A missing link in cholesterol absorption: Cholesterol is important for general health, but too much can build up in artery walls and cause cardiovascular disease. Low-density lipoprotein cholesterol (LDL-C) is often referred to as “bad cholesterol”; keeping LDL-C within stringent limits is recommended to reduce the risk of heart attack and stroke. Zhang et al. discovered that some individuals have an inherited frameshift mutation in the LIMA1 gene (also known as EPLIN or SREBP3 ). The gene has not been linked to lipid metabolism before, but altered LIMA1 was found to maintain low plasma LDL-C by reducing the absorption of cholesterol through the intestine. Pharmacological targeting through the LIMA1 pathway might thus provide a strategy to improve heart health. Science , this issue p. 1087

Background: AsCas12a and LbCas12a nucleases are reported to be promising tools for genome engineering with protospacer adjacent motif (PAM) TTTV as the optimal. However, the C-containing PAM (CTTV, TCTV, TTCV, etc.) recognition by Cas12a might induce extra off-target edits at these non-canonical PAM sites. Results: Here, we identify a novel Cas12a nuclease CeCas12a from Coprococcus eutactus, which is a programmable nuclease with genome-editing efficiencies comparable to AsCas12a and LbCas12a in human cells. Moreover, CeCas12a is revealed to be more stringent for PAM recognition in vitro and in vivo followed by very low off-target editing rates in cells. Notably, CeCas12a renders less off-target edits located at C-containing PAM at multiple sites compared to LbCas12a and AsCas12a, as assessed by targeted sequencing methods. Conclusions: Our study shows that CeCas12a nuclease is active in human cells and the stringency of PAM recognition could be an important factor shaping off-target editing in gene editing. Thus, CeCas12a provides a promising candidate with distinctive characteristics for research and therapeutic applications.

BACKGROUND: Low-density lipoprotein (LDL) is internalized into cells mainly through LDLR (LDL receptor)–mediated endocytosis. In an acidic endosome, LDLR is released from LDL and recycles back to the cell surface, whereas LDL is left in the endosome and degraded in the lysosome. Circulating PCSK9 (proprotein convertase subtilisin/kexin 9) binds with LDLR and is internalized into the endosome, similar to LDL. In an acidic endosome, LDLR fails to disassociate from PCSK9, and both proteins are degraded in the lysosome. PCSK9 inhibitors are widely used for treating hypercholesterolemia. However, how PCSK9 diverts LDLR to the lysosome for degradation remains elusive. Some patients are resistant to PCSK9 inhibitors, for unknown reasons. METHODS: Both in vitro and in vivo approaches were used to investigate the molecular and cellular mechanisms of PCSK9-mediated LDLR degradation. LDLR containing FH sequence variations was expressed in Ldlr knockout mice and knockout HuH7 cells to evaluate their response to PCSK9 and PCSK9 inhibitors. RESULTS: Acidic pH induces a conformational change in LDLR extracellular domain and promotes its interaction with SNX17 (sorting nexin 17) through the intracellular domain. Knocking down SNX17 abolishes LDLR recycling and causes accelerated degradation in the lysosome. PCSK9 prevents the acidic pH–induced conformational change in LDLR and blocks its interaction with SNX17. Knocking down SNX17 abolishes PCSK9-mediated LDLR degradation. Any FH sequence variations that disrupt LDLR recycling are unresponsive to PCSK9 or PCSK9 inhibitors, even though they can internalize LDL. CONCLUSIONS: PCSK9 promotes LDLR degradation by preventing SNX17-mediated LDLR recycling. Patients with sequence variations in FH leading to defects in LDLR recycling are resistant to PCSK9 inhibitors. Genetic diagnosis and alternative drugs independent of LDLR will be needed for treatment of these patients.