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PubMed · 39527204

Targeted Forward Genetics: Saturating Mutational Analyses of Specific Target Loci Within the Genome.

Reine U Protacio·Wayne P Wahls
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Abstract

Precise allele replacement by homologous recombination (also known as "gene targeting" or "genome editing") allows scientists to engineer altered DNA sequences, insertions, or deletions at specific locations in the genome. Such reverse genetics provides powerful tools to elucidate the structure and function of regulatory DNA elements, genes, RNAs, and proteins within their natural, endogenous context. Here, we describe in detail the methodology for Targeted Forward Genetics (TFG), which supports population-scale, saturating screens of allele replacements spanning thousands of base pairs at a specific target locus in the genome. The overall approach and detailed protocols, developed for the fission yeast Schizosaccharomyces pombe, are extensible to other organisms in which gene targeting is feasible.

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SchizosaccharomycesGene TargetingGenome, FungalDNA Mutational AnalysisHomologous RecombinationAllelesGene EditingGenetic LociAllele replacementForward geneticsGene targetingGenetic screenHomologous recombinationMutagenesisReverse geneticsSelectionade6ura4Journal ArticleResearch Support, N.I.H., Extramural

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Reine U Protacio, Wayne P Wahls. 2025. Targeted Forward Genetics: Saturating Mutational Analyses of Specific Target Loci Within the Genome.. https://doi.org/10.1007/978-1-0716-4168-2_16

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