGTGGGGAGGGAGGTAGAGGGGTGATGTGGGGAGTACGCTGCAGGGCCTCACTCCTTTTGCAGA

[0475] CCACAGTCCATGCCATCACTGCCACCCAGAAGACTGTGGATGGCCCCTCCGGGAAACTGTGGCG

[0476] TGATGGCCGCGGGGCTCTCCAGAACATCATCCCTGCCTCTACTGGCGCTGCCAAGGCTGTGGGC

[0477] AAGGTCATCCCTGAGCTGAACGGGAAGCTCACTGGCATGGCCTTCCGTGTCCCCACTGCCAACG

[0478] TGTCAGTGGTGGACCTGACCTGCCGTCTAGAAAAACCTGCCAAATATGATGACATCAAGAAGGT

[0479] GGTGAAGCAGGCGTCGGAGGGCCCCCTCAAGGGCATCCTGGGCTACACTGAGCACCAGGTGGTC

[0480] TCCTCTGACTTCAACAGCGACACCCACTCCTCCACCTTTGACGCTGGGGCTGGCATTGCCCTCA

[0481] ACGACCACTTTGTCAAGCTCATTTCCTGGTATGTGGCTGGGGCCAGAGACTGGCTCTTAAAAAG

[0482] TGCAGGGTCTGGCGCCCTCTGGTGGCTGGCTCAGAAAAAGGGCCCTGACAACTCTTTACATCTT

[0483] CTAGGTATGACAACGAGTTCGGATATAGCAATAGAGTGGTCGATCTGATGGCTCATATGGCTAG

[0484] CAAAGAGGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAAC

[0485] CCTGGACCTATGGCGCTGCCGGTGACGGCCTTACTCTTGCCTCTCGCTCTCCTGCTGCATGCTG

[0486] CTCGCCCTGACTACAAGGATGACGATGATAAGCAGGTGCAGCTTCAGCAGTCTGGGCCAGGCCT

[0487] CGTCAAGCCCTCTCAGACCCTGAGCCTGACCTGTGCCATCTCCGGCGACTCCGTATCATCTAAC

[0488] AGCGCGGCCTGGAACTGGATCCGGCAGTCACCGTCCCGCGGACTCGAGTGGCTGGGGCGCACTT

[0489] ACTACCGCTCCAAATGGTACAACGACTATGCCGTGAGCGTGAAGTCCCGCATCACAATTAATCC

[0490] CGACACCTCCAAAAACCAGTTCTCCTTGCAGCTCAACAGTGTTACCCCCGAGGACACCGCCGTG

[0491] TACTACTGTGCTCGCGAGGTGACAGGAGACCTGGAAGATGCCTTCGACATCTGGGGCCAGGGCA

[0492] CCATGGTAACCGTGTCCTCCGGTGGCGGAGGATCCGATATTCAGATGACCCAGAGCCCTTCTTC

[0493] CCTGTCCGCTTCCGTGGGAGACAGGGTCACCATCACCTGCCGCGCCTCGCAGACCATTTGGTCC

[0494] TACCTGAACTGGTACCAGCAGCGCCCGGGGAAGGCGCCGAACCTGCTGATTTACGCGGCGTCTT

[0495] CGCTACAAAGCGGCGTTCCCAGCAGGTTTTCGGGCCGCGGCTCTGGTACTGACTTCACCCTCAC

[0496] TATCTCCTCCCTGCAGGCCGAGGACTTTGCTACGTACTACTGTCAGCAGAGCTACAGCATCCCC

[0497] CAGACATTCGGCCAGGGAACCAAATTAGAGATCAAGGCCGCAGCCGGCACGGAGGCTGCGGCTA

[0498] AGGAAGCGGCCGCGAAGGAGGCAGCTGCGAAGGACATCCAGATGACCCAGACCACCAGTAGTCT

[0499] TAGCGCATCGCTGGGGGACCGCGTGACCATCTCCTGTCGCGCGAGCCAAGACATCTCTAAGTAC

[0500] CTTAACTGGTACCAGCAGAAGCCGGACGGCACTGTGAAGCTGTTGATCTACCACACGTCCCGGC TGCATTCGGGTGTCCCCTCTCGGTTCAGCGGCAGTGGTTCCGGGACCGACTACTCTCTAACCAT CTCTAACCTGGAGCAAGAGGATATCGCCACCTATTTCTGTCAACAGGGAAATACGCTGCCCTAC ACCTTCGGCGGTGGCACCAAGCTAGAGATCACCGGCTCCACCTCTGGCTCCGGTAAGCCGGGCT CTGGCGAGGGATCTACCAAGGGCGAAGTTAAGCTGCAGGAGAGCGGTCCCGGCCTGGTGGCTCC TTCACAGAGTCTGAGTGTCACCTGCACTGTCAGCGGCGTGTCCCTGCCAGACTACGGCGTCAGT TGGATACGACAGCCGCCCCGCAAAGGTTTGGAGTGGCTGGGTGTGATCTGGGGCAGCGAGACCA CAT AT T AC AAC T C T G C T T T AAAT C C C G C C T GAC TAT CAT C AAGGAT AC T C T AG T C C C AG G T GTTCCTGAAAATGAATTCGCTGCAGACTGATGACACCGCGATCTACTATTGCGCCAAGCACTAC TACTACGGTGGGTCCTACGCCATGGATTATTGGGGCCAGGGCACCAGCGTGACTGTAAGTTCTA TTGAAGTGATGTATCCCCCACCCTATCTTGACAACGAGAAGTCGAATGGCACCATCATCCATGT GAAGGGCAAGCACTTGTGCCCATCGCCCCTGTTCCCGGGACCAAGCAAGCCCTTCTGGGTCCTG GTCGTGGTGGGCGGCGTTCTGGCGTGCTACTCCCTACTGGTGACTGTGGCCTTCATCATCTTCT GGGTGCGCTCGAAGAGATCTAGGCTCCTCCACTCCGACTACATGAACATGACTCCTCGTAGGCC CGGGCCTACCCGCAAGCACTACCAGCCCTATGCCCCTCCGCGGGACTTCGCCGCATACCGCTCC CGCGTGAAATTTTCTCGCAGCGCGGACGCCCCTGCCTACCAGCAGGGTCAGAACCAGCTGTACA ATGAGCTGAACCTGGGCCGGCGCGAGGAGTACGACGTGCTGGACAAACGCCGTGGCCGTGACCC GGAGATGGGCGGAAAGCCGCGTAGAAAGAACCCTCAGGAGGGCCTTTACAACGAGTTACAGAAG GACAAGATGGCTGAGGCCTACAGCGAGATTGGTATGAAGGGAGAACGCCGCCGTGGCAAAGGCC ACGACGGCCTTTATCAGGGCTTGTCCACAGCTACCAAAGACACTTACGACGCACTGCATATGCA GGCCCTGCCCCCGCGCCGACGTAAGCGCGGTTCTGGGGAGGGCCGAGGCTCCCTGCTGACCTGC GGGGATGTGGAGGAGAATCCAGGCCCTATGTACAGGATGCAACTACTGTCTTGCATTGCTCTGT CGCTTGCACTGGTGACCAACAGCAACTGGGTCAACGTGATCAGCGACCTGAAGAAGATTGAGGA TCTGATCCAGTCTATGCACATTGATGCTACGCTGTACACCGAGTCCGATGTGCACCCCTCTTGT AAGGTCACTGCCATGAAGTGTTTTCTGCTGGAGCTGCAGGTCATATCACTCGAGAGCGGGGACG CTAGCATCCACGATACCGTGGAGAACCTCATCATCCTGGCCAACAACTCCCTGTCGTCGAATGG CAACGTGACTGAGAGCGGGTGCAAGGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTC CTGCAGTCCTTTGTCCACATCGTGCAGATGTTTATTAACACGTCGTGATAGTAAGCGGCCGCGT CGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCA TCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTT CCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGG GGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTG GGCTCTATGGATTTGGCTACAGCAACAGGGTGGTGGACCTCATGGCCCACATGGCCTCCAAGGA G T AAGAC C C C T G GAC C AC C AGC C C C AGC AAGAG C AC AAGAG GAAGAGAGAGAC C C T C AC T GC T G GGGAGTCCCTGCCACACTCAGTCCCCCACCACACTGAATCTCCCCTCCTCACAGTTGCCATGTA GACCCCTTGAAGAGGGGAGGGGCCTAGGGAGCCGCACCTTGTCATGTACCATCAATAAAGTACC CTGTGCTCAACCAGTTACTTGTCCTGTCTTATTCTAGGGTCTGGGGCAGAGGGGAGGGAAGCTG GGCTTGTGTCAAGGTGAGACATTCTTGCTGGGGAGGGACCTGGTATGTTCTCCTCAGACTGAGG GTAGGGCCTCCAAACAGCCTTGCTTGCTTCGAGAACCATTTGCTTCCCGCTCAGACGTCTTGAG TGCTACAGGAAGCTGGCACCACTACTTCAGAGAACAAGGCCTTTTCCTCTCCTCGCTCCAGTCC TAGGCTATCTGCTGTTGGCCAAACATGGAAGAAGCTATTCTGTGGGCAGCCCCAGGGAGGCTGA CAGGTGGAGGAAGTCAGGGCTCGCACTGGGCTCTGACGCTGACTGGTTAGTGGAGCTCAGCCTG GAGCTGAGCTGCAGCGGGCAATTCCAGCTTGGCCTCCGCAGCTGTGAGGTCTTGAGCACGTGCT CTATTGCCCATGGCCAAGCTTGCATGGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACA AAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCC CCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCC TTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGT AGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTT ATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCC ACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGC CTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTT

[0501] CGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTT

[0502] GTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTA

[0503] CTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTT

[0504] GCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTG

[0505] CAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGG

[0506] AAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGC

[0507] CGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAG

[0508] GCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAG

[0509] GCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTT

[0510] GTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTA

[0511] CTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGA

[0512] ATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACAT

[0513] AGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCT

[0514] TACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTT

[0515] TACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATA

[0516] AGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATC AGGGTTATTGTCTCATGAGCGGATACATA

[0517] SEQ ID NO: 119 - GAPDH-CAR.Mesothelin-anti-PDl ScFv-B7H3-TriKE KI template plasmid

[0518] CGCGTCGCGAGGCCATATGGCTGAGGCTCCCACCTTTCTCATCCAAGACTGGCTCCTCCCTGCC

[0519] GGGGCTGCGTGCAACCCTGGGGTTGGGGGTTCTGGGGACTGGCTTTCCCATAATTTCCTTTCAA

[0520] GGTGGGGAGGGAGGTAGAGGGGTGATGTGGGGAGTACGCTGCAGGGCCTCACTCCTTTTGCAGA

[0521] CCACAGTCCATGCCATCACTGCCACCCAGAAGACTGTGGATGGCCCCTCCGGGAAACTGTGGCG

[0522] TGATGGCCGCGGGGCTCTCCAGAACATCATCCCTGCCTCTACTGGCGCTGCCAAGGCTGTGGGC

[0523] AAGGTCATCCCTGAGCTGAACGGGAAGCTCACTGGCATGGCCTTCCGTGTCCCCACTGCCAACG

[0524] TGTCAGTGGTGGACCTGACCTGCCGTCTAGAAAAACCTGCCAAATATGATGACATCAAGAAGGT

[0525] GGTGAAGCAGGCGTCGGAGGGCCCCCTCAAGGGCATCCTGGGCTACACTGAGCACCAGGTGGTC

[0526] TCCTCTGACTTCAACAGCGACACCCACTCCTCCACCTTTGACGCTGGGGCTGGCATTGCCCTCA

[0527] ACGACCACTTTGTCAAGCTCATTTCCTGGTATGTGGCTGGGGCCAGAGACTGGCTCTTAAAAAG

[0528] TGCAGGGTCTGGCGCCCTCTGGTGGCTGGCTCAGAAAAAGGGCCCTGACAACTCTTTACATCTT

[0529] CTAGGTATGACAACGAGTTCGGATATAGCAATAGAGTGGTCGATCTGATGGCTCATATGGCTAG

[0530] CAAAGAGGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAAC

[0531] CCTGGACCTATGGCGCTGCCTGTGACAGCTCTGCTCCTGCCTCTTGCGCTGCTGCTGCATGCCG

[0532] CCAGGCCAGGTAAGCCCATCCCTAACCCGCTGCTGGGCCTCGACTCCACCGGAAGTGGGGGCAC

[0533] CGGCTCACAGGTCCAGCTGCAGCAGTCTGGGCCGGAACTCGAGAAGCCCGGGGCTAGCGTTAAG

[0534] ATCAGTTGCAAGGCGTCGGGCTACTCCTTCACCGGCTACACCATGAACTGGGTTAAACAGTCCC

[0535] ATGGAAAGTCGCTGGAGTGGATAGGCCTAATCACCCCGTACAACGGTGCATCCTCCTACAACCA

[0536] GAAGTTTCGGGGTAAGGCCACCTTAACCGTGGACAAGTCTTCCAGCACCGCCTACATGGATCTC

[0537] CTCTCCTTGACCTCCGAGGACAGCGCCGTCTACTTCTGTGCACGTGGCGGTTACGACGGCCGTG

[0538] GTTTCGATTATTGGGGCCAGGGCACCACAGTAACCGTCTCTAGTGGCGGAGGTGGCTCTGGAGG

[0539] CGGTGGATCTAGTGGTGGCGGATCAGACATCGAGCTCACTCAGTCTCCAGCCATCATGAGTGCC

[0540] AGTCCTGGCGAGAAGGTGACGATGACCTGCTCCGCCTCCTCATCTGTTAGCTACATGCACTGGT

[0541] ACCAGCAGAAGAGCGGTACTTCCCCCAAGCGGTGGATCTACGACACCAGTAAGCTGGCTTCCGG

[0542] AGTCCCCGGCAGGTTTAGCGGATCGGGCTCGGGCAATTCCTACTCGTTAACTATCTCGTCCGTG GAGGCCGAGGATGACGCTACCTACTACTGTCAGCAGTGGTCTAAGCATCCGCTGACGTACGGAG CCGGGACCAAGCTGGAGATTAAGGCGTCTATTGAGGTGATGTACCCCCCCCCGTACCTGGACAA CGAGAAATCCAATGGGACCATCATCCACGTTAAGGGCAAGCACCTGTGCCCATCCCCGCTCTTC CCTGGGCCCTCTAAGCCCTTCTGGGTCCTAGTGGTGGTCGGTGGAGTGCTGGCGTGCTACTCTC TGCTGGTGACAGTGGCGTTCATAATCTTCTGGGTGAGGTCTAAACGCTCCCGCCTGCTGCACTC GGATTACATGAATATGACTCCGCGCCGTCCCGGCCCCACTCGCAAGCACTACCAGCCGTACGCC CCCCCCCGCGACTTTGCCGCTTACCGCTCCCGTGTGAAATTTTCCCGCTCCGCCGATGCCCCGG CTTATCAGCAGGGACAGAACCAGCTGTACAACGAGCTCAACCTGGGACGCAGGGAGGAGTACGA TGTCCTGGACAAGCGGAGGGGACGAGATCCAGAGATGGGAGGAAAGCCTCGACGCAAGAATCCT CAGGAGGGCCTTTATAACGAGTTGCAGAAGGACAAAATGGCCGAGGCATACAGCGAGATCGGTA TGAAGGGCGAGAGACGCCGCGGGAAGGGCCACGACGGCCTGTACCAGGGGCTCTCTACCGCGAC AAAAGACACCTATGACGCCTTACATATGCAGGCCCTTCCACCCCGCAGACGCAAGCGCGGCTCC GGAGAAGGCCGTGGTTCTCTTTTGACCTGCGGGGACGTGGAAGAGAACCCCGGCCCGATGGTGT TGCAAACTCAGGTGTTCATCAGTCTCCTTTTGTGGATTAGCGGCGCATACGGAGATTACAAGGA CGACGATGACAAAGAAATCGTGCTGACGCAGTCCCCCGCGACCCTCTCCTTATCTCCAGGCGAG CGCGCCACTCTTTCCTGCCGAGCCTCTAAAGGCGTGAGCACCTCCGGCTACTCCTACTTGCATT GGTACCAACAGAAGCCCGGTCAGGCCCCCCGCCTGCTTATCTACCTGGCGTCGTATCTGGAGTC GGGCGTGCCAGCTCGCTTCTCCGGCTCGGGTTCCGGGACCGATTTCACACTGACAATCAGCTCG CTGGAGCCTGAGGACTTTGCCGTGTACTACTGTCAGCACTCTCGCGACCTGCCTCTAACCTTCG GAGGTGGCACCAAGGTGGAGATCAAGCGCGGGTCAACCTCGGGTAGCGGCAAGCCCGGGAGCGG TGAGGGCAGCACCAAGGGCCAAGTGCAGCTGGTACAGTCTGGCGTGGAGGTGAAGAAGCCTGGC GCAAGCGTCAAAGTGTCTTGCAAGGCTTCCGGCTACACCTTCACCAACTATTACATGTATTGGG TGCGCCAGGCGCCCGGCCAAGGCCTGGAGTGGATGGGCGGCATTAATCCCTCAAATGGAGGGAC T AAC T T C AAC GAGAAAT T T AAGAAC C GO G T GAG C C T GAG AAC T GAG T C C T C T AC GAC GAG C G C C TACATGGAGCTGAAATCCCTGCAGTTCGATGACACCGCTGTTTACTACTGTGCCCGCCGAGATT ACCGCTTCGATATGGGCTTTGACTACTGGGGTCAGGGCACCACTGTGACGGTTTCCTCCCGGAG AAAGCGGGGTAGTGGCGCTACCAACTTCTCTCTGCTGAAGCAGGCCGGTGATGTGGAGGAAAAT CCGGGTCCCATGGACATGCGTGTGCTGGCGCAGCTGCTGGGCTTGCTACTGCTGTGCTTCCCTG GCGCTCGCTGCCAGGTCCAGCTGGTGGAGAGCGGCGGTGGCCTGGTCCAGCCTGGCGGTTCTCT GCGCCTGTCGTGCGCCGCGTCCGGGCTGACCTTCTCGTCCTACAACATGGGCTGGTTCCGCCAG GCCCCCGGCCAGGGGCTGGAGGCTGTCGCAAGCATCACCTGGTCTGGTCGCGACACCTTCTATG CGGACTCCGTGAAGGGACGCTTCACCATTTCTCGTGATAACTCCAAGAATACCCTGTACCTTCA GATGAACTCACTGCGGGCAGAGGACACTGCTGTCTACTACTGCGCCGCCAACCCGTGGCCAGTA GCCGCGCCCCGGTCTGGGACCTATTGGGGCCAGGGTACGCTGGTGACCGTGTCCTCTTCTGGTG GAGGCGGGAGCGGAGGCGGCGGTAGTGGTGGAGGAGGCTCTGGCGGTGGTGGAAGCGGCAACTG GGTCAACGTCATTTCCGACTTGAAAAAGATAGAGGACCTCATTCAGTCCATGCATATTGACGCC ACCCTGTACACTGAGAGCGACGTGCACCCGTCCTGTAAAGTTACAGCCATGAAGTGTTTTTTAC TGGAACTGCAAGTCATCTCTCTGGAGAGCGGTGATGCTAGCATCCACGATACAGTGGAGAACTT GATCATCCTGGCTAACAACAGCTTATCTAGCAATGGCAACGTGACGGAGTCTGGGTGCAAGGAG TGTGAGGAGCTGGAGGAGAAGAATATCAAGGAGTTCTTGCAGAGCTTCGTGCACATCGTGCAGA TGTTTATCAACACCAGCGCCGAGGCTGCCAAAGAGGCCGCTAAAGAAGCGGCCAAAGAAGCCGC CAAGGCTCTGGAGGCGGAGGCAGCAAAGGAGGCTGCTAAGGAAGCTGCTAAGGAGGCCGCCAAG GCGCAGGTACAGCTCGTCGAGTCCGGGGGTGGCCTGGTTCAACCGGGAGGCAGCCTGCGCCTGT CCTGTGCTGCTAGCGGCTTCACCTTCTCCTCCTATTGGATGTACTGGGTGCGCCAGACCCCTGG GAAAGGCCTTGAGTGGGTCTCGACCATCAATCGCGACGGCTCCGCCACCTGGTACGCGGACTCC GTAAAGGGCCGTTTCACCATCTCTCGTGACAACGCGAAGAACACTGGTTACTTGCAGATGAACA GTCTCAAGCCGGACGACACAGCGGTGTATTACTGCGTGTCCGACCCGGACAACTATTCTAGCGA CGAGATGGTGCCCTATTGGGGCCAGGGCACCCAGGTGACCGTGAGTTCTGTGGACGAGCACCAC CACCACCACCACTGATAGTAAGCGGCCGCGTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATC AGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTG ACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTC TGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGA AGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGATTTGGCTACAGCAACAGGGTGG TGGACCTCATGGCCCACATGGCCTCCAAGGAGTAAGACCCCTGGACCACCAGCCCCAGCAAGAG CACAAGAGGAAGAGAGAGACCCTCACTGCTGGGGAGTCCCTGCCACACTCAGTCCCCCACCACA CTGAATCTCCCCTCCTCACAGTTGCCATGTAGACCCCTTGAAGAGGGGAGGGGCCTAGGGAGCC GCACCTTGTCATGTACCATCAATAAAGTACCCTGTGCTCAACCAGTTACTTGTCCTGTCTTATT CTAGGGTCTGGGGCAGAGGGGAGGGAAGCTGGGCTTGTGTCAAGGTGAGACATTCTTGCTGGGG AGGGACCTGGTATGTTCTCCTCAGACTGAGGGTAGGGCCTCCAAACAGCCTTGCTTGCTTCGAG AACCATTTGCTTCCCGCTCAGACGTCTTGAGTGCTACAGGAAGCTGGCACCACTACTTCAGAGA ACAAGGCCTTTTCCTCTCCTCGCTCCAGTCCTAGGCTATCTGCTGTTGGCCAAACATGGAAGAA GCTATTCTGTGGGCAGCCCCAGGGAGGCTGACAGGTGGAGGAAGTCAGGGCTCGCACTGGGCTC TGACGCTGACTGGTTAGTGGAGCTCAGCCTGGAGCTGAGCTGCAGCGGGCAATTCCAGCTTGGC CTCCGCAGCTGTGAGGTCTTGAGCACGTGCTCTATTGCCCATGGCCAAGCTTGCATGGTTTTTC CATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACC CGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCC GACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAT AGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACG AACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGT AAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTA GGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTG GTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAA ACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAA G GAT C T C AAGAAGAT C C T T T GAT C T T T T C T C T AC C AT G C T T AAT C G T GAG G C AC C T AT C T C AGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATA CGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTC CAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTT ATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAAT AGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGG CTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAA AGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTC ATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGA CTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCC GGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAA CGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCA CTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAAC AGGAAGGCAAAAT GCCGCAAAAAAGGGAATAAGGGCGACACGGAAAT GT T GAATAC T CATAC T C TTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATA

[0543] SEQ ID NO: 120 - anti-CD22 / 19 CAR (CAR22.19) DNA sequence

[0544] ATGGCGCTGCCGGTGACGGCCTTACTCTTGCCTCTCGCTCTCCTGCTGCATGCTGCTCGCCCTG ACTACAAGGATGACGATGATAAGCAGGTGCAGCTTCAGCAGTCTGGGCCAGGCCTCGTCAAGCC CTCTCAGACCCTGAGCCTGACCTGTGCCATCTCCGGCGACTCCGTATCATCTAACAGCGCGGCC TGGAACTGGATCCGGCAGTCACCGTCCCGCGGACTCGAGTGGCTGGGGCGCACTTACTACCGCT CCAAATGGTACAACGACTATGCCGTGAGCGTGAAGTCCCGCATCACAATTAATCCCGACACCTC CAAAAACCAGTTCTCCTTGCAGCTCAACAGTGTTACCCCCGAGGACACCGCCGTGTACTACTGT GCTCGCGAGGTGACAGGAGACCTGGAAGATGCCTTCGACATCTGGGGCCAGGGCACCATGGTAA CCGTGTCCTCCGGTGGCGGAGGATCCGATATTCAGATGACCCAGAGCCCTTCTTCCCTGTCCGC TTCCGTGGGAGACAGGGTCACCATCACCTGCCGCGCCTCGCAGACCATTTGGTCCTACCTGAAC TGGTACCAGCAGCGCCCGGGGAAGGCGCCGAACCTGCTGATTTACGCGGCGTCTTCGCTACAAA GCGGCGTTCCCAGCAGGTTTTCGGGCCGCGGCTCTGGTACTGACTTCACCCTCACTATCTCCTC CCTGCAGGCCGAGGACTTTGCTACGTACTACTGTCAGCAGAGCTACAGCATCCCCCAGACATTC GGCCAGGGAACCAAATTAGAGATCAAGGCCGCAGCCGGCACGGAGGCTGCGGCTAAGGAAGCGG CCGCGAAGGAGGCAGCTGCGAAGGACATCCAGATGACCCAGACCACCAGTAGTCTTAGCGCATC GCTGGGGGACCGCGTGACCATCTCCTGTCGCGCGAGCCAAGACATCTCTAAGTACCTTAACTGG TACCAGCAGAAGCCGGACGGCACTGTGAAGCTGTTGATCTACCACACGTCCCGGCTGCATTCGG GTGTCCCCTCTCGGTTCAGCGGCAGTGGTTCCGGGACCGACTACTCTCTAACCATCTCTAACCT GGAGCAAGAGGATATCGCCACCTATTTCTGTCAACAGGGAAATACGCTGCCCTACACCTTCGGC GGTGGCACCAAGCTAGAGATCACCGGCTCCACCTCTGGCTCCGGTAAGCCGGGCTCTGGCGAGG GATCTACCAAGGGCGAAGTTAAGCTGCAGGAGAGCGGTCCCGGCCTGGTGGCTCCTTCACAGAG TCTGAGTGTCACCTGCACTGTCAGCGGCGTGTCCCTGCCAGACTACGGCGTCAGTTGGATACGA CAGCCGCCCCGCAAAGGTTTGGAGTGGCTGGGTGTGATCTGGGGCAGCGAGACCACATATTACA ACTCTGCTTTAAAATCCCGCCTGACTATCATCAAGGATAACTCTAAGTCCCAGGTGTTCCTGAA AATGAATTCGCTGCAGACTGATGACACCGCGATCTACTATTGCGCCAAGCACTACTACTACGGT GGGTCCTACGCCATGGATTATTGGGGCCAGGGCACCAGCGTGACTGTAAGTTCTATTGAAGTGA T G T AT C C C C C AC C C T AT C T T GACAAC GAGAAG T C GAAT G G C AC C AT C AT C C AT G T GAAG G GC AA GCACTTGTGCCCATCGCCCCTGTTCCCGGGACCAAGCAAGCCCTTCTGGGTCCTGGTCGTGGTG GGCGGCGTTCTGGCGTGCTACTCCCTACTGGTGACTGTGGCCTTCATCATCTTCTGGGTGCGCT CGAAGAGATCTAGGCTCCTCCACTCCGACTACATGAACATGACTCCTCGTAGGCCCGGGCCTAC CCGCAAGCACTACCAGCCCTATGCCCCTCCGCGGGACTTCGCCGCATACCGCTCCCGCGTGAAA TTTTCTCGCAGCGCGGACGCCCCTGCCTACCAGCAGGGTCAGAACCAGCTGTACAATGAGCTGA ACCTGGGCCGGCGCGAGGAGTACGACGTGCTGGACAAACGCCGTGGCCGTGACCCGGAGATGGG CGGAAAGCCGCGTAGAAAGAACCCTCAGGAGGGCCTTTACAACGAGTTACAGAAGGACAAGATG GCTGAGGCCTACAGCGAGATTGGTATGAAGGGAGAACGCCGCCGTGGCAAAGGCCACGACGGCC TTTATCAGGGCTTGTCCACAGCTACCAAAGACACTTACGACGCACTGCATATGCAGGCCCTGCC CCCGCGC

[0545] SEQ ID NO: 121 - anti-CD22 / 19 CAR (CAR22.19) amino acid sequence

[0546] MALPVTALLLPLALLLHAARPDYKDDDDKQVQLQQSGPGLVKPSQTLSLTCAI SGDSVSSNSAA WNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRI T INPDTSKNQFSLQLNSVTPEDTAVYYC AREVTGDLEDAFDIWGQGTMVTVSSGGGGSDIQMTQSPSSLSASVGDRVTI TCRASQT IWSYLN WYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLT I SSLQAEDFATYYCQQSYS IPQTF GQGTKLE IKAAAGTEAAAKEAAAKEAAAKDIQMTQTTSSLSASLGDRVTISCRASQDI SKYLNW YQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTI SNLEQEDIATYFCQQGNTLPYTFG GGTKLE I TGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIR QPPRKGLEWLGVIWGSETTYYNSALKSRLT I IKDNSKSQVFLKMNSLQTDDTAI YYCAKHYYYG GSYAMDYWGQGTSVTVSS IEVMYPPPYLDNEKSNGT I IHVKGKHLCPSPLFPGPSKPFWVLVW GGVLACYSLLVTVAFI I FWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVK FSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM AEAYSE IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0547] SEQ ID NO: 122 - CISH left homology arm ATCATCTAACAGCGCGGCCTGGAACTGGATCCGGCAGTCACCGTCCCGCGGACTCGAGTGGCTG GGGCGCACTTACTACCGCTCCAAATGGTACAACGACTATGCCGTGAGCGTGAAGTCCCGCATCA CAATTAATCCCGACACCTCCAAAAACCAGTTCTCCTTGCAGCTCAACAGTGTTACCCCCGAGGA CACCGCCGTGTACTACTGTGCTCGCGAGGTGACAGGAGACCTGGAAGATGCCTTCGACATCTGG GGCCAGGGCACCATGGTAACCGTGTCCTCCGGTGGCGGAGGATCCGATATTCAGATGACCCAGA GCCCTTCTTCCCTGTCCGCTTCCGTGGGAGACAGGGTCACCATCACCTGCCGCGCCTCGCAGAC CATTTGGTCCTACCTGAACTGGTACCAGCAGCGCCCGGGGAAGGCGCCGAACCTGCTGATTTAC GCGGCGTCTTCGCTACAAAGCGGCGTTCCCAGCAGGTTTTCGGGCCGCGGCTCTGGTACTGACT TCACCCTCACTATCTCCTCCCTGCAGGCCGAGGACTTTGCTACGTACTACTGTCAGCAGAGCTA CAGCATCCCCCAGACATTCGGCCAGGGAACCAAATTAGAGATCAAGGCCGCAGCCGGCACGGAG GCTGCGGCTAAGGAAGCGGCCGCGAAGGAGGCAGCTGCGAAGGACATCCAGATGACCCAGACCA CCAGTAGTCTTAGCGCATCGCTGGGGGACCGCGTGACCATCTCCTGTCGCGCGAGCCAAGACAT CTCTAAGTACCTTAACTGGTACCAGCAGAAGCCGGACGGCACTGTGAAGCTGTTGATCTACCAC ACGTCCCGGCTGCATTCGGGTGTCCCCTCTCGGTTCAGCGGCAGTGGTTCCGGGACCGACTACT CTCTAACCATCTCTAACCTGGAGCAAGAGGATATCGCCACCTATTTCTGTCAACAGGGAAATAC GCTGCCCTACACCTTCGGCGGTGGCACCAAGCTAGAGATCACCGGCTCCACCTCTGGCTCCGGT AAGCCGGGCTCTGGCGAGGGATCTACCAAGGGCGAAGTTAAGCTGCAGGAGAGCGGTCCCGGCC TGGTGGCTCCTTCACAGAGTCTGAGTGTCACCTGCACTGTCAGCGGCGTGTCCCTGCCAGACTA CGGCGTCAGTTGGATACGACAGCCGCCCCGCAAAGGTTTGGAGTGGCTGGGTGTGATCTGGGGC AG C GAGAC C AC AT AT T AC AAC T C T G C T T T AAAAT C C C GC C T GAC TAT CAT C AAG GAT AAC T C T A AGTCCCAGGTGTTCCTGAAAATGAATTCGCTGCAGACTGATGACACCGCGATCTACTATTGCGC CAAGCACTACTACTACGGTGGGTCCTACGCCATGGATTATTGGGGCCAGGGCACCAGCGTGACT GTAAGTTCTATTGAAGTGATGTATCCCCCACCCTATCTTGACAACGAGAAGTCGAATGGCACCA TCATCCATGTGAAGGGCAAGCACTTGTGCCCATCGCCCCTGTTCCCGGGACCAAGCAAGCCCTT CTGGGTCCTGGTCGTGGTGGGCGGCGTTCTGGCGTGCTACTCCCTACTGGTGACTGTGGCCTTC ATCATCTTCTGGGTGCGCTCGAAGAGATCTAGGCTCCTCCACTCCGACTACATGAACATGACTC CTCGTAGGCCCGGGCCTACCCGCAAGCACTACCAGCCCTATGCCCCTCCGCGGGACTTCGCCGC ATACCGCTCCCGCGTGAAATTTTCTCGCAGCGCGGACGCCCCTGCCTACCAGCAGGGTCAGAAC CAGCTGTACAATGAGCTGAACCTGGGCCGGCGCGAGGAGTACGACGTGCTGGACAAACGCCGTG GCCGTGACCCGGAGATGGGCGGAAAGCCGCGTAGAAAGAACCCTCAGGAGGGCCTTTACAACGA GTTACAGAAGGACAAGATGGCTGAGGCCTACAGCGAGATTGGTATGAAGGGAGAACGCCGCCGT GGCAAAGGCCACGACGGCCTTTATCAGGGCTTGTCCACAGCTACCAAAGACACTTACGACGCAC TGCATATGCAGGCCCTGCCCCCGCGCCGACGTAAGCGCGGTTCTGGGGAGGGCCGAGGCTCCCT GCTGACCTGCGGGGATGTGGAGGAGAATCCAGGCCCTATGTACAGGATGCAACTACTGTCTTGC

[0548] ATTGCTCTGTCGCTTGCACTGGTGACCAACAGCAACTGGGTCAACGTGATCAGCGACCTGAAGA AGATTGAGGATCTGATCCAGTCTATGCACATTGATGCTACGCTGTACACCGAGTCCGATGTGCA CCCCTCTTGTAAGGTCACTGCCATGAAGTGTTTTCTGCTGGAGCTGCAGGTCATATCACTCGAG AGCGGGGACGCTAGCATCCACGATACCGTGGAGAACCTCATCATCCTGGCCAACAACTCCCTGT CGTCGAATGGCAACGTGACTGAGAGCGGGTGCAAGGAGTGCGAGGAACTGGAAGAGAAGAACAT CAAAGAGTTCCTGCAGTCCTTTGTCCACATCGTGCAGATGTTTATTAACACGTCGTGATAATAG GCGGCCGCGTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAG TTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCC ACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTC TGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGG GGATGCGGTGGGCTCTATGGGGAATCTGGTGAGTCTGAGGGGGGAGGCAGGCCTTTTCCTGAGT AGTCTGGTGGGAGAAGCTTAGTTCTGCCTTTGAGTCTGATTTGGGAGGCATAGCCCGGCCCAGG AGAGTCTGATGGGAGAGGCACAGCCCTCTCTAGGGAAGCGTGAAGCTGGAGGCACTGCCTTGAC TTGCACTGGATTGGTTACCCCAGCTAATTTCCACTATGTCCCTTGGCCCCCTCTGCACTTGCCT AGGCTGGTATTGGGGTTCCATTACGGCCAGCGAGGCCCGACAACACCTGCAGAAGATGCCAGAA GGCACGTTCTTAGTACGTGACAGCACGCACCCCAGCTACCTGTTCACGCTGTCAGTGAAAACCA

[0549] CTCGTGGCCCCACCAATGTACGCATTGAGTATGCCGACTCCAGCTTCCGTCTGGACTCCAACTG CTTGTCCAGGCCACGCATCCTGGCCTTTCCGGATGTGGTCAGCCTTGTGCAGCACTATGTGGCC TCCTGCACTGCTGATACCCGAAGCGACAGCCCCGATCCTGCTCCCACCCCGGCCCTGCCTATGC

[0550] CTAAGGAGGATGCGCCTAGTGACCCAGCACTGCCTGCTCCTCCACCAGCCACTGCTGTACACCT AAAACTGGTGCAGCCCTTTGTACGCAGAAGCAGTGCCCGCAGCCTGCAACACCTGTGCCGCCTT GTCATCAACCGTCTGGCCCATGGCCAAGCTTGCATGGTTTTTCCATAGGCTCCGCCCCCCTGAC

[0551] GAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACC AGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATA CCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTC

[0552] AGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACC GCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACT GGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTG

[0553] AAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGC CAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGG TGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTG

[0554] ATCTTTTCTACTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTC ATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGC CCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACC

[0555] AGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTAT TAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCC ATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCC

[0556] AACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCC TCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCAT AATTCTCTTACTGTCATGCCATCCGTAAG TGCTTTTCTGTGACTGGTGAGT CTCA CCAAGT

[0557] CATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATAC CGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTC TCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTT

[0558] CAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAA AAAGGGAATAAGGGCGACACGGAAAT GT T GAATAC T CATAC T C T T CC T T T T T CAATAT TAT T GA AGCAT T TAT CAGGGT TAT T GTC T CAT GAGCGGATACATA

[0559] SEQ ID NO: 127 - PFKFB4 left homology arm

[0560] GGCCCACAGCTGGGAGGAGGGGCCACCAGGCTCTGGGGCACATAAGACAGCCTCAGAGAGCTAA GGGAAGTTTCAGATTCCCTATTCCTCAGGCCTGAGACTCCCTGAGCCCGACAGGCCAAGGATGG GACTGAGCTCTGCAGTTTGGTGGGGTCCTTCTATAGCTGAGGGCTTCAGGAGGCCCTGTACCCC

[0561] AGTTCTGAGGGGACAGACCAGGCTGCAGACCAGGAAATGCCCATAGGCTTCTGGGGCACCTCTG ACACCTGCGTGCCAACACTTTAAACTAGGAAGGAGGCACCCCCAGGAGTTGAACCCCTTCCCTG CCCCACAGATGAGGATAAAATCCCCAAGCCAGGGGTTCCTCCTCAATTGACTCAGCCCTGAGAT

[0562] AGGAGGTGGGAGTGGCCTTGCTTTGGGTATTGCCTGACCTGGCTGCAGTGGAGCAGGAAACGAG GTGGCATCCTCCACCGCACCTTTGTTCTATTGTCCATCCACCCGCTATGTGGCACGAAGCTCTT GGCCATGTATTCTTAGGGAAGCTTGTGGTCTAGAGTCTTGCGGCTGTGGCTGCTACAGTGAGAA

[0563] TGTGAAGCCTGATGAACCACAGTGATGAGGACGGCAGACAGCAGGCATCTCATCTATTCCTGCC ATGGAGTGAACGCTCTGTGTTTTTATTGTACAGAACGTGGACATCTCAAGACCTCCAGAG SEQ ID NO: 128 - PFKFB4 right homology arm

[0564] GAAGCCCTTGTCACGGTGCCTGCTCACCAGTGACCATGTTCATCCACTGTGACCACTAGGCAGG

[0565] CACTGCTCTCTGCAGAGGGGGTCATTCCAGGCCCTCCAGTGTGTGTGATAGTCACCATGCCATG

[0566] CAGGGATATTCTTGAAGCCACACATGGCTGGCGGAACCCAGAGCCCCCACCCCAGCCCACCTGG

[0567] CTCTTTGTTGACAGTCGGCG CAAGGTTGTGCGTGGCTCCTGACCTGCTGCTAAGAGTCACTTG

[0568] ACCAGACTGCATCTGCATGGGCTGCGCGGAGGTTGCCCAGCCCCAGTTTCTTCCGGCGCAGCTC

[0569] TTAGGTGTTCACTCTCGCCAGCTCAGTTGGCTTTGTGAAGTGTGAAACCCTACAATGTGAAAGG

[0570] AAAGTGCTTGCTGTGATGTTCCTACTGTGGCCCAGCTGCCCAGCATGGACCTGGTGACTCTCCA

[0571] CAGGGCCTCTACCATCCTCTCTGTGGCCACTTCCTGAGCCAGAGGCCAGGTCTTCATGGGGCCC

[0572] TGAGCTTCTGCTGCCTCTGGTGAGAGGGAGAGCCCTTCCCATCCTTACCCACCAGGAACTAGAG

[0573] CCCCAACCACAGCAGATGCTTCCTGGGCAGCCACTGGCCAGGCCGTTGTATCCATGTCACCCTT

[0574] AGTTGTGGGCATTCATGAAAGCAATGCGCTTGCTTCAGCACATTGGGATGCACAGAAACG

[0575] SEQ ID NO: 129 - PFKFB4 dsHDRT F primer

[0576] GGCCCACAGCTGGGAGGAGGGGCCACCAGGCTCTGGGGCACATAAG

[0577] SEQ ID NO: 130 - PFKFB4 dsHDRT R primer

[0578] CGTTTCTGTGCATCCCAATGTGCTG

[0579] SEQ ID NO: 131 - PFKFB4-IL12-tNGFR KI template plasmid

[0580] CGCGTCGCGAGGCCATATGGGGCCCACAGCTGGGAGGAGGGGCCACCAGGCTCTGGGGCACATA

[0581] AGACAGCCTCAGAGAGCTAAGGGAAGTTTCAGATTCCCTATTCCTCAGGCCTGAGACTCCCTGA

[0582] GCCCGACAGGCCAAGGATGGGACTGAGCTCTGCAGTTTGGTGGGGTCCTTCTATAGCTGAGGGC

[0583] TTCAGGAGGCCCTGTACCCCAGTTCTGAGGGGACAGACCAGGCTGCAGACCAGGAAATGCCCAT

[0584] AGGCTTCTGGGGCACCTCTGACACCTGCGTGCCAACACTTTAAACTAGGAAGGAGGCACCCCCA

[0585] GGAGTTGAACCCCTTCCCTGCCCCACAGATGAGGATAAAATCCCCAAGCCAGGGGTTCCTCCTC

[0586] AATTGACTCAGCCCTGAGATAGGAGGTGGGAGTGGCCTTGCTTTGGGTATTGCCTGACCTGGCT

[0587] GCAGTGGAGCAGGAAACGAGGTGGCATCCTCCACCGCACCTTTGTTCTATTGTCCATCCACCCG

[0588] CTATGTGGCACGAAGCTCTTGGCCATGTATTCTTAGGGAAGCTTGTGGTCTAGAGTCTTGCGGC

[0589] TGTGGCTGCTACAGTGAGAATGTGAAGCCTGATGAACCACAGTGATGAGGACGGCAGACAGCAG

[0590] GCATCTCATCTATTCCTGCCATGGAGTGAACGCTCTGTGTTTTTATTGTACAGAACGTGGACAT

[0591] CTCAAGACCTCCAGAGGAGGCTCTGGTGACTGTCCCCGCGCACCAGGGCTCCGGTGCCACCAAC

[0592] TTCAGCCTGCTCAAGCAGGCCGGGGACGTGGAGGAGAACCCGGGCCCTATGTGCCACCAGCAGC

[0593] TAGTCATAAGTTGGTTCTCCCTGGTGTTCTTAGCGTCACCTCTGGTGGCTATTTGGGAGCTCAA

[0594] AAAGGACGTGTACGTGGTCGAGCTCGACTGGTACCCTGACGCCCCCGGGGAGATGGTGGTTCTG

[0595] ACATGCGACACGCCCGAGGAGGACGGCATAACCTGGACCCTTGACCAGAGCAGCGAGGTGCTGG

[0596] GCTCGGGCAAGACCCTTACCATCCAGGTAAAGGAGTTCGGCGACGCTGGACAGTACACCTGTCA

[0597] CAAGGGCGGCGAGGTGCTGAGCCACTCTCTGTTGCTGCTCCACAAAAAGGAAGACGGCATCTGG

[0598] TCTACAGACATCCTGAAGGACCAGAAGGAGCCAAAGAACAAGACCTTCCTGCGGTGCGAGGCTA

[0599] AAAACTATTCTGGTAGATTCACCTGCTGGTGGCTCACTACCATCTCTACTGATCTCACCTTCTC

[0600] TGTGAAGTCATCCAGGGGGAGCTCGGACCCTCAGGGTGTGACCTGCGGTGCGGCTACCCTGAGC

[0601] GCGGAGCGCGTCCGTGGTGACAACAAGGAGTACGAGTACTCCGTGGAATGTCAGGAGGACAGTG

[0602] CCTGCCCAGCCGCTGAGGAATCGCTGCCCATCGAAGTGATGGTGGACGCCGTCCATAAGTTGAA

[0603] GTACGAAAACTACACCTCCTCCTTCTTTATCCGGGACATCATCAAGCCCGACCCCCCCAAGAAC

[0604] CTGCAACTGAAACCTCTGAAAAACTCACGCCAAGTAGAGGTATCTTGGGAGTACCCCGATACCT

[0605] GGTCGACCCCCCACAGCTATTTCTCTCTTACTTTCTGCGTGCAGGTGCAGGGCAAGTCCAAGAG

[0606] GGAGAAGAAGGATCGCGTGTTCACCGATAAAACCAGTGCCACTGTCATCTGTCGTAAAAATGCT TCCATCTCTGTGAGAGCCCAGGACCGCTACTACTCTTCCAGTTGGAGCGAATGGGCATCTGTCC

[0607] CCTGTTCTGGAGGCGGCGGCGGAGGCTCCCGCAACCTGCCAGTTGCTACCCCTGACCCGGGAAT

[0608] GTTTCCTTGCCTTCACCACTCACAGAACCTACTCCGCGCTGTTTCTAACATGCTGCAGAAGGCG

[0609] CGCCAGACTCTGGAGTTCTACCCTTGCACTTCCGAAGAGATTGACCACGAGGACATCACCAAGG

[0610] ACAAAACGAGTACTGTCGAGGCTTGCCTCCCCCTGGAACTGACTAAGAACGAGAGCTGTCTCAA

[0611] CTCCCGCGAGACTAGTTTTATCACTAATGGGAGCTGCCTGGCCAGCCGCAAGACCTCCTTTATG

[0612] ATGGCCCTGTGCCTGTCCTCCATTTACGAGGATTTGAAAATGTATCAGGTGGAGTTCAAGACTA

[0613] TGAACGCCAAGCTCCTGATGGATCCAAAGCGTCAAATTTTCCTGGACCAGAACATGTTGGCAGT

[0614] AATCGACGAGCTGATGCAGGCACTGAACTTCAACAGTGAGACCGTCCCTCAGAAGTCCAGCCTG

[0615] GAGGAGCCCGACTTTTACAAGACCAAGATCAAGCTGTGCATCCTTTTGCACGCCTTCCGCATCC

[0616] GCGCTGTGACAATTGACAGGGTGATGTCTTACCTCAACGCGTCTCGCCGCAAGCGGGGCTCCGG

[0617] CGAGGGTCGAGGATCCCTGTTAACCTGCGGGGATGTGGAGGAGAATCCAGGCCCCATGGGAGCG

[0618] GGTGCGACCGGCAGGGCCATGGATGGCCCGCGACTGCTGTTGCTGCTGCTGCTGGGCGTGTCCT

[0619] TAGGGGGCGCCAAGGAGGCTTGCCCAACAGGCCTGTACACACATAGCGGCGAGTGTTGTAAAGC

[0620] ATGCAACCTGGGCGAGGGTGTTGCCCAACCATGCGGAGCCAACCAGACTGTCTGCGAACCTTGC

[0621] CTAGACAGCGTGACCTTCTCCGACGTGGTGTCCGCGACCGAGCCCTGTAAGCCCTGCACCGAGT

[0622] GCGTGGGCTTGCAGTCTATGTCCGCTCCCTGCGTGGAGGCCGATGACGCCGTGTGCCGATGTGC

[0623] CTACGGCTATTACCAGGATGAGACCACTGGCCGTTGCGAGGCCTGCCGAGTTTGTGAGGCCGGC

[0624] TCGGGTCTCGTGTTTTCTTGCCAGGACAAGCAGAACACCGTGTGCGAGGAGTGCCCAGACGGCA

[0625] CCTACTCCGACGAGGCCAACCATGTGGACCCGTGTCTGCCGTGTACTGTCTGTGAGGACACGGA

[0626] GCGGCAGCTGCGCGAGTGCACTCGCTGGGCGGACGCCGAGTGCGAGGAGATCCCTGGGCGCTGG

[0627] ATCACCCGCTCGACCCCCCCTGAGGGCTCCGATAGCACGGCGCCCTCCACTCAGGAGCCGGAAG

[0628] CTCCTCCTGAACAGGACCTGATCGCCTCCACCGTGGCTGGCGTCGTCACAACCGTGATGGGCTC

[0629] TTCCCAGCCGGTGGTGACCCGCGGCACCACAGATAACTTGATCCCGGTGTACTGCTCTATTCTG

[0630] GCAGCGGTGGTCGTTGGGCTGGTTGCCTACATCGCGTTCAAACGGTGGAACTCCTGATAATAGG

[0631] CGGCCGCGTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGT

[0632] TGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCA

[0633] CTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCT

[0634] GGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGG

[0635] GATGCGGTGGGCTCTATGGGAAGCCCTTGTCACGGTGCCTGCTCACCAGTGACCATGTTCATCC

[0636] ACTGTGACCACTAGGCAGGCACTGCTCTCTGCAGAGGGGGTCATTCCAGGCCCTCCAGTGTGTG

[0637] TGATAGTCACCATGCCATGCAGGGATATTCTTGAAGCCACACATGGCTGGCGGAACCCAGAGCC

[0638] CCCACCCCAGCCCACCTGGCTCTTTGTTGACAGTCGGCGACAAGGTTGTGCGTGGCTCCTGACC

[0639] TGCTGCTAAGAGTCACTTGACCAGACTGCATCTGCATGGGCTGCGCGGAGGTTGCCCAGCCCCA

[0640] GTTTCTTCCGGCGCAGCTCTTAGGTGTTCACTCTCGCCAGCTCAGTTGGCTTTGTGAAGTGTGA

[0641] AACCCTACAATGTGAAAGGAAAGTGCTTGCTGTGATGTTCCTACTGTGGCCCAGCTGCCCAGCA

[0642] TGGACCTGGTGACTCTCCACAGGGCCTCTACCATCCTCTCTGTGGCCACTTCCTGAGCCAGAGG

[0643] CCAGGTCTTCATGGGGCCCTGAGCTTCTGCTGCCTCTGGTGAGAGGGAGAGCCCTTCCCATCCT

[0644] TACCCACCAGGAACTAGAGCCCCAACCACAGCAGATGCTTCCTGGGCAGCCACTGGCCAGGCCG

[0645] TTGTATCCATGTCACCCTTAGTTGTGGGCATTCATGAAAGCAATGCGCTTGCTTCAGCACATTG

[0646] GGATGCACAGAAACGCCCATGGCCAAGCTTGCATGGTTTTTCCATAGGCTCCGCCCCCCTGACG

[0647] AGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCA

[0648] GGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATAC

[0649] CTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCA

[0650] GTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCG

[0651] CTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTG

[0652] GCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGA AGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCC AGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGT GGT T T T T T T GT T T GCAAGCAGCAGAT TACGCGCAGAAAAAAAGGAT C T CAAGAAGAT CC T T T GA TCTTTTCTACTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCA TCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCC CCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCA GCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATT AATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCA TTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCA ACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCT CCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATA ATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTC ATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACC GCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCT CAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTC AGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAA AAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAA GCAT T TAT CAGGGT TAT T GT CT CAT GAGCGGATACATA

[0653] SEQ ID NO: 132 - PFKFB4-CAR.B7H3-sIL15 KI template plasmid

[0654] CGCGTCGCGAGGCCATATGGGGCCCACAGCTGGGAGGAGGGGCCACCAGGCTCTGGGGCACATA AGACAGCCTCAGAGAGCTAAGGGAAGTTTCAGATTCCCTATTCCTCAGGCCTGAGACTCCCTGA GCCCGACAGGCCAAGGATGGGACTGAGCTCTGCAGTTTGGTGGGGTCCTTCTATAGCTGAGGGC TTCAGGAGGCCCTGTACCCCAGTTCTGAGGGGACAGACCAGGCTGCAGACCAGGAAATGCCCAT AGGCTTCTGGGGCACCTCTGACACCTGCGTGCCAACACTTTAAACTAGGAAGGAGGCACCCCCA GGAGTTGAACCCCTTCCCTGCCCCACAGATGAGGATAAAATCCCCAAGCCAGGGGTTCCTCCTC AATTGACTCAGCCCTGAGATAGGAGGTGGGAGTGGCCTTGCTTTGGGTATTGCCTGACCTGGCT GCAGTGGAGCAGGAAACGAGGTGGCATCCTCCACCGCACCTTTGTTCTATTGTCCATCCACCCG CTATGTGGCACGAAGCTCTTGGCCATGTATTCTTAGGGAAGCTTGTGGTCTAGAGTCTTGCGGC TGTGGCTGCTACAGTGAGAATGTGAAGCCTGATGAACCACAGTGATGAGGACGGCAGACAGCAG GCATCTCATCTATTCCTGCCATGGAGTGAACGCTCTGTGTTTTTATTGTACAGAACGTGGACAT CTCAAGACCTCCAGAGGAGGCTCTGGTGACTGTCCCCGCGCACCAGCGTCGCAAACGTGGAAGC GGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTATGG CGCTGCCGGTGACGGCCTTACTCTTGCCTCTCGCTCTCCTGCTGCATGCTGCTCGCCCTGACTA CAAGGATGACGATGATAAGCAGGTTCAGCTAGTGGAGTCTGGAGGCGGCCTCGTGCAGCCAGGT GGTTCTCTCCGCCTGTCTTGTGCCGCAAGCGGCTTTACTTTTAGCAGCTACTGGATGTATTGGG TGCGCCAGACTCCTGGCAAAGGGCTCGAGTGGGTCAGTACCATCAACCGCGATGGCTCGGCTAC CTGGTACGCGGACAGCGTGAAGGGCCGCTTCACCATCTCTCGCGACAACGCGAAAAACACGGGC TACCTCCAGATGAATTCGCTGAAGCCTGATGATACGGCGGTATACTACTGCGTGTCCGACCCGG ACAACTACAGCTCGGACGAGATGGTCCCCTATTGGGGCCAGGGCACCCAGGTGACAGTCTCCTC C G T GGAC GAGAT T GAAG TGATGTATCCCC C C CCTATCTT GAC AAC G GAAG T C GAAT G G C AC C ATCATCCATGTGAAGGGCAAGCACTTGTGCCCATCGCCCCTGTTCCCGGGACCAAGCAAGCCCT TCTGGGTCCTGGTCGTGGTGGGCGGCGTTCTGGCGTGCTACTCCCTACTGGTGACTGTGGCCTT CATCATCTTCTGGGTGCGCTCGAAGAGATCTAGGCTCCTCCACTCCGACTACATGAACATGACT CCTCGTAGGCCCGGGCCTACCCGCAAGCACTACCAGCCCTATGCCCCTCCGCGGGACTTCGCCG CATACCGCTCCCGCGTGAAATTTTCTCGCAGCGCGGACGCCCCTGCCTACCAGCAGGGTCAGAA CCAGCTGTACAATGAGCTGAACCTGGGCCGGCGCGAGGAGTACGACGTGCTGGACAAACGCCGT CTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCT TCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAA AAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTG AAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATA

[0655] SEQ ID NO: 133 - PFKFB4 coding region restore amino acid sequence

[0656] EALVTVPAHQ

[0657] SEQ ID NO: 134 - PFKFB4 coding region restore DNA sequence

[0658] GAGGCTCTGGTGACTGTCCCCGCGCACCAG

[0659] SEQ ID NO: 135 - anti-B7H3 CAR (CAR.B7H3) DNA sequence

[0660] ATGGCGCTGCCGGTGACGGCCTTACTCTTGCCTCTCGCTCTCCTGCTGCATGCTGCTCGCCCTG

[0661] ACTACAAGGATGACGATGATAAGCAGGTTCAGCTAGTGGAGTCTGGAGGCGGCCTCGTGCAGCC

[0662] AGGTGGTTCTCTCCGCCTGTCTTGTGCCGCAAGCGGCTTTACTTTTAGCAGCTACTGGATGTAT TGGGTGCGCCAGACTCCTGGCAAAGGGCTCGAGTGGGTCAGTACCATCAACCGCGATGGCTCGG CTACCTGGTACGCGGACAGCGTGAAGGGCCGCTTCACCATCTCTCGCGACAACGCGAAAAACAC GGGCTACCTCCAGATGAATTCGCTGAAGCCTGATGATACGGCGGTATACTACTGCGTGTCCGAC CCGGACAACTACAGCTCGGACGAGATGGTCCCCTATTGGGGCCAGGGCACCCAGGTGACAGTCT CCTCCGTGGACGAGATTGAAGTGATGTATCCCCCACCCTATCTTGACAACGAGAAGTCGAATGG

[0663] CACCATCATCCATGTGAAGGGCAAGCACTTGTGCCCATCGCCCCTGTTCCCGGGACCAAGCAAG CCCTTCTGGGTCCTGGTCGTGGTGGGCGGCGTTCTGGCGTGCTACTCCCTACTGGTGACTGTGG CCTTCATCATCTTCTGGGTGCGCTCGAAGAGATCTAGGCTCCTCCACTCCGACTACATGAACAT GACTCCTCGTAGGCCCGGGCCTACCCGCAAGCACTACCAGCCCTATGCCCCTCCGCGGGACTTC GCCGCATACCGCTCCCGCGTGAAATTTTCTCGCAGCGCGGACGCCCCTGCCTACCAGCAGGGTC AGAACCAGCTGTACAATGAGCTGAACCTGGGCCGGCGCGAGGAGTACGACGTGCTGGACAAACG

[0664] CCGTGGCCGTGACCCGGAGATGGGCGGAAAGCCGCGTAGAAAGAACCCTCAGGAGGGCCTTTAC AACGAGTTACAGAAGGACAAGATGGCTGAGGCCTACAGCGAGATTGGTATGAAGGGAGAACGCC GCCGTGGCAAAGGCCACGACGGCCTTTATCAGGGCTTGTCCACAGCTACCAAAGACACTTACGA CGCACTGCATATGCAGGCCCTGCCCCCGCGCCGACGTAAGCGCGGTTCTGGGGAGGGCCGAGGC TCCCTGCTGACCTGCGGGGATGTGGAGGAGAATCCAGGCCCTATGTACAGGATGCAACTACTGT CTTGCATTGCTCTGTCGCTTGCACTGGTGACCAACAGCAACTGGGTCAACGTGATCAGCGACCT

[0665] GAAGAAGATTGAGGATCTGATCCAGTCTATGCACATTGATGCTACGCTGTACACCGAGTCCGAT

[0666] GTGCACCCCTCTTGTAAGGTCACTGCCATGAAGTGTTTTCTGCTGGAGCTGCAGGTCATATCAC TCGAGAGCGGGGACGCTAGCATCCACGATACCGTGGAGAACCTCATCATCCTGGCCAACAACTC CCTGTCGTCGAATGGCAACGTGACTGAGAGCGGGTGCAAGGAGTGCGAGGAACTGGAAGAGAAG AAC AT CAAAGAG T T C 0 T G C AG T C C T T T G T C C AC AT C G T G C AGAT G T T T AT TAAC AC G T C G

[0667] SEQ ID NO: 136 - anti-B7H3 CAR (CAR.B7H3) amino acid sequence

[0668] MALPVTALLLPLALLLHAARPDYKDDDDKQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMY WVRQTPGKGLEWVSTINRDGSATWYADSVKGRFT I SRDNAKNTGYLQMNSLKPDDTAVYYCVSD PDNYSSDEMVPYWGQGTQVTVSSVDE IEVMYPPPYLDNEKSNGTI IHVKGKHLCPSPLFPGPSK PFWVLVWGGVLACYSLLVTVAFI I FWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDF AAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY NELQKDKMAEAYSE IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Claims

What is claimed is:

1. A method of treating an NK cell to induce a gene edit, the method comprising: contacting the NK cell with a transfection composition comprising: poly-L-glutamic acid (y-PGA), a single-stranded oligo deoxynucleotide (ssODN) enhancer, or a combination thereof; contacting the NK cell with an editing composition comprising: a DNA-PK inhibitor, and an HD AC inhibitor; contacting the NK cell with a repair template; electroporating the NK cell; and contacting the NK cell with a recovery composition comprising DNase I.

2. The method of claim 1, wherein the NK cell comprises a feeder-expanded NK cell.

3. The method of claim 1, wherein the NK cell has been previously cryopreserved.

4. The method of claim 1, further comprising contacting the cell with editing reagents.

5. The method of claim 4, wherein the editing reagents comprise Cas9 and a guide RNA, such as a single guide RNA (sgRNA).

6. The method of any preceding claim, wherein the recovery composition further comprises N-acetyl-cysteine, L-glutathione, or a combination thereof.

7. The method of any preceding claim, wherein the recovery composition further comprises emricasan, trans-ISRIB, or a combination thereof.

8. The method of any preceding claim, wherein the DNA-PK inhibitor comprises M3814 orAZD-7648.

9. The method of any preceding claim, wherein the HDAC inhibitor comprises Trichostatin A.

10. The method of any preceding claim, wherein the repair template comprises plasmid DNA, single-stranded DNA, or double-stranded DNA.

11. The method of claim 10, wherein the repair template comprises a tCTS.

12. The method of any preceding claim, wherein at least 100 NK cells are treated.

13. The method of claim 12, wherein at least 50% of the treated NK cells comprise the gene edit following treatment.

14. A method of increasing the efficiency of gene editing in an NK cell comprising contacting the NK cell with poly-L-glutamic acid (PGA) and gene editing reagents.

15. An edited NK cell prepared using the method of any preceding claim.

16. The edited NK cell of claim 15, wherein the NK cell is derived from a human NK cell.

17. The edited NK cell of claim 16, wherein the NK cell comprises an inserted DNA sequence at the human CISH locus or the human CD96 locus.

18. The edited NK cell of claim 17, wherein the NK cell comprises an inserted DNA sequence at one or more of the TOMM70, CTCF, ACTG1, MAP4, EEF1G, YWHAQ, HNRNPA1, EN01, or GAPDH loci.

19. An editing composition for use in gene editing an NK cell comprising Trichostatin A and a DNA-PK inhibitor comprising AZD-7648, M3814, or a combination thereof.

20. A method of using the editing composition of claim 19 comprising contacting an NK cell with gene editing reagents and the editing composition.

21. A recovery composition for use in gene editing an NK cell comprising DNasel; an apoptosis inhibitor comprising emricasan, and / or trans-ISRIB inhibitor; and an antioxidant comprising N-acetylcysteine, L-glutathione, or a combination thereof.

22. A method of using the recovery composition of claim 21 comprising contacting an NK cell with gene editing reagents and the recovery composition.

23. A cell therapy reagent comprising an edited NK cell produced using the method of any preceding claim.

24. The cell therapy reagent of claim 23, wherein the edited NK cell comprises a chimeric antigen receptor (CAR)-NK cell.

25. The cell therapy reagent of claim 24, wherein the CAR targets CD 19, CD22, or both CD 19 and CD22.

26. A method of preparing an engineered NK cell comprising a fluorescent reporter at an endogenous locus, the method comprising: contacting an NK cell with a transfection composition comprising: poly-L-glutamic acid (y-PGA), a single-stranded oligo deoxynucleotide (ssODN) enhancer, or a combination thereof; contacting the NK cell with an editing composition comprising: a DNA-PK inhibitor, and an HD AC inhibitor; contacting the NK cell with a repair template including a nucleic acid sequence encoding a fluorescent reporter; electroporating the NK cell; and contacting the NK cell with a recovery composition comprising DNase I.

27. The method of claim 26, wherein the endogenous locus comprises the TOMM70, CTCF, ACTG1, MAP4, EEF1G, YWHAQ, HNRNPA1, EN01, or GAPDH locus.

28. The method of claim 26, wherein the endogenous locus comprises a hypoxia-sensitive locus, such as the PFKB4 locus.

29. The method of claim 26, wherein the repair template includes a nucleic acid sequence having at least 80% identity to a region of the endogenous locus.

30. A method of treating an NK cell to introduce an exogenous nucleic acid, the method comprising: contacting the NK cell with a transfection composition comprising: poly-L-glutamic acid (y-PGA), a single-stranded oligo deoxynucleotide (ssODN) enhancer, or a combination thereof; contacting the NK cell with an editing composition comprising: a DNA-PK inhibitor, and an HD AC inhibitor; contacting the NK cell with an exogenous nucleic acid; electroporating the NK cell; and contacting the NK cell with a recovery composition comprising DNase I.

31. The method of claim 30, wherein the exogenous nucleic acid comprises DNA.

32. The method of claim 31, wherein the exogenous nucleic acid comprises a DNA plasmid.

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