CRISPR-mediated tyr knockout to establish a visual knock-in assay. a Schematic illustration of CRISPR/Cas9-mediated gene editing of tyr. First to knock out (KO) gene function with a 25 nt deletion within the first exon and then knock-in (KI) rescue of this gene using a repair template. b Target sites and T7E1 assays of tyr and tyr^25del/25del loci. PAMs are marked with red. Ctl represents PCR products without T7E1 digestion. WT denotes PCR products from uninjected embryos with T7E1 digestion. Tyr or tyr^25del/25del denotes PCR products from injected embryos with T7E1 digestion. c T-cloning and Sanger sequencing identify tyr^25del/25del in F2 zebrafish. Upper row shows wild type (WT) sequence. Open reading frame codons are demarked in green frame. 25 bp deletion in homozygous tyr mutants is marked with blue in upper row, which leads to a frameshift mutation (marked with red in lower row). d Lateral views of larvae at 2 dpf (scale bar = 1 mm) and adult (scale bar = 10 mm): wild type (upper row) and tyr^25de/l25del (lower row).

A genetic assay for comparing the efficiency of homology-directed repair using tyr mutant. a Table of template design schematics (left), attributes of the template (middle) and proportion of observed pigmented embryos in the tyr^25de/l25del model (right). Embryos are analysed at 2 dpf after co-injection of zCas9 mRNA, tyr^25de/l25del gRNA together with repair template. Number of embryos evaluated (n) exceeded 100 for each condition. b Phenotypic evaluation of embryos at 2 dpf into three groups according to number of pigmented cells: low rescue (1–20 pigmented cells), medium rescue (21–40 pigmented cells) and high rescue (more than 40 pigmented cells). Scale bar = 1 mm. c Statistics of HDR efficiency induced by different repair templates. zLOST: long single stranded template 299 bp, ssODN: single strand DNA oligonucleotides 105 bp, cdsDNA: circular double stranded DNAs 1527 bp (with two gRNA sites at both ends of the homologous arms), Ctl: without repair template. Number of embryos assessed (n) is shown for each group. X²-test (***p < 0.001). d Sequence analysis confirming that the larvae contained a correctly repaired tyr locus by zLOST. Correct insertion by HDR (green), PAM region (blue), target sites (underlined), Indels (red) are indicated.

Zebrafish genome editing at three other target sites by zLOST

a Restriction enzyme-based method design of three target sites. Target sequence (black), PAM region (blue), target modification sites (red), and restriction site (underlined) are indicated. b Restriction enzymes are used to digest the amplified region of the target genes. T = th, N = nop56, R = rps14. The “positive embryos” groups are highlighted by asterisk. c Sequencing results of the th, nop56 and rps14 loci. Patterns of DNA modification observed in independent embryos pool. Note: △1 and △2 mean the presence of additional undesirable mutations outside of the shown sequence window.

NGS analysis of precise point mutation introduction to the genes th, nop56 and rps14

Total percentages of defined sequence reads classes at knock-in sites of th(a), nop56(b) and rps14(c) genes as engineered with three types of repair template (cdsDNA, ssODN and zLOST). All the reads are divided into four classes: WT, others, correct_HDR and incorrect_HDR. Incorrect_HDR indicates the reads containing target modification sites, but with extra undesirable amino acid changes. d Representative examples of different classes of th, nop56 and rps14 HDR knock-ins: Correct_HDR and Incorrect_HDR.

zLOST enables mimicking of human disease related mutations in zebrafish

Alignment of human patients and desired zebrafish mutations to model human Barber-Say syndrome (BSS) or Diamond-Blackfan anaemia (DBA), schematic outlines of the gene editing strategy and sequencing of the resulting twist2 and rpl18 zebrafish loci. a Diagram of the mutation associated with human BSS. The substituted target base is marked in red, which means a p.E78Q amino-acid change in the zebrafish homologue precisely mimics the p.E75Q mutation found in human patients. b and d Design principles of HDR templates that contain a non-synonymous mutation of the sequence close to the PAM site in addition to synonymous nucleotide changes that create a Coding-bar used for genotyping that utilizes a de novo endonuclease restriction site. Sequencing result at the twist2 and rpl18 zebrafish loci targeted by the zLOST system. The Coding-bar includes a restriction endonuclease (PflFI) site 5′-GACNNNGTC-3′ in twist2 and a restriction endonuclease (PvuI) site 5′-CGATCG-3′ in rpl18. c Diagram depicting the mutation associated with human DBA, mimicking the p.L51S mutation at rpl18 locus found in patients.