Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Here, we describe a set of resources to streamline reporter gene knock-ins in zebrafish and demonstrate the broader utility of the method in mammalian cells. Our approach uses short homology of 24–48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at high frequency at a double strand break in the targeted gene. Our vector series, pGTag (plasmids for Gene Tagging), contains reporters flanked by a universal CRISPR sgRNA sequence which enables in vivo liberation of the homology arms. We observed high rates of germline transmission (22–100%) for targeted knock-ins at eight zebrafish loci and efficient integration at safe harbor loci in porcine and human cells. Our system provides a straightforward and cost-effective approach for high efficiency gene targeting applications in CRISPR and TALEN compatible systems.

In this study, we used comparative genomics and developmental genetics to identify epigenetic regulators driving oncogenesis in a zebrafish retinoblastoma 1 (rb1) somatic-targeting model of RB1 mutant embryonal brain tumors. Zebrafish rb1 brain tumors caused by TALEN or CRISPR targeting are histologically similar to human central nervous system primitive neuroectodermal tumors (CNS-PNETs). Like the human oligoneural OLIG2+/SOX10+ CNS-PNET subtype, zebrafish rb1 tumors show elevated expression of neural progenitor transcription factors olig2, sox10, sox8b and the receptor tyrosine kinase erbb3a oncogene. Comparison of rb1 tumor and rb1/rb1 germline mutant larval transcriptomes shows that the altered oligoneural precursor signature is specific to tumor tissue. More than 170 chromatin regulators were differentially expressed in rb1 tumors, including overexpression of chromatin remodeler components histone deacetylase 1 (hdac1) and retinoblastoma binding protein 4 (rbbp4). Germline mutant analysis confirms that zebrafish rb1, rbbp4 and hdac1 are required during brain development. rb1 is necessary for neural precursor cell cycle exit and terminal differentiation, rbbp4 is required for survival of postmitotic precursors, and hdac1 maintains proliferation of the neural stem cell/progenitor pool. We present an in vivo assay using somatic CRISPR targeting plus live imaging of histone-H2A.F/Z-GFP fusion protein in developing larval brain to rapidly test the role of chromatin remodelers in neural stem and progenitor cells. Our somatic assay recapitulates germline mutant phenotypes and reveals a dynamic view of their roles in neural cell populations. Our study provides new insight into the epigenetic processes that might drive pathogenesis in RB1 brain tumors, and identifies Rbbp4 and its associated chromatin remodeling complexes as potential target pathways to induce apoptosis in RB1 mutant brain cancer cells.

22 23Wierson, Welker, Almeida et al. GeneWeld: a method for efficient targeted integration directed by short homology 2 1 Wierson et al. describe a targeted integration strategy, called GeneWeld, and a vector 2 series for gene tagging, pGTag, which promote highly efficient and precise targeted integration 3 in zebrafish, pig fibroblasts, and human cells. This approach establishes an effective genome 4 engineering solution that is suitable for creating knock-in mutations for functional genomics and 5 gene therapy applications. The authors describe high rates of germline transmission (50%) for 6 targeted knock-ins at eight different zebrafish loci and efficient integration at safe harbor loci in 7 porcine and human cells. Abstract 11Choices for genome engineering and integration involve high efficiency with little or no 12 target specificity or high specificity with low activity. Here, we describe a targeted integration 13 strategy, called GeneWeld, and a vector series for gene tagging, pGTag (plasmids for Gene 14Tagging), which promote highly efficient and precise targeted integration in zebrafish embryos, 15 pig fibroblasts, and human cells utilizing the CRISPR/Cas9 system. Our work demonstrates that 16 in vivo targeting of a genomic locus of interest with CRISPR/Cas9 and a donor vector containing 17 as little as 24 to 48 base pairs of homology directs precise and efficient knock-in when the 18 homology arms are exposed with a double strand break in vivo. Our results suggest that the 19 length of homology is not important in the design of knock-in vectors but rather how the 20 homology is presented to a double strand break in the genome. Given our results targeting 21 multiple loci in different species, we expect the accompanying protocols, vectors, and web 22 interface for homology arm design to help streamline gene targeting and applications in 23 CRISPR and TALEN compatible systems. 25Keywords

Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Multiple design strategies for zebrafish gene targeting have previously been reported with widely varying frequencies for germline recovery of integration alleles. The GeneWeld protocol and pGTag (plasmids for Gene Tagging) vector series provide a set of resources to streamline precision gene targeting in zebrafish. Our approach uses short homology of 24-48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at a CRISPR/Cas induced DNA double-strand break. The pGTag vectors contain reporters flanked by a universal CRISPR sgRNA sequence to liberate the targeting cassette in vivo and expose homology arms for homologydriven integration. Germline transmission rates for precision-targeted integration alleles range 22-100%.Our system provides a streamlined, straightforward, and cost-effective approach for high-efficiency gene targeting applications in zebrafish.

We previously reported efficient precision targeted integration of reporter DNA in zebrafish and human cells using CRISPR/Cas9 and short regions of homology. Here, we apply this strategy to isolate zebrafish Cre recombinase drivers whose spatial and temporal restricted expression mimics endogenous genes. A 2A-Cre recombinase transgene with 48 bp homology arms was targeted into proneural genes ascl1b, olig2 and neurod1. We observed high rates of germline transmission ranging from 10 to 100% (2/20 olig2; 1/5 neurod1; 3/3 ascl1b). The transgenic lines Tg(ascl1b-2A-Cre)is75, Tg(olig2-2A-Cre)is76, and Tg(neurod1-2A-Cre)is77 expressed functional Cre recombinase in the expected proneural cell populations. Somatic targeting of 2A-CreERT2 into neurod1 resulted in tamoxifen responsive recombination in the nervous system. The results demonstrate Cre recombinase expression is driven by the native promoter and regulatory elements of the targeted genes. This approach provides a straightforward, efficient, and cost-effective method to generate cell type specific zebrafish Cre and CreERT2 drivers, overcoming challenges associated with promoter-BAC and transposon mediated transgenics.