Moreno-Mateos MA et al. (2017), CRISPR-Cpf1 mediates efficient homology-directe...

XB-ART-54347

Nat Commun 2017 Dec 08;81:2024. doi: 10.1038/s41467-017-01836-2.

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CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing.

Moreno-Mateos MA , Fernandez JP , Rouet R , Vejnar CE , Lane MA , Mis E , Khokha MK , Doudna JA , Giraldez AJ .

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Cpf1 is a novel class of CRISPR-Cas DNA endonucleases, with a wide range of activity across different eukaryotic systems. Yet, the underlying determinants of this variability are poorly understood. Here, we demonstrate that LbCpf1, but not AsCpf1, ribonucleoprotein complexes allow efficient mutagenesis in zebrafish and Xenopus. We show that temperature modulates Cpf1 activity by controlling its ability to access genomic DNA. This effect is stronger on AsCpf1, explaining its lower efficiency in ectothermic organisms. We capitalize on this property to show that temporal control of the temperature allows post-translational modulation of Cpf1-mediated genome editing. Finally, we determine that LbCpf1 significantly increases homology-directed repair in zebrafish, improving current approaches for targeted DNA integration in the genome. Together, we provide a molecular understanding of Cpf1 activity in vivo and establish Cpf1 as an efficient and inducible genome engineering tool across ectothermic species.

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Genes referenced: alb kidins220 pam slc45a2

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	Fig. 1. LbCpf1–crRNA RNP complexes are a robust genome editing system in zebrafish. a Diagram illustrating three crRNAs (orange) targeting slc45a2 and tyr exon 1 in zebrafish. b Schematic illustrating the experimental set-up to analyze CRISPR-Cpf1-mediated mutations in zebrafish. Three crRNAs (a) were either mixed with mRNA coding for AsCpf1 or LbCpf1 or assembled into RNP complexes with their corresponding purified proteins and injected in one-cell-stage embryos. c Phenotypic evaluation of crRNAs (30 pg/crRNA) and mRNA (100 pg) injections. Stacked barplots showing the percentage of mosaic (gray) and phenotypically wild-type (WT; black) embryos 48 h post fertilization (hpf) after injection. d Phenotypes obtained after the injection of the LbCpf1–crRNA RNP complexes targeting slc45a2 showing different levels of mosaicism compared to the WT. Lateral views (scale bar, 0.5 mm) and insets of the eyes (scale bar, 0.25 mm) of 48 hpf embryos are shown. e Phenotypic evaluation of Cpf1–crRNA RNP complexes injections targeting slc45a2 (albino). Stacked barplots showing the percentage of alb-like (white), mosaic (gray), and phenotypically WT (black) embryos 48 hpf after injection using different amounts (fmol) of RNP complexes. Number of embryos evaluated (n) is shown for each condition. f Phenotypes obtained after the injection of the LbCpf1–crRNA RNP complexes targeting tyr showing different levels of mosaicism compared to the WT. Lateral views (scale bar, 0.5 mm) and insets of the eyes (scale bar, 0.25 mm) of 48 hpf embryos are shown. g Phenotypic evaluation of Cpf1–crRNA RNP complexes targeting tyrosinase (tyr). Stacked barplots showing the percentage of tyr-like (white), severe mutant (light gray), mild mutant (dark gray), and phenotypically WT (black) embryos 48 hpf after injection using different amounts (fmol) of RNP complexes. Number of embryos evaluated (n) is shown for each condition
	Fig. 2. Temperature is a key factor modulating Cpf1 activity in vitro and in vivo. a Schema illustrating different temperature incubations after Cpf1–crRNA RNP complex injections targeting slc45a2 (alb) and tyr. b Phenotypic evaluation of AsCpf1–crRNA RNP complex (10 fmol) injections at different temperature incubations (T). Stacked barplots showing the percentage of tyr-like (white), severe mutant (light gray), mild mutant (dark gray), and phenotypically WT (black) embryos 48 hpf after injection. Number of embryos evaluated (n) is shown for each condition. χ 2-test (**p < 0.0001). c A representative picture showing 48-hpf-old embryos obtained after AsCpf1–crRNA RNP complex injections targeting tyr at different temperature incubations. Scale bar, 1.25 mm. d Phenotypic evaluation of LbCpf1–crRNA RNP complex (10 fmol) injections at different temperature incubations (T). Stacked barplots showing the percentage of alb/tyr-like (white), mosaic mutants (gray), and phenotypically WT (black) embryos 48 hpf after injection. Number of embryos evaluated (n) is shown for each condition. χ 2-test (p < 0.0001). e A representative picture showing 48-hpf-old embryos obtained after LbCpf1–crRNA RNP complex injections targeting slc45a2 at different temperature incubations. Scale bar, 1.25 mm. f Schematic illustrating different incubation conditions (0, 4, 8, or 24 h at 34 °C, then at 28 °C) after AsCpf1–crRNA RNP complex (10 fmol) injections targeting tyr in zebrafish (top). Phenotypic evaluation of AsCpf1–crRNA RNP complex injections targeting tyr in the conditions described above (bottom). Stacked barplots showing the percentage of tyr-like (white), severe mutant (light gray), mild mutant (dark gray), and phenotypically WT (black) embryos 48 hpf after injection. Number of embryos evaluated (n) is shown for each condition. χ 2-test (p < 0.0001). g Schematic illustrating different incubation conditions: 8 h at 28 °C, 16 h at 34 °C, and then 24 h at 28 °C (34 °C 8–24 hpf) or 24 h at 28 °C, then 24 h at 34 °C (34 °C 24–48 hpf) after AsCpf1–crRNA RNP complex (10 fmol) injections targeting tyr in zebrafish (top). Phenotypic evaluation of AsCpf1–crRNA RNP complex injections targeting tyr in the conditions described above (bottom). Stacked barplots showing the percentage of severe mutant (light gray), mild mutant (dark gray), and phenotypically WT (black) embryos 48 hpf after injection. Number of embryos evaluated (n) is shown for each condition. χ 2-test (**p < 0.0001)
	Fig. 3. Catalytic dead SpCas9 (SpdCas9) proximal targeting increases Cpf1 activity. a Schematic diagram of proxy-CRISPR approach. Temperature may control Cpf1 activity to access and/or unwind genomic DNA, impeding AsCpf1 to access the genomic target in vivo at 28 °C. When SpdCas9 binds in the proximity of the AsCpf1 target, it facilitates the availability of AsCpf1 to cleave the inaccessible target. b Diagram illustrating crRNA 1 (orange) and a proximal sgRNA (purple) targeting tyr exon 1 (Supplementary Fig. 10c and Supplementary Data 1) in zebrafish (top). AsCpf1–crRNA and/or SpdCas9-sgRNA RNP complexes were injected into one-cell-stage embryos and then incubated at 28 °C for 48 h (bottom). c Phenotypic evaluation of the experiment described in (b). Stacked barplots showing the percentage of severe mutant (light gray), mild mutant (dark gray), and phenotypically WT (black) embryos 48 hpf after injection. Number of embryos evaluated (n) is shown for each condition. χ 2-test (**p < 0.0001). d Diagram illustrating crRNA 2 (orange) and sgRNA 2 (purple) targeting alb exon 1 (Supplementary Data 1, Supplementary Figs. 3f and 8e) in zebrafish (top). LbCpf1–crRNA and/or SpdCas9-sgRNA RNP complexes were injected into one-cell-stage embryos and then incubated at 28 °C for 48 h (bottom). e Phenotypic evaluation of the experiment described in (d). Stacked barplots showing the percentage of alb-like (white), mosaic mutants (gray) and phenotypically WT (black) embryos 48 hpf after injection. Number of embryos evaluated (n) is shown for each condition. χ 2-test (**p < 0.0001)
	Fig. 4. LbCpf1-mediated homology-directed repair. a Schematic illustrating LbCpf1–crRNA RNP complex and SpCas9–sgRNA RNP complex interaction with their respective DNA targets. crRNA and sgRNA-DNA annealing occurs on the target strand (blue), and PAM sequences are in the non-target strand (red). b crRNA (pink line) and sgRNA (orange line) overlapping target sequences in the albino locus used for this analysis. c Schema illustrating different donor ssDNA (#1–6) complementary to either the target strand (t) or non-target strand (nt) and with symmetric or asymmetric homology arms used in combination with LbCpf1–crRNA. PAM sequence was modified (TgaV/ActB) to prevent new editing post-HDR. An optimized ssDNA donor (#8) described for SpCas9-induced HDR19 and its complementary version (#7) were used in combination with SpCas9–sgRNA RNP complexes as references for comparison (bottom). d qPCR quantification showing % of HDR from individual embryos when using LbCpf1 and different ssDNA donors in comparison to SpCas9. % of HDR: amount of integrated DNA per total amount of genomic DNA per embryo (see Methods for details). Results are shown as the averages ± S.D. of the means from 16 embryos in two independent experiments (n = 8 embryos per experiment). Positive embryos: number of embryos per condition showing a detectable qPCR amplification signal. The data were analyzed by Kruskal–Wallis test followed by Dunn’s post test for significance vs. control condition (#8), p < 0.01, *P < 0.001

References [+] :

Bazzini, Codon identity regulates mRNA stability and translation efficiency during the maternal-to-zygotic transition. 2016, Pubmed, Xenbase

	Fig. 1. LbCpf1–crRNA RNP complexes are a robust genome editing system in zebrafish. a Diagram illustrating three crRNAs (orange) targeting slc45a2 and tyr exon 1 in zebrafish. b Schematic illustrating the experimental set-up to analyze CRISPR-Cpf1-mediated mutations in zebrafish. Three crRNAs (a) were either mixed with mRNA coding for AsCpf1 or LbCpf1 or assembled into RNP complexes with their corresponding purified proteins and injected in one-cell-stage embryos. c Phenotypic evaluation of crRNAs (30 pg/crRNA) and mRNA (100 pg) injections. Stacked barplots showing the percentage of mosaic (gray) and phenotypically wild-type (WT; black) embryos 48 h post fertilization (hpf) after injection. d Phenotypes obtained after the injection of the LbCpf1–crRNA RNP complexes targeting slc45a2 showing different levels of mosaicism compared to the WT. Lateral views (scale bar, 0.5 mm) and insets of the eyes (scale bar, 0.25 mm) of 48 hpf embryos are shown. e Phenotypic evaluation of Cpf1–crRNA RNP complexes injections targeting slc45a2 (albino). Stacked barplots showing the percentage of alb-like (white), mosaic (gray), and phenotypically WT (black) embryos 48 hpf after injection using different amounts (fmol) of RNP complexes. Number of embryos evaluated (n) is shown for each condition. f Phenotypes obtained after the injection of the LbCpf1–crRNA RNP complexes targeting tyr showing different levels of mosaicism compared to the WT. Lateral views (scale bar, 0.5 mm) and insets of the eyes (scale bar, 0.25 mm) of 48 hpf embryos are shown. g Phenotypic evaluation of Cpf1–crRNA RNP complexes targeting tyrosinase (tyr). Stacked barplots showing the percentage of tyr-like (white), severe mutant (light gray), mild mutant (dark gray), and phenotypically WT (black) embryos 48 hpf after injection using different amounts (fmol) of RNP complexes. Number of embryos evaluated (n) is shown for each condition
	Fig. 2. Temperature is a key factor modulating Cpf1 activity in vitro and in vivo. a Schema illustrating different temperature incubations after Cpf1–crRNA RNP complex injections targeting slc45a2 (alb) and tyr. b Phenotypic evaluation of AsCpf1–crRNA RNP complex (10 fmol) injections at different temperature incubations (T). Stacked barplots showing the percentage of tyr-like (white), severe mutant (light gray), mild mutant (dark gray), and phenotypically WT (black) embryos 48 hpf after injection. Number of embryos evaluated (n) is shown for each condition. χ 2-test (**p < 0.0001). c A representative picture showing 48-hpf-old embryos obtained after AsCpf1–crRNA RNP complex injections targeting tyr at different temperature incubations. Scale bar, 1.25 mm. d Phenotypic evaluation of LbCpf1–crRNA RNP complex (10 fmol) injections at different temperature incubations (T). Stacked barplots showing the percentage of alb/tyr-like (white), mosaic mutants (gray), and phenotypically WT (black) embryos 48 hpf after injection. Number of embryos evaluated (n) is shown for each condition. χ 2-test (p < 0.0001). e A representative picture showing 48-hpf-old embryos obtained after LbCpf1–crRNA RNP complex injections targeting slc45a2 at different temperature incubations. Scale bar, 1.25 mm. f Schematic illustrating different incubation conditions (0, 4, 8, or 24 h at 34 °C, then at 28 °C) after AsCpf1–crRNA RNP complex (10 fmol) injections targeting tyr in zebrafish (top). Phenotypic evaluation of AsCpf1–crRNA RNP complex injections targeting tyr in the conditions described above (bottom). Stacked barplots showing the percentage of tyr-like (white), severe mutant (light gray), mild mutant (dark gray), and phenotypically WT (black) embryos 48 hpf after injection. Number of embryos evaluated (n) is shown for each condition. χ 2-test (p < 0.0001). g Schematic illustrating different incubation conditions: 8 h at 28 °C, 16 h at 34 °C, and then 24 h at 28 °C (34 °C 8–24 hpf) or 24 h at 28 °C, then 24 h at 34 °C (34 °C 24–48 hpf) after AsCpf1–crRNA RNP complex (10 fmol) injections targeting tyr in zebrafish (top). Phenotypic evaluation of AsCpf1–crRNA RNP complex injections targeting tyr in the conditions described above (bottom). Stacked barplots showing the percentage of severe mutant (light gray), mild mutant (dark gray), and phenotypically WT (black) embryos 48 hpf after injection. Number of embryos evaluated (n) is shown for each condition. χ 2-test (**p < 0.0001)
	Fig. 3. Catalytic dead SpCas9 (SpdCas9) proximal targeting increases Cpf1 activity. a Schematic diagram of proxy-CRISPR approach. Temperature may control Cpf1 activity to access and/or unwind genomic DNA, impeding AsCpf1 to access the genomic target in vivo at 28 °C. When SpdCas9 binds in the proximity of the AsCpf1 target, it facilitates the availability of AsCpf1 to cleave the inaccessible target. b Diagram illustrating crRNA 1 (orange) and a proximal sgRNA (purple) targeting tyr exon 1 (Supplementary Fig. 10c and Supplementary Data 1) in zebrafish (top). AsCpf1–crRNA and/or SpdCas9-sgRNA RNP complexes were injected into one-cell-stage embryos and then incubated at 28 °C for 48 h (bottom). c Phenotypic evaluation of the experiment described in (b). Stacked barplots showing the percentage of severe mutant (light gray), mild mutant (dark gray), and phenotypically WT (black) embryos 48 hpf after injection. Number of embryos evaluated (n) is shown for each condition. χ 2-test (**p < 0.0001). d Diagram illustrating crRNA 2 (orange) and sgRNA 2 (purple) targeting alb exon 1 (Supplementary Data 1, Supplementary Figs. 3f and 8e) in zebrafish (top). LbCpf1–crRNA and/or SpdCas9-sgRNA RNP complexes were injected into one-cell-stage embryos and then incubated at 28 °C for 48 h (bottom). e Phenotypic evaluation of the experiment described in (d). Stacked barplots showing the percentage of alb-like (white), mosaic mutants (gray) and phenotypically WT (black) embryos 48 hpf after injection. Number of embryos evaluated (n) is shown for each condition. χ 2-test (**p < 0.0001)
	Fig. 4. LbCpf1-mediated homology-directed repair. a Schematic illustrating LbCpf1–crRNA RNP complex and SpCas9–sgRNA RNP complex interaction with their respective DNA targets. crRNA and sgRNA-DNA annealing occurs on the target strand (blue), and PAM sequences are in the non-target strand (red). b crRNA (pink line) and sgRNA (orange line) overlapping target sequences in the albino locus used for this analysis. c Schema illustrating different donor ssDNA (#1–6) complementary to either the target strand (t) or non-target strand (nt) and with symmetric or asymmetric homology arms used in combination with LbCpf1–crRNA. PAM sequence was modified (TgaV/ActB) to prevent new editing post-HDR. An optimized ssDNA donor (#8) described for SpCas9-induced HDR19 and its complementary version (#7) were used in combination with SpCas9–sgRNA RNP complexes as references for comparison (bottom). d qPCR quantification showing % of HDR from individual embryos when using LbCpf1 and different ssDNA donors in comparison to SpCas9. % of HDR: amount of integrated DNA per total amount of genomic DNA per embryo (see Methods for details). Results are shown as the averages ± S.D. of the means from 16 embryos in two independent experiments (n = 8 embryos per experiment). Positive embryos: number of embryos per condition showing a detectable qPCR amplification signal. The data were analyzed by Kruskal–Wallis test followed by Dunn’s post test for significance vs. control condition (#8), p < 0.01, *P < 0.001