Abstract
CRISPR/Cas9 technology has transformed mouse genome editing with unprecedented precision, efficiency, and ease; however, the current practice of microinjecting CRISPR reagents into pronuclear-stage embryos remains rate-limiting. We thus developed CRISPR ribonucleoprotein (RNP) electroporation of zygotes (CRISPR-EZ), an electroporation-based technology that outperforms pronuclear and cytoplasmic microinjection in efficiency, simplicity, cost, and throughput. In C57BL/6J and C57BL/6N mouse strains, CRISPR-EZ achieves 100% delivery of Cas9/single-guide RNA (sgRNA) RNPs, facilitating indel mutations (insertions or deletions), exon deletions, point mutations, and small insertions. In a side-by-side comparison in the high-throughput KnockOut Mouse Project (KOMP) pipeline, CRISPR-EZ consistently outperformed microinjection. Here, we provide an optimized protocol covering sgRNA synthesis, embryo collection, RNP electroporation, mouse generation, and genotyping strategies. Using CRISPR-EZ, a graduate-level researcher with basic embryo-manipulation skills can obtain genetically modified mice in 6 weeks. Altogether, CRISPR-EZ is a simple, economic, efficient, and high-throughput technology that is potentially applicable to other mammalian species.
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Acknowledgements
We thank A. Lee and B. Lee for technical assistance. We thank C. Di Biagio for help in making the protocol video. We thank C. Jeans and the University of California, Berkeley (UC Berkeley) California Institute for Quantitative Biosciences (QB3) Macrolab for purified Cas9 protein used during testing. We thank J. Doudna, D. Carrol, and J. Corn for stimulating discussions, and N. Anchell, P. Dao, K. Jager, and D. Young for zygote collection, electroporation, and embryo transfer. We thank K.N. Grimsrud for veterinary oversight. L.H. was supported by a Howard Hughes Medical Institute (HHMI) Faculty Scholar award, a Bakar Fellow award at UC Berkeley, several grants from the National Institutes of Health (NIH; R01GM114414, R01CA139067, 2R01CA139067, 1R21HD088885 and R21HD088885), and a research scholar award from the American Cancer Society. A.J.M. was supported by a F32 postdoctoral fellowship from the NIH (CA192636-03). S.C. was supported by a CCRC predoctoral fellowship and a Siebel predoctoral fellowship. J.A.W. and K.C.K.L. acknowledge support from two NIH grants (UM1 OD023221 and U42 OD011175) and a grant from the American College of Laboratory Animal Medicine.
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Contributions
A.J.M. conceived the original idea that led to the development of the CRISPR-EZ technology, drafted the manuscript and methods, analyzed the raw experimental data, and produced all figures and tables. S.C. and A.J.M. trained the staff at the Mouse Biology Program (MBP) at UC Davis to perform CRISPR-EZ experiments, optimized electroporation conditions and produced a video for all experimental procedures. S.C. conceived of the Tyr editing scheme for optimizing the CRISPR-EZ protocol, and drafted and revised the manuscript. B.J.W. contributed to sgRNA design, RNP preparation, genotype screening, data analysis, and manuscript preparation. J.A.W. and K.C.K.L. contributed to the study design, collaboration setup, and paper revision. L.H. contributed to the experimental design, initiated and established the collaboration with MBP at UC Davis, and worked closely with A.J.M. and S.C. to interpret the data and to draft and revise the manuscript.
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Integrated supplementary information
Supplementary Figure 1 CRISPR-EZ enables high-throughput genome editing.
(a) No appreciable differences are observed in editing efficiency (measured by mouse coat color, left) or viability (measured by live birth rate, right) when performing CRISPR-EZ using a pool of 35, 60 or 100 C57B/6J pronuclear embryos. (b) Since Tyr deficiency causes albinism in edited mice, the extent of albinism correlates the extent of editing that disrupted Tyr function. Representative image is shown for the edited mice from experiments in A, (100 Pooled Embryos). (c) The size of deletion affects editing efficiency using CRISPR-EZ. Deletions in the range of 1030-2672 bp (n=6) exhibit reduced efficiency compared to those ranging from 383-955 bp (n=16). Data are means ± SD across all genes. ** P < 0.01; *** P < 0.001; **** P < 0.0001; n.s., not significant (All P-values were calculated on a basis of an unpaired one-tailed Student’s t test.)
Supplementary Figure 2 Successful examples of targeted exon deletions by CRISPR-EZ.
Experimental design and representative genotyping results are shown for several successful CRISPR-EZ projects that aim to employ up to two pairs of sgRNAs to mediate deletion of key protein-coding exon(s) (red exons). Successfully targeted genes by CRISPR-EZ include Copb1 (a), Xpo5 (b), Kpna2 (c), Ipo7 (d), Slc25a18 (e), Mcts1 (f), Unc5cl (g) and Wdr82 (h). QIAxcel fragment analysis image of PCR genotyping results as well as chromatograms of validated sequence at the edited sites are shown accompanying each diagram. NTC, non-template control; WT, wild type.
Supplementary Figure 3 Successful examples of targeted exon deletions by CRISPR-EZ that are refractory to microinjection.
Experimental designs are shown genes in which CRISPR-EZ and Microinjection methodologies were directly tested against one another. Successfully targeted genes by CRISPR-EZ include Gsg1l (a), Fubp1 (b), Clcnkb (c), Cdnp2 (d), Ddx6 (e), Cfap57 (f), Gndp2 (g) and Sfmbt2 (h). In the case of Clcnkb, three guides were used as indicated in (c). QIAxcel fragment analysis image of PCR genotyping results as well as chromatograms of validated sequence at the edited sites are shown accompanying each diagram. Schematics without QIAxcel fragment analysis and chromatograms are pending and underway. NTC, non-template control; WT, wild type.
Supplementary Figure 4 Timing of events during the first cell cycle of a fertilized mouse embryo.
Diagram illustrating the timing of critical events during the first cell cycle of a fertilized mouse embryo, including morphological changes, approximate timing of cell cycle stage and molecular hallmarks that lead up to the first cleavage event. As the exact timing of insemination is not known, a reasonable suggestion would be to regard hour 0 as midnight. The first zygotic cell cycle culminates approximately 21 hours post-fertilization, allowing a short window as the ideal timing for CRISPR-EZ editing. NHEJ is the predominate repair mechanism active during G1, S and G2 phase of cell cycle while HDR repair demonstrates peak activity in late S and G2 phases, when sister chromatin is most accessible. Thus, the ideal timing to deliver CRISPR editing machinery for both NHEJ and HDR is before entry into the first zygotic S-phase, prior to DNA replication, which occurs approximately 10-11 hours post-fertilization. This would roughly translate as conducting CRISPR-EZ between 7-11 AM. In light of these timelines, pre-assembled Cas9/sgRNA RNPs have several advantages over cas9 mRNA/sgRNA, as the RNPs catalyze rapid editing on a diploid pronucleus genome, while the absence of sustained Cas9 translation minimizes potential off-target effects. With our optimized CRISPR-EZ protocol, the majority of pronuclear embryos have just begun to enter S-Phase when the Cas9 RNPs are delivered by electroporation. Consistent with Kim et al 2014, our experimentally-derived kinetics within the embryo suggest that the first signs of editing occur 2-4 hours post-electroporation, around the time when HDR is anticipated to be active. Thus, the CRISPR-EZ protocol is optimized towards facilitating both NHEJ and HDR outcomes.
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Supplementary Figure 5 Preparing a mouth pipette.
(a) Glass transfer pipets are made by carefully bringing the center of a glass capillary tube into a medium height flame. (b) As the glass capillary tube begins to melt, quickly lift the tube out of the flame and then pull the tube outward about 2-3 inches. (c) The pulled tube is then snapped in the middle, producing two glass transfer pipets. (d) The glass transfer pipet is then inserted into the clear pipet holder of the mouth pipet.
Supplementary Figure 6 Diagram illustrating how to set up an embryo culture dish.
(a) Top and side views of a 35 mm plate used for embryo culture are shown, ∼20-30 μl of KSOM+BSA droplets are evenly distributed with a handheld pipet. These droplets are then carefully overlaid with mineral oil delivered by serological pipet. Droplets will maintain their integrity due to different viscosity than the overlaid mineral oil. The plate is then allowed to equilibrate for at least 4 hours (preferably overnight) in a cell culture incubator. Embryos are then placed into droplets by piercing the mineral oil with a glass mouth pipet so as to minimally perturb the equilibrated culturing environment. (b) Photographic images of embryo culture plates are shown.
Supplementary Figure 7 Diagram illustrating how to set up an embryo wash dish.
Top and side views of a 60 mm plate used for embryo washes are shown, 50 μl of M2+BSA droplets are distributed with a handheld pipet. The pattern shown is to maximize the number of wash droplets per plate. The plate can be left in 37°C incubator conditions prior to use so that embryos are not exposed to non-physiological temperatures for very long.
Supplementary Figure 8 Potential issues during sgRNA quality control.
Bioanalyzer traces illustrate typical experimental issues observed during sgRNA synthesis and quality control. (a) Loading marker; (b) Primer dimers or severe sgRNA degradation; (c) Multiple sgRNA synthesis products, possibly due to mis-priming; (d) An example of sgRNA that exhibits a slight shoulder forming from the main peak. While this profile is slightly deviated from that of a perfect sgRNA synthesis, their editing efficiency is comparable (e) Insufficient heat denaturation of good quality sgRNAs prior to loading; (f) A representative profile for an ideal sgRNA synthesis.
Supplementary Figure 9 Potential issues during embryo collection and culture.
Representative images of newly ovulated oocytes and embryos. Viable embryos display a distinct oolema boundary that is easily distinguished from the zona pellucida (green arrows). Inviable embryos take on an amorphous appearance where it is difficult to identify a specific cellular boundary (red arrows). Even at the highest magnification shown (40x), it is difficult to discern oocytes from fertilized pronucleus stage embryos by the presence of male and female pronucleus. Scale Bars: 50 μm.
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Modzelewski, A., Chen, S., Willis, B. et al. Efficient mouse genome engineering by CRISPR-EZ technology. Nat Protoc 13, 1253–1274 (2018). https://doi.org/10.1038/nprot.2018.012
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DOI: https://doi.org/10.1038/nprot.2018.012
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