Chemical reprogramming enhances homology-directed genome enhancing in zebrafish embryos

A system for quantitative in vivo evaluation of HDR

To ascertain a easy and quantitative readout for HDR, we centered on the fast-muscle fibers within the zebrafish trunk. We devised a gene alternative technique whereby eBFP2 expressed particularly within the fast-muscle fibers of a steady transgenic zebrafish is disrupted by the insertion of tdTomato, changing the muscle fiber from blue to pink fluorescence (Fig. 1a). The donor template included a CRISPR/Cas9 goal web site on the tdTomato insertion web site within the homology arm that was shared with the eBFP2 genomic goal locus (Fig. 1b) since this method was reported to extend HDR effectivity24. When a single information RNA (sgRNA) concentrating on eBFP2 was co-injected with Cas9 protein, CRISPR/Cas9 linearized donor DNA serves as a template for HDR-mediated gene enhancing (Fig. 1b). To check the feasibility of this method, we used a double transgenic zebrafish pressure that expressed eBFP2 below the management of the fast-muscle acta1 promoter29 and eGFP below the management of the slow-muscle smyhc1 promoter30 (Fig. 1c, d). Zebrafish gradual muscle is a single layer of parallel fibers that encase the fish beneath the pores and skin, rendering them accessible to fast and correct quantitation by fluorescence microscopy (Fig. 1e). To judge the effectivity of the sgRNA, we focused a area that’s an identical between eBFP2 and eGFP (Fig. 1b) and confirmed the lack of eGFP fluorescence in a mosaic sample throughout particular person slow-muscle cells at 72 h publish fertilization (hpf) (Fig. 1f, g). Related lack of eBFP2 expression in fast-muscle cells was noticed (Supplementary Film 1). Subsequent, we examined whether or not a donor DNA fragment encoding the pink fluorescent protein tdTomato flanked by a 303 bp left homology arm (LHA) and 1022 bp proper homology arm (RHA) might be inserted into the acta1:eBFP2 transgene (Fig. 1b). We noticed pink fluorescent sign in particular person fast-muscle fibers that confirmed lack of eBFP2 expression (Supplementary Film 2), demonstrating profitable insertion of the tdTomato transgene into the eBFP2 locus (and due to this fact inflicting loss in expression of eBFP2). Management injections with out eBFP2 sgRNA didn’t generate any pink fluorescent sign (Supplementary Film Three). Our knowledge are per integration of the tdTomato transgene right into a CRISPR/Cas9-dependent genomic lesion at low effectivity ( ± pink fibers per embryo). Importantly, this assay offers fast quantification of in vivo genomic enhancing at single-cell decision.

Fig. 1Fig. 1

Overview of the in vivo homology-directed restore (HDR) detection system. a Schematic illustration of the visible HDR readout. b The one information RNA (sgRNA)-Cas9 complicated targets the an identical sequence in eGFP and eBFP2. Embryos are co-injected with HDR donor template to combine the tdTomato transgene into the acta1-eBFP2 locus. c Confocal sections displaying smyhc1:eGFP and acta1-eBFP2 expression and d merged pictures. Scale bars: 75 µm. e Cross-sectional illustration of a zebrafish embryo displaying gradual and quick muscle mass. f Expression of smyhc1-eGFP in slow-muscle fibers (Three dpf). g Mosaic lack of smyhc1-eGFP expression in slow-muscle fibers (Three dpf) of embryos injected with a CRISPR (clustered frequently interspaced quick palindromic repeat)/Cas9 complicated concentrating on eGFP. Scale bars: 500 µm, important picture and 100 µm, inset

Small-molecule enhancement of HDR effectivity

Inhibition of NHEJ and stimulation of HDR by small molecules seems to have context-specific outcomes for exact genome editing1,2,13,14,15. We used the fast-muscle fiber assay described above as a take a look at mattress to establish the optimum small-molecule therapy for enhancing HDR in zebrafish. Transgenic acta1:eBFP2 embryos had been co-injected with SCR7, RS-1, or NU7441 (Fig. 2a–c), at a variety of doses as much as the solubility restrict of every drug. Not one of the remedies affected embryo survival (Supplementary Fig. 1). Moreover, injection of donor DNA alone didn’t generate any pink fibers (Fig. 2e), highlighting the specificity of the assay. Subsequently, we proceeded to quantify the whole variety of muscle fibers expressing tdTomato within the trunk of every embryo (n ≥ 40 zebrafish per situation; Fig. 2f). SCR7 administration had no impact on pink fiber quantity in comparison with the dimethyl sulfoxide (DMSO) car management (Fig. 2a). On the highest examined doses, RS-1 confirmed a modest however statistically important improve within the variety of pink fibers (Fig. 2b; DMSO, four.eight ±; 15 µM, 7.2 ± Three.7, p = zero.02; 30 µM, 7.Three ± 5.Three, p = zero.01). In distinction, NU7441 administration confirmed a dramatic improve (Fig. 2f, g), with the maximal impact at 50 µM (Fig. 2c, DMSO, ±; 50 µM, 53.7 ± 22.1, p < zero.0001). We didn't observe an additional improve within the variety of pink fibers by including RS-1 to the optimum NU7441 dose (Fig. second).

Fig. 2Fig. 2

Chemical enhancement of CRISPR (clustered frequently interspaced quick palindromic repeat)/Cas9-mediated homology-directed restore (HDR) effectivity. Results of a SCR7, b NU7441, c RS-1 on the effectivity of CRISPR/Cas9-mediated tdTomato expression in zebrafish embryos. d Co-injection of optimized concentrations of NU7441 and RS-1 in zebrafish embryos. Error bars signify normal error of the imply. e–g Fluorescent microscopy of zebrafish embryos injected with e tdTomato donor template, f CRISPR/Cas9 with tdTomato donor template, or g CRISPR/Cas9 with tdTomato donor template and optimized focus of NU7441 at 50 µM. Scale bars: 250 µm

Qualitative evaluation masks the results of HDR stimulation

Highlighting the significance of quantitative single fiber evaluation, qualitative evaluation (presence vs. absence of pink fibers) didn’t reveal an HDR-stimulating impact for any of the drug remedies (Supplementary Fig. 1). For instance, a comparability of qualitative evaluation of DMSO management group with out medicine, RS-1 at 30 µM and NU7441 at 50 µM show comparable charges of pink fiber induction (Supplementary Fig. 1; DMSO, 69.5%, n = 130; RS-1 30 µM, 75.5%, n = 352; Nu 50 µM,, n = 314). Nonetheless, amongst the animals that exhibited pink fluorescence, we noticed a distinction within the variety of pink fibers between the management and drug-treated embryos that was per the one fiber evaluation. For instance, the DMSO management embryos that exhibited pink fast-muscle fibers (69% of complete) had a mean of ± fibers per embryo, whereas NU7441 (50 µM) administered embryos that exhibited pink fast-muscle fibers (80% of complete) had a mean of 53.7 ± 22.1 fibers per embryo. We conclude that qualitative assays cut back the dynamic vary of whole-embryo assays and may masks the results HDR-stimulating circumstances.

Zebrafish-specific optimizations for Cas9-mediated HDR

To optimize Cas9-mediated HDR effectivity in zebrafish, we first in contrast round and linear plasmid DNA donors. We discovered no distinction between donor kind in DMSO controls (Supplementary Fig. 2B; linear, four.2 ± zero.7, n = 25; round, four.9 ± zero.5, n = 34). Nonetheless, co-injection with round plasmid DNA yielded a considerably larger variety of pink fast-muscle fibers than linearized plasmid DNA in NU7441 (50 µM)-administered embryos (Supplementary Fig. 2B; linear, ± Three.Three, n = 30; round, ± Three.2, n = 43, p < zero.zero01). Our outcomes additionally indicated that linearized plasmid DNA present extra toxicity in comparison with round plasmid DNA in all experimental teams (Supplementary Fig. 2B). You will need to word that total HDR effectivity is relative to the reducing effectivity of CRISPR/Cas9, which is extremely variable throughout totally different loci. On this experiment, we examined six totally different sgRNA concentrating on eBFP2 (Supplementary Desk 1) and used the sgRNA displaying the very best effectivity (sg_eBFP2_04).

The fast embryonic growth of zebrafish poses a problem for genome enhancing. The post-fertilization cell cycle size is lower than an hour in fertilized zebrafish embryos31 in comparison with 16–20 h in mice32. This fast proliferation offers a 20 min experimental window during which to inject DNA into single-cell eggs (~20–40 min publish fertilization), and due to this fact it’s conceivable that variability within the timing of the DNA injection inside the cell division cycle on this quick time interval might contribute to mosaicism and low germline transmission charges in zebrafish. We examined whether or not slowing the primary cell division of zebrafish embryos, by incubation on ice, enhanced Cas9-mediated HDR effectivity by permitting extra time for the formation CRISPR/Cas9 meeting adopted by DNA restore by HDR. Our outcomes confirmed ice incubation for 15–20 min considerably enhances HDR effectivity in NU7441 and NU7441/RS-1 administered embryos with solely minimal influence on growth and survival (Supplementary Fig. 3A–B; NU7441 50 µM RT, 36.6 ± 2.6, n = 54; NU7441 50 µM ice, 50.1 ± Three.6, n = 43, p = zero.0019). These outcomes confirmed Cas9-mediated HDR might be elevated 1.5-fold to 2-fold by slowing embryo growth quickly. Subsequently, we performed all additional experiments utilizing the low-temperature injection protocol.

Phenotypic and molecular evaluation of integration occasions

The transmission of the acta1:eBFP2 transgene is per a single-copy Tol2-mediated insertion within the zebrafish genome29. Subsequently, donor integration ought to get rid of eBFP2 expression in tdTomato-expressing cells. As predicted for HDR-mediated integration, in NU7441-injected embryos we noticed a mutually unique sample of pink and blue fluorescence in particular person fast-muscle fibers (Fig. 3a–f). Moreover, the homology arms had been important for concentrating on as a result of no integration into the slow-muscle smyhc:eGFP transgene, which contained an an identical CRISPR/Cas9 goal web site, was noticed (Fig. 3a–f). To additional affirm that tdTomato integration requires homology with the goal web site, we used a validated sgRNA concentrating on the endogenous zebrafish golden locus26, which is crucial for pigmentation. We didn’t observe any pink fast-muscle fibers regardless of disrupting pigmentation (Supplementary Fig. 4A–C). Subsequently, we conclude that tdTomato is just not effectively built-in into non-homologous double-stranded breaks.

Fig. ThreeFig. 3

Single fiber evaluation of homology-directed restore (HDR) occasions. Cross-sectional in vivo imaging of a zebrafish embryo (Three dpf) co-injected with NU7441 at 50 µM and RS-1 at 30 µM. a Merged picture displaying mutually unique expression of eBFP2 and tdTomato in particular person fast-muscle fibers (yellow arrows). c, e Particular person channels displaying eBFP2 expression (c) and tdTomato (e). Adjoining slow-muscle fibers specific eGFP. b, d, f Increased magnification confocal sections of areas indicated in a, c, e. Scale bars: 50 µm

Subsequent, we designed PCR-based assays to research whether or not blue to pink colour switching in fast-muscle fibers is mediated by exact HDR (Fig. 4a–c). Primers 306 and 307 generate a 1981 bp PCR amplicon (Band 1), which is barely amplified when the donor-derived tdTomato coding sequence is inserted downstream of the transgenic acta1:eBFP2 promoter (Fig. 4c). We noticed a band of the anticipated dimension (Band 1) in embryos with pink fast-muscle fibers, which was not noticed in controls missing the eBFP2-targeting sgRNA (Fig. 4d), indicating that amplifcation requires genomic integration. To research whether or not the variety of pink fast-muscle fibers corresponds to the proportion of edited alleles, we used a second set of PCR primers (306 and 308) to concurrently amplify the unique (Band Three, 497 bp) and edited (Band 2, 2172 bp) trangenic acta1 alleles (Fig. 4a–c). NU7441 elevated the depth of the edited Band 2 amplicon in comparison with controls (Fig. 4e). Quantification of particular person embryos revealed a 7.7-fold improve within the depth of Band 2 amplicon in NU7441-injected embryos (Supplementary Fig. 5), which was much like the impact on pink fast-muscle fiber quantity (9.Three-fold, Fig. 2c).

Fig. fourFig. 4

Molecular evaluation of homology-directed restore (HDR)-mediated integration occasions. a–c Primer design for PCR amplicons and sequencing evaluation of HDR occasions. DNA fragments from a the acta1:eBFP2 transgene, b the tdTomato donor template, and c the expected CRISPR (clustered frequently interspaced quick palindromic repeat)/Cas9-mediated HDR integration of tdTomato are depicted displaying the positions of primers 306, 307, and 308. d PCR evaluation utilizing primers 306/307. The primer 306 binding web site is just not current within the donor sequence, whereas the primer 307 binding is exclusive to the donor template. Thus, PCR amplification of a 1981bp band is barely anticipated in embryos with focused integration occasions. e PCR evaluation utilizing primers 306/308. Primer 308 binds to an eBFP2 sequence that’s current in each the acta1:eBFP2 transgene and the tdTomato donor. Subsequently, the 306/308 primer pair can generate two merchandise: a 497 bp amplicon (no template integration) and a 2172 bp amplicon (HDR-mediated integration of the tdTomato transgene). f Sequence alignment of the 5′ finish of genome-edited clones. (Magenta) Pam web site, (blue) sgRNA binding web site, and (pink) built-in donor fragment. g Sequence alignment of the three′ finish of clones that confirmed exact HDR in f

Enhanced germline transmission of exact HDR-edited alleles

To find out whether or not the edited alleles had been generated by exact HDR, we sequenced each ends of the DSB from tdTomato-positive embryos. Three out of 10 edited alleles confirmed seamless integration of transgene (Fig. 4f, g). That is per our outcomes from scanning 3D-rendered y and z stacks of embryos for exact alternative of blue fluorescent sign with pink fluorescence in fast-muscle fibers. (Supplementary Films four and 5).

Lastly, to look at whether or not transient chemical reprogramming elevated the germline transmission price of HDR-edited alleles, we raised injected embryos to maturity and screened their progeny for the expression of tdTomato in fast-muscle fibers. Germline transmission was analyzed in outcrosses with wild-type (non-transgenic) breeding companions (Supplementary Desk 2). Within the DMSO management group, only one/16 fish produced tdTomato-expressing embryos (42/120 embryos from the one germline-transmitting fish), whereas Three/6 fish within the NU7441 group produced tdTomato-expressing embryos (19/24, 81/110, and 68/75 embryos from the three germline-transmitting fish: χ2p = zero.018 in comparison with DMSO management). As predicted from the one fiber evaluation, the addition of RS-1 didn’t improve the germline transmission price as in comparison with NU7441 alone (four/10 fish produced 61.5% (n = 200) tdTomato-expressing embryos; χ2p = zero.034 in comparison with DMSO management).

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