20nucleotide

A 20-nucleotide sequence, often written as 20-nt, is a string of twenty nucleotides in a nucleic acid such as DNA or RNA. Nucleotides are the basic building blocks, consisting of a sugar, a phosphate group, and a nitrogenous base (adenine, thymine or uracil, cytosine, and guanine). The 20-nt length permits a vast number of possible sequences (4^20, about 1.1 trillion), which underpins their use in diverse biological applications. The practical properties of a 20-nt stretch depend on its composition, including the proportion of guanine and cytosine nucleotides, which influences binding stability and melting temperature.

In genome editing, the 20-nt length is especially notable as the spacer portion of many single-guide RNAs (sgRNAs) used with CRISPR-Cas systems. The 20-nt spacer base-pairs with a complementary DNA target, guiding the Cas enzyme to the intended site for modification. The remainder of the sgRNA provides structural interaction with the Cas protein. The 20-nt length is a common standard because it offers a balance between target specificity and practical design considerations, though different CRISPR systems and applications may use spacers of varying lengths.

Beyond CRISPR, 20-nt sequences are widely used as synthetic DNA or RNA oligonucleotides in research and diagnostics, serving as primers, probes, or adapters. In any context, design considerations include sequence uniqueness within a genome, GC content, potential secondary structures, and on-target versus off-target effects, all of which influence performance and reliability.