Materials and Methods

Key sequence features of CRISPR RNA for dual-guide CRISPR-Cas9 ribonucleoprotein complexes assembled with wild-type or HiFi Cas9

MATERIALS AND METHODSZebrafish husbandryZebrafish (Danio rerio) were bred and maintained under standard laboratory conditions on a 14 h/10 h light/dark cycle. All zebrafish experiments were conducted in accordance with the Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology of Japan, using protocols approved by the Animal Experiments Committee of Kochi University of Technology.Preparation of CRISPR-Cas9 RNP complexesCRISPR-Cas9 RNP complexes were prepared using crRNAs (Alt-R CRISPR-Cas9 crRNA) (listed in Supplementary Table S1), tracrRNA (Alt-R CRISPR-Cas9 tracrRNA-ATTO 550), and Cas9 protein (Alt-R S.p. Cas9 Nuclease V3 and HiFi Cas9 Nuclease V3) purchased from IDT, and used according to the manufacturer's protocol (Zebrafish embryo microinjection: Ribonucleoprotein delivery using the Alt-R CRISPR-Cas9 System). In brief, 100 μM crRNA and 100 μM tracrRNA were mixed in Nuclease-Free Duplex Buffer (IDT) to create a 3 μM gRNA solution. The solution was heated at 95°C for 5 min and then cooled to room temperature. The 3 μM gRNA solution was combined with an equimolar amount of 3 μM Cas9 diluted in Cas9 working buffer (20 mM HEPES pH 7.5, 150 mM KCl) and incubated at 37°C for 10 min to assemble 1.5 μM RNP complex.Preparation of HiFi Cas9 mRNApCS2-HiFi Cas9 was constructed by inserting a HiFi Cas9-encoding DNA fragment from pX330-Flag-HiFi SpCas9 (a gift from Ervin Welker, Addgene plasmid #126778) into the pCS2 vector. pCS2-HiFi Cas9 was linearized with NotI and used as a template for mRNA synthesis using AmpliCap SP6 High Yield Message Maker Kit (CELLSCRIPT). mRNA was then purified with RNA clean & concentrator (Zymo Research). HiFi Cas9 mRNA solution (200 ng/μl) was combined with the same volume of the 3 μM gRNA solution and used for microinjection.Microinjection and genomic DNA preparationTypically, 1 nl of 1.5 μM CRISPR-Cas9 RNP complex was microinjected into the yolk of 1-cell stage embryos of TL zebrafish, delivering 1.5 fmol of RNP complex. When HiFi Cas9 mRNA was used, 2 nl of solution containing 100 ng/μl HiFi Cas9 mRNA and 1.5 μM gRNA were typically microinjected, delivering 200 pg of Cas9 mRNA and 3 fmol of gRNA. Injected embryos were raised at 28°C until 24 h post-fertilization (hpf) and genomic DNA was prepared from pools of embryos using 20 μl per embryo of DNA extraction buffer (low EDTA) (10 mM Tris–HCl pH 8, 0.1 mM EDTA, 0.2% Triton X-100, 200 μg/ml Proteinase K). After incubation at 55°C for 2–3 h with occasional vortexing and heating at 95°C for 10 min to inactivate Protease K, crude genomic DNA solution was used directly in polymerase chain reaction (PCR) assays.Assessment of indel frequencies using TIDE and ICE toolsQuantification of indel frequencies was performed using the tracking-of-indels-by-decomposition (TIDE) algorithm ( and inference-of-CRISPR-edits (ICE) method (, both of which were developed to analyze indels using Sanger sequence traces generated from PCR amplicons of target DNA regions (28,29). PCR primers were designed to amplify a fragment of approximately 600 bp surrounding the CRISPR-Cas9 target site (Supplementary Table S2). PCR was performed using Taq DNA Polymerase (New England Biolabs, NEB) in 30 μl of reaction mixture containing 1× ThermoPol Reaction Buffer, 200 μM dNTPs, 0.5 μM forward and reverse primers, 0.15 embryo equivalent of genomic DNA, 0.75 units of Taq DNA Polymerase, and 1× red sucrose solution (10% sucrose, 0.17 mM cresol red). PCR conditions were as follows: initial denaturation at 95°C for 30 s; 30 cycles of denaturation at 95°C for 15 s, annealing at 60°C or 65°C for 30 s, and extension at 68°C for 45 s; final extension at 68°C for 5 min. After confirming amplification of PCR products by agarose gel electrophoresis, PCR products were purified using NucleoSpin Gel and PCR Clean-up (Macherey-Nagel) or KAPA Pure Beads (Kapa Biosystems). Purified PCR products were Sanger sequenced using primers listed in Supplementary Table S2. Sanger sequencing traces from genomic DNAs prepared from CRISPR-Cas9 injected and uninjected control embryos were then used for TIDE (version 3.2.0; alignment window (bp) = 25, Indel size range = 25) and ICE (v2) to assess indel frequencies. In vitro CRISPR-Cas9 cleavage assayPurified PCR products that were amplified from TL zebrafish genomic DNA with primers for the TIDE and ICE assays were used as cleavage assay substrates. The in vitro cleavage reaction was performed according to the IDT protocol (Alt-R CRISPR-Cas9 System: In vitro cleavage of target DNA with ribonucleoprotein complex) in 10 μl of reaction mixture containing Cas9 Nuclease Reaction Buffer (20 mM HEPES pH 6.5, 100 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA), 100 nM CRISPR-Cas9 RNP complex, and 5 nM PCR products. The reaction mixture was incubated at 37°C for 20 min. The cleavage reaction was terminated by adding 1 μl of 20 mg/ml Proteinase K and incubating at 56°C for 10 min. The DNA substrate was subjected to agarose gel electrophoresis, and the gel was stained with 1× dsGreen (Lumiprobe) and visualized with a Fusion imaging system (Vilber Lourmat). Band intensities were quantified using ImageJ (v1.53) and cleavage efficiency was calculated by quantifying band intensities of uncleaved fragments.Sequence feature analysis by kpLogoSequence features of crRNAs were analyzed with the kpLogo (k-mer probability logo) tool using the locally installed program with default option settings (46) (, v1.1). Protospacer and PAM sequences of the tested 51 crRNAs that were weighted by cleavage frequencies obtained with ICE were used as input data. In vivo plasmid cleavage assayGenomic DNA fragments of the TL zebrafish strain that encompass target sites of crRNAs (otx2b_AA/AB, pax2a_AJ and sox19a-KO_4) were amplified using KOD-Plus-Neo DNA polymerase (Toyobo) with the primers listed in Supplementary Table S2. These fragments were cloned into the pUC19 vector at EcoRI and either HindIII or PstI restriction sites and used as CRISPR-Cas9 substrates. Mutant target plasmids for mutant crRNAs were constructed by site-directed mutagenesis using overlap extension PCR, in which PCRs were performed using KOD-One PCR Master Mix (Toyobo) with specific primers containing the intended mutations (Supplementary Table S2) and the wild-type target plasmids as templates.1 nl of the 10 ng/μl plasmid DNA (10 pg) was first microinjected into the cytoplasm of 1-cell stage embryos and 2 nl of 1.5 μM RNP complex (3 fmol) were subsequently microinjected into the yolk of plasmid-injected embryos. Injected embryos were raised at 28°C for 24 h and DNAs were prepared from both the nuclear and cytoplasmic fractions (47). In brief, 20 embryos were dechorionated using tweezers in E2 embryo medium (48) and washed 3x in 1 ml of ice-cold egg lysis buffer (125 mM NaCl, 5 mM MgCl2, 100 mM glycine, 20 mM HEPES pH 7.6) on ice. Embryos were homogenized with a micro pestle in 20 μl of egg lysis buffer on ice. Homogenate was centrifuged at 10 000 × g for 8 min at 4°C, and the supernatant was used as a cytoplasmic fraction. The pellet that was resuspended in 200 μl of ice-cold egg lysis buffer was underlaid with 200 μl of ice-cold 1 M sucrose/PBS and centrifuged at 5000 × g for 8 min at 4°C. This process was repeated once and the pellet was used as a nuclear fraction. DNA extraction buffer (low EDTA) was added to both fractions to achieve 0.05 embryo equivalent/μl and lysates were incubated at 55°C for 2–3 h with occasional mixing and heated at 95°C for 10 min to inactivate Proteinase K. Indel frequencies were assessed using TIDE, as described above, in which a fragment of approximately 600 bp surrounding the CRISPR-Cas9 target site on the plasmid was amplified using primers specific for the plasmid backbone sequence (Supplementary Table S2).CRISPR-Cas9 gRNA design toolsCRISPR-Cas9 gRNA design tools used in this study are listed in Supplementary Table S3, which summarizes their availability, experimental conditions to generate data (organism/cell types and gRNA types), and modelling methods.

Article TitleKey sequence features of CRISPR RNA for dual-guide CRISPR-Cas9 ribonucleoprotein complexes assembled with wild-type or HiFi Cas9


Probability value-based CRISPR-kp scores for crRNAs of interest can be obtained using the Excel spreadsheets (Supplementary Table S8) by replacing protospacer plus PAM sequences of the crRNAs.

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