All-in-One Adeno-associated Virus Delivery and Genome Editing by Neisseria meningitidis Cas9 in vivo

Abstract

Clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) have recently opened a new avenue for gene therapy. Cas9 nuclease guided by a single-guide RNA (sgRNA) has been extensively used for genome editing. Currently, three Cas9 orthologs have been adapted for in vivo genome engineering applications: SpyCas9, SauCas9 and CjeCas9. However, additional in vivo editing platforms are needed, in part to enable a greater range of sequences to be accessed via viral vectors. Here, we present an additional in vivo editing platform using Neisseria meningitidis Cas9 (NmeCas9). NmeCas9 is compact, edits with high accuracy, and possesses a distinct PAM, making it an excellent candidate for safe gene therapy applications. Here we demonstrate that NmeCas9 can be used to target the Pcsk9 and Hpd genes in mice. Using tail vein hydrodynamic-based delivery of NmeCas9 plasmid to target the Hpd gene, we successfully reprogrammed the tyrosine degradation pathway in Hereditary Tyrosinemia Type I mice. More importantly, we delivered NmeCas9 with its single-guide RNA in a single recombinant adeno-associated vector (rAAV) to target Pcsk9, resulting in lower cholesterol levels in mice. This all-in-one vector yielded >35% gene modification after two weeks of vector administration, with minimal off-target cleavage in vivo. Our findings indicate that NmeCas9 can facilitate future efforts to correct disease-causing mutations by expanding the targeting scope of RNA-guided nucleases.3 SignificanceWe and others have shown that NmeCas9 is an efficient Cas9 nuclease with a high degree of specificity in cultured human cells. NmeCas9 has a unique PAM that expands the targeting range of Cas9-based genome editing. Since NmeCas9 is more compact than SpyCas9, its delivery with its guide-RNA in a single recombinant adeno-associated virus becomes a viable option. Furthermore, NmeCas9 has an unusually low off-target profile. These characteristics make NmeCas9 an ideal candidate for in vivo genome editing, including therapeutic gene knockouts, as we demonstrate in the mouse liver. We anticipate that successful in vivo delivery of this accurate and effective Cas9 can advance therapeutic editing in humans.

Self-inactivating, all-in-one AAV vectors for precision Cas9 genome editing via homology-directed repair in vivo

“…Development of a gene-editing rAAV construct with a minimized Nme2Cas9 payload. We previously engineered ~4.7-kb rAAV gene editing vectors that deliver Nme1Cas9 or Nme2Cas9 and a 145-nucleotide (nt) sgRNA 24,27 . Both Cas9 transgenes were fused with four nuclear localization signals (NLSs) and a triple-HA epitope tag.…”

“…A hallmark of this disease in mice is the gradual loss of body weight when the mice are not maintained on water supplemented with the drug 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), which blocks the tyrosine catabolism pathway upstream of FAH, preventing the buildup of the hepatotoxic metabolites that result from loss of FAH 69 . Knocking out HPD by excising exons 3 and 4 can rescue the lethal phenotypes of HT-I mice by blocking the tyrosine catabolic pathway upstream of FAH 27,70 . In addition to the expected segmental DNA deletion, we also anticipated detecting inversions and small indels at one or both of the two target sites 31,63,71 .…”

“…The reduced sizes are important for some modes of in vivo delivery, especially viral vectors such as AAV, which has great potential as a delivery modality but is severely constrained in cargo size (<5 kb in most cases) 26 . Delivery of most of these compact Cas9s within single-vector AAVs, in which both the Cas9 and its sgRNA are packaged into the same virion, has been validated in vivo [22][23][24][27][28][29] . AAV vectors encoding SauCas9 along with two sgRNAs 30,31 , as well as CjeCas9 with three sgRNAs 32 , have also been reported, enabling the introduction of multiplexed edits or segmental deletions.…”

“…AAV vectors encoding SauCas9 along with two sgRNAs 30,31 , as well as CjeCas9 with three sgRNAs 32 , have also been reported, enabling the introduction of multiplexed edits or segmental deletions. A few of the smaller Cas9s are also less prone to off-target cellular editing than wild-type SpyCas9 23,24,27,33,34 , even with guides that support efficient on-target editing, likely because of reduced enzymatic efficiencies that disproportionately dampen activity at near-cognate sites 35 .…”

Anti-CRISPR AcrIIA5 Potently Inhibits All Cas9 Homologs Used for Genome Editing

“…While many different Cas9 proteins exist in nature, only a few are commonly used for genome engineering applications. These include the type II-A Cas9 proteins derived from Streptococcus pyogenes (SpyCas9) and Staphylococcus aureus (SauCas9) (Colella et al, 2017;Ran et al, 2015) and the type II-C Cas9 proteins from Neisseria meningitidis (Nme1Cas9) and Campylobacter jejuni (CjeCas9) (Ibraheim et al, 2018;Kim et al, 2017;Lee et al, 2016;Mir et al, 2018b;Zhang et al, 2015). These Cas9 homologs vary in features such as protospacer adjacent motif (PAM) specificity, size, and off-target activity, which makes each more or less advantageous for particular genome-editing applications.…”