Cas9 Orthologs Research Articles

Immune-orthogonal orthologues of AAV capsids and of Cas9 circumvent the immune response to the administration of gene therapy.

Protein-based therapeutics can activate the adaptive immune system and lead to the production of neutralizing antibodies and to cytotoxic-T-cell-mediated clearance of the treated cells. Here, we show that the sequential use of immune-orthogonal orthologues of the CRISPR-associated protein 9 (Cas9) and of adeno-associated viruses (AAVs) eludes adaptive immune responses and enables effective gene editing from repeated dosing. We compared total sequence similarities and predicted binding strengths to class-I and class-II major-histocompatibility-complex proteins for 284 DNA-targeting and 84 RNA-targeting CRISPR effectors, and for 167 AAV VP1-capsid-protein orthologues. We predict the absence of cross-reactive immune responses for 79% of the DNA-targeting Cas orthologs, which we validate for three Cas9 orthologs in mice, yet anticipate broad immune cross-reactivity among the AAV serotypes. We also show that efficacious in vivo gene editing is uncompromised when using multiple dosing with orthologues of AAVs and Cas9 in mice previously immunized against the AAV vector and the Cas9 payload. Multiple dosing with protein orthologues may allow for sequential regimens of protein therapeutics that circumvent pre-existing immunity or induced immunity.

Biochemical characterization of RNA-guided ribonuclease activities for CRISPR-Cas9 systems.

The majority of bacteria and archaea rely on CRISPR-Cas systems for RNA-guided, adaptive immunity against mobile genetic elements. The Cas9 family of type II CRISPR-associated DNA endonucleases generates programmable double strand breaks in the CRISPR-complementary DNA targets flanked by the PAM motif. Nowadays, CRISPR-Cas9 provides a set of powerful tools for precise genome manipulation in eukaryotes and prokaryotes. Recently, a few Cas9 orthologs have been reported to possess intrinsic CRISPR-guided, sequence-specific ribonuclease activities. These discoveries fundamentally expanded the targeting capability of CRISPR-Cas9 systems, and promise to provide new CRISPR tools to manipulate specific cellular RNA transcripts. Here we present a detailed method for the biochemical characterization of Cas9's RNA-targeting potential.

Active fusions of Cas9 orthologs

Many recent epigenetic studies utilize the advantages of CRISPR/dCas9 based tools in linking certain epigenetic modification with gene expression regulation. Various multifactorial diseases often contain changed epigenetic signatures at many loci, so tools for simultaneously targeting different loci would significantly facilitate the understanding of disease pathogenesis. We tested different dCas9 orthologs (dCjCas9, dNmCas9, dSt1Cas9, dFnCas9, dSaCas9 and dSpCas9) in C-terminal fusion with DNMT3A effector domain to find candidates that potentiate effector domain to perform its function at the target site. We demonstrated that nuclear localization signals (NLS) at both termini of fusion constructs is crucial for both proper nuclear import of such large constructs as well as for maximization of targeted DNA methylation activity. We identified SpCas9, SaCas9 and CjCas9 as potential candidates for the fusion constructs. With further optimization of the SaCas9 ortholog, due to less complex PAM requirements in contrast to CjCas9, we showed that N-terminal fusion with DNMT3A (dSaCas9-DNMT3A) is optimal to exert targeted DNA methylation activity comparable to the dSpCas9-DNMT3A construct. N-terminal fusions showed better results for both Cas9 orthologs, SaCas9 and SpCas9, so it can be used as universal approach for linking different effector domains in order to obtain highly active fusions.

The next generation of CRISPR-Cas technologies and applications.

The prokaryote-derived CRISPR-Cas genome editing systems have transformed our ability to manipulate, detect, image and annotate specific DNA and RNA sequences in living cells of diverse species. The ease of use and robustness of this technology have revolutionized genome editing for research ranging from fundamental science to translational medicine. Initial successes have inspired efforts to discover new systems for targeting and manipulating nucleic acids, including those from Cas9, Cas12, Cascade and Cas13 orthologues. Genome editing by CRISPR-Cas can utilize non-homologous end joining and homology-directed repair for DNA repair, as well as single-base editing enzymes. In addition to targeting DNA, CRISPR-Cas-based RNA-targeting tools are being developed for research, medicine and diagnostics. Nuclease-inactive and RNA-targeting Cas proteins have been fused to a plethora of effector proteins to regulate gene expression, epigenetic modifications and chromatin interactions. Collectively, the new advances are considerably improving our understanding of biological processes and are propelling CRISPR-Cas-based tools towards clinical use in gene and cell therapies.

CRISPR/Cas9-based epigenome editing: An overview of dCas9-based tools with special emphasis on off-target activity.

Molecular tools for gene regulation and epigenome editing consist of two main parts: the targeting moiety binding a specific genomic locus and the effector domain performing the editing or regulatory function. The advent of CRISPR-Cas9 technology enabled easy and flexible targeting of almost any locus by co-expression of a small sgRNA molecule, which is complementary to the target sequence and forms a complex with Cas9, directing it to that particular target. Here, we review strategies for recruitment of effector domains, used in gene regulation and epigenome editing, to the dCas9 DNA-targeting protein. To date, the most important CRISPR-Cas9 applications in gene regulation are CRISPR activation or interference, while epigenome editing focuses on targeted changes in DNA methylation and histone modifications. Several strategies for signal amplification by recruitment of multiple effector domains deserve special focus. While some approaches rely on altering the sgRNA molecule and extending it with aptamers for effector domain recruitment, others use modifications to the Cas9 protein by direct fusions with effector domains or by addition of an epitope tag, which also has the ability to bind multiple effector domains. A major barrier to the widespread use of CRISPR-Cas9 technology for therapeutic purposes is its off-target effect. We review efforts to enhance CRISPR-Cas9 specificity by selection of Cas9 orthologs from various bacterial species and their further refinement by introduction of beneficial mutations. The molecular tools available today enable a researcher to choose the best balance of targeting flexibility, activity amplification, delivery method and specificity.

Structural basis for the promiscuous PAM recognition by Corynebacterium diphtheriae Cas9

The RNA-guided DNA endonuclease Cas9 cleaves double-stranded DNA targets bearing a protospacer adjacent motif (PAM) and complementarity to an RNA guide. Unlike other Cas9 orthologs, Corynebacterium diphtheriae Cas9 (CdCas9) recognizes the promiscuous NNRHHHY PAM. However, the CdCas9-mediated PAM recognition mechanism remains unknown. Here, we report the crystal structure of CdCas9 in complex with the guide RNA and its target DNA at 2.9 Å resolution. The structure reveals that CdCas9 recognizes the NNRHHHY PAM via a combination of van der Waals interactions and base-specific hydrogen bonds. Moreover, we find that CdCas9 exhibits robust DNA cleavage activity with the optimal 22-nucleotide length guide RNAs. Our findings highlight the mechanistic diversity of the PAM recognition by Cas9 orthologs, and provide a basis for the further engineering of the CRISPR-Cas9 genome-editor nucleases.

Cell-specific CRISPR-Cas9 activation by microRNA-dependent expression of anti-CRISPR proteins.

The rapid development of CRISPR–Cas technologies brought a personalized and targeted treatment of genetic disorders into closer reach. To render CRISPR-based therapies precise and safe, strategies to confine the activity of Cas(9) to selected cells and tissues are highly desired. Here, we developed a cell type-specific Cas-ON switch based on miRNA-regulated expression of anti-CRISPR (Acr) proteins. We inserted target sites for miR-122 or miR-1, which are abundant specifically in liver and cardiac muscle cells, respectively, into the 3′UTR of Acr transgenes. Co-expressing these with Cas9 and sgRNAs resulted in Acr knockdown and released Cas9 activity solely in hepatocytes or cardiomyocytes, while Cas9 was efficiently inhibited in off-target cells. We demonstrate control of genome editing and gene activation using a miR-dependent AcrIIA4 in combination with different Streptococcus pyogenes (Spy)Cas9 variants (full-length Cas9, split-Cas9, dCas9-VP64). Finally, to showcase its modularity, we adapted our Cas-ON system to the smaller and more target-specific Neisseria meningitidis (Nme)Cas9 orthologue and its cognate inhibitors AcrIIC1 and AcrIIC3. Our Cas-ON switch should facilitate cell-specific activity of any CRISPR–Cas orthologue, for which a potent anti-CRISPR protein is known.

Unified energetics analysis unravels SpCas9 cleavage activity for optimal gRNA design

While CRISPR/Cas9 is a powerful tool in genome engineering, the on-target activity and off-target effects of the system widely vary because of the differences in guide RNA (gRNA) sequences and genomic environments. Traditional approaches rely on separate models and parameters to treat on- and off-target cleavage activities. Here, we demonstrate that a free-energy scheme dominates the Cas9 editing efficacy and delineate a method that simultaneously considers on-target activities and off-target effects. While data-driven machine-learning approaches learn rules to model particular datasets, they may not be as transferrable to new systems or capable of producing new mechanistic insights as principled physical approaches. By integrating the energetics of R-loop formation under Cas9 binding, the effect of the protospacer adjacent motif sequence, and the folding stability of the whole single guide RNA, we devised a unified, physical model that can apply to any cleavage-activity dataset. This unified framework improves predictions for both on-target activities and off-target efficiencies of spCas9 and may be readily transferred to other systems with different guide RNAs or Cas9 ortholog proteins.

Diverse Mechanisms of CRISPR-Cas9 Inhibition by Type IIC Anti-CRISPR Proteins

Anti-CRISPR proteins (Acrs) targeting CRISPR-Cas9 systems represent natural "off switches" for Cas9-based applications. Recently, AcrIIC1, AcrIIC2, and AcrIIC3 proteins were found to inhibit Neisseria meningitidis Cas9 (NmeCas9) activity in bacterial and human cells. Here we report biochemical and structural data that suggest molecular mechanisms of AcrIIC2- and AcrIIC3-mediated Cas9 inhibition. AcrIIC2 dimer interacts with the bridge helix of Cas9, interferes withRNA binding, and prevents DNA loading into Cas9. AcrIIC3 blocks the DNA loading step through binding to a non-conserved surface of the HNH domain ofCas9. AcrIIC3 also forms additional interactions with the REC lobe of Cas9 and induces thedimerization of the AcrIIC3-Cas9 complex. While AcrIIC2 targets Cas9 orthologs from different subtypes, albeit with different efficiency, AcrIIC3 specifically inhibits NmeCas9. Structure-guided changes inNmeCas9 orthologs convert them into anti-CRISPR-sensitive proteins. Our studies provide insights intoanti-CRISPR-mediated suppression mechanisms and guidelines for designing regulatory tools in Cas9-based applications.

Improving CRISPR-Cas9 Genome Editing Efficiency by Fusion with Chromatin-Modulating Peptides.

Bacterial-derived CRISPR-Cas9 nucleases have become a common tool in genome engineering. However, the editing efficiency by even the best-crafted Cas9 nucleases varies considerably with different genomic sites, and efforts to explore the vast natural Cas9 diversity have often met with mixed or little success. Here, we show that modification of the widely used Streptococcus pyogenes Cas9 by fusion with chromatin-modulating peptides (CMPs), derived from high mobility group proteins HMGN1 and HMGB1, histone H1, and chromatin remodeling complexes, improves its activity by up to several fold, particularly on refractory target sites. We further show that this CMP fusion strategy (termed CRISPR-chrom) is also effective in improving the activities of smaller Cas9 nucleases from Streptococcus pasteurianus and Campylobacter jejuni, as well as four newly characterized Cas9 orthologs from Bacillus smithii, Lactobacillus rhamnosus, Mycoplasma canis, and Parasutterella excrementihominis. Our findings suggest that this CRISPR-chrom strategy can be used to improve established Cas9 nucleases and facilitate exploration of novel Cas9 orthologs for genome modification.

CRISPR-Cas Systems Reduced to a Minimum

In two recent studies in Molecular Cell, Wright etal. (2019) report complete spacer integration by a Cas1mini-integrase and Edraki etal. (2019) describe accurate genome editing by a small Cas9 ortholog with less stringent PAM requirements.

Identification of preexisting adaptive immunity to Cas9 proteins in humans.

The CRISPR-Cas9 system is a powerful tool for genome editing, which allows the precise modification of specific DNA sequences. Many efforts are underway to use the CRISPR-Cas9 system to therapeutically correct human genetic diseases1-6. The most widely used orthologs of Cas9 are derived from Staphylococcus aureus and Streptococcus pyogenes5,7. Given that these two bacterial species infect the human population at high frequencies8,9, we hypothesized that humans may harbor preexisting adaptive immune responses to the Cas9 orthologs derived from these bacterial species, SaCas9 (S. aureus) and SpCas9 (S. pyogenes). By probing human serum for the presence of anti-Cas9 antibodies using an enzyme-linked immunosorbent assay, we detected antibodies against both SaCas9 and SpCas9 in 78% and 58% of donors, respectively. We also found anti-SaCas9 T cells in 78% and anti-SpCas9 T cells in 67% of donors, which demonstrates a high prevalence of antigen-specific T cells against both orthologs. We confirmed that these T cells were Cas9-specific by demonstrating a Cas9-specific cytokine response following isolation, expansion, and antigen restimulation. Together, these data demonstrate that there are preexisting humoral and cell-mediated adaptive immune responses to Cas9 in humans, a finding that should be taken into account as the CRISPR-Cas9 system moves toward clinical trials.

Structural insights into a high fidelity variant of SpCas9

The RNA-guided endonucleases of the CRISPR-Cas9 system, including the most widely used Cas9 from Streptococcus pyogenes (SpCas9), are becoming a robust genome editing tool in model organisms and hold immense promise for therapeutic applications. Many strategies have been employed to overcome the limitations caused by SpCas9’s off-target effects and its stringent requirement for the protospacer adjacent motif (PAM) sequence. However, the structural mechanisms underlying these strategies remain undefined. Here, we present crystal structure of a SpCas9 variant, xCas9 3.7 that has broad PAM compatibility and high DNA targeting specificity, in complex with a single-guide RNA and its double-stranded DNA targets. Structural comparison revealed that salt bridge-stabilized R1335 is critical for the stringent selection of PAM sequence by SpCas9. Unrestricted rotamerization of this residue by the E1219V mutation in xCas9 3.7 lessens the stringency for PAM recognition and allows SpCas9 to recognize multiple PAM sequences as further supported by biochemical data. Compared to those in wild-type (WT) SpCas9, REC2 and REC3 domains in xCas9 3.7 undergo striking conformational changes, leading to reduced contact with DNA substrate. SpCas9 mutants engineered to display less interaction with DNA and have conformationally more flexible REC2 and REC3 domains display enhanced specificity for DNA substrates in both biochemical and cellular assays. Taken together, our findings reveal the structural mechanisms underlying the broadened PAM compatibility and high DNA fidelity of xCas9 3.7, which can assist rational engineering of more efficient SpCas9 variants and probably other Cas9 orthologs.

A pipeline for characterization of novel Cas9 orthologs.

In recent years, Cas9 has revolutionized the genome-editing field and enabled a broad range of applications from basic biology to biotechnology and medicine. Cas9 specificity is dictated by base pairing of the guide RNA to the complementary DNA strand, however to initiate hybridization, a short protospacer adjacent motif (PAM) sequence is required in the vicinity of the target sequence. The PAM is recognized by the Cas9 protein and varies between Cas9s. There are thousands of type II CRISPR-Cas9 sequences available in sequence databases. To characterize the PAM recognition diversity provided by Cas9 orthologs, we developed a phylogeny-guided bioinformatics approach and streamlined our experimental procedures for Cas9 expression and RNP complex assembly using cell lysates and in vitro translation mixtures. This approach could be easily adapted for the characterization of other CRISPR-Cas nucleases that require PAM sequences and generate double-strand breaks following target recognition.

Potent Cas9 Inhibition in Bacterial and Human Cells by AcrIIC4 and AcrIIC5 Anti-CRISPR Proteins.

In their natural settings, CRISPR-Cas systems play crucial roles in bacterial and archaeal adaptive immunity to protect against phages and other mobile genetic elements, and they are also widely used as genome engineering technologies. Previously we discovered bacteriophage-encoded Cas9-specific anti-CRISPR (Acr) proteins that serve as countermeasures against host bacterial immunity by inactivating their CRISPR-Cas systems (A. Pawluk, N. Amrani, Y. Zhang, B. Garcia, et al., Cell 167:1829-1838.e9, 2016, https://doi.org/10.1016/j.cell.2016.11.017). We hypothesized that the evolutionary advantages conferred by anti-CRISPRs would drive the widespread occurrence of these proteins in nature (K. L. Maxwell, Mol Cell 68:8-14, 2017, https://doi.org/10.1016/j.molcel.2017.09.002; A. Pawluk, A. R. Davidson, and K. L. Maxwell, Nat Rev Microbiol 16:12-17, 2018, https://doi.org/10.1038/nrmicro.2017.120; E. J. Sontheimer and A. R. Davidson, Curr Opin Microbiol 37:120-127, 2017, https://doi.org/10.1016/j.mib.2017.06.003). We have identified new anti-CRISPRs using the same bioinformatic approach that successfully identified previous Acr proteins (A. Pawluk, N. Amrani, Y. Zhang, B. Garcia, et al., Cell 167:1829-1838.e9, 2016, https://doi.org/10.1016/j.cell.2016.11.017) against Neisseria meningitidis Cas9 (NmeCas9). In this work, we report two novel anti-CRISPR families in strains of Haemophilus parainfluenzae and Simonsiella muelleri, both of which harbor type II-C CRISPR-Cas systems (A. Mir, A. Edraki, J. Lee, and E. J. Sontheimer, ACS Chem Biol 13:357-365, 2018, https://doi.org/10.1021/acschembio.7b00855). We characterize the type II-C Cas9 orthologs from H. parainfluenzae and S. muelleri, show that the newly identified Acrs are able to inhibit these systems, and define important features of their inhibitory mechanisms. The S. muelleri Acr is the most potent NmeCas9 inhibitor identified to date. Although inhibition of NmeCas9 by anti-CRISPRs from H. parainfluenzae and S. muelleri reveals cross-species inhibitory activity, more distantly related type II-C Cas9s are not inhibited by these proteins. The specificities of anti-CRISPRs and divergent Cas9s appear to reflect coevolution of their strategies to combat or evade each other. Finally, we validate these new anti-CRISPR proteins as potent off-switches for Cas9 genome engineering applications.IMPORTANCE As one of their countermeasures against CRISPR-Cas immunity, bacteriophages have evolved natural inhibitors known as anti-CRISPR (Acr) proteins. Despite the existence of such examples for type II CRISPR-Cas systems, we currently know relatively little about the breadth of Cas9 inhibitors, and most of their direct Cas9 targets are uncharacterized. In this work we identify two new type II-C anti-CRISPRs and their cognate Cas9 orthologs, validate their functionality in vitro and in bacteria, define their inhibitory spectrum against a panel of Cas9 orthologs, demonstrate that they act before Cas9 DNA binding, and document their utility as off-switches for Cas9-based tools in mammalian applications. The discovery of diverse anti-CRISPRs, the mechanistic analysis of their cognate Cas9s, and the definition of Acr inhibitory mechanisms afford deeper insight into the interplay between Cas9 orthologs and their inhibitors and provide greater scope for exploiting Acrs for CRISPR-based genome engineering.

NmeCas9 is an intrinsically high-fidelity genome-editing platform

BackgroundThe development of CRISPR genome editing has transformed biomedical research. Most applications reported thus far rely upon the Cas9 protein from Streptococcus pyogenes SF370 (SpyCas9). With many RNA guides, wildtype SpyCas9 can induce significant levels of unintended mutations at near-cognate sites, necessitating substantial efforts toward the development of strategies to minimize off-target activity. Although the genome-editing potential of thousands of other Cas9 orthologs remains largely untapped, it is not known how many will require similarly extensive engineering to achieve single-site accuracy within large genomes. In addition to its off-targeting propensity, SpyCas9 is encoded by a relatively large open reading frame, limiting its utility in applications that require size-restricted delivery strategies such as adeno-associated virus vectors. In contrast, some genome-editing-validated Cas9 orthologs are considerably smaller and therefore better suited for viral delivery.ResultsHere we show that wildtype NmeCas9, when programmed with guide sequences of the natural length of 24 nucleotides, exhibits a nearly complete absence of unintended editing in human cells, even when targeting sites that are prone to off-target activity with wildtype SpyCas9. We also validate at least six variant protospacer adjacent motifs (PAMs), in addition to the preferred consensus PAM (5′-N4GATT-3′), for NmeCas9 genome editing in human cells.ConclusionsOur results show that NmeCas9 is a naturally high-fidelity genome-editing enzyme and suggest that additional Cas9 orthologs may prove to exhibit similarly high accuracy, even without extensive engineering.

Gene Editing of BTK in Acute Myeloid Leukaemia Using CRISPR-Cas9

Acute myeloid leukemia (AML) blood cancer is found primarily in adults and the elderly, being the third biggest blood cancer killer. In the last decades there has been some improved rates in younger patients, but little improvement in older patients. AML survival rates are the worst of all blood cancers, therefore finding new therapeutic targets to treat AML is crucial.Bruton's tyrosine kinase (BTK) is involved in the signalling of multiple receptors including growth factor receptors, cytokine receptors, G-protein coupled receptors, antigen receptors and integrins. BTK in turn activates many of the major downstream signalling pathways that control cell migration, adhesion, survival and proliferation. BTK is best known for its role in B-cell receptor signalling, but BTK is also expressed and constitutively active in AML cell lines and AML patient blasts. Targeting BTK in B-cell malignancies with the BTK inhibitor ibrutinib has shown clinical effectiveness and tolerability in recent patient trials.Ibrutinib is orally active and covalently binds at the Cys-481 in the ATP-binding domain of BTK, to irreversibly block its enzymatic activity. A proportion of CLL and MCL patients treated with ibrutinib relapse during the therapy, with secondary resistance developing against ibrutinib. This resistance is mainly due to a BTK cysteine to serine mutation at position 481 (C481S) which disrupts ibrutinib's covalent binding to BTK.As little is known about BTK's role and therapeutic potential in AML, we aimed to study the effects and functional consequences of pharmacological inhibition of BTK using ibrutinib. We furthermore generated the BTK-C481S mutation in AML cell lines using the CRISPR-Cas9 gene-editing system. Shown here for the first time is a gene-edited mutation introduced into a human leukemia cell clone that has been purified to homogeneity. We explored the functional impact of BTK-C481S mutation on ibrutinib sensitivity, and mutation-induced adaptation of the phosphoproteome and kinome in AML cells.HEK-293T cells were used for validation purposes to find the most efficient knock-in strategy. We tested the most commonly used CRISPR endonuclease spCas9 as well as two other Cas9 orthologs called SaCas9 and AsCas12a. The designed spacer sequences within the sgRNA that target the gene of interest were delivered either via plasmid or in vitro transcripts. A repair template for homology directed repair (HDR) was designed containing the necessary base changes to change the amino acid at position 481 in BTK from cysteine to serine. The HDR template was delivered via transfection as single-stranded oligodeoxynucleotide (ssODN) or incorporated into plasmid. All strategies tested were successful but due to the poor transfectability of AML cell lines, we used lentiviral delivery to introduce BTK-C481S mutation into AML cell lines.The BTK-C481S mutation was introduced in THP-1 and OCI-AML3 cells. Mutant-positive cells were found via clonal selection and Sanger sequencing. Wild-type (WT) as well as mutant cells were treated with various ibrutinib concentrations ranging from 1 nM to 1 µM and analysed for BTK expression/phosphorylation via Western blot, with effects on cell proliferation, viability and cell cycle measured via flow cytometry. We also performed phosphoproteomics and kinome assays comparing untreated as well as treated (30 nM ibrutinib, 2h) THP-1 WT and THP-1 BTK-C481S cells.Western blot results showed that the effective concentration required for BTK inhibition is 30-fold higher in BTK-C481S mutant compared to WT cells (300 nM vs 10 nM). Wash-out experiments revealed that ibrutinib binding can be reversed in BTK-C481S mutant cells but not WT cells. Little effect of ibrutinib on proliferation and viability were seen comparing BTK-C481S mutant and WT AML cells.These signalling adaptations in BTK-C481S cells are consistent with changes observed in ibrutinib-resistant leukemia patient samples. DisclosuresSlupsky:Verastem: Research Funding.

Long-Term Effects of In Vivo Genome Editing in the Mouse Retina Using Campylobacter jejuni Cas9 Expressed via Adeno-Associated Virus

Genome editing with CRISPR systems provides an unprecedented opportunity to modulate cellular responses in pathological conditions by inactivating undruggable targets, such as transcription factors. Previously, we demonstrated that the smallest Cas9 ortholog characterized to date, from Campylobacter jejuni (CjCas9) targeted to Hif1a and delivered in an adeno-associated virus (AAV) vector, effectively suppressed pathological choroidal neovascularization in the mouse retina. Before implementation of CjCas9 as an invivo therapeutic modality, it is essential to investigate the long-term effects of target gene disruption via AAV-mediated delivery of CjCas9 invivo. In this study, histologic and electroretinographic analyses demonstrated that CjCas9 targeted to Hif1a did not induce any definite toxicity in the retina, although the target gene was mutated with a frequency ranging from 45% to 79% in retinal or retinal pigment epithelial cells. Importantly, at 14months after injection, no indels were detected at potential off-target sites identified using Digenome-seq and Cas-OFFinder, suggesting that long-term expression of CjCas9 does not aggravate off-target effects. Taken together, our results show that intravitreal injection of AAV encoding CjCas9 targeted to Hif1a effectively induced and maintained mutations in retinal tissues for more than 1 year and did not affect retinal histologic integrity or functions.

All-in-one adeno-associated virus delivery and genome editing by Neisseria meningitidis Cas9 in vivo

BackgroundClustered, 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: Streptococcus pyogenes Cas9 (SpyCas9), Staphylococcus aureus Cas9 (SauCas9), and Campylobacter jejuni (CjeCas9). However, additional in vivo editing platforms are needed, in part to enable a greater range of sequences to be accessed via viral vectors, especially those in which Cas9 and sgRNA are combined into a single vector genome.ResultsHere, we present in vivo editing using Neisseria meningitidis Cas9 (NmeCas9). NmeCas9 is compact, edits with high accuracy, and possesses a distinct protospacer adjacent motif (PAM), making it an excellent candidate for safe gene therapy applications. We find 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 reprogram the tyrosine degradation pathway in Hereditary Tyrosinemia Type I mice. More importantly, we deliver NmeCas9 with its sgRNA 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.ConclusionsOur findings indicate that NmeCas9 can enable the editing of disease-causing loci in vivo, expanding the targeting scope of RNA-guided nucleases.

Heavily and fully modified RNAs guide efficient SpyCas9-mediated genome editing

RNA-based drugs depend on chemical modifications to increase potency and to decrease immunogenicity in vivo. Chemical modification will likely improve the guide RNAs involved in CRISPR-Cas9-based therapeutics as well. Cas9 orthologs are RNA-guided microbial effectors that cleave DNA. Here, we explore chemical modifications at all positions of the crRNA guide and tracrRNA cofactor. We identify several heavily modified versions of crRNA and tracrRNA that are more potent than their unmodified counterparts. In addition, we describe fully chemically modified crRNAs and tracrRNAs (containing no 2′-OH groups) that are functional in human cells. These designs will contribute to Cas9-based therapeutics since heavily modified RNAs tend to be more stable in vivo (thus increasing potency). We anticipate that our designs will improve the use of Cas9 via RNP and mRNA delivery for in vivo and ex vivo purposes.