Optimizing Cas9 and sgrna concentrations for increasing mutation efficiency. Nature Methods: doi: /nmeth.3360

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1 Supplementary Figure 1 Optimizing Cas9 and sgrna concentrations for increasing mutation efficiency.

2 (a) Wild-type, 2-dpf slc24a5 b1 embryos and a series of slc24a5 CRISPR-injected embryos; anterior is to the left. Wild-type pigmentation is lost in slc24a5 ( golden ) mutants. Injection of Cas9-encoding mrna and an sgrna targeting slc24a5 resulted in embryos with mosaic loss of eye pigmentation (a ). (b) Model of the slc24a5 CRISPR target site with arrows denoting PCR primers used for sequence analysis of the locus. (c) Sequencing from embryos injected with 100/1,200 pg of slc24a5 sgrna/cas9-encoding mrna. Deletions are denoted by dashes (-), and insertions are in bold. The underlined sequence denotes the NGG motif used by Cas9. (d) Quantitation of eye pigmentation as assessed by mean pixel gray value of the eye with varying amounts of Cas9-encoding mrna and a constant 100 pg of slc24a5 sgrna. Each open triangle represents an individual animal. (e,f) Quantitation of toxicity seen in injected embryos for varying amounts of Cas9-encoding mrna and a constant 100 pg of slc24a5 sgrna (e) or a constant 1,200 pg of Cas9 with varying slc24a5 sgrna (f). Toxicity encompassed embryo death, edema, localized cell death and general developmental defects. In d, N = 24 for each condition; error bars denote s.e.m. In e and f, N > 77 embryos for each point.

3 Supplementary Figure 2 Optimized CRISPR can recapitulate known mutant defects in injected embryos. (a) Model of pk1b and vangl2 sgrna target sites. Underlined sequence denotes the NGG motif used byy Cas9. (b) Images are 20-μm dorsal-view projections of the hindbrain at 2 dpf. Anterior is up. Scale bar, 200 μm. Larvae are transgenic for Tg(isl1:GFP)rw0, which marks the branchiomotor neurons. A subset of o these, the facial branchiomotor neurons (shown), undergo a stereotypical migration from rhombomere 4 (r4) to r6. Mutants in the planar cell polarity pathway (pk1b and vangl2) cause a failure in this migration. These phenotypes can be recapitulated in injected CRISPR F 0 embryos. (c) Quantitation of migrationn defects. (d) Lateral views of 1-dpf embryos. Scale bar, 1 mm. vangl2 mutants have defects in anterior- posterior axis elongation due to defects in mesodermal convergence and extension. This can bee recapitulated in injected CRISPR F 0 embryos. (e) Quantitation of convergent extension defect. In c and e, e N > 24 embryos for each bar.

4 Supplementary Figure 3 Optimizing Cas9 and sgrna concentrations for electrical-synapse phenotypes. (a) Quantitationn of mosaic electrical-synapse loss in injected embryos for varying amounts of gjd1aa sgrna injected with 1,200 pg of Cas9-encoding mrna. (b) Quantitation of toxicity seen in injectedd embryos for varying amounts of gjd1a sgrna injected with 1,200 pg of Cas9-encoding mrna. In a, N = 24 embryos for each bar. In b, N > 86 embryos for each point.

5 Supplementary Figure 4 Analysis of sgrnas and genomic alterations in CRISPR injected embryos revealed by targeted deep sequencing of screen targets.

6 (a) Comparison of GC content versus indel frequency. Individual targets are denoted by open squares. The solid line is a linear trend line of the data (R 2 = ). (b) Analysis of sgrna indel efficiency based on binding the non-coding (, N = 10) or coding (+, N = 32) strand. Box-and-whisker plot denotes median, quartiles, and extremes of the data. (c) Heat map for positions 2 to N of the sgrna showing the relative increase in indel efficiency for each specific position. N is the first position of the PAM sequence. Color scale represents increased (orange) or decreased (cyan) indel efficiency relative to average. (d) Percentage of indels that caused out-of-frame changes to the target sequence. Individual targets are denoted by open squares. (e) Histogram of deletion positions relative to the NGG site bound by Cas9. Data are included from all targets aligned to the NGG. (f) Distribution of deletion sizes. Data are combined from all targets. The x-axes of e and f are log scales.

7 Supplementary Figure 5 Direct genotype-phenotype association for tjp1b and electrical-synapse loss.

8 (a) Each triangle represents an individual animal injected with 16.5/1,200 pg tjp1b_l sgrna/cas9- encoding mrna. Spinal cords were analyzed for the loss of synapses and heads were assayed for mutational efficiency on the basis of qpcr analysis of the tjp1b locus. We found that heads and spinal cords had indistinguishable mutational efficiencies (heads, 0.59 ± 0.04; tail, 0.61 ± 0.05; P = 0.84; values are average ± s.e.m. compared by Student s t-test). The square of the mutational efficiency is plotted on the x-axis on the basis of the expectation that phenotypes are expected to be due to biallelic loss of a gene. There is a strong correlation of genotype to phenotype (R 2 = 0.98). The lines represent the expected genotype-phenotype association, assuming random mutagenesis and that deleterious alleles occur only with out-of-frame mutations (dotted line, y = (2/3) 2 x) or both in-frame and out-of-frame mutations (solid line, y = (3/3) 2 x). The fact that the phenotypic rate approaches 100% suggests either that in-frame deletions are deleterious or that heterozygous loss of tjp1b effects synapse formation. Note that we found that the qpcr genotypic analysis underestimated the mutational efficiency by 6% 15% (see Online Methods). This underestimation would result in an increased slope of genotype-phenotype association plotted here. (b d) Images are 15-μm dorsal-view projections of two spinal cord segments at 5 dpf. Anterior is to the left. Scale bar, 10 μm. Larvae were stained for Connexin36 (Cx36, white) to mark the electrical synapses and for neurofilaments (RMO44, red) to mark neuronal processes, including M and CoLo. Individual Cx36 channel is shown in neighboring panel. Heterozygous loss of tjp1b has no effect on M synapse formation, whether the germline-transmitted mutation is out-of-frame (b, tjp1b fh449/+ ) or in-frame (c, tjp1b fh451/+ ). However, trans-heterozygous tjp1b mutants carrying an out-of-frame and an in-frame mutation (tjp1b fh449/fh451 ) lose electrical synapses, confirming that in-frame mutations are deleterious at this locus (d). In a, N = 32 embryos.

9 Supplementary Figure 6 Retesting sgrnas with degradation. low indel rates from the screen revealed thatt most were due too (a) qpcr quantitation of mutational efficiency in injected embryos for each of the failed target sgrnass from the screen and a newly synthesized version. Eachh was individually injected at 100/1,200 pgg sgrna/cas9-encoding mrna. Mutation efficiency was assessed by qpcr and was done in triplicate; error bars denote s.e.m. (b) nrxn1b_s contained two polymorphisms in the designed sgrna as comparedd to the genomic sequence of the fish into which it was injected. These differences likely reflect its failure too induce indels. In a, each bar represents five embryos pooled in three replicates; error bars denote s.e.m.

10 Supplementary Figure 7 Visual method for design, synthesis, quality control and injection of sgrnas and Cas9-encoding mrna into zebrafish embryos. See the Online Methods for details on each step.

11 Screen Targets Gene ENSDARG# ENSDART# chr exon sgrna Score NGS Forward Primer NGS Reverse Primer NGS Efficiency gjd1a ENSDARG ENSDART GAGAATGGACTATATTGGAGAGG 91 GGCTTTCAAGAGGAGCCCCAAGAAG CATGCACTCTGTGGAGTGTGTGTGTG gjd1b GACCATTTTAGAGCGCCTCCTGG 96 GCCATCGGCGGAACTATTTTTA TTCCGATCATAGTAGAGTGCTGCTG gjd2a GAGAATGGACCATACTAGAGAGG 93 AGACGCGAAACAGTTGCTCGCATT CCAGAAGCCAATCGCCCTTAGTCAG 22.6 gjd2b ENSDARG ENSDART GACAATTCTCGAGCGTCTCCTGG 98 CCACAACTGCGGACACTGAGAGGAA ATCTTCTACTCCGAAAACGCCGCAAA 0.99* cnsta ENSDARG ENSDART GGCAGAGCGACGGCCCTGTAAGG 97 CATGCACTGCTCTCAGAAAGCCTGA AGCAGCCGGTTGTGGTTGTCATCA cnstb ENSDARG ENSDART GGAGTCCGAAAGCAGGAAATCGG 91 TGCGTAAGCGACAGAAGGGTGAAGA CCGCGTTCATGACTTCTGTTTCTGG nlgn1 ENSDARG ENSDART GTGCTGCGTAGGGAACCCCCAGG 99 GCCTGGATCTGCCATTCTTGTGTTG ACTGAGTTGCGTTGCGGATTTCTGA nlgn2a ENSDARG ENSDART GGGCTATGCCCACAATTCAGAGG 98 CCCATTCCTGTCCTCCCTATTCTTGC AGTGCCAGGATCGATGCGTTGACAT 38.7 nlgn2b ENSDARG ENSDART GGTGACCATAGGGTGCTTGGCGG 97 AGACGGTTCGCTCCTGTCCACGAC GCGTAGGGGACACCCAAGTACTGCT nlgn3a ENSDARG ENSDART GGGACCAGTGGACCAGTACCTGG 97 GGGCAAGGCTACTACCCCACAGTCA TGTGAATGTTTTGTGGGCACACTGG nlgn3b ENSDARG ENSDART GGGACCTGTGGATCAATATCTGG 93 GTCTGTGGGCAAACAGGAGGAAAGC TGCTCGTGTGGTTGATGTGGTCAGT nlgn4a ENSDARG ENSDART GGATTGTGCTTGCGGCTGCGTGG 94 TGGGTTGTACCTTGGCTTTGGAGGA TCATTGGGCAGAGGTGTTTTCAAGC 41.3 nlgn4b ENSDARG ENSDART GAAGCTCCGCGGGCTCCGTGTGG 96 TCACCATGATCCAGCACATCCTCAG AGCGAACTGTGTGGCGTTCCTGAT nrxn1a_l ENSDARG ENSDART GGGGCCAGCATGGAGTTCACTGG 96 ACAATGAGCTTCTCAATGCGGAACG CCATTGTGGATGAGCAGTTCCAAGA nrxn1b_l ENSDARG ENSDART GGATCTAGTCTCAGGTTTACAGG 94 GCAACAATAACATCTGGTGCCACCTT AGGCCATTCAGATGTGTGGTTTTCA nrxn2a_l ENSDARG ENSDART GTCACCGGCCTTCGTTTGGCTGG 97 TGCAGTGGATTAAGGGAAACGGAACA TCCACCATCATCCAGGTACAGCACA 0.4* nrxn2b_l ENSDARG ENSDART GTGGGCACGGTACTCTCGGTGGG 99 TCATCATCAGCGAGTGTGAGATGGA TCCAGAGATCAGCAGCTCCAGGAAA nrxn3a_l ENSDARG ENSDART GTAAACTCCAGGCCCAAGCAGGG 93 TGACCTTTGAACCAAAGCGAATTCAA GCCGCCATCGTCAAAGTACAGGATT nrxn3b_l ENSDARG ENSDART GGTTTGGGCCTGGAGTTCACAGG 93 TGTAGCATACGCTCTGGCTACCATGC CTGCACAGTCCACGCTAAAATGCAA nrxn1a_s ENSDARG ENSDART GGCTGCCACCGCTGACTTTGGGG 94 CAGGTGGTTTGAGGACACCCCTCTT TGGCTGTCGAATAAGCTACGCACCA nrxn1b_s ENSDARG ENSDART GGAGATCATCACTCGGCGTTTGG 98 CCGGTGTGCTGAAGTGTGTGTTTGT TACTGTTGTACCCACCCTGCCATGC 1.85** nrxn2a_s ENSDARG ENSDART GGCGCTATCTCTGGCCTTGTTGG 97 TGATTGATATGGGCACCGCTCTTTG TGGAAGTGATGGACATGGTGTGTGG nrxn2b_s ENSDARG ENSDART GGAGCTACAGCCGGGGCTTGTGG 93 TCCAGAGATCAGCAGCTCCAGGAAA GCGTGGTGCTCAGACTGGAGGATAC nrxn3a_s ENSDARG ENSDART GATAATTCAGTGGTCGCCGCTGG 98 AGTGTGAGTCGGGTGTGCGTTATGC ATATGGAAATGGGTGAAGCGTGGTG nrxn3b_s ENSDARG ENSDART GGTGCGTGTTCGGAGCCGTATGG 99 AACGGACGTCAGGTTCTGCTCACCT CAGGTGATGGTGGTGCTGTGTTTCA tjp1a_l ENSDARG ENSDART GCATACGGTGACTCTTCACAGGG 92 CACCTCACGCCATTAACAAAGAGGA GCCCTCATCAGATTATGAAGTGTGAA tjp1a_b ENSDARG ENSDART GGAAAGTGTCGGATTGAGGCTGG 96 CAGGTGATGGTGGTGCTGTGTTTCA CCTGAGTATCTGGTCTCCCTCCTCCA tjp1b_l ENSDARG ENSDART GAGTGCAGCAATGGATGAGACGG 82 CCTTCAGGCCGTGTGGGAATGTAAC TCTGCTCTGGGACTCCAAACACACA 25.3 tjp1b_b ENSDARG ENSDART GTGGGCTTGAGGCTCGCTGGGGG 93 CATGGCCCGTTTCCACTGCAATAA GGTCTCCCTCTTCCAGGCCCTCTT 52.1 tjp2a ENSDARG ENSDART GGCTTTGGCCTAGCGGTGTCTGG 96 AAGATCGGAAAAACCCTCCACATCAA TCACAGGTTGAGGGCTGTGTTGAAA tjp2b ENSDARG ENSDART GTTTGGATTGTCCCGGCCGCCGG 98 CTAGCTGTTTGCACGTCCCCATGAA TGAACCGTACAAGAGCAGTCCATCG tjp3 ENSDARG ENSDART GATTCCCACCGGTCCCGCCATGG 94 ATTCCAAGGTGGGCTTTGGTTTTGC GCGTCCTACGGGCAAGAAATCACAT cntn1a GAAGCCCGACTCACACTACCTGG 99 ACCAGCCAGTGCGGCTTATCTGAG TGCAGCTGAGGGACTTACGGGACTT cntn1b ENSDARG ENSDART GATGAGCACTTCAGCCTGGTTGG 93 GGCGGGGCTTACCCATAGCTCAAT CCAAACTTGACGTTGGCCTCTTTGC cntn2 ENSDARG ENSDART GGAGCGAGCCTGGCAGCTCAGGG 94 TTTTTGGTAGGTGGGGATGTGTGTG GCAAGTAAAGGTCGTGTTTTGTTCG cntn3a ENSDARG ENSDART GTGTCCTGCTCGACGAATGAGGG 95 TCCAAAGAGATGCGGTTCAGTATAGC GGAGTATCACCATCACTGCGGAGAA cntn3b ENSDARG ENSDART GTGTAATTCCCGACGTCGGACGG 95 TGAGTACCCTCACTTCGTCCAGCAA TGACTGTGAAAAGCCCTTTGATGTT 1.28* cntn4 ENSDARG ENSDART GTACCCATCAATCGTGAAGCAGG 97 TTCGGGGCAGTAGATTGACCTCTTTC TTGGCGGCCCTTGTACTTGTGTTTT 4.97* cntn5 ENSDARG ENSDART GCAAGCGAAGTCATCGATTATGG 96 TTGCTTTTGCAGTTGGCTGATCAAT GGGTTCGAGATTACTCACAGGCAAA 0.8* cntn6 ENSDARG ENSDART GGCCGTCCGAGTGTCGCTGTTGG 95 TGGCCAGTTATTTCCCTCCCTTTGA TTCCCCACATCTGACGTCTCCACCT 30.3 cntnap1 ENSDARG ENSDART GGGGAGGAAGTATCGTATTATGG 97 ATTTTCTGATGACGCAGGGAGCAGT TACCATGTTGCCTCCTTTCATGACG 44.7 CNTNAP2 ENSDARG ENSDART GTATCTGCCCTGCGTTGCGATGG 94 TGCGGTGTGCACTAAAGCATCGTAA GTCCTGCCGATAGGGTTTCCAGTTG 34.6 cntnap2a ENSDARG ENSDART GGGGTGATTCTGCACGGAGAGGG 91 CAGGGGGTGATCTCTTACCGCTTCA TGGAACCATTGATGCCAATAAAGCA cntnap2b ENSDARG ENSDART GAATGAAGTCGGGGTTGCCCAGG 94 GGAACGCTGCTTCTTTCAACACACC GGGACTTTAAAGCGCAGTTCCTCACA cntnap4 ENSDARG ENSDART GTCTCCAGCCAGGGCCGATACGG 98 TCAATTGTGTGGATTCCGGCATTTC CCTATGCTGTCTTCCTGGCGGTACG 74 CNTNAP5 ENSDARG ENSDART GGACCTACGGGATAGGTTGAAGG 99 AGCGAGTGCCTGAATTGTGAAGAGAA ACGGTCTCCAGGTTCGTCCAGAGTC cntnap5a ENSDARG ENSDART GGACAGGTCTCCTTGGCTGCAGG 96 GGGGGCTTTAAATGACAAACAGTGG GGAGCATGTAAGCACTGACCCAGTC cntnap5b ENSDARG ENSDART GGTCACAGCTGTGGCGACCCAGG 97 AGCAACGCTGGAATTGCTCACTCCT TGTCCTCCTGTCTGAACTGCTTCCA Off- Targets for NGS Name ENSDARG# ENSDART# chr exon Potential Site Score Forward NGS Primer Reverse NGS Primer NGS Efficiency gjd1a_off AGAATGGACGGTATTGGAGGAGG 1.4 CCAATCACGTCCATTCTCCTCTCACA CGCACACACACACACACACACACAC 1.63 gjd1a_off GAACATGGACTATATTAGAGCGG 1.2 TGCATCAACAGAGTATTGAGCAAAGG CAGCGATCTGTGGAGCAATGAAAG 0.17 gjd1a_off GAGGATGTGATATATTGGAGAGG 0.8 ACGCTGAGGTCAGCAACAGACTTGC TTGTGCATGAGAGGAGCTGTTCGTG 0.16 gjd2a_off GGTAATGGAGGATACTAGAGAGG 0.7 TGTTTCTACCACTGATCGCATCACA GCGCTGATGTTTTCTCCTCGGTATC 0.25 tjp1b_b_off AAGCGCTTGTGGCTCGCTGGAGG 1.3 GCAAATGAAAAAGAAAGCCAAACCA AGAACAGGCAGCAATCATTCGGTTC 0.36 tjp1b_l_off TAGTGCAAAAATGGATGAGAGGG 1.6 AAAAAGAATGCCTTTTTATGTCACTG TGCTCTGTTAAACATCATTTGGGAAA 3.17 tjp1b_l_off GCGGGCAGCAGTGGATGAGATGG 1.5 GACAGTGCGCTGGTTGAACCGTGT AGGAAATTAGGCTGAGCCGGCAGAA 0.46 Other Targets Name ENSDARG# ENSDART# chr exon grna Score Forward Primer Reverse Primer gjd1a_02 ENSDARG ENSDART GACTATATTGGAGAGGTTGCTGG 89 AGGTCGGCAGGGAAGTGAGAGT TAGAGCTCTTACCTGCCGATCA slc24a5 ENSDARG ENSDART GGTCTCTCGCAGGATGTTGCTGG 94 ACTAAAACCAACATGTTTGTGT AATTGTTTACCCAGAAATGCAG pk1b ENSDARG ENSDART GCACCGGACATGGACTCGAGAGG 92 vangl2 ENSDARG ENSDART GCGTCCAGCAGTGGTTCTCCAGG 97 CCCGATCATCTCCGCGTGTGG AGCAGAGACAGCAGCAGCAGA sox10 ENSDARG ENSDART GGAGCGCCCGACGAGTGACCAGG 99 GAAATGAGTCCCGGGGTGTCGG CATCTGAGACTGCTGACCGGGC pkd2 ENSDARG ENSDART GGGTCTCATCGGGCACTCCGAGG 97 TCCTCCACCGCATGAAGGCATC CTCCATGCCTGTCTGGAGCTGG hmcn1 ENSDARG ENSDART GGACATCTTCTTTTGGGTCCTGG 92 ACTTCGCCGTTTTTTCTTGGTCTCA AACGCTAAGGTGGAGGCTCCCT edn1 ENSDARG ENSDART GATGTCAGCATGGTCAGAACTGG 94 TTTAAATACATACCAAAGTCAAAAA GTTTTACTGGACTACTACTGGA slc12a2 ENSDARG ENSDART GCTCTGGCTCGGAGTCGGACCGG 98 CTCTCTGCGCCCGAACCTGATG AAGCCGACAGTGGTTTCACCGT gfp GGGCACGGGCAGCTTGCCGGTGG TTCAGCGTGTCCGGCGAGG TAGGTCAGGGTGGTCACGAGGG qpcr Primers Name Forward Primer Reverse Primer "ON" Primer Forward NGS primers have 5' tag: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG gjd1a AGGTCGGCAGGGAAGTGAGAGT TAGAGCTCTTACCTGCCGATCA GAGAATGGACTATATTGGAG Reverse NGS primers have 5' tag: GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG gjd1a_02 AGGTCGGCAGGGAAGTGAGAGT TAGAGCTCTTACCTGCCGATCA gjd2b CGCATTTGATTTCACACGGTTCTC CACTTTTTCCCCTCACCTCCCAAT GACAATTCTCGAGCGTCTCC *5 Targets failed due to sgrna degradation cntn3b TTCTTAGAACCAACGGTGTCG TGAGTACCCTCACTTCGTCCA GTGTAATTCCCGACGTCGGA ** Target failed due to polymorphisms in genome cntn4 GGGGCACTAGATTGACCTCTT TCACGACACAGGTGTAGTTTCC cntn5 GCTGATCAATGGAACGAATGT CAATGCTGTTTTCAGCTCTGC nrxn1b_s GCTGAAGTGTGTGTTTGTGCT AAATCATGCCCACAGACAGAG GACTATATTGGAGAGGTTGC GTACCCATCAATCGTGAAGC GCAAGCGAAGTCATCGATTA GGAGATCATCACTCGGCGTT nrxn2a_l AAGGAAGGATTAGGAGCTTGC GGCCTGGTAAACCATCAAACT GTCACCGGCCTTCGTTTGGC

12 Supplementary Table 1 Gene, sgrna sequence, primer sequences, and efficiency data for all targets investigated in the paper.