Lessons in genome engineering: opportunities, tools and pitfalls

CRISPR/Cas technology allows the creation of double strand breaks and hence loss of function mutations at any location in the genome. This technology is now routine for many organisms and cell lines. Here we describe how CRISPR/Cas can be combined with other DNA manipulation techniques (e.g. homology-based repair, site-specific integration and Cre or FLP-mediated recombination) to create sophisticated tools to measure and manipulate gene activity. In one class of applications, a single site-specific insertion generates a transcriptional reporter, a loss-of function allele, and a tagged allele. In a second class of modifications, essential sequences are deleted and replaced with an integrase site, which serves as a platform for the creation of custom reporters, transcriptional drivers, conditional alleles and regulatory mutations. We describe how these tools and protocols can be implemented easily and efficiently. Importantly, we also highlight unanticipated failures, which serve as cautionary tales, and suggest mitigating measures. Our tools are designed for use in Drosophila but the lessons we draw are likely to be widely relevant. AUTHOR SUMMARY The genome contains all the information that an organism needs to develop and function throughout its life. One of the goal of genetics is to decipher the role of all the genes (typically several thousands for an animal) present in the genome. One approach is to delete each gene and assay the consequences. Deletion of individual genes is now readily achieved with a technique called CRISPR/Cas9. However, simple genetic deletion provides limited information. Here we describe strains and DNA vectors that streamline the generation of more sophisticated genetic tools. We describe general means of creating alleles (genetic variants) that enable gene activity to be measured and experimentally modulated in space and time. Although the tools we describe are universally applicable, each gene requires special consideration. Based on our experience of successes and failures, we suggest measures to maximise the chances that engineered alleles serve their intended purpose. Although our methods are designed for use in Drosophila, they could be adapted to any organism that is amenable to CRISPR/Cas9 genome modification.

[1]  D. G. Gibson,et al.  Enzymatic assembly of DNA molecules up to several hundred kilobases , 2009, Nature Methods.

[2]  Gerald M Rubin,et al.  Using translational enhancers to increase transgene expression in Drosophila , 2012, Proceedings of the National Academy of Sciences.

[3]  C. Nüsslein-Volhard,et al.  Mutations affecting segment number and polarity in Drosophila , 1980, Nature.

[4]  Sean B. Carroll,et al.  Integration of positional signals and regulation of wing formation and identity by Drosophila vestigial gene , 1996, Nature.

[5]  Y. Bellaïche,et al.  Transmission of cytokinesis forces via E-cadherin dilution and actomyosin flows , 2017, Nature.

[6]  C. Alexandre,et al.  Accelerated homologous recombination and subsequent genome modification in Drosophila , 2013, Development.

[7]  S. Bullock,et al.  Creating Heritable Mutations in Drosophila with CRISPR-Cas9. , 2016, Methods in molecular biology.

[8]  J. Vlak,et al.  Role of the 3' untranslated region of baculovirus p10 mRNA in high-level expression of foreign genes. , 1999, The Journal of general virology.

[9]  C. Lobe,et al.  Conditional genome alteration in mice , 1998, BioEssays : news and reviews in molecular, cellular and developmental biology.

[10]  Grzegorz Sienski,et al.  Efficient CRISPR/Cas9 Plasmids for Rapid and Versatile Genome Editing in Drosophila , 2014, G3: Genes, Genomes, Genetics.

[11]  E. Bier,et al.  The Drosophila rhomboid gene mediates the localized formation of wing veins and interacts genetically with components of the EGF-R signaling pathway. , 1993, Genes & development.

[12]  A. Martinez Arias,et al.  Roles of wingless in patterning the larval epidermis of Drosophila. , 1991, Development.

[13]  R. Palmer,et al.  Godzilla-dependent transcytosis promotes Wingless signalling in Drosophila wing imaginal discs , 2016, Nature Cell Biology.

[14]  C. Desplan,et al.  Power tools for gene expression and clonal analysis in Drosophila , 2011, Nature Methods.

[15]  A. Gould,et al.  Fat cells reactivate quiescent neuroblasts via TOR and glial Insulin relays in Drosophila , 2011, Nature.

[16]  Mario R. Capecchi,et al.  Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century , 2005, Nature Reviews Genetics.

[17]  Cyrille Alexandre.  Cuticle preparation of Drosophila embryos and larvae. , 2008, Methods in molecular biology.

[18]  M. Peifer,et al.  Roles of the C terminus of Armadillo in Wingless signaling in Drosophila. , 1999, Genetics.

[19]  S. Streichan,et al.  EGFR signaling coordinates patterning with cell survival during Drosophila epidermal development , 2018, bioRxiv.

[20]  K. Basler,et al.  Dpp controls growth and patterning in Drosophila wing precursors through distinct modes of action , 2017, eLife.

[21]  Melissa M. Harrison,et al.  CRISPR‐Cas9 Genome Editing in Drosophila , 2015, Current protocols in molecular biology.

[22]  S. Carroll,et al.  Organization of wing formation and induction of a wing-patterning gene at the dorsal/ventral compartment boundary , 1994, Nature.

[23]  Y. Dong,et al.  Systematic functional analysis of the Caenorhabditis elegans genome using RNAi , 2003, Nature.

[24]  J. Rossant,et al.  Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos , 2018, Nature Biotechnology.

[25]  Simon L. Bullock,et al.  Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila , 2014, Proceedings of the National Academy of Sciences.

[26]  C. Alexandre,et al.  Patterning and growth control by membrane-tethered Wingless , 2013, Nature.

[27]  J. Sekelsky.  DNA Repair in Drosophila: Mutagens, Models, and Missing Genes , 2017, Genetics.