High-density oligo libraries and CRISPR-Cas9 technology have revolutionized large-scale genetic screens, primarily in cell culture. Here, we present novel platforms for multicellular systems that utilize compact gRNA designs to efficiently retrieve specific oligos from comprehensive arrayed libraries via a small set of orthogonal primers by dial-out PCR. Applying our strategy to C. elegans, we developed two genome-wide CRISPR-Cas9 libraries: one encodes crRNAs and homology regions for templated insertions, whereas another enables in vitro sgRNA transcription for RNP-based editing. Each library targets 189,390 genomic sites arrayed in four 384-well plates with 120 oligos per well, with each oligo accessible using 12 forward and 10 reverse primers. We employed individual 300 bp oligos to insert up to 7.1 kb that trap and tag endogenous genes, balancing the lethality of csr-1, myo-2, and egl-19 and verifying AlphaFold2-predicted linker regions within the myosin heavy-chain B protein. We used oligos that span gene loci to generate PCR-detectable deletions in GPCRs and to conduct a gene-scale mutagenesis screen on unc-119 to produce novel variants, including a temperature-sensitive allele. Our platforms set a precedent for similar advancements across multicellular systems and integrate with various CRISRP-based genetic perturbations, offering adaptable frameworks for creating cost-effective genetic resources in diverse biological models.
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