Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency

Abstract

Versatile and precise genome modifications are needed to create a wider range of adoptive cellular therapies^1-5. Here we report two improvements that increase the efficiency of CRISPR-Cas9-based genome editing in clinically relevant primary cell types. Truncated Cas9 target sequences (tCTSs) added at the ends of the homology-directed repair (HDR) template interact with Cas9 ribonucleoproteins (RNPs) to shuttle the template to the nucleus, enhancing HDR efficiency approximately two- to fourfold. Furthermore, stabilizing Cas9 RNPs into nanoparticles with polyglutamic acid further improves editing efficiency by approximately twofold, reduces toxicity, and enables lyophilized storage without loss of activity. Combining the two improvements increases gene targeting efficiency even at reduced HDR template doses, yielding approximately two to six times as many viable edited cells across multiple genomic loci in diverse cell types, such as bulk (CD3⁺) T cells, CD8⁺ T cells, CD4⁺ T cells, regulatory T cells (Tregs), γδ T cells, B cells, natural killer cells, and primary and induced pluripotent stem cell-derived⁶ hematopoietic stem progenitor cells (HSPCs).

Figure 1.. Truncated Cas9 Targets Sequences (tCTS) in HDR templates increase large non-viral knockin efficiency.

(a) Enzymatically active Cas9-NLS RNPs can bind truncated Cas9 target sequences (tCTS) added to the ends of an HDR template. (b) “In” facing orientation of the tCTSs (PAM facing in towards the center of the inserted sequence vs “out” away from the insert) on the edges of both the 5’ and 3’ homology arms improved knockin efficiency of a new TCRα-TCRβ specificity at the endogenous TRAC locus. *P<0.05 (Mann-Whitney test vs no tCTS control). (c) Representative flow cytometry plots showed improved targeting efficiency across target genomic loci with the tCTS modifications compared to an unmodified dsDNA HDR template. (d) The tCTS modifications improved targeting efficiencies of large knockins across eight genomic loci tested in both CD4+ and CD8+ T cells. Note CD4-GFP expression was not observed at relevant levels in CD8+ T cells, as expected. *P<0.05 (Two-way paired T test with Holm-Sidak multiple comparison correction). (e) Multiplexed electroporation of GFP and RFP knockin templates to the RAB11A locus where neither, one, or both templates had a tCTS modification revealed direct competitive knockin advantage of ‘shuttle’ system compared to unmodified dsDNA template (technical replicates from n=2 donors). (f) The tCTS modification improved multiplexed dual knockin at different genomic loci as well as bi-allelic knockin at a single target locus (technical replicates from n=2 donors). (g) Full improvement of knockin efficiencies with the tCTS modifications (but not with unmodified dsDNA HDR templates) were dependent upon the presence of a NLS on Cas9 protein (multiple technical replicates from n=2 donors). (h) Decreased viability was seen with the tCTS modifications at lower DNA concentrations compared to unmodified dsDNA HDR template. The relative rates of HDR (b, d, e, g), multiplexed HDR (f), or viability (h) with the tCTS shuttle are displayed normalized to unmodified dsDNA HDR template (b, d-g) or to no electroporation controls (h) in n=4 biologically independent blood donors (For some templates different healthy donors were used) (b, c, d) or from n=2 biologically independent blood donors with multiple technical replicates shown to convey variance (e-h). Center lines indicate mean (b, d), error bars indicate standard deviation (d). HDR efficiency was measured 4 days post electroporation and viability (total number of live cells relative to no electroporation control) at 2 days post electroporation.

Figure 2:. Stabilizing Cas9 RNP nanoparticles with anionic polymers improves editing outcomes.

(a) Photograph 15 minutes after mixing gRNA and Cas9 protein incubated at 37C to form RNPs. Cas9 RNPs prepared at low molar ratio of gRNA:protein appeared cloudy and rapidly settled out of solution. Representative photograph of 3 repeated independent experiments. (b) Multiple polymers were screened for the ability to enhance CD4 gene knock-out editing when mixed with RNPs formulated at 1:1 gRNA:protein ratio then electroporated into primary human CD4+ T cells. Loss of surface CD4 expression at 3 days assessed by flow cytometry is normalized to unenhanced editing efficiency (RNP 1:1 without any additive) on the y-axis, and the live cell count is normalized to mock non-electroporated (NT) cells on the x-axis. Negatively charged polymers are shaded blue: poly(L-glutamic acid) (PGA), heparin sulfate (Hep), hyaluronic acid 150 kDa (HA-150), poly(acrylic acid) at 5 kDa (PAA-5) 25 kDa (PAA-25) or 250 kDa (PAA-250), poly(L-aspartic acid) (PLD), ssODNenh. Neutral polymers are shaded green: poly(ethylene glycol) 35 kDa (PEG-35), and positively charged polymers are shaded orange: polyethyleneimine 25 kDa (PEI), poly(L-arginine) 15–70 kDa (PLA), poly(L-lysine) 15–30 kDa (PLL), poly(L-ornithine) 30–70 kDa (PLO), and protamine sulfate (PS). PGA (blue circles outlined in red) with chemical structure shown inset above data point that corresponds to 100mg/mL concentration. Each polymer sample was tested at serial dilutions to avoid potential dose-dependent cytotoxicity falsely masking impact on editing efficiency, and each concentration is depicted as an individual point that is an average for two different blood donors. (c) Cas9 RNPs at 0.5 molar ratio of gRNA:protein (prepared same as in (a)) could be further dispersed with addition of PGA or ssODNenh, whereas dilution with water alone had no visible benefit. Representative photograph of three repeated independent experiments. (d) PGA and ssODNenh stabilized and reduced the size of RNP nanoparticles. Cas9 RNPs prepared at 2:1 molar ratio of gRNA:protein alone (RNP) or mixed with PGA or ssODNenh were assessed for hydrodynamic particle size by dynamic light scattering. Z-average particle size is shown for n=2 independent preparations (individual sample size distributions and peaks shown in Supplementary Fig. 8). (e) Multiple anionic polymers boosted knockin editing efficiency. Polymers mixed with Cas9 RNPs prepared at a 2:1 gRNA:protein ratio were further mixed with 1ug unmodified dsDNA HDR templates (targeting insertion of an N-terminal fusion of GFP to Rab11a), electroporated into CD4+ T cells, and editing efficiency were assessed by flow cytometry at day 3. The relative rates of HDR is displayed compared to unmodified dsDNA HDR template without enhancer. Data shown for each of n=2 biologically independent blood donors. (f) PGA-stabilized Cas9 RNPs prepared at a 2:1 ratio gRNA:protein markedly improved knockin editing in primary human Bulk (CD3+) T cells targeting a C-terminal fusion of GFP to clathrin and (g) improve viability of electroporated cells (compared to untreated cells). Data shown for each of n=2 biologically independent blood donors. (h) Cas9 RNPs prepared at a 2:1 ratio gRNA:protein without or with PGA or ssODNenh were mixed with 1ug of unmodified dsDNA HDR template targeting an N-terminal fusion of GFP to RAB11A, lyophilized overnight, stored dry at −80C, then later reconstituted in water prior to electroporating into primary human CD3+ (Pan) T cells. PGA-stabilized Cas9 nanoparticles were protected through lyophilization and reconstitution and retained activity for robust knockin editing. Three technical replicates shown for n=1 blood donor, representative of two repeated independent experiments.

Figure 3:. PGA-stabilized Cas9 RNP and tCTS-modified-HDR templates improved knockin gene editing outcomes across a variety of genetic loci and clinically-relevant immune cell types.

(a-b) Cas9 RNPs were prepared at a 2:1 ratio gRNA:protein with or without PGA polymer and mixed with high doses (2–4 ug) of regular dsDNA or tCTS-modified HDR template targeting knockin at multiple genomic loci: transgenic NY-ESO 1 tumor antigen TCR into the TRAC locus, or GFP fusion at the N- or C- of RAB11A, CD4, TUBA1B, ACTB, FBL, or CLTA genes. The combination of PGA-stabilized Cas9 RNP nanoparticles and ‘shuttle’ tCTS-modified-HDR template both improved relative frequency of HDR (a) and resulted in higher yield of successfully edited cells (b). (c-e) Cas9 RNPs were prepared at 2:1 ratio gRNA:protein with or without PGA polymer and mixed with low doses (0.5 – 1 ug) of unmodified dsDNA or ‘shuttle’ tCTS-modified HDR templates targeting knockin of GFP or mCherry to the N-terminus of Clathrin. The PGA-stabilized Cas9 RNP nanoparticles and tCTS-modified HDR templates improved editing efficiency in a variety of primary human immune cell types as visualized in representative flow cytometry plots (after gating for live cells and respective cell type-specific surface markers) (c) or expressed as relative frequency of GFP or mCherry+ positive cells (d), and resulted in higher yield of number of successfully edited cells (e). The relative rates of HDR (a,d) or edited cell recovery (b,e) are displayed normalized to unmodified dsDNA HDR template without enhancer for each given gene locus (a-b) or cell type (d-e). Data in shown for each of n=2 (for CD4, CD8, BulkT, Treg, γδ-T, B cell, HSC, iPS-CD34) or n=3 (for NK) biologically independent blood donors. Data in (c) representative of two repeated independent experiments.