Manipulation of DNA by CRISPR-Cas enzymes requires the recognition of a protospacer-adjacent motif (PAM), limiting target site recognition to a subset of sequences. To remove this constraint, we engineered variants of Streptococcus pyogenes Cas9 (SpCas9) to eliminate the NGG PAM requirement. We developed a variant named SpG that is capable of targeting an expanded set of NGN PAMs, and we further optimized this enzyme to develop a near-PAMless SpCas9 variant named SpRY (NRN and to a lesser extent NYN PAMs). SpRY nuclease and base-editor variants can target almost all PAMs, exhibiting robust activities on a wide range of sites with NRN PAMs in human cells and lower but substantial activity on those with NYN PAMs. Using SpG and SpRY, we generated previously inaccessible disease-relevant genetic variants, supporting the utility of high-resolution targeting across genome editing applications.

Dominant gain-of-function mechanisms in Huntington's disease (HD) suggest selective silencing of mutant HTT produces robust therapeutic benefits. Here, capitalizing on exonic PAM-Altering SNP (PAS), we developed an allele-specific CRISPR-Cas9 strategy to permanently inactivate mutant HTT through nonsensemediated decay (NMD). Comprehensive sequence/haplotype analysis identified SNP-generated NGG PAM sites on exons of common HTT haplotypes in HD subjects, revealing a clinically relevant PAS-based mutantspecific CRISPR-Cas9 strategy. Alternative allele of rs363099 (29th exon) eliminates the NGG PAM site on the most frequent normal HTT haplotype in HD, permitting mutant-specific CRISPR-Cas9 therapeutics in a predicted ~20% of HD subjects with European ancestry. Our rs363099-based CRISPR-Cas9 showed perfect allele specificity and good targeting efficiencies in patient-derived cells. Dramatically reduced mutant HTT mRNA and complete loss of mutant protein suggest that our allele-specific CRISPR-Cas9 strategy inactivate mutant HTT through NMD. In addition, GUIDE-seq analysis and subsequent validation experiments supported high levels of on-target gene specificity. Together, our data demonstrated a significant target population, complete mutant specificity, decent targeting efficiency in patient-derived cells, and minimal off-target effects on protein-coding genes, proving the concept of PAS-based allele-specific NMD-CRISPR-Cas9 and supporting its therapeutic potential in HD.

Purpose of reviewTo summarize recent advances with respect to the use of genome editing to modify blood lipid levels in vivo.Recent findingsGenome-editing technologies have been successfully used to target the PCSK9 gene in the livers of nonhuman primates and significantly reduce blood LDL cholesterol levels.SummaryMultiple proof-of-concept nonhuman primate studies raise the prospect of genome editing empowering ‘one-and-done’ therapies for the treatment of dyslipidemic patients.

Among the best-established causal risk factors for cardiovascular disease is the blood concentration of low-density lipoprotein cholesterol (LDL-C). Proprotein convertase subtilisin/kexin type 9 ( PCSK9 ), an antagonist to the LDL receptor, has emerged as a promising therapeutic target for the prevention of coronary heart disease. Current PCSK9 inhibitors are administered via subcutaneous injection, and their effects are short-lived. An alternative “one-and-done” strategy using genome editing to disrupt PCSK9 at the DNA level has been demonstrated in preclinical animal models, including non-human primates. However, concerns about the permanence and irreversibility of the genomic changes have been raised and might limit the acceptance of the therapies. Recently, a set of CRISPR-based epigenome editing tools, CRISPRoff and CRISPRon, were reported to regulate gene expression via site-directed methylation and demethylation of gene promoters, respectively. We hypothesized that these epigenome editing tools could durably and reversibly induce methylation changes in the PCSK9 promoter and thereby modulate its expression. We first screened CRISPRoff guide RNAs (gRNAs) targeting the PCSK9 promoter, individually and in dual combinations, for their ability to reduce PCSK9 expression in the human HuH-7 hepatoma cell line. We then performed long-term experiments with the lead candidate gRNAs. We found that these CRISPRoff gRNAs induced profound increases in methylation at CpG dinucleotides in the PCSK9 promoter, with up to 80% decreases in PCSK9 expression. These methylation increases and gene expression decreases have endured through >56 cell divisions so far, with only mild attenuation over time. Using the same gRNAs with CRISPRon in cells previously treated with CRISPRoff, we observed moderate decreases in methylation and increases in PCSK9 expression. Having established these effects in vitro , we are similarly assessing the durability and reversibility of epigenome editing using the lead gRNAs in a PCSK9 -humanized mouse model. Overall, this work provides a proof of concept of precise gene regulation via methylation and demethylation and suggests a potential new therapeutic approach for protection against coronary heart disease.

BackgroundHepatic knockdown of the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene or the angiopoietin-like 3 (ANGPTL3) gene has been demonstrated to reduce blood low-density lipoprotein cholesterol (LDL-C) levels, and hepatic knockdown of the angiotensinogen (AGT) gene has been demonstrated to reduce blood pressure. Genome editing can productively target each of these three genes in hepatocytes in the liver, offering the possibility of durable “one-and-done” therapies for hypercholesterolemia and hypertension. However, concerns around making permanent gene sequence changes via DNA strand breaks might hinder acceptance of these therapies. Epigenome editing offers an alternative approach to gene inactivation, via silencing of gene expression by methylation of the promoter region, but the long-term durability of epigenome editing remains to be established.MethodsWe assessed the ability of epigenome editing to durably reduce the expression of the humanPCSK9, ANGPTL3, andAGTgenes in HuH-7 hepatoma cells. Using the CRISPRoff epigenome editor, we identified guide RNAs that produced efficient gene knockdown immediately after transfection. We assessed the durability of gene expression and methylation changes through serial cell passages.ResultsCells treated with CRISPRoff andPCSK9guide RNAs were maintained for up to 124 cell doublings and demonstrated durable knockdown of gene expression and increased CpG dinucleotide methylation in the promoter, exon 1, and intron 1 regions. In contrast, cells treated with CRISPRoff andANGPTL3guide RNAs experienced only transient knockdown of gene expression. Cells treated with CRISPRoff andAGTguide RNAs also experienced transient knockdown of gene expression; although initially there was increased CpG methylation throughout the early part of the gene, this methylation was geographically heterogeneous—transient in the promoter, and stable in intron 1.ConclusionsThis work demonstrates precise and durable gene regulation via methylation, supporting a new therapeutic approach for protection against cardiovascular disease via knockdown of genes such asPCSK9. However, the durability of knockdown with methylation changes is not generalizable across target genes, likely limiting the therapeutic potential of epigenome editing compared to other modalities.