<span class="paragraphSection"><div class="boxTitle">Abstract</div>Currently available random and untargeted DNA mutagenesis techniques are limited by both the number of consecutive nucleotides that can be mutated and by the type of accessible mutations. These methodologies also create multiple different mutated sites within each DNA sequence-of-interest, which significantly confounds any precise and high-throughput phenotype-to-genotype mapping. Here, we describe two unique and cell-independent DNA mutagenesis methods that enable either a single random and small-scale (1–30 nt) duplication, deletion, or insertion of an entire DNA motif (RADDIM), or nucleotide-constrained mutagenesis of random DNA regions spanning &gt;8 consecutive nucleotides (NSM). By utilizing these mechanistically unique methods, we randomly duplicated and deleted cryptic regulatory DNA elements in two yeast promoters (pACT1 and pTEF1) to change their transcriptional expression. We randomly mutated the protein structure of an inactivated β-lactamase (TEM-1) to restore its enzymatic function by generating multiple, consecutive in-frame InDels. We also selectively mutated the AT-content and introduced TATA-box–like sequences and homopolymeric mutations, within random DNA regions. Collectively, RADDIM and NSM allow for an unprecedented level of bespoke DNA mutagenesis at random DNA locations, expanding the toolkit for genetic engineering, directed evolution, and the functional mapping of novel protein structures and cryptic regulatory DNA motifs.</span>