Cas9 Activity Research Articles

Disrupting off-target Cas9 activity in the liver.

Off-target genome editing in the liver can be reduced by using lipid nanoparticles to deliver oligonucleotides that disrupt the secondary structure of single-guide RNAs as well as short interfering RNAs targeting Cas9 mRNA.

CRISPR/Cas9 On- and Off-Target Activity Using Correlative Force and Fluorescence Single-Molecule Microscopy.

The discovery of CRISPR/Cas9 as an easily programmable endonuclease heralds a new era of genetic manipulation. With this comes the prospect of novel gene therapy approaches, and the potential to cure previously untreatable genetic diseases. However, reports of spurious off-target editing by CRISPR/Cas9 pose a significant hurdle to realizing this potential. A deeper understanding of the factors that affect Cas9 specificity is vital for development of safe and efficient therapeutics. Here, we describe methods for the use of optical tweezers combined with confocal fluorescence microscopy and microfluidics for the analysis of on- and off-target activity of Cas9 activity.

Cytosine extensions optimize case activity for telomere length regulation: Implications for CRISPR-based therapies – A short communication

Telomeres are nucleoprotein structures that play a crucial role in maintaining genomic stability, and their length determines cellular lifespan. Telomere shortening is linked to cellular senescence and an increased risk of cancer. The CRISPR-Cas9 system has emerged as a tool for genome engineering and telomere length regulation. However, several factors, including chromatin accessibility, the efficiency of Double-Stranded Break (DSB) repair and the specificity of the sgRNA/Cas9 complex, limit the efficiency of Cas9-mediated telomere length regulation. Recent studies have demonstrated the use of modified Cas9 nucleases, such as Cas9-NG, and the development of modified sgRNAs to improve the efficiency of Cas9-mediated telomere length regulation. In this study, Bhattacharyya, et al. investigated the optimization of Cas9 activity through the addition of cytosine (C) extensions to the 5’ end of sgRNAs. They found that C extensions significantly increased Cas9 activity at telomeres and demonstrated that the optimal length of C extensions was three Cs. The addition of C extensions did not affect the specificity of the sgRNA/Cas9 complex, as assessed by the frequency of off-target DSBs. These findings have important implications for the development of CRISPR-Cas9-based therapies for telomere-related diseases. Further studies are needed to confirm these findings and optimize the use of C extensions in different cell types and disease contexts.

Inhibition mechanisms of CRISPR-Cas9 by AcrIIA17 and AcrIIA18.

Mobile genetic elements such as phages and plasmids have evolved anti-CRISPR proteins (Acrs) to suppress CRISPR-Cas adaptive immune systems. Recently, several phage and non-phage derived Acrs including AcrIIA17 and AcrIIA18 have been reported to inhibit Cas9 through modulation of sgRNA. Here, we show that AcrIIA17 and AcrIIA18 inactivate Cas9 through distinct mechanisms. AcrIIA17 inhibits Cas9 activity through interference with Cas9-sgRNA binary complex formation. In contrast, AcrIIA18 induces the truncation of sgRNA in a Cas9-dependent manner, generating a shortened sgRNA incapable of triggering Cas9 activity. The crystal structure of AcrIIA18, combined with mutagenesis studies, reveals a crucial role of the N-terminal β-hairpin in AcrIIA18 for sgRNA cleavage. The enzymatic inhibition mechanism of AcrIIA18 is different from those of the other reported type II Acrs. Our results add new insights into the mechanistic understanding of CRISPR-Cas9 inhibition by Acrs, and also provide valuable information in the designs of tools for conditional manipulation of CRISPR-Cas9.

Single nucleotide substitutions effectively block Cas9 and allow for scarless genome editing in Caenorhabditis elegans.

In Caenorhabditis elegans, germline injection of Cas9 complexes is reliably used to achieve genome editing through homology-directed repair of Cas9-generated DNA breaks. To prevent Cas9 from targeting repaired DNA, additional blocking mutations are often incorporated into homologous repair templates. Cas9 can be blocked either by mutating the PAM sequence that is essential for Cas9 activity or by mutating the guide sequence that targets Cas9 to a specific genomic location. However, it is unclear how many nucleotides within the guide sequence should be mutated, since Cas9 can recognize “off-target” sequences that are imperfectly paired to its guide. In this study, we examined whether single-nucleotide substitutions within the guide sequence are sufficient to block Cas9 and allow for efficient genome editing. We show that a single mismatch within the guide sequence effectively blocks Cas9 and allows for recovery of edited animals. Surprisingly, we found that a low rate of edited animals can be recovered without introducing any blocking mutations, suggesting a temporal block to Cas9 activity in C. elegans. Furthermore, we show that the maternal genome of hermaphrodite animals is preferentially edited over the paternal genome. We demonstrate that maternally provided haplotypes can be selected using balancer chromosomes and propose a method of mutant isolation that greatly reduces screening efforts postinjection. Collectively, our findings expand the repertoire of genome editing strategies in C. elegans and demonstrate that extraneous blocking mutations are not required to recover edited animals when the desired mutation is located within the guide sequence.

Effective Genome Editing in Leishmania (Viannia) braziliensis Stably Expressing Cas9 and T7 RNA Polymerase.

Until 2015, loss-of-function studies to elucidate protein function in Leishmania relied on gene disruption through homologous recombination. Then, the CRISPR/Cas9 revolution reached these protozoan parasites allowing efficient genome editing with one round of transfection. In addition, the development of LeishGEdit, a PCR-based toolkit for generating knockouts and tagged lines using CRISPR/Cas9, allowed a more straightforward and effective genome editing. In this system, the plasmid pTB007 is delivered to Leishmania for episomal expression or integration in the β-tubulin locus and for the stable expression of T7 RNA polymerase and Cas9. In South America, and especially in Brazil, Leishmania (Viannia) braziliensis is the most frequent etiological agent of tegumentary leishmaniasis. The L. braziliensis β-tubulin locus presents significant sequence divergence in comparison with Leishmania major, which precludes the efficient integration of pTB007 and the stable expression of Cas9. To overcome this limitation, the L. major β-tubulin sequences, present in the pTB007, were replaced by a Leishmania (Viannia) β-tubulin conserved sequence generating the pTB007_Viannia plasmid. This modification allowed the successful integration of the pTB007_Viannia cassette in the L. braziliensis M2903 genome, and in silico predictions suggest that this can also be achieved in other Viannia species. The activity of Cas9 was evaluated by knocking out the flagellar protein PF16, which caused a phenotype of immobility in these transfectants. Endogenous PF16 was also successfully tagged with mNeonGreen, and an in-locus complementation strategy was employed to return a C-terminally tagged copy of the PF16 gene to the original locus, which resulted in the recovery of swimming capacity. The modified plasmid pTB007_Viannia allowed the integration and stable expression of both T7 RNA polymerase and Cas9 in L. braziliensis and provided an important tool for the study of the biology of this parasite.

Fine-Tuning Cas9 Activity with a Cognate Inhibitor AcrIIA4 to Improve Genome Editing in Streptomyces.

Efficient enabling technology is required for synthetic biology in Streptomyces due to its natural product reservoir. Though the CRISPR-Cas9 system is powerful for genome editing in this genus, the proposed Cas9 toxicity has limited its application. Here on the basis of previous inducible Cas9 expression at the transcriptional and translational levels coupled with atpD overexpression, a Cas9 cognate inhibitor AcrIIA4 was further introduced to fine-tune the Cas9 activity. In both laboratory and industrial Streptomyces species, we showed that, compared to the constitutively expressed Cas9, incorporating AcrIIA4 increased the conjugation efficiency from 700- to 7000-fold before induction, while a comparable 65%-90% editing efficiency was obtained even on multiple loci for simultaneous deletion after Cas9 expression was induced, along with no significant off-targets. Thus, AcrIIA4 could be a modulator to control Cas9 activity to significantly improve genome editing, and this new toolkit would be widely adaptable and fasten genetic engineering in Streptomyces.

Transcriptional activation with Cas9 activator nanocomplexes rescues Alzheimer's disease pathology

CRISPR/Cas9-mediated gene activation is a potential therapeutic strategy that does not induce double-strand break (DSB) DNA damage. However, in vivo gene activation via a Cas9 activator remains a challenge, currently limiting its therapeutic applications. We developed a Cas9 activator nanocomplex that efficiently activates an endogenous gene in the brain in vivo, suggesting its possible application in novel therapeutics. We demonstrated a potential treatment application of the Cas9 activator nanocomplex by activating Adam10 in the mouse brain without introducing insertions and deletions (inDels). Remarkably, in vivo activation of Adam10 with the Cas9 activator nanocomplex improved cognitive deficits in an Alzheimer's disease (AD) mouse model. These results demonstrate the therapeutic potential of Cas9 activator nanocomplexes for a wide range of neurological diseases.

A CRISPR Activation Screen Identifies an Atypical Rho GTPase That Enhances Zika Viral Entry.

Zika virus (ZIKV) is a re-emerging flavivirus that has caused large-scale epidemics. Infection during pregnancy can lead to neurologic developmental abnormalities in children. There is no approved vaccine or therapy for ZIKV. To uncover cellular pathways required for ZIKV that can be therapeutically targeted, we transcriptionally upregulated all known human coding genes with an engineered CRISPR–Cas9 activation complex in human fibroblasts deficient in interferon (IFN) signaling. We identified Ras homolog family member V (RhoV) and WW domain-containing transcription regulator 1 (WWTR1) as proviral factors, and found them to play important roles during early ZIKV infection in A549 cells. We then focused on RhoV, a Rho GTPase with atypical terminal sequences and membrane association, and validated its proviral effects on ZIKV infection and virion production in SNB-19 cells. We found that RhoV promotes infection of some flaviviruses and acts at the step of viral entry. Furthermore, RhoV proviral effects depend on the complete GTPase cycle. By depleting Rho GTPases and related proteins, we identified RhoB and Pak1 as additional proviral factors. Taken together, these results highlight the positive role of RhoV in ZIKV infection and confirm CRISPR activation as a relevant method to identify novel host–pathogen interactions.

Photoactivatable nanoCRISPR/Cas9 System Based on crRNA Reversibly Immobilized on Carbon Nanoparticles.

Here, we proposed a new approach to engineering a photoactivatable CRISPR/Cas9 gene-editing system. The novel nanoCRISPR/Cas9 system is based on the use of auxiliary photocleavable oligodeoxyribonucleotides (PC-DNAs) complementary to crRNA. PC-DNAs contained up to three UV-sensitive linkers made of 1-(2-nitrophenyl)-1,2-ethanediol inside the oligonucleotide chain. Immobilizing PC-DNAs on the surface of carbon nanoparticles through 3′-terminal pyrene residue provided sufficient blocking of crRNA (and corresponding Cas9 activity) before UV irradiation and allows for crRNA release after UV irradiation at 365 nm, which restores Cas9 activity. We optimized the length of blocking photocleavable oligonucleotide, number of linkers, time of irradiation, and the type of carbon nanoparticles. Based on the results, we consider the nanoCRISPR/Cas9 system involving carbon-encapsulated iron nanoparticles the most promising. It provides the greatest difference of functional activity before/after irradiation and can be used in prospective for magnetic field-controlled delivery of CRISPR system into the target cells or tissues and spatiotemporal gene editing induced by UV irradiation.

Conformational control of Cas9 by CRISPR hybrid RNA-DNA guides mitigates off-target activity in T cells

The off-target activity of the CRISPR-associated nuclease Cas9 is a potential concern for therapeutic genome editing applications. Although high-fidelity Cas9 variants have been engineered, they exhibit varying efficiencies and have residual off-target effects, limiting their applicability. Here, we show that CRISPR hybrid RNA-DNA (chRDNA) guides provide an effective approach to increase Cas9 specificity while preserving on-target editing activity. Across multiple genomic targets in primary human Tcells, we show that 2'-deoxynucleotide (dnt) positioning affects guide activity and specificity in a target-dependent manner and that this can be used to engineer chRDNA guides with substantially reduced off-target effects. Crystal structures of DNA-bound Cas9-chRDNA complexes reveal distorted guide-target duplex geometry and allosteric modulation of Cas9 conformation. These structural effects increase specificity by perturbing DNA hybridization and modulating Cas9 activation kinetics to disfavor binding and cleavage of off-target substrates. Overall, these results pave the way for utilizing customized chRDNAs in clinical applications.

Genome dependent Cas9/gRNA search time underlies sequence dependent gRNA activity

CRISPR-Cas9 is a powerful DNA editing tool. A gRNA directs Cas9 to cleave any DNA sequence with a PAM. However, some gRNA sequences mediate cleavage at higher efficiencies than others. To understand this, numerous studies have screened large gRNA libraries and developed algorithms to predict gRNA sequence dependent activity. These algorithms do not predict other datasets as well as their training dataset and do not predict well between species. Here, to better understand these discrepancies, we retrospectively examine sequence features that impact gRNA activity in 44 published data sets. We find strong evidence that gRNA sequence dependent activity is largely influenced by the ability of the Cas9/gRNA complex to find the target site rather than activity at the target site and that this drives sequence dependent differences in gRNA activity between different species. This understanding will help guide future work to understand Cas9 activity as well as efforts to identify optimal gRNAs and improve Cas9 variants.

Engineering chloroplast development in rice through cell-specific control of endogenous genetic circuits.

SummaryThe engineering of C4 photosynthetic activity into the C3 plant rice has the potential to nearly double rice yields. To engineer a two‐cell photosynthetic system in rice, the rice bundle sheath (BS) must be rewired to enhance photosynthetic capacity. Here, we show that BS chloroplast biogenesis is enhanced when the transcriptional activator, Oryza sativa Cytokinin GATA transcription factor 1 (OsCGA1), is driven by a vascular specific promoter. Ectopic expression of OsCGA1 resulted in increased BS chloroplast planar area and increased expression of photosynthesis‐associated nuclear genes (PhANG), required for the biogenesis of photosynthetically active chloroplasts in BS cells of rice. A further refinement using a DNAse dead Cas9 (dCas9) activation module driven by the same cell‐type specific promoter, directed enhanced chloroplast development of the BS cells when gRNA sequences were delivered by the dCas9 module to the promoter of the endogenous OsCGA1 gene. Single gRNA expression was sufficient to mediate the transactivation of both the endogenous gene and a transgenic GUS reporter fused with OsCGA1 promoter. Our results illustrate the potential for tissue‐specific dCas9‐activation and the co‐regulation of genes needed for multistep engineering of C4 rice.

Structural Investigation of Self-Assembly and Target Binding of Anti-CRISPR AcrIIC2.

Anti-CRISPR (Acr) proteins are phage-borne inhibitors of the CRISPR-Cas immune system in archaea and bacteria. AcrIIC2 from prophages of Neisseria meningitidis disables the nuclease activity of type II-C Cas9, such that dimeric AcrIIC2 associates with the bridge helix (BH) region of Cas9 to compete with guide RNA loading. AcrIIC2 in solution readily assembles into oligomers of variable lengths, but the oligomeric states are not clearly understood. In this study, we investigated the dynamic assembly of AcrIIC2 oligomers, and identified key interactions underlying the self-association. We report that AcrIIC2 dimers associate into heterogeneous high-order oligomers with the equilibrium dissociation constant K D ∼8 μM. Oligomerization is driven by electrostatic interactions between charged residues, and rational mutagenesis produces a stable AcrIIC2 dimer with intact Cas9 binding. Remarkably, the BH peptide of Cas9 is unstructured in solution, and undergoes a coil-to-helix transition upon AcrIIC2 binding, revealing a unique folding-upon-binding mechanism for Acr recognition.

Encounters between Cas9/dCas9 and G-Quadruplexes: Implications for Transcription Regulation and Cas9-Mediated DNA Cleavage.

Using the nuclease-dead Cas9 (dCas9), we targeted in cellulo a G-rich sequence, which contains multiple potentially G-quadruplex (GQ) forming sites, within the human tyrosine hydroxylase (TH) promoter. We demonstrate that transcription can be up or down regulated by targeting different parts of this G-rich sequence. Our results suggest that TH transcription levels correlate with stability of different GQs formed by this sequence and targeting them with dCas9 can modulate their stability. Unlike alternative approaches, regulating TH expression by targeting the promoter GQs with dCas9 enables a specific and potentially transient control and does not require mutations in the sequence. We also investigated whether the presence of GQs in target sequences impacts DNA cleavage activity of Cas9. We discovered significant reduction in cleavage activity when the vicinity of a high-stability GQ was targeted. Furthermore, this reduction is significantly more prominent for the G-rich strand compared to the complementary C-rich strand.

Highly efficient C-to-T and A-to-G base editing in a Populus hybrid.

CRISPR-Cas based genome editing technologies hold great potential for genetic research and bioengineering in trees. CRISPR-Cas9 was previously demonstrated to be a highly efficient genome editing system in poplar (Zhou et al., 2015). However, these applications only used the nuclease activity of Cas9 for targeted mutagenesis. By contrast, Cas9-derived base editors can install specific base changes in a plant genome.

Targeting G-quadruplex Forming Sequences with Cas9.

Clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins, particularly Cas9, have provided unprecedented control on targeting and editing specific DNA sequences. If the target sequences are prone to folding into noncanonical secondary structures, such as G-quadruplex (GQ), the conformational states and activity of the CRISPR–Cas9 complex may be influenced, but the impact has not been assessed. Using single molecule FRET, we investigated structural characteristics of the complex formed by CRISPR–Cas9 and target DNA, which contains a potentially GQ forming sequence (PQS) in either the target or the nontarget strand (TS or NTS). We observed different conformational states and dynamics depending on the stability of the GQ and the position of PQS. When PQS was in NTS, we observed evidence for GQ formation for both weak and stable GQs. This is consistent with R-loop formation between TS and crRNA releasing NTS from Watson–Crick pairing and facilitating secondary structure formation in it. When PQS was in TS, R-loop formation was adequate to maintain a weak GQ in the unfolded state but not a GQ with moderate or high stability. The observed structural heterogeneity within the target dsDNA and the R-loop strongly depended on whether the PQS was in TS or NTS. We propose these variations in the complex structures to have functional implications for Cas9 activity.

Seeing is believing: a visualization toolbox to enhance selection efficiency in maize genome editing.

The emerging and promising biotechnology methodology of CRISPR/Cas9 gene-editing is revolutionizing crop improvement. However, the low-efficiency, time-consuming, and labor-intensive nature of positive or/and negative selections during or after transformation and subsequent identification of mutations are major challenges for its agricultural application, from upstream (high-throughput) mutant screening to downstream commercial production (He and Zhao, 2020). Although visual markers, and in particular fluorescent markers including green fluorescent protein (GFP) and red fluorescent protein (RFP), have been employed for the rapid visualization of transgenic material (Qi et al., 2020), the integration of cost-effective and non-invasive CRISPR-separation gene-editing tools is still in its infancy but remains critical for crop genetics and breeding (Callaway, 2018). Furthermore, special light sources are required to visualize fluorescent signals, which increases the cost and inconvenience of the application of fluorescence markers, especially in field conditions. We have developed a visualization toolbox, ViMeBox (VIsual Maize Editing toolBox), for the selection of positive transformants in maize (Zea mays). In the ViMeBox system, the vector expressing Cas9 includes a gene cassette containing a visible marker expressed from a tissue-specific promoter; Cas9-free kernels that are easy to separate by the visible marker are also undergoing gene editing. ViMeBox offers two advantages: 1) It enables enhanced expression of DsRED2, which makes seeds containing Cas9 visible to the naked eye in natural light, and does not affect genome editing efficiency or plant development. 2) It is effective for different seed tissues, for example, using embryo-specific promoters or promoters preferentially expressed in the embryo or aleurone. Furthermore, ViMeBox has potential applications in diverse additional scenarios.

Multiplex Targeted Epigenome Editing Utilizing CRSPR/Cas9 for Potent Anti-Inflammatory Gene Therapy in Lung Transplant

<h3>Purpose</h3> Flexible modulation of gene expressions in the donor lung holds great promise in lung transplant. We hypothesized that activation of multiple anti-inflammatory genes would synergistically induce more potent effects. CRISPR/Cas9 system has strength in multiplexing due to its simple mechanism of the programmable RNA-guided DNA targeting. This study investigated the efficacy of simultaneous activation of the IL-10 and IL-1 receptor antagonist (IL1RN) genes, both of which elicit anti-inflammatory effects, using CRISPR/Cas9-mediated epigenome editing <i>in vitro</i>. <h3>Methods</h3> gRNAs were designed to target the promoter regions of the IL-10 or IL1RN genes and expressed along with the Cas9 activator (Fig. A) in the rat lung epithelial cell line (L2). The concentrations of IL-10 and IL1RN in the culture supernatant were measured 48 hours after plasmid transfection (n=3). Cells were assessed in four groups; No gRNA (expressing Cas9 activator alone), IL-10 activating (expressing Cas9 activator and two gRNAs for the IL-10 gene), IL-10+IL1RN activating (expressing Cas9 activator and two gRNAs for the IL-10 gene and two gRNAs for the IL1RN gene), and negative control (no plasmid). <h3>Results</h3> In L2 cells (Fig. B), the IL-10 activating group showed increased IL-10 (2353 ± 551 pg/ml) compared to nearly undetectable in the no gRNA and negative control groups. The IL-10+IL1RN activating group increased IL1RN (769 ± 104 pg/ml vs 8 ± 9.5 pg/ml, p=0.0023) compared to the IL-10 activating group, accompanied by a retained increase in IL-10 (2047 ± 493 pg/ml). Similar observations were found in primary lung fibroblasts cells (data not shown) supporting the efficacy of this strategy in diverse cell types. <h3>Conclusion</h3> We have achieved simultaneous activation of the IL-10 and IL1RN genes through epigenome editing using CRISPR/Cas9 <i>in vitro</i>. This study demonstrates the potential of manipulating the expression of multiple genes using DNA targeting via CRISPR/Cas9 for engineering optimized organs for transplant.

CRISPR/Cas9-Mediated Epigenome Editing of the IL-10 Gene for Targeted Whole Organ Gene Therapy for Lung Transplant

<h3>Purpose</h3> Anti-inflammatory and immunomodulatory gene therapy has shown promise in lung transplant. CRISPR/Cas9 technology unleashed the possibility of making gene therapy persistent by radically altering gene expression through precise DNA targeting. We envisioned activation of the endogenous IL-10 gene, which codes for this anti-inflammatory cytokine, using epigenome editing via CRISPR/Cas9 in the lung. This study investigated the efficacy and specificity in vitro and in rat lungs. <h3>Methods</h3> Adenoviral vectors were developed expressing a Cas9 activator and either one or two gRNAs targeting the promoter region of the IL-10 gene in the genome (Fig. A). Lewis rats were divided into three groups (Fig. B, n=5-10), trans-bronchially administered adenovirus (1 × 10⁸ PFU/rat) under standard transplant triple immunosuppression, and assessed after 72 hours. For specificity, primary lung fibroblasts treated with adenovirus were analyzed at 48 hours using chromatin immunoprecipitation (ChIP) of Cas9 followed by qPCR designed against the on- and off-target regions. <h3>Results</h3> The two-gRNA group showed significantly increased IL-10 gene expression compared to the diluent group (relative expression: 19.0±10.8 vs 1.3±1.2, p=0.001) and the single gRNA group (2.9±0.78, p=0.0009) (Fig. C). There was no significant increase in TNFα or IL-6 gene expression or Wet/Dry ratio in the two-gRNA group compared to the diluent group (Fig. D, E) suggesting minimal vector-related inflammation. ChIP-qPCR demonstrated enriched binding of Cas9 to on-target sites (Fig. F, fold enrichment; 17.5±1.7, p=0.02, 18.5±5.8, p=0.02, respectively) compared to the control region whereas the binding to off-target sites were not significantly enriched. <h3>Conclusion</h3> We have achieved targeted in vivo whole organ activation of the IL-10 gene via CRISPR/Cas9-mediated epigenome editing in the lung. This study demonstrates the feasibility of this strategy for optimizing and immunologically enhancing donor organs for transplant.