UCSF

Mismatch-tolerant DNA recognition by Cas9-single-guide RNA (sgRNA) complexes creates off-target concerns for CRISPR transcriptional regulation. We evaluated first-order nullomers as synthetic addresses. The 15-base-pair Streptococcus pyogenes Cas9 target/PAM word and all 45 single-substitution neighbors were absent from T2T-CHM13, placing the target at least two substitutions from every reference word. A lentiviral massively parallel reporter assay tested 268 nullomers in 400 HepG2-characterized cis-regulatory elements. Most showed no clear directional change; the remaining effects depended on the scaffold. Cognate targeting increased CRISPRa output in all 15 inactive-CRE/nullomer combinations on both assay days. CRISPRi was context dependent: 11 of 15 active-CRE/nullomer combinations were lower than Safe2 on both days, and four changed direction. Per-CRE P values are exploratory because technical wells were nested within two assay days; Safe2 also differed in spacer sequence and length. In target-absent HepG2 dCas9-SAM cells, Nullomers A, B and C were associated with 4, 6 and 73 strong-effect genes. Nullomer A supported inducible-caspase-9 reporter loss and F8 transgene regulation. Cognate targeting produced 2.20-fold more F8 messenger RNA than Safe2, and a doxycycline-responsive guide promoter supported dose- and withdrawal-associated control. First-order nullomers can support CRISPR regulation in reporter and transgene contexts, but whether they are safer or more specific than matched conventional targets remains untested.