Targeted Forward Genetics: Population-Scale Analyses of Allele Replacements Spanning Thousands of Base Pairs in Fission Yeast.

ABSTRACT: Precise allele replacement (genome editing), without unwanted changes to the genome, provides a powerful tool to define the functions of DNA elements and encoded factors in their normal biological context. While CRISPR is now used extensively for gene targeting, its utility for precise allele replacement at population scale is limited because: (A) there is a strict requirement for a correctly positioned PAM motif to introduce recombinogenic dsDNA breaks (DSBs); (B) efficient replacements only occur very close to the DSBs; and (C) indels and off-target changes are frequently generated. Here we show, using a saturated mutation library with about 15,000 alleles of the ade6 gene of Schizosaccharomyces pombe, that pop-in, pop-out allele replacement circumvents these problems. Two

SUBMITTER: Storey AJ 

PROVIDER: S-EPMC6893178 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature