scholarly journals NmeCas9 is an intrinsically high-fidelity genome editing platform

2017 ◽  
Author(s):  
Nadia Amrani ◽  
Xin D. Gao ◽  
Pengpeng Liu ◽  
Alireza Edraki ◽  
Aamir Mir ◽  
...  
Keyword(s):  
Genome Editing ◽  
Human Cells ◽  
High Fidelity ◽  
Wild Type ◽  
Adeno Associated Virus ◽  
Reading Frame ◽  
Cas9 Protein ◽  
Mammalian Genomes ◽  
Editing Enzyme ◽  
Target Activity

ABSTRACTBackgroundThe 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, wild-type 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 (e.g. mammalian) genomes. In addition to its off-targeting propensity, SpyCas9 is encoded by a relatively large (~4.2 kb) 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 (e.g. from Staphylococcus aureus, Campylobacter jejuni, Geobacillus stearothermophilus and Neisseria meningitidis) are considerably smaller and therefore better suited for viral delivery.ResultsHere we show that wild-type NmeCas9, when programmed with guide sequences of natural length (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.

scholarly journals Enhanced proofreading governs CRISPR-Cas9 targeting accuracy

2017 ◽  
Author(s):  
Janice S. Chen ◽  
Yavuz S. Dagdas ◽  
Benjamin P. Kleinstiver ◽  
Moira M. Welch ◽  
Lucas B. Harrington ◽  
...  
Keyword(s):  
Genome Editing ◽  
Single Molecule ◽  
Catalytic Domain ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Human Cells ◽  
High Fidelity ◽  
Cas9 Nuclease ◽  
Target Activity ◽  
Targeting Accuracy

The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing1-4. High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity were unknown5-9. Using single-molecule Förster resonance energy transfer (smFRET) experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state10 when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target mismatches and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we identified residues within REC3 involved in mismatch sensing and designed a new hyper-accurate Cas9 variant (HypaCas9) that retains robust on-target activity in human cells. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing.

scholarly journals Ultra-deep sequencing reveals no evidence of oncogenic mutations or enrichment by ex vivo CRISPR/Cas9 genome editing in human hematopoietic stem and progenitor cells

2021 ◽  
Author(s):  
M. Kyle Cromer ◽  
Valentin V. Barsan ◽  
Erich Jaeger ◽  
Mengchi Wang ◽  
Jessica P. Hampton ◽  
...  
Keyword(s):  
Genome Editing ◽  
Deep Sequencing ◽  
Limit Of Detection ◽  
Ex Vivo ◽  
High Fidelity ◽  
Seed Region ◽  
Hematopoietic Stem ◽  
Oncogenic Mutations ◽  
Ex Vivo Culture ◽  
Target Activity

As CRISPR-based therapies enter the clinic, evaluation of the safety remains a critical and still active area of study. While whole genome sequencing is an unbiased method for identifying somatic mutations introduced by ex vivo culture and genome editing, this methodology is unable to attain sufficient read depth to detect extremely low frequency events that could result in clonal expansion. As a solution, we utilized an exon capture panel to facilitate ultra-deep sequencing of >500 tumor suppressors and oncogenes most frequently altered in human cancer. We used this panel to investigate whether transient delivery of high-fidelity Cas9 protein targeted to three different loci (using guide RNAs (gRNAs) corresponding to sites at AAVS1, HBB, and ZFPM2) at day 4 and day 10 timepoints post-editing resulted in the introduction or enrichment of oncogenic mutations. In three separate primary human HSPC donors, we identified a mean of 1,488 variants per Cas9 treatment (at <0.07% limit of detection). After filtering to remove germline and/or synonymous changes, a mean of 3.3 variants remained per condition, which were further reduced to six total mutations after removing variants in unedited treatments. Of these, four variants resided at the predicted off-target site in the myelodysplasia-associated EZH2 gene that were subject to ZFPM2 gRNA targeting in Donors 2 and 3 at day 4 and day 10 timepoints. While Donor 1 displayed on-target cleavage at ZFPM2, we found no off-target activity at EZH2. Sanger sequencing revealed a homozygous single nucleotide polymorphism (SNP) at position 14bp distal from the Cas9 protospacer adjacent motif in EZH2 that eliminated any detectable off-target activity. We found no evidence of exonic off-target INDELs with either of the AAVS1 or HBB gRNAs. These findings indicate that clinically relevant delivery of high-fidelity Cas9 to primary HSPCs and ex vivo culture up to 10 days does not introduce or enrich for tumorigenic variants and that even a single SNP outside the seed region of the gRNA protospacer is sufficient to eliminate Cas9 off-target activity with this method of delivery into primary, repair competent human HSPCs.

Genome editing can now be carried out in an isogenic setting. It can be effectively transmitted to somatic tissues in mice, but not to humans. Despite these doubts, CRIS has great potential as a medical promise.

2021 ◽  
Author(s):  
Moataz Dowaidar
Keyword(s):  
Genome Editing ◽  
Viral Vectors ◽  
Cell Types ◽  
Polymorphic Ventricular Tachycardia ◽  
Cardiovascular Research ◽  
Cas9 Protein ◽  
Somatic Tissues ◽  
Somatic Gene ◽  
Delivery Mechanism ◽  
Editing Enzyme

As a result of genome editing, the field of cardiovascular research and treatmentis changing. Related variants can now be introduced into patient-derived cellsand tested in an isogenic environment. To evaluate the safety and efficacy ofgene-editing therapeutics, cellular phenotyping of genome-edited iPSC-derivedcardiomyocytes and other cell types will be invaluable. Parallel to theseadvances, viral vectors and nanoparticles can be used to efficiently edit genes inthe liver and core. Somatic gene editing has been used to treathypercholesterolemia, hypertriglyceridemia, WPW syndrome,catecholaminergic polymorphic ventricular tachycardia, and Duchennemuscular dystrophy in animal models. While these early achievements arepromising, they have also shown major challenges. Off-target editing withCRISPR/Cas systems will be influenced by the same editing enzyme,architecture, target cell type, and delivery mechanism. It is possible to transmitefficiently to somatic tissues in mice, but not all delivery pathways scale well tohumans. On-target unintentional editing operations, such as major deletions andinsertions, require further analysis and risk evaluation. Immunity, as well as thesubsequent development of an immune response to the Cas9 protein extractedfrom bacteria, will necessitate caution. Despite these reservations, CRISPR/Cashas tremendous therapeutic promise, and it is expected to enhance research andmedical treatment in the future.

scholarly journals CRISPR/Cas “non-target” sites inhibit on-target cutting rates

2020 ◽  
Author(s):  
Eirik A. Moreb ◽  
Mitchell Hutmacher ◽  
Michael D. Lynch
Keyword(s):  
Genome Editing ◽  
Target Site ◽  
High Fidelity ◽  
List Type ◽  
Dependent Manner ◽  
Guide Rna ◽  
Protospacer Adjacent Motif ◽  
Target Sites ◽  
Dose Dependent ◽  
Target Activity

AbstractCRISPR/Cas systems have become ubiquitous for genome editing in eukaryotic as well as bacterial systems. Cas9 associated with a guide RNA (gRNA) searches DNA for a matching sequence (target site) next to a protospacer adjacent motif (PAM) and once found, cuts the DNA. The number of PAM sites in the genome are effectively a non-target pool of inhibitory substrates, competing with the target site for the Cas9/gRNA complex. We demonstrate that increasing the number of non-target sites for a given gRNA reduces on-target activity in a dose dependent manner. Furthermore, we show that the use of Cas9 mutants with increased PAM specificity towards a smaller subset of PAMs (or smaller pool of competitive substrates) improves cutting rates. Decreasing the non-target pool by increasing PAM specificity provides a path towards improving on-target activity for slower high fidelity Cas9 variants. These results demonstrate the importance of competitive non-target sites on Cas9 activity and, in part, may help to explain sequence and context dependent activities of gRNAs. Engineering improved PAM specificity to reduce the competitive non-target pool offers an alternative strategy to engineer Cas9 variants with increased specificity and maintained on-target activity.HighlightsThe pool of non-target PAM sites inhibit Cas9/gRNA on-target activitynon-target PAM inhibition is dose dependentnon-target PAM inhibition is a function of gRNA sequencenon-target PAM inhibition is a function of Cas9 levels

scholarly journals Prime editing enables precise genome editing in mouse liver and retina

2021 ◽  
Author(s):  
Hyewon Jang ◽  
Jeong Hong Shin ◽  
Dong Hyun Jo ◽  
Jung Hwa Seo ◽  
Goosang Yu ◽  
...  
Keyword(s):  
Genome Editing ◽  
Pigment Epithelium ◽  
Double Strand Breaks ◽  
Wild Type ◽  
Adeno Associated Virus ◽  
Strand Breaks ◽  
Trans Splicing ◽  
Low Levels ◽  
Hereditary Tyrosinemia ◽  
Target Effects

Prime editing can induce any small-sized genetic change without donor DNA or double strand breaks. However, it has not been investigated whether prime editing is possible in postnatal animals. Here we delivered prime editors 2 and 3 into a mouse model of hereditary tyrosinemia, a genetic liver disease, using hydrodynamic injection, which corrected the disease-causing mutation and rescued the phenotype. We also achieved prime editing in the retina and retina pigment epithelium in wild-type mice by delivering prime editor 3 using trans-splicing adeno-associated virus. Deep sequencing showed that unintended edits at or near the target site or off-target effects were not detectable except for low levels (0% to 1.2%) of indels when PE3, but not PE2, was used. Our study suggests that precise, prime editor-mediated genome editing is possible in somatic cells of adult animals.

scholarly journals Discovery and engineering of small SlugCas9 with broad targeting range and high specificity and activity

2020 ◽  
Author(s):  
Ziying Hu ◽  
Chengdong Zhang ◽  
Shuai Wang ◽  
Jingjing Wei ◽  
Miaomiao Li ◽  
...  
Keyword(s):  
High Activity ◽  
Genome Editing ◽  
Basic Research ◽  
High Specificity ◽  
Clinical Applications ◽  
Mammalian Genome ◽  
High Fidelity ◽  
Staphylococcus Lugdunensis ◽  
Adeno Associated Virus ◽  
Comparable Activity

AbstractThe compact CRISPR/Cas9 system, which can be delivered by adeno-associated virus (AAV), is a promising platform for therapeutic applications. However, current compact Cas9 nucleases have limited activity, targeting scope and specificity. Here, we identified three compact SaCas9 orthologs, Staphylococcus lugdunensis Cas9 (SlugCas9), Staphylococcus lutrae Cas9 (SlutrCas9) and Staphylococcus haemolyticus Cas9 (ShaCas9), for mammalian genome editing. Interestingly, SlugCas9 recognizes a simple NNGG PAM and displays comparable activity to SaCas9. We further generated a SlugCas9-SaCas9 chimeric nuclease, which has both high specificity and high activity. We lastly engineered SlugCas9 with mutations to generate a high fidelity variant that maintains high specificity without compromising on-target editing efficiency. Our study offers important minimal Cas9 tools that are ideal for both basic research and clinical applications.

scholarly journals Improving the specificity of adenine base editor using high-fidelity Cas9

2019 ◽  
Author(s):  
Ruisha Hong ◽  
Sidi Ma ◽  
Feng Wang
Keyword(s):  
Gene Therapy ◽  
Streptococcus Pyogenes ◽  
Genomic Dna ◽  
High Fidelity ◽  
Wild Type ◽  
Sequencing Data ◽  
Deep Sequencing Data ◽  
Target Sites ◽  
Target Activity ◽  
Adenine Base

ABSTRACTAdenine base editor (ABE) mediates the conversion of A to G in genomic DNA. In human, approximately 47.8% of known pathogenic SNPs can be corrected by A to G conversion, indicating that ABE have tremendous potential in gene therapy. However, the off-target activity of ABE limits its clinical application. ABE off-target activity in DNA is depended on the bonding of Streptococcus pyogenes Cas9 (SpCas9) on off-target sites [1, 2]. Therefore, using high-fidelity Cas9 should be able to improve the specificity of ABE. Based on this, we replaced the wild-type SpCas9 in ABE7.10 with four high-fidelity Cas9s to improve its specificity. The analysis of target deep sequencing data demonstrate that the specificity of e-ABE is substantially improved compared to conventional ABE7.10 in four test sites. But the broad editing window of ABE hampers its application, ABE needs to be optimized to get variants with narrow editing window.

scholarly journals Engineered Campylobacter jejuni Cas9 variant with enhanced activity

2021 ◽  
Author(s):  
Ryoya Nakagawa ◽  
Soh Ishiguro ◽  
Sae Okazaki ◽  
Hideto Mori ◽  
Mamoru Tanaka ◽  
...  
Keyword(s):  
Genome Editing ◽  
Campylobacter Jejuni ◽  
Genome Engineering ◽  
Cytosine Deaminase ◽  
Human Cells ◽  
Adeno Associated Virus ◽  
Cleavage Activity ◽  
Protospacer Adjacent Motif ◽  
Its Gene

Abstract The RNA-guided DNA endonuclease Cas9 is a versatile genome-editing tool. However, the molecular weight of the commonly used Streptococcus pyogenes Cas9 is relatively large. Consequently, its gene cannot be efficiently packaged into an adeno-associated virus vector, thereby limiting its applications for therapeutic genome editing. Here, we biochemically characterized the compact Cas9 from Campylobacter jejuni (CjCas9) and found that CjCas9 has a previously unrecognized preference for the N3VRYAC protospacer adjacent motif. We thus rationally engineered a CjCas9 variant (enCjCas9), which exhibits enhanced cleavage activity and a broader targeting range both in vitro and in human cells, as compared with CjCas9. Furthermore, a nickase version of enCjCas9, but not CjCas9, fused with a cytosine deaminase mediated C-to-T conversions in human cells. Overall, our findings expand the CRISPR-Cas toolbox for therapeutic genome engineering.

scholarly journals Rationally engineered Staphylococcus aureus Cas9 nucleases with high genome-wide specificity

2019 ◽  
Vol 116 (42) ◽  
pp. 20969-20976 ◽  
Author(s):  
Yuanyan Tan ◽  
Athena H. Y. Chu ◽  
Siyu Bao ◽  
Duc Anh Hoang ◽  
Firaol Tamiru Kebede ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Genome Editing ◽  
Wild Type ◽  
Adeno Associated Virus ◽  
Genome Wide ◽  
Protospacer Adjacent Motif ◽  
Targeted Deep Sequencing ◽  
Targeting Accuracy ◽  
Target Effects

RNA-guided CRISPR-Cas9 proteins have been widely used for genome editing, but their off-target activities limit broad application. The minimal Cas9 ortholog from Staphylococcus aureus (SaCas9) is commonly used for in vivo genome editing; however, no variant conferring high genome-wide specificity is available. Here, we report rationally engineered SaCas9 variants with highly specific genome-wide activity in human cells without compromising on-target efficiency. One engineered variant, referred to as SaCas9-HF, dramatically improved genome-wide targeting accuracy based on the genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) method and targeted deep sequencing analyses. Among 15 tested human endogenous sites with the canonical NNGRRT protospacer adjacent motif (PAM), SaCas9-HF rendered no detectable off-target activities at 9 sites, minimal off-target activities at 6 sites, and comparable on-target efficiencies to those of wild-type SaCas9. Furthermore, among 4 known promiscuous targeting sites, SaCas9-HF profoundly reduced off-target activities compared with wild type. When delivered by an adeno-associated virus vector, SaCas9-HF also showed reduced off-target effects when targeting VEGFA in a human retinal pigmented epithelium cell line compared with wild type. Then, we further altered a previously described variant named KKH-SaCas9 that has a wider PAM recognition range. Similarly, the resulting KKH-HF remarkably reduced off-target activities and increased on- to off-target editing ratios. Our finding provides an alternative to wild-type SaCas9 for genome editing applications requiring exceptional genome-wide precision.

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