Instantaneous visual genotyping and facile site-specific transgenesis via CRISPR-Cas9 and phiC31 integrase

ABSTRACT Here, we introduce ‘TICIT’, targeted integration by CRISPR-Cas9 and integrase technologies, which utilizes the site-specific DNA recombinase – phiC31 integrase – to insert large DNA fragments into CRISPR-Cas9 target loci. This technique, which relies on first knocking in a 39-basepair phiC31 landing site via CRISPR-Cas9, enables researchers to repeatedly perform site-specific transgenesis at the exact genomic location with high precision and efficiency. We applied this approach to devise a method for the instantaneous determination of a zebrafish's genotype simply by examining its color. When a zebrafish mutant line must be propagated as heterozygotes due to homozygous lethality, employing this method allows facile identification of a population of homozygous mutant embryos even before the mutant phenotypes manifest. Thus, it should facilitate various downstream applications, such as large-scale chemical screens. We demonstrated that TICIT could also create reporter fish driven by an endogenous promoter. Further, we identified a landing site in the tyrosinase gene that could support transgene expression in a broad spectrum of tissue and cell types. In sum, TICIT enables site-specific DNA integration without requiring complex donor DNA construction. It can yield consistent transgene expression, facilitate diverse applications in zebrafish, and may be applicable to cells in culture and other model organisms.


SpCas9 protein gRNA ssODN
Microinjection of one cell-stage zebrafish embryos Step 1: Generation of genomic attP landing sites Step 2: Generation of allele-tracking reporter fish Founder screen (F 0 ) Identify attP knock-in F 1 fish Collect fin biopsy and perform: Collect fin biopsy and use PCR to identify F 1 fish carrying the correct integration.Outcross F 1 fish to the wild-type fish and assess the ratio of fluorescent F 2 embryos.Lyse some fluorescent F 2 embryos and use PCR to confirm that the F 1 fish do not harbor any random integration.

Fig. S4. The workflow for the generation of allele-tracking reporter zebrafish using TICIT.
The flowchart outlines the procedures for generating allele-tracking reporter zebrafish.Table S5.Fluorescent PCR fragment length analysis for gRNA efficiency.For each gRNA, two pools of embryos (10 embryos per pool) were analyzed.Samples K5, K6, G7, and G8 are uninjected control embryos.PCR products were run on ABI 3730xl DNA Analyzer at MGH DNA core.Fragment sizes, peak heights, and peak areas were determined using GeneMapper v4.0.The fragments correspond to the wild-type alleles are highlighted in yellow.gRNA efficiencies were determined by comparing the abundance of the wild-type alleles over the sum of all peaks in the same samples based on peak intensity.For attP knock-in, kcnh6a_2 and gfap_1 gRNAs were used.
Fig. S2.GFP immunohistochemistry (IHC) staining of wild-type adult fish.Histology sections of wild-type adult fish stained with H&E or anti-EGFP antibody (IHC).These samples were harvested, processed, stained, and imaged simultaneously with the samples shown in Figure 6.All images were acquired under 400X magnification.
For introducing T7 promoter sequence 2nd PCR for detecting attP KI at the gfap site PCR for decting attL at the tyr_1 site PCR for decting attR at the tyr_1 site PCR for decting attL at the gfap site PCR for decting attR at the gfap site 2nd PCR for detecting attP KI at the tyr_2 site PCR for next-generation sequencing to detect attP KI at the tyr_2 site 1st PCR for detecting attP KI at the kcnh6a site 2nd PCR for detecting attP KI at the kcnh6a site 1st PCR for detecting attP KI at the gfap site 1st PCR for detecting attP KI at the tyr_1 site 2nd PCR for detecting attP KI at the tyr_1 site PCR for next-generation sequencing to detect attP KI at the tyr_1site 1st PCR for detecting attP KI at the tyr_2 site

Table S1 . SpCas9 target sites tested or used in this study.
In the 'Target site sequence', PAM sequence is underlined.In the 'gRNA spacer sequence', an extra 'G' (shown in red) is added to faciliate efficient in vitro transcription.For attP knock-in, kcnh6a_2 and gfap_1 gRNAs were used.

Table S2 .
Oligonucleotides used for gRNA construction.In 'Sequnce', T7 or SP6 promoter sequences are shown in green, and the sequences complementary to the C9 or C9E constant oligo are shown in blue.

Table S4 . Sequences of the single-stranded oligonucleotides (ssODNs) used for attP knock-in experiments.
The ssODNs were chemically synthesized and two phosphorothioate linkages (denoted by asterisks) were added to both termini to enhance stability.The knock-in sequences are shown in bold.The attP sequences are shown in bold uppercase letters.The bold lowercase letters were added to avoid stop codons in the reading frames.