Precision genome editing for model organisms has revolutionized functional analysis and validation of a wide variety of molecular systems. To date, the capacity to insert single-copy transgenes into the model nematode Caenorhabditis eleganshas focused on utilizing either transposable elements or CRISPR-based safe harbor strategies. These methods require plate-level screening processes to avoid selecting heritable extrachromosomal arrays or rely on co-CRISPR markers to identify knock-in events. As a result, verification of transgene insertion requires anti-array selection screening methods and PCR genotyping. These approaches also rely on cloning plasmids for the addition of transgenes. Here, we present a novel safe harbor CRISPR-based integration strategy that utilizes engineered insertion locations containing a synthetic guide RNA target and a split-selection system to eliminate false positives from array formation, thereby providing integration-specific selection. This approach allows the experimenter to confirm an integration event has taken place without molecular validation or anti-array screening methods and is capable of producing integrated transgenic lines in as little as five days post-injection. To further increase the speed of generating transgenic lines, we also utilized the C. elegans native microhomology-based recombination, to assemble transgenes in-situ, removing the cloning step. We show that complete transgenes can be made and inserted into our split-selection safe harbor locations starting from PCR products, providing a clone-free and molecular-validation-free strategy for single-copy transgene integration. Overall, this combination of approaches provides an economical and rapid system for generating highly reproducible complex transgenics in C. elegans.

24Precision genome editing for model organisms has revolutionized functional analysis and 25 validation of a wide variety of molecular systems. To date, the capacity to insert transgenes into 26 the model nematode Caenorhabditis elegans has focused on utilizing either transposable 27 elements or CRISPR-based safe harbor strategies. These methods require laborious screening 28 processes that often result in false positives from heritable extrachromosomal arrays or rely on 29 co-CRISPR markers to identify likely edited individuals. As a result, verification of transgene 30 insertion requires anti-array selection screening methods or extensive PCR genotyping 31 respectively. These approaches also rely on cloning plasmids for the addition of transgenes. 32Here, we present a novel safe harbor CRISPR-based integration strategy that utilizes engineered 33 insertion locations containing a synthetic guide RNA target and a split-selection system to 34 eliminate false positives from array formation, thereby providing integration-specific selection. 35This approach allows the experimenter to confirm an integration event has taken place without 36 molecular validation or anti-array screening methods, and is capable of producing integrated 37 transgenic lines in as little as five days post-injection. To further increase the speed of generating 38 transgenic lines, we also utilized the C. elegans native homology-based formation of extra-39 chromosomal arrays to assemble transgenes in-situ, removing the cloning step. We show that 40 complete transgenes can be made and inserted into our split-selection safe harbor locations 41 starting from PCR products, providing a clone-free and molecular-validation-free strategy for 42 single-copy transgene integration. Overall, this combination of approaches provides an 43 economical and rapid system for generating highly reproducible complex transgenics in C. 44 elegans. 45 46 3 Introduction: 47