Harnessing non-Watson–Crick’s base pairing to enhance CRISPR effectors cleavage activities and enable gene editing in mammalian cells

Genomic DNA of the cyanophage S-2L virus is composed of 2-aminoadenine (Z), thymine (T), guanine (G), and cytosine (C), forming the genetic alphabet ZTGC, which violates Watson–Crick base pairing rules. The Z-base has an extra amino group on the two position that allows the formation of a third hydrogen bond with thymine in DNA strands. Here, we explored and expanded applications of this non-Watson–Crick base pairing in protein expression and gene editing. Both ZTGC-DNA (Z-DNA) and ZUGC-RNA (Z-RNA) produced in vitro show detectable compatibility and can be decoded in mammalian cells, including Homo sapiens cells. Z-crRNA can guide CRISPR-effectors SpCas9 and LbCas12a to cleave specific DNA through non-Watson–Crick base pairing and boost cleavage activities compared to A-crRNA. Z-crRNA can also allow for efficient gene and base editing in human cells. Together, our results help pave the way for potential strategies for optimizing DNA or RNA payloads for gene editing therapeutics and give insights to understanding the natural Z-DNA genome.