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FIGURE 1
A workflow for amplicon sequencing analysis of on-target regions using the MiSeq platform. (a) Amplification of on-target regions using a gene-specific primer set harboring unique barcode and Illumina overhang adaptor sequences. (b) Workflow for sequencing of pooled amplicons from multiple crispants in a single-indexed library on a shared MiSeq run
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FIGURE 2
Mutation profiles of on-target sites in crispants. Mapped massive reads of on-target regions from Xenopus tropicalis tyrosinase (tyr, n = 10) and oculocutaneous albinism II (oca2, n = 5) crispants are visualized through Integrate Genomics Viewer (IGV). Total mapped reads (reads) and sample sizes (n) are shown in each image. Deletions, insertions and base substitutions are indicated by bars of black, purple and four other colors (red, blue, green and ocher), respectively. Protospacer adjacent motif (PAM), protospacer sequences and the putative double-strand break (DSB) site are indicated by red bar, blue bar and lightning motif, respectively
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FIGURE 3
Overview of CLiCKAR analysis. CLiCKAR requires only two types of input data: single-end sequencing data or joined paired-end sequencing data (fastq or fastq.gz files) and query table (csv file), as described in Table S1. Pooled amplicon data are demultiplexed by recognizing each barcoded sequence in Step 1. An output file from Step 1 (demultiplexed_fastq.dat) is subjected to Step 2 to align to each reference sequence. After the assignment of CIGAR strings to each read, subtypes of mutations are counted and frameshift mutation rates are calculated within a set target window around the double-strand break (DSB) site. Graphical reports (mutation_rate.pdf and indel_position.pdf) and data tables (mutation_ratio.csv and count.xlsx) regarding the mutation profile in each crispant are obtained as output
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FIGURE 4
Representative output files of CLiCKAR. (a) An example of demultiplexing results after Step 1. In total, 103,495 reads from 30 Xenopus tropicalis crispants targeting five different genes (oca2, pax6, slc45a2, tyr and tyrp1) were divided into individual crispants. They were distinguished by each color in this bar graph. These amplicon sequence data were obtained from a single-indexed library. Read counts are shown for each target gene. (b) An example of an indel position report in a tyr crispant. Vertical and horizontal axes indicate the number and position of indels relative to the reference sequence, respectively. Predicted double-strand break (DSB) site is indicated by a red vertical line. (c) An example of a graphical report regarding the frameshift mutation rate in tyr crispants (n = 10). Blue, green and red colors indicate percentages of out-of-frame mutation, in-frame mutation and wild-type allele, respectively. (d) An example of a data table in a tyr crispant. Column A: Sequence of mutant alleles within the target window, columns B–C: read counts and frequencies of mutant alleles within the target window, column D: CIGAR strings, columns E–F: insertion and deletion counts, column G: decisions of in- or out-of-frame mutation and column H: full-length sequences of each mutant allele
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FIGURE 5
Example of verification of genotype–phenotype correlation using our genotyping workflow in crispant assay. (Upper panels) Representative phenotypes of wild-type embryos and tyrosinase-related protein 1 (tyrp1), solute carrier family 45 member 2 (slc45a2) and paired box 6 (pax6) X. tropicalis crispants generated by Cas9 RNP. High-magnification images of eyes from dotted line boxes are shown at the lower right corners in each target gene. slc42a2 crispants lost pigmentation in retinal pigmented epithelium (RPE). tyrp1 crispants showed brown colored eyes. pax6 crispants exhibited severe eye malformation. (Lower panels) Frameshift mutation rates in slc42a2, tyrp1 and pax6 crispants. Each genotype is shown corresponding to each crispant phenotype in the upper panels (A-E). Blue, green and red colors indicate percentages of out-of-frame mutation (Out), in-frame mutation (In) and wild-type allele (WT), respectively
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Figure S1. Mutation profiles of on-target sites in X. tropicalis crispants. Mapped massive
reads of on-target regions from tyrp1, slc45a2 and pax6 crispants. Mapped reads are
visualised through IGV. Total mapped reads (reads) and sample sizes (n) are shown in
each image. Deletions, insertions and base substitutions are indicated by bars of black,
purple and four other colours (red, blue, green and ochre), respectively. PAM, protospacer
sequences and the putative DSB site are indicated by red bar, blue bar and lightning motif,
respectively.
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Figure S2. Graphical report regarding frameshift mutation rates in oca2 crispants of X.
tropicalis (n = 5). Green, blue and red colours indicate percentages of in-frame mutation
(In), out-of-frame mutation (Out) and wild-type allele (WT), respectively.
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Figure S3. Phenotypic variations of X. tropicalis crispants in Figure 5. In slc42a2,
crispants were classified into four groups: severe, near complete loss of pigmentation in
retinal pigmented epithelium (RPE); moderate, more than half of pigmentation lost; weak,
less than half of pigmentation lost; and normal, no alteration in pigmentation. In tyrp1,
crispants showing brown coloured eyes were counted (brown eye). In pax6, crispants
showing eye malformation in both eyes or one eye were counted. Total sgRNA/Cas9
injected or uninjected (control) embryos (N) are indicated at the top of each graph. Most
crispants showed severe phenotypes.
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Figure S4. Detection of large deletion in a Pleurodeles waltl crispant. Amplicon
sequencing data of the shh limb-specific enhancer, MFCS1/ZRS, on-target site was reanalyzed by CLiCKAR (Suzuki et al., 2018). Wild allele of amplicon length is 435 bp.
Note that 120 bp large deletion around the on-target site was detected by CLiCLAR (red).
In this case, length of the mutant allele was 315 bp. The target window was set as default
+/− 10 bp.
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Figure S5. Comparison of genotyping results among three web tools. CRISPResso, CasAnalyzer and CLiCKAR were benchmarked using amplicon sequence data from a tyrp1
X. tropicalis crispant. Cas-Analyzer cannot report the frameshift rate; therefore, it was
calculated based on the numbers of deletions and insertions within the output sequence.
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