Compositions, systems, and methods for epigenetic regulation of proprotein convertase subtilisin/kexin type 9 (PCSK9) gene expression
Provided in some aspects are epigenetic-modifying DNA-targeting systems, such as CRISPR-Cas/guide RNA (gRNA) systems for the transcriptional repression of genes to promote a cellular phenotype that leads to reduction of low-density lipoprotein (LDL). In some embodiments, the epigenetic-modifying DNA-targeting systems bind to or target a target site of at least one gene or regulatory element thereof that regulate LDL. In some embodiments, the systems are multiplexed systems that bind to or target a target site in at least two genes or regulatory elements thereof. Also provided herein are methods and uses related to the provided epigenetic-modifying DNA targeting systems in connection with treatments for cardiovascular disease and familial hypercholesterolemia.
1. An epigenetic-modifying DNA-targeting system comprising
(a) a fusion protein comprising, in the following order from the N-to C-terminus:
(i) a DNA-methyltransferase (DNMT) 3A/L domain comprising the amino acid sequence set forth in SEQ ID NO: 199;
(ii) a dead Streptococcus pyogenes Clustered Regularly Interspaced Short Palindromic Repeats associated protein 9 (dSpCas9) comprising the amino acid sequence set forth in SEQ ID NO: 207, and
(iii) a Krüppel associated box (KRAB) domain comprising the amino acid sequence set forth in SEQ ID NO:290,
wherein the fusion protein comprises one or more nuclear localization signals (NLS), wherein an NLS is between the DNMT3A/L domain and the dSpCas9 protein, and an NLS is between the SpCas9 protein and the KRAB domain; and
(b) a gRNA comprising the sequence set forth in SEQ ID NO: 66.
2. The epigenetic-modifying DNA-targeting system of claim 1 , wherein the NLS between the DNMT3A/L domain and the dSpCas9 protein has the amino acid sequence PKKKRKV (SEQ ID NO: 234).
3. The epigenetic-modifying DNA-targeting system of claim 1 , wherein the NLS between the dSpCas9 protein and the KRAB domain has the amino acid sequence KRPAATKKAGQAKKKK (SEQ ID NO: 249).
4. The epigenetic-modifying DNA-targeting system of claim 1 , further comprising an NLS at the C-terminus of the fusion protein.
5. The epigenetic-modifying DNA-targeting system of claim 4 , wherein the NLS at the C-terminus of the fusion protein has the amino acid sequence PKKKRKV (SEQ ID NO: 234).
6. An epigenetic-modifying DNA-targeting system comprising
(a) a fusion protein comprising the amino acid sequence set forth in SEQ ID NO: 278; and
(b) a gRNA comprising the sequence set forth in SEQ ID NO:66.
7. The epigenetic-modifying DNA-targeting system of claim 6 , wherein the gRNA comprises the sequence set forth in SEQ ID NO:129.
8. The epigenetic-modifying DNA-targeting system of claim 1 , wherein the gRNA comprises the sequence set forth in SEQ ID NO:129.