High-fidelity Cas9 Research Articles

Multiplex nucleotide editing by high-fidelity Cas9 variants with improved efficiency in rice

BackgroundApplication of the CRISPR/Cas9 system or its derived base editors enables targeted genome modification, thereby providing a programmable tool to exploit gene functions and to improve crop traits.ResultsWe report that PmCDA1 is much more efficient than rAPOBEC1 when fused to CRISPR/Cas9 nickase for the conversion of cytosine (C) to thymine (T) in rice. Three high-fidelity SpCas9 variants, eSpCas9(1.1), SpCas9-HF2 and HypaCas9, were engineered to serve with PmCDA1 (pBEs) as C-to-T base editors. These three high-fidelity editors had distinct multiplex-genome editing efficiencies. To substantially improve their base-editing efficiencies, a tandemly arrayed tRNA-modified single guide RNA (sgRNA) architecture was applied. The efficiency of eSpCas9(1.1)-pBE was enhanced up to 25.5-fold with an acceptable off-target effect. Moreover, two- to five-fold improvement was observed for knock-out mutation frequency by these high-fidelity Cas9s under the direction of the tRNA-modified sgRNA architecture.ConclusionsWe have engineered a diverse toolkit for efficient and precise genome engineering in rice, thus making genome editing for plant research and crop improvement more flexible.

High-fidelity endonuclease variant HypaCas9 facilitates accurate allele-specific gene modification in mouse zygotes

CRISPR/Cas9 has been widely used for the efficient generation of genetically modified animals; however, this system could have unexpected off-target effects. In the present study, we confirmed the validity of a high-fidelity Cas9 variant, HypaCas9, for accurate genome editing in mouse zygotes. HypaCas9 efficiently modified the target locus while minimizing off-target effects even in a single-nucleotide mismatched sequence. Furthermore, by applying HypaCas9 to the discrimination of SNP in hybrid strain-derived zygotes, we accomplished allele-specific gene modifications and successfully generated mice with a monoallelic mutation in an essential gene. These results suggest that the improved accuracy of HypaCas9 facilitates the generation of genetically modified animals.

Optimized CRISPR guide RNA design for two high-fidelity Cas9 variants by deep learning

Highly specific Cas9 nucleases derived from SpCas9 are valuable tools for genome editing, but their wide applications are hampered by a lack of knowledge governing guide RNA (gRNA) activity. Here, we perform a genome-scale screen to measure gRNA activity for two highly specific SpCas9 variants (eSpCas9(1.1) and SpCas9-HF1) and wild-type SpCas9 (WT-SpCas9) in human cells, and obtain indel rates of over 50,000 gRNAs for each nuclease, covering ~20,000 genes. We evaluate the contribution of 1,031 features to gRNA activity and develope models for activity prediction. Our data reveals that a combination of RNN with important biological features outperforms other models for activity prediction. We further demonstrate that our model outperforms other popular gRNA design tools. Finally, we develop an online design tool DeepHF for the three Cas9 nucleases. The database, as well as the designer tool, is freely accessible via a web server, http://www.DeepHF.com/.

Boosting activity of high-fidelity CRISPR/Cas9 variants using a tRNAGln-processing system in human cells

CRISPR/Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. High-fidelity Cas9 variants have been identified; however, they often have reduced activity, constraining their utility, which presents a major challenge for their use in research applications and therapeutics. Here we developed a tRNAGln-processing system to restore the activity of multiple high-fidelity Cas9 variants in human cells, including SpCas9-HF1, eSpCas9, and xCas9. Specifically, acting on previous observations that small guide RNAs (sgRNAs) harboring an extra A or G (A/G) in the first 5' nucleotide greatly affect the activity of high-fidelity Cas9 variants and that tRNA-sgRNA fusions improve Cas9 activity, we investigated whether a GN20 sgRNA fused to different tRNAs (G-tRNA-N20) could restore the activity of SpCas9 variants in human cells. Using flow cytometry, a T7E1 assay, deep sequencing-based DNA cleavage activity assays, and HEK-293 cells, we observed that a tRNAGln-sgRNA fusion system enhanced the activity of Cas9 variants, which could be harnessed for efficient correction of a pathogenic mutation in the retinoschisin 1 (RS1) gene, resulting in 6- to 8-fold improved Cas9 activity. We propose that the tRNA-processing system developed here specifically for human cells could facilitate high-fidelity Cas9-mediated human genome-editing applications.

Company Profile: Aldevron Expands to Facilitate Emerging Therapies.

Company Profile: Aldevron Expands to Facilitate Emerging Therapies.

Highly Efficient and Marker-free Genome Editing of Human Pluripotent Stem Cells by CRISPR-Cas9 RNP and AAV6 Donor-Mediated Homologous Recombination.

Genome editing of human pluripotent stem cells (hPSCs) provides powerful opportunities for invitro disease modeling, drug discovery, and personalized stem cell-based therapeutics. Currently, only small edits can be engineered with high frequency, while larger modifications suffer from low efficiency and a resultant need for selection markers. Here, we describe marker-free genome editing in hPSCs using Cas9 ribonucleoproteins (RNPs) in combination with AAV6-mediated DNA repair template delivery. We report highly efficient and bi-allelic integration frequencies across multiple loci and hPSC lines, achieving mono-allelic editing frequencies of up to 94% at the HBB locus. Using this method, we show robust bi-allelic correction of homozygous sickle cell mutations in a patient-derived induced PSC (iPSC) line. Thus, this strategy shows significant utility for generating hPSCs with large gene integrations and/or single-nucleotide changes at high frequency and without the need for introducing selection genes, enhancing the applicability of hPSC editing for research and translational uses.

Gene correction for SCID-X1 in long-term hematopoietic stem cells

Gene correction in human long-term hematopoietic stem cells (LT-HSCs) could be an effective therapy for monogenic diseases of the blood and immune system. Here we describe an approach for X-linked sSevere cCombined iImmunodeficiency (SCID-X1) using targeted integration of a cDNA into the endogenous start codon to functionally correct disease-causing mutations throughout the gene. Using a CRISPR-Cas9/AAV6 based strategy, we achieve up to 20% targeted integration frequencies in LT-HSCs. As measures of the lack of toxicity we observe no evidence of abnormal hematopoiesis following transplantation and no evidence of off-target mutations using a high-fidelity Cas9 as a ribonucleoprotein complex. We achieve high levels of targeting frequencies (median 45%) in CD34+ HSPCs from six SCID-X1 patients and demonstrate rescue of lymphopoietic defect in a patient derived HSPC population in vitro and in vivo. In sum, our study provides specificity, toxicity and efficacy data supportive of clinical development of genome editing to treat SCID-Xl.

Optimizing genome editing strategy by primer-extension-mediated sequencing

Efficient and precise genome editing is essential for clinical applications and generating animal models, which requires engineered nucleases with high editing ability while low off-target activity. Here we present a high-throughput sequencing method, primer-extension-mediated sequencing (PEM-seq), to comprehensively assess both editing ability and specificity of engineered nucleases. We showed CRISPR/Cas9-generated breaks could lead to chromosomal translocations and large deletions by PEM-seq. We also found that Cas9 nickase possessed lower off-target activity while with some loss of target cleavage ability. However, high-fidelity Cas9 variants, including both eCas9 and the new FeCas9, could significantly reduce the Cas9 off-target activity with no obvious editing retardation. Moreover, we found AcrIIA4 inhibitor could greatly reduce the activities of Cas9, but off-target loci were not so effectively suppressed as the on-target sites. Therefore, PEM-seq fully evaluating engineered nucleases could help choose better genome editing strategy at given loci than other methods detecting only off-target activity.

Structural and Functional Insights into CRISPR/Cas9 Catalytic Activation and Specificity Enhancement

CRISPR/Cas9 has been repurposed as a powerful genome editing tool, with immense potential toward therapeutic applications. Despite recent advances in understanding DNA recognition and cleavage by Cas9, consistent structural and functional information about the catalytic state of the HNH nuclease domain remains to be elucidated. On the basis of our recent work (Nature Scientific Reports, 7:17271), we here report a new Cas9 active state complex structure discovered by molecular dynamics simulations and validated by site-directed mutagenesis experiments. In this structure, the HNH domain is poised for cleaving the target DNA strand using a canonical catalytic triad as seen in phage T4 Endonuclease VII. Guided by the derived new structural information, we rationally designed and tested a library of new Cas9 variants to improve Cas9 specificities. Our ultimate goal is to offer several high-fidelity Cas9 enzymes that can be broadly used in basic research and medical therapy.

High-fidelity CRISPR/Cas9- based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis

While suppression of specific genes through aberrant promoter methylation contributes to different diseases including organ fibrosis, gene-specific reactivation technology is not yet available for therapy. TET enzymes catalyze hydroxymethylation of methylated DNA, reactivating gene expression. We here report generation of a high-fidelity CRISPR/Cas9-based gene-specific dioxygenase by fusing an endonuclease deactivated high-fidelity Cas9 (dHFCas9) to TET3 catalytic domain (TET3CD), targeted to specific genes by guiding RNAs (sgRNA). We demonstrate use of this technology in four different anti-fibrotic genes in different cell types in vitro, among them RASAL1 and Klotho, both hypermethylated in kidney fibrosis. Furthermore, in vivo lentiviral delivery of the Rasal1-targeted fusion protein to interstitial cells and of the Klotho-targeted fusion protein to tubular epithelial cells each results in specific gene reactivation and attenuation of fibrosis, providing gene-specific demethylating technology in a disease model.

A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells.

Translation of the CRISPR/Cas9 system to human therapeutics holds high promise. Specificity remains a concern, however, especially when modifying stem cell populations. We show that existing rationally-engineered Cas9 high fidelity variants have reduced on-target activity using the therapeutically relevant ribonucleoprotein (RNP) delivery method. Therefore, we devised an unbiased bacterial screen to isolate variants that retain activity in the RNP format. Introduction of a single point mutation, R691A (HiFi Cas9), retained high on-target activity while reducing off-target editing. HiFi Cas9 induces robust AAV6-mediated gene targeting at five therapeutically-relevant loci (HBB, IL2RG, CCR5, HEXB, TRAC) in human CD34+ hematopoietic stem and progenitor cells (HSPCs) as well as primary T-cells. We also show that the HiFi Cas9 mediates high-level correction of the sickle cell disease (SCD)-causing Glu6Val mutation in SCD patient derived HSPCs. We anticipate that HiFi Cas9 will have wide utility for both basic science and therapeutic genome editing applications.

PO-459 Unravelling endoglin as a potential therapeutic target for the treatment of uveal melanoma

IntroductionUveal Melanoma(UM) is a rare deadly tumour where, despite highly aggressive management of the primary disease, approximately 50% of patients develop metastases. Therapies directed towards systemic/metastatic disease have failed in the clinic.Endoglin (ENG),a co-receptor of TGF-β,plays an important role in tissue remodelling and cancer.A soluble form is produced upon proteolytic cleavage by matrix metalloprotease MMP14.Our work focuses on the role of ENG/MMP14, how they influence UM metastasis, and their potential as therapeutic targets.Material and methodsNormalised gene expression data with patient follow-up from 80 primary UM tumours was downloaded from The Cancer Genome Atlas (TCGA) NIH Data Portal.mRNA/protein from UM and Ewing Sarcoma cell lines were isolated, and ENG/MMP14 levels were evaluated by q-RT-PCR/western blot. Immunohistochemistry analysis of ENG/MMP14 expression was performed in 2 independent patient cohorts.MTT assays were performed to evaluate proliferation inhibition after exposure to OMTX503 anti-ENG Antibody Drug Conjugate (ADC).CRISPR technology with high fidelity Cas9 was used to knockout MMP14/ENG, which was validated by mismatch-cleavage assays and by the loss of protein expression.Results and discussionsBioinformatics analysis showed that a higher expression of ENG correlated with worst disease-free survival (DFS)(p=0.00023), whereas MMP14 showed a similar trend without reaching statistical significance(p=0.077).These results differ from skin melanoma DFS(p=0.74 for ENG,p=0.22 for MMP14).Pathway enrichment analysis showed that higher ENG expression is associated with a higher abundance of CD8+T-cells (p<0.002),endothelial cells(p<0.003) and fibroblasts(p<0.001).In the 2 patient cohorts, differential levels of ENG/MMP14 were detected.In vitro, cell line MUM2B expressed higher levels of ENG/MMP14.UM cell lines were highly sensitive to treatment with ADC OMTX503, with IC50 in the sub-nM range.Sensitivity was dependent on ENG expression.Knockout of MMP14 resulted in lack of protein expression in the single cell derived clones.The effect on ENG shedding and malignant features upon lack of MMP14 is currently being analysed.ConclusionENG/MMP14 are expressed in UM cell lines and in patient tumour samples.Results suggest that higher levels of ENG are associated with a more aggressive phenotype.ENG is an attractive target, and ENG-targeting biologics such as OMTX503 show promising in vitro activity.In-depth evaluation of the role of ENG/MMP14 in UM is ongoing.

Abstract LB-040: New conditional Kras-alleles generated by CRISPR-based genome editing to model tumor initiation

Abstract KRAS is the most frequently mutated oncogene in human cancers, and specific cancer types show a clear bias in the types and frequency of Kras alterations. However, we do not have a clear understanding of how distinct Kras mutations dictate tumor biology and response to therapy. The generation of conditional animal models, such as Lox-Stop-Lox (LSL) KrasG12D and LSL-KrasG12Vgeo mice, have provided important tools to dissect the impact of Kras mutation in tumor development, but these models alone do not recapitulate the spectrum of Kras alterations in human cancer. To better reflect the Kras mutational landscape in human cancers, we have developed a series of critical LSL-Kras mouse models that recapitulate common Kras alterations observed in colorectal (G13D), pancreatic (G12R), and lung cancer (G12C), allowing the direct interrogation of the effects of these Kras mutations on disease phenotype and treatment response. To do this, we targeted embryonic stem cells (ESCs) carrying the LSL-KrasG12D allele, by co-transfection of a modified high-fidelity Cas9 (Cas9-HFz), a LSL-Kras-targeted sgRNA, and an HDR template carrying the new mutation. While Cas9-HFz displayed a slight decrease in homology-directed repair (HDR) efficiency in comparison with wildtype Cas9, it enabled high specificity editing of only the LSL allele. Targeted deep sequencing revealed a significant reduction in ‘off-target' events in wildtype Kras locus in Cas9-HFz transfected samples compared to unmodified Cas9. We next generated transgenic mice by blastocyst injection and produced mice carrying each LSL-Kras variant, a pancreas specific Cre (p48-Cre) and a far-red fluorescent Cre-reporter (mKate2). We are now analyzing the impact of specific Kras alterations in tumor initiation in distinct tissues such as the pancreas, colon and lung. Together with available LSL-KrasG12D and LSL-KrasG12V already available, our new alleles will serve as critical tools for better dissect the genetic factors that dictate disease initiation, progression, and response to therapy. Citation Format: Maria Paz Zafra, Emma M. Schatoff, Teng Han, Lukas E. Dow. New conditional Kras-alleles generated by CRISPR-based genome editing to model tumor initiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-040.

CRISPR off-target analysis in genetically engineered rats and mice.

Despite widespread use of CRISPR, comprehensive data on the frequency and impact of Cas9-mediated off-targets in modified rodents are limited. Here we present deep-sequencing data from 81 genome-editing projects on mouse and rat genomes at 1,423 predicted off-target sites, 32 of which were confirmed, and show that high-fidelity Cas9 versions reduced off-target mutation rates in vivo. Using whole-genome sequencing data from ten mouse embryos, treated with a single guide RNA (sgRNA), and from their genetic parents, we found 43 off-targets, 30 of which were predicted by an adapted version of GUIDE-seq.

Abstract A18: Generation of new conditional Kras-alleles by CRISPR-based genome editing

Abstract KRAS is the most frequently mutated oncogene in human cancers, and specific cancer types show a clear bias in the types and frequency of Kras alterations. However, we do not have a clear understanding of how distinct Kras mutations dictate tumor biology and response to therapy. The generation of conditional animal models, such as Lox-Stop-Lox (LSL) KrasG12D and LSL-KrasG12Vgeo mice, has provided important tools to dissect the impact of Kras mutation in tumor development, but these models alone do not recapitulate the spectrum of Kras alterations in human cancer. To better reflect the Kras mutational landscape in human cancers, we have developed a series of critical LSL-Kras mouse models that recapitulate common Kras alterations observed in colorectal (G13D), pancreatic (G12R), and lung cancer (G12C), allowing the direct interrogation of the effects of these Kras mutations on disease phenotype and treatment response. To do this, we targeted embryonic stem cells (ESCs) carrying the LSL-KrasG12D allele, by co-transfection of a modified high-fidelity Cas9 (Cas9-HFz), a LSL-Kras-targeted sgRNA, and an HDR template carrying the new mutation. While Cas9-HFz displayed a slight decrease in homology-directed repair (HDR) efficiency in comparison with wild-type Cas9, it enabled high specificity editing of only the LSL allele. Targeted deep sequencing revealed a significant reduction in “off-target” events in wild-type Kras locus in Cas9-HFz transfected samples compared to unmodified Cas9. We next generated transgenic mice by blastocyst injection and produced mice carrying each LSL-Kras variant, a pancreas-specific Cre (p48-Cre), and a far-red fluorescent Cre-reporter (mKate2). We are now analyzing the impact of specific Kras alterations in tumor initiation in distinct tissues such as the pancreas, colon, and lung. Together with available LSL-KrasG12D and LSL-KrasG12V already available, our new alleles will serve as critical tools for better dissecting the genetic factors that dictate disease initiation, progression, and response to therapy. Citation Format: Maria Paz Zafra, Emma M. Schatoff, Teng Han, Lukas E. Dow. Generation of new conditional Kras-alleles by CRISPR-based genome editing [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A18.

Expanding the CRISPR/Cas9 toolkit for Pichia pastoris with efficient donor integration and alternative resistance markers.

Komagataella phaffii (syn. Pichia pastoris) is one of the most commonly used host systems for recombinant protein expression. Achieving targeted genetic modifications had been hindered by low frequencies of homologous recombination (HR). Recently, a CRISPR/Cas9 genome editing system has been implemented for P. pastoris enabling gene knockouts based on indels (insertion, deletions) via non‐homologous end joining (NHEJ) at near 100% efficiency. However, specifically integrating homologous donor cassettes via HR for replacement studies had proven difficult resulting at most in ∼20% correct integration using CRISPR/Cas9. Here, we demonstrate the CRISPR/Cas9 mediated integration of markerless donor cassettes at efficiencies approaching 100% using a ku70 deletion strain. The Ku70p is involved in NHEJ repair and lack of the protein appears to favor repair via HR near exclusively. While the absolute number of transformants in the Δku70 strain is reduced, virtually all surviving transformants showed correct integration. In the wildtype strain, markerless donor cassette integration was also improved up to 25‐fold by placing an autonomously replicating sequence (ARS) on the donor cassette. Alternative strategies for improving donor cassette integration using a Cas9 nickase variant or reducing off targeting associated toxicity using a high fidelity Cas9 variant were so far not successful in our hands in P. pastoris. Furthermore we provide Cas9/gRNA expression plasmids with a Geneticin resistance marker which proved to be versatile tools for marker recycling. The reported CRSIPR‐Cas9 tools can be applied for modifying existing production strains and also pave the way for markerless whole genome modification studies in P. pastoris.

An episomal vector-based CRISPR/Cas9 system for highly efficient gene knockout in human pluripotent stem cells

Human pluripotent stem cells (hPSCs) represent a unique opportunity for understanding the molecular mechanisms underlying complex traits and diseases. CRISPR/Cas9 is a powerful tool to introduce genetic mutations into the hPSCs for loss-of-function studies. Here, we developed an episomal vector-based CRISPR/Cas9 system, which we called epiCRISPR, for highly efficient gene knockout in hPSCs. The epiCRISPR system enables generation of up to 100% Insertion/Deletion (indel) rates. In addition, the epiCRISPR system enables efficient double-gene knockout and genomic deletion. To minimize off-target cleavage, we combined the episomal vector technology with double-nicking strategy and recent developed high fidelity Cas9. Thus the epiCRISPR system offers a highly efficient platform for genetic analysis in hPSCs.

Organoid technologies meet genome engineering.

Three-dimensional (3D) stem cell differentiation cultures recently emerged as a novel model system for investigating human embryonic development and disease progression in vitro, complementing existing animal and two-dimensional (2D) cell culture models. Organoids, the 3D self-organizing structures derived from pluripotent or somatic stem cells, can recapitulate many aspects of structural organization and functionality of their in vivo organ counterparts, thus holding great promise for biomedical research and translational applications. Importantly, faithful recapitulation of disease and development processes relies on the ability to modify the genomic contents in organoid cells. The revolutionary genome engineering technologies, CRISPR/Cas9 in particular, enable investigators to generate various reporter cell lines for prompt validation of specific cell lineages as well as to introduce disease-associated mutations for disease modeling. In this review, we provide historical overviews, and discuss technical considerations, and potential future applications of genome engineering in 3D organoid models.

328. Targeting Specificity of Cas9 Nucleases and Tools for the Selection of High Fidelity Cas9 Targets

328. Targeting Specificity of Cas9 Nucleases and Tools for the Selection of High Fidelity Cas9 Targets