# Rice University evolves SlugCas9 variants for NNAN PAMs and edits APOC3 and PCSK9 sites in cells

- Published: 2026-11-05 13:12 UTC
- Section: Life & Health › Biotechnology
- Tags: SlugCas9, NNAN PAM, compact Cas9, Sequence Display, base editing, APOC3, PCSK9, genome editing, Han Xiao, Nature Communications
- URL: https://realtech.media/news/rt-7ac178f3a8fe2d18af75e078_rice_university_evolves/

**Variants of the compact Cas9 nuclease SlugCas9 recognize PAMs that the native enzyme binds weakly. Tested in cultured mammalian cells only, not in animals or patients.**

![Rice University evolves SlugCas9 variants for NNAN PAMs and edits APOC3 and PCSK9 sites in cells](https://sgzwpqqelzmajkcubgkl.supabase.co/storage/v1/object/public/rt-media/2026/11/e3a46123c1b5cbd6.jpg)
*Maggie Bartlett, NHGRI, Wikimedia Commons, Public domain*

A team at Rice University led by chemistry professor Han Xiao has reported variants of the compact nuclease SlugCas9 (Staphylococcus lugdunensis Cas9) that act on PAMs of the NNAN family: NNAA, NNAT, NNAC and NNAG. The study was published on 30 October 2026 in Nature Communications; the first author is graduate student Linqi Cheng. The variants improved base editing at selected sites in cultured mammalian cells, including sites in the APOC3 and PCSK9 genes. [1]

A protospacer adjacent motif (PAM) is a short DNA stretch that Cas9 must recognize before it can edit an adjacent target matching the guide RNA. According to the authors, PAM requirements constrain the targeting scope of CRISPR-Cas9 and limit applications that need precise nuclease positioning. Broad-PAM and near-PAMless Cas9 variants have widened the editable genome, but robust targeting across some PAM families, NNAN in particular, remains difficult. [1] Compact Cas9 enzymes are easier to deliver but recognize fewer targets.

## Sequence Display and SlugCas9 activity data

The researchers used Sequence Display to produce multiplexed, large-scale sequence–activity datasets for SlugCas9 across the four NNAN PAMs. The datasets quantify how individual and combined mutations change nuclease activity in each PAM context. [1] The authors used them to build detailed activity landscapes and select variants. Xiao said the team found promising candidates within a week.

The resulting variants, the authors report, show enhanced and broader activity across NNAN PAMs. They recognize PAMs beyond the G-compatible range of native SlugCas9. [1]

## Editing APOC3 and PCSK9 in cultured cells

APOC3 and PCSK9 are genes linked to coronary heart disease risk. According to Cheng, the sites chosen for editing carry PAMs that earlier SlugCas9 versions could not bind reliably. With the evolved variants, base editing at these selected sites improved in cultured mammalian cells. [1] The authors say the introduced changes are potentially protective and are associated with a lower risk of coronary heart disease.

The material reports results for selected endogenous sites in cell culture. It contains no data on animals or patients. The authors describe the work as a strategy for reprogramming PAM preference in compact Cas9 nucleases and present Sequence Display as a general platform for evolving genome editors toward difficult-to-access PAM spaces. [1]

## Sources

1. [Nature Communications](https://doi.org/10.1038/s41467-026-79035-1)
2. [EurekAlert](https://www.eurekalert.org/news-releases/1150493)

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Source: RealTech, realtech.media — https://realtech.media/news/rt-7ac178f3a8fe2d18af75e078_rice_university_evolves/