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 recent years, Setaria viridis has been developed as a model plant to better understand the C4 photosynthetic pathway in major crops. With the increasing availability of genomic resources for S. viridis research, highly efficient genome editing technologies are needed to create genetic variation resources for functional genomics. Here, we developed a protoplast assay to rapidly optimize the multiplexed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system in S. viridis. Targeted mutagenesis efficiency was further improved by an average of 1.4-fold with the exonuclease, Trex2. Distinctive mutation profiles were found in the Cas9_Trex2 samples, with 94% of deletions larger than 10 bp, and essentially no insertions at all tested target sites. Further analyses indicated that 52.2% of deletions induced by Cas9_Trex2, as opposed to 3.5% by Cas9 alone, were repaired through microhomology-mediated end joining (MMEJ) rather than the canonical non-homologous end joining DNA repair pathway. Combined with a robust Agrobacterium-mediated transformation method with more than 90% efficiency, the multiplex CRISPR/Cas9_-Trex2 system was demonstrated to induce targeted mutations in two tightly linked genes, svDrm1a and svDrm1b, at a frequency ranging from 73% to 100% in T0 plants. These mutations were transmitted to at least 60% of the transgene-free T1 plants, with 33% of them containing bi-allelic or homozygous mutations in both genes. This highly efficient multiplex CRISPR/Cas9_Trex2 system makes it possible to create a large mutant resource for S. viridis in a rapid and high throughput manner, and has the potential to be widely applicable in achieving more predictable and deletion-only MMEJ-mediated mutations in many plant species.

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.

CRISPR–Cas9-mediated genome editing has been widely adopted for basic and applied biological research in eukaryotic systems. While many studies consider DNA sequences of CRISPR target sites as the primary determinant for CRISPR mutagenesis efficiency and mutation profiles, increasing evidence reveals the substantial role of chromatin context. Nonetheless, most prior studies are limited by the lack of sufficient epigenetic resources and/or by only transiently expressing CRISPR–Cas9 in a short time window. In this study, we leveraged the wealth of high-resolution epigenomic resources in Arabidopsis (Arabidopsis thaliana) to address the impact of chromatin features on CRISPR–Cas9 mutagenesis using stable transgenic plants. Our results indicated that DNA methylation and chromatin features could lead to substantial variations in mutagenesis efficiency by up to 250-fold. Low mutagenesis efficiencies were mostly associated with repressive heterochromatic features. This repressive effect appeared to persist through cell divisions but could be alleviated through substantial reduction of DNA methylation at CRISPR target sites. Moreover, specific chromatin features, such as H3K4me1, H3.3, and H3.1, appear to be associated with significant variation in CRISPR–Cas9 mutation profiles mediated by the non-homologous end joining repair pathway. Our findings provide strong evidence that specific chromatin features could have substantial and lasting impacts on both CRISPR–Cas9 mutagenesis efficiency and DNA double-strand break repair outcomes.

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