Powerful Gene Editing Tool Research Articles

CRISPR/Cas System in Human Genetic Diseases

Gene editing involves altering a particular sequence within an organism’s genome using gene-editing techniques. Efficiently and accurately insert, delete or replace genes to alter their genetic information and phenotypic characteristics. DNA nuclease-based gene editing technology has advanced rapidly, from the first-generation editing system ZFNs, the second-generation TALENs to the third-generation CRISPR/Cas9 system, the efficiency of gene editing has been continuously improved, the cost has been gradually reduced, and the application scope has been expanding. The new classification system categorizes CRISPR-Cas proteins into Class I and II as the two main classes. Class I encompasses several subtypes, including Type I, III, and IV, all form complexes through the coordinated function of multiple Cas proteins. While Class II includes Type II, Type V, and Type VI, which all rely on single large Cas protein to perform all response. By designing single guide RNA (sgRNA), CRISPR can be used to target any gene sequence intended to be edited, achieving the wanted therapeutic outcome in treatment of inherited genetic disease. Although potential for off-target leads to undesired genetic alterations even result in oncogenesis, CRISPR-Cas still works as powerful therapy and gene editing tool in all aspects. Future research requires us to solve off-target issue. This review systematically introduces CRISPR-Cas systems as promising therapeutic strategy towards various genetic disease, explaining molecular mechanism and classification of CRISPR and differences among them.

Progress on CRISPR-Cas gene editing technology in sheep production.

Gene editing is a kind of genetic engineering technology that can modify the genome. In recent years, with the rapid development of molecular biotechnology, the clustered regularly interspaced short palindromic repeats associated protein system has been widely used as a powerful gene editing tool due to its high efficiency, accuracy and flexibility. The CRISPR-Cas system makes a significant contribution to different aspects of livestock production by introducing site-specific modifications such as insertions, deletions or single base replacements at specific genomic sites. In terms of sheep production applications, by establishing animal models that improve production economic traits and disease resistance, the function of key genes can be studied to accelerate the improvement of traits, thereby accelerating the improvement of traits. In this review, we summarize the mechanism and function of CRISPR-Cas system and its application in the production of reproductive traits, meat use traits, wool production traits, lactation traits and disease resistance traits of sheep and the establishment of sheep animal models.

CRISPR-CAS System: Current Applications and Future Prospect

The CRISPR-Cas system is a revolutionary technology that has transformed gene editing and has the potential to revolutionize many fields. It is a bacterial immune system that can be programmed to recognize and cleave specific DNA sequences using Cas proteins and guide RNAs. This technology has numerous biotechnological and bioengineering applications and is simpler to design with high targeting efficiency and multiplex editing capability compared to older gene editing technologies. This review aims to explore the general application, impact on molecular biology and medicine, limitations, and future potential of the CRISPR-Cas system. With its potential to revolutionize medicine, agriculture, and environmental science, the CRISPR-Cas system is a powerful gene editing tool that offers hope for a wide range of applications.

PAM‐Engineered Toehold Switches as Input‐Responsive Activators of CRISPR‐Cas12a for Sensing Applications

AbstractThe RNA‐programmed CRISPR effector protein Cas12a has emerged as a powerful tool for gene editing and molecular diagnostics. However, additional bio‐engineering strategies are required to achieve control over Cas12a activity. Here, we show that Toehold Switch DNA hairpins, presenting a rationally designed locked protospacer adjacent motif (PAM) in the loop, can be used to control Cas12a in response to molecular inputs. Reconfiguring the Toehold Switch DNA from a hairpin to a duplex conformation through a strand displacement reaction provides an effective means to modulate the accessibility of the PAM, thereby controlling the binding and cleavage activities of Cas12a. Through this approach, we showcase the potential to trigger downstream Cas12a activity by leveraging proximity‐based strand displacement reactions in response to target binding. By utilizing the trans‐cleavage activity of Cas12a as a signal transduction method, we demonstrate the versatility of our approach for sensing applications. Our system enables rapid, one‐pot detection of IgG antibodies and small molecules with high sensitivity and specificity even within complex matrices. Besides the bioanalytical applications, the switchable PAM‐engineered Toehold Switches serve as programmable tools capable of regulating Cas12a‐based targeting and DNA processing in response to molecular inputs and hold promise for a wide array of biotechnological applications.

PAM-Engineered Toehold Switches as Input-Responsive Activators of CRISPR-Cas12a for Sensing Applications.

The RNA-programmed CRISPR effector protein Cas12a has emerged as a powerful tool for gene editing and molecular diagnostics. However, additional bio-engineering strategies are required to achieve control over Cas12a activity. Here, we show that Toehold Switch DNA hairpins, presenting a rationally designed locked protospacer adjacent motif (PAM) in the loop, can be used to control Cas12a in response to molecular inputs. Reconfiguring the Toehold Switch DNA from a hairpin to a duplex conformation through a strand displacement reaction provides an effective means to modulate the accessibility of the PAM, thereby controlling the binding and cleavage activities of Cas12a. Through this approach, we showcase the potential to trigger downstream Cas12a activity by leveraging proximity-based strand displacement reactions in response to target binding. By utilizing the trans-cleavage activity of Cas12a as a signal transduction method, we demonstrate the versatility of our approach for sensing applications. Our system enables rapid, one-pot detection of IgG antibodies and small molecules with high sensitivity and specificity even within complex matrices. Besides the bioanalytical applications, the switchable PAM-engineered Toehold Switches serve as programmable tools capable of regulating Cas12a-based targeting and DNA processing in response to molecular inputs and hold promise for a wide array of biotechnological applications.

Challenges with Newly Approved CRISPR Gene Technologies

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology is a powerful gene-editing tool that allows for precise modification of DNA. While CRISPR has shown immense potential in laboratory and preclinical studies, the development of CRISPR-based therapies for human use is still in the early stages. However, on December 08, the Food and Drug Administration (FDA) of the United States of America approved two milestone treatments, Casgevy and Lyfgenia, representing the first cell-based gene therapies for the treatment of sickle cell disease (SCD) in patients 12 years and older. Several CRISPR-Cas9 therapies that work on the same principle are in clinical trials as treatments for a range of diseases. However, there are still significant concerns with the use of this new technology. One major concern with CRISPR technology is the potential for off-target effects, where unintended changes may occur in locations other than the target gene. The introduction of CRISPR components into the body may also trigger an immune response. Several laboratories are investigating ways to mitigate immune reactions and improve the delivery of CRISPR components. The method of delivering CRISPR components into cells is also important for safety. Further research into different delivery methods, such as viral vectors or nanoparticles, would help optimize efficiency while minimizing potential side effects. The long-term effects of CRISPR-based interventions are an area of ongoing research. Understanding the stability of CRISPR-induced changes over time and potential consequences is crucial for assessing safety. Ethical considerations are important to discussions about CRISPR use, particularly when it comes to editing the human germline (sperm, eggs, embryos), as changes made in these cells would be heritable. The potential for unintended consequences and ethical implications necessitates careful consideration. It is important to consider that although CRISPR holds great promise, the technology is relatively young, and research is ongoing to address safety concerns. The scientific community and regulatory bodies are actively working to ensure that CRISPR technologies are developed and implemented responsibly. Continuous advancements, ongoing research, and open communication will be essential to addressing safety challenges associated with CRISPR.

Cas9 is mostly orthogonal to human systems of DNA break sensing and repair.

CRISPR/Cas9 system is а powerful gene editing tool based on the RNA-guided cleavage of target DNA. The Cas9 activity can be modulated by proteins involved in DNA damage signalling and repair due to their interaction with double- and single-strand breaks (DSB and SSB, respectively) generated by wild-type Cas9 or Cas9 nickases. Here we address the interplay between Streptococcus pyogenes Cas9 and key DNA repair factors, including poly(ADP-ribose) polymerase 1 (SSB/DSB sensor), its closest homolog poly(ADP-ribose) polymerase 2, Ku antigen (DSB sensor), DNA ligase I (SSB sensor), replication protein A (DNA duplex destabilizer), and Y-box binding protein 1 (RNA/DNA binding protein). None of those significantly affected Cas9 activity, while Cas9 efficiently shielded DSBs and SSBs from their sensors. Poly(ADP-ribosyl)ation of Cas9 detected for poly(ADP-ribose) polymerase 2 had no apparent effect on the activity. In cellulo, Cas9-dependent gene editing was independent of poly(ADP-ribose) polymerase 1. Thus, Cas9 can be regarded as an enzyme mostly orthogonal to the natural regulation of human systems of DNA break sensing and repair.

Lipid Nanoparticle-Mediated Gene Editing of Human Primary T Cells and Off-Target Analysis of the CRISPR-Cas9 Indels

Background and Aims: The CRISPR/Cas9 system has emerged as a powerful tool for gene editing of primary cells. In our previous work, we demonstrated a novel lipid nanoparticle (LNP) reagent for the multi-step engineering of gene-edited CAR T cells. We showed high cell viabilities and potent CAR-T mediated killing, even after multiple genetic manipulations. Here, we extend this work by assessing potential off-target editing effects in both LNP-treated T cells and T-cells where the CRISPR reagents were delivered by electroporation. Further, we evaluated multiple Cas9 variants and guide RNA targets. Methods: LNPs encapsulating wild type or high fidelity S.p. Cas9 mRNA and various TRAC and CD52 targeted guide RNAs (sgRNAs) were produced using our scalable NanoAssemblrTM microfluidics platform. Concurrently, electroporation was performed to deliver equivalent cargoes. Purified primary T cells were cultured, activated, and expanded in serum-free media in plates, flasks, or small bioreactors. The LNPs were added to cells by direct addition for gene editing. Gene expression and cell viability were measured using flow cytometry or colorimetric assays. Multi-target performance of CRISPR-Cas9 editing was evaluated through rhAmpSeq-based targeted next-generation sequencing (NGS) and indels analyzed for with CRISPRAltRations. Results: TCR or CD52 targeted Cas9 mRNA-LNP addition or electroporation yielded high single and double knockout efficiencies. High-throughput NGS analysis showed strong agreeance to flow cytometry for on-target analysis. We tested a range of sgRNA targets, wild-type and high-fidelity Cas9 mRNAs, and determined off-target editing for all targets and variants investigated. Similar results were obtained when comparing different LNP batch sizes (micro to milligram RNA) and cell culture vessels (0.1 to 45 million cells), demonstrating scalability of both LNP production and cell treatment. Conclusions: The results from this study further support the utility RNA-LNPs for the genetic engineering of primary T cells. The simple and gentle nature of LNP cell treatment allows for multiple genetic engineering steps for simultaneous expression and deletion of proteins for the next cell therapies. These LNPs can be easily manufactured from small-scale screening of RNA libraries to rapid scale-up for clinical translation.

High-efficiency targeted transgene integration via primed micro-homologues

Due to the difficulties in precisely manipulating DNA repair pathways, high-fidelity targeted integration of large transgenes triggered by double-strand breaks is inherently inefficient. Here, we exploit prime editors to devise a robust knock-in (KI) strategy named primed micro-homologues-assisted integration (PAINT), which utilizes reverse-transcribed single-stranded micro-homologues to boost targeted KIs in different types of cells. The improved version of PAINT, designated PAINT 3.0, maximizes editing efficiency and minimizes off-target integration, especially in dealing with scarless in-frame KIs. Using PAINT 3.0, we target a reporter transgene into housekeeping genes with editing efficiencies up to 80%, more than 10-fold higher than the traditional homology-directed repair method. Moreover, the use of PAINT 3.0 to insert a 2.5-kb transgene achieves up to 85% KI frequency at several therapeutically relevant genomic loci, suggesting its potential for clinical applications. Finally, PAINT 3.0 enables high-efficiency non-viral genome targeting in primary T cells and produces functional CAR-T cells with specific tumor-killing ability. Thus, we establish that the PAINT method is a powerful gene editing tool for large transgene integrations and may open new avenues for cell and gene therapies and genome writing technologies.

Generation of Centromere-Associated Protein-E <em>CENP-E<sup>-/-</sup></em> Knockout Cell Lines using the CRISPR/Cas9 System

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has emerged as a powerful tool for precise and efficient gene editing in a variety of organisms. Centromere-associated protein-E (CENP-E) is a plus-end-directed kinesin required for kinetochore-microtubule capture, chromosome alignment, and spindle assembly checkpoint. Although cellular functions of the CENP-E proteins have been well studied, it has been difficult to study the direct functions of CENP-E proteins using traditional protocols because CENP-E ablation usually leads to spindle assembly checkpoint activation, cell cycle arrest, and cell death. In this study, we have completely knocked out the CENP-E gene in human HeLa cells and successfully generated the CENP-E-/- HeLa cells using the CRISPR/Cas9 system. Three optimized phenotype-based screening strategies were established, including cell colony screening, chromosome alignment phenotypes, and the fluorescent intensities of CENP-E proteins, which effectively improve the screening efficiency and experimental success rate of the CENP-E knockout cells. Importantly, CENP-E deletion results in chromosome misalignment, the abnormal location of the BUB1 mitotic checkpoint serine/threonine kinase B (BubR1) proteins, and mitotic defects. Furthermore, we have utilized the CENP-E knockout HeLa cell model to develop an identification method for CENP-E-specific inhibitors. In this study, a useful approach to validate the specificity and toxicity of CENP-E inhibitors has been established. Moreover, this paper presents the protocols of CENP-E gene editing using the CRISPR/Cas9 system, which could be a powerful tool to investigate the mechanisms of CENP-E in cell division. Moreover, the CENP-E knockout cell line would contribute to the discovery and validation of CENP-E inhibitors, which have important implications for antitumor drug development, studies of cell division mechanisms in cell biology, and clinical applications.

Efficient CRISPR/Cas9-mediated gene editing in mammalian cells by the novel selectable traffic light reporters

CRISPR/Cas9 is a powerful tool for gene editing in various cell types and organisms. However, it is still challenging to screen genetically modified cells from an excess of unmodified cells. Our previous studies demonstrated that surrogate reporters can be used for efficient screening of genetically modified cells. Here, we developed two novel traffic light screening reporters, puromycin-mCherry-EGFP (PMG) based on single-strand annealing (SSA) and homology-directed repair (HDR), respectively, to measure the nuclease cleavage activity within transfected cells and to select genetically modified cells. We found that the two reporters could be self-repaired coupling the genome editing events driven by different CRISPR/Cas nucleases, resulting in a functional puromycin-resistance and EGFP selection cassette that can be afforded to screen genetically modified cells by puromycin selection or FACS enrichment. We further compared the novel reporters with different traditional reporters at several endogenous loci in different cell lines, for the enrichment efficiencies of genetically modified cells. The results indicated that the SSA-PMG reporter exhibited improvements in enriching gene knockout cells, while the HDR-PMG system was very useful in enriching knock-in cells. These results provide robust and efficient surrogate reporters for the enrichment of CRISPR/Cas9-mediated editing in mammalian cells, thereby advancing basic and applied research.

Archaeal and bacteriophage derived Cas proteins demonstrate memory T cell response in humans

Abstract The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas system is a powerful gene editing tool with clinical applications in the pipeline. In vivodelivery of Cas proteins could induce immune responses which would limit the utility of the technology. Previously, we and others have demonstrated pre-existing immunity to Cas9 proteins derived from Staphylococcus aureus(Sa) and Streptococcus pyogenes(Sp) in human populations. As these Cas proteins are of microbial origin this finding was not surprising. There are numerous naturally occurring alternatives to Cas9 and many of these are being leveraged for gene editing. Alternatives to Cas9 that have successfully been used in gene editing technologies include Cas12a (derived from Acidaminococcus sp), Cas14a (derived from DPANN, a super-phylum of archaea) and Casϕ (derived from a huge bacteriophage). It has been postulated that humans would not have pre-existing immunity to some of these Cas proteins. In the comparative study described here, we evaluated pre-existing antibodies and memory T-cell responses to Cas9, Cas12a, Cas14a and Casϕ from the same 18 healthy donors for all proteins. Pre-existing antibodies were identified using ELISA and memory T-cell responses were measured using the ELISpot assay. All four Cas proteins tested positive for both assays in at least some donors. It has been speculated that pre-existing immunity to Cas9 proteins is due to human exposure to the pathogenic bacteria they are derived from. Consequently, Cas14a and Casϕ should not be associated with pre-existing immunity. However, our results show that anti-Cas14a and anti-Casϕ antibodies were detected in ~50% of the study population and these donors also showed memory T-cell responses.

CRISPR/Cas9-Mediated Gene Knockout in Cells and Tissues Using Lentivirus.

CRISPR-Cas9 has become a powerful and popular gene editing tool. However, successful application of this tool in the lab can still be quite daunting to many newcomers to molecular biology, mostly because it is a relatively lengthy process involving multiple steps with variations of each step. Here, we provide a reliable, stepwise, and newcomer-friendly protocol to knock out a target gene in wild-type human fibroblasts. This protocol involves sgRNA design using CRISPOR, construction of an "all-in-one" vector expressing both sgRNA and Cas9 using Golden Gate cloning, streamlined production of high-titer lentiviruses in 1 week after molecular cloning, and transduction of cells to generate a knockout cell pool. We further introduce a protocol for lentiviral transduction of ex vivo mouse embryonic salivary epithelial explants. In summary, our protocol is useful for new researchers to apply CRISPR-Cas9 to generate stable gene knockout cells and tissue explants using lentivirus. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: sgRNA design Basic Protocol 2: Cloning sgRNA in plasmid vector containing Cas9 encoding sequence using golden gate cloning Basic Protocol 3: Lentivirus packaging Basic Protocol 4: Lentivirus transduction of cells Basic Protocol 5: Lentivirus transduction of salivary gland epithelial buds.

Advances in the RNA-targeting CRISPR-Cas systems

The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated proteins) system is an adaptive immune system of bacteria and archaea against phages, plasmids and other exogenous genetic materials. The system uses a special RNA (CRISPR RNA, crRNA) guided endonuclease to cut the exogenous genetic materials complementary to crRNA, thus blocking the infection of exogenous nucleic acid. According to the composition of the effector complex, CRISPR-Cas system can be divided into two categories: class 1 (including type Ⅰ, Ⅳ, and Ⅲ) and class 2 (including type Ⅱ, Ⅴ, and Ⅵ). Several CRISPR-Cas systems have been found to have very strong ability to specifically target RNA editing, such as type Ⅵ CRISPR-Cas13 system and type Ⅲ CRISPR-Cas7-11 system. Recently, several systems have been widely used in the field of RNA editing, making them a powerful tool for gene editing. Understanding the composition, structure, molecular mechanism and potential application of RNA-targeting CRISPR-Cas systems will facilitate the mechanistic research of this system and provide new ideas for developing gene editing tools.

CRISPR/Cas9-mediated knock-in in ebony gene using a PCR product donor template in Drosophila

CRISPR/Cas9 technology has been a powerful tool for gene editing in Drosophila, particularly for knocking in base-pair mutations or a variety of gene cassettes into endogenous gene loci. Among the Drosophila community, there has been a concerted effort to establish CRISPR/Cas9-mediated knock-in protocols that decrease the amount of time spent on molecular cloning. Here, we report the CRISPR/Cas9-mediated insertion of a ∼50 base-pair sequence into the ebony gene locus, using a linear double-stranded DNA (PCR product) donor template. By circumventing the cloning step of the donor template, our approach suggests the PCR product as a useful, alternative knock-in donor format.

Edición del genoma: CRISPR

CRISPR has gained a lot of attention recently as a result of a debate among scientists about the possibility of genetically modifying the human germ line and the ethical implications of doing so. However, CRISPR is not just a method for editing the genomes of embryonic cells, as the public discussion might have implied; is a powerful, efficient, and reliable tool for gene editing in any organism, and has attracted significant attention and use among biologists for a variety of purposes.

RNA-Mediated Nuclease Genome Editing System Type II: A Mini Review

The CRISPR /CAS system is a modern genome editing tool derived from a bacterium as it functions as an active immune system to prevent the bacteria from the viruses and plasmids. In the earlier years before the discovery of the CRISPR system, there were many other tools to perform genome editing but due to their low specificity and reliability, they were not considered an efficient tool. The discovery of CRISPR/CAS9 system overcomes these limitations and considered as a highly specific and efficient technique in the field of genome editing or DNA alteration. The purpose of studying the CRISPR/CAS system is to develop a powerful gene-editing tool that can make every possible gene editing and also to prevent the genomic defect in a certain group of organisms. The CRISPR/CAS9 system uses an RNA molecule which is a main functional part of the CRISPR through which a bacterial cell can recognize and cleave/destroy the foreign viral elements that enter the bacterial cell using a certain group of restriction nuclease enzymes isolated from different bacteria. Several repairing mechanisms in the cell have been used to ligate the degraded viral sequences as these repair mechanisms are error-prone and generate frame shift mutations in the sequence. As a result, the foreign viral components and their expressions were reduced or deleted. The purpose of this review is to understand the mechanism of bacterial CRISPR/CAS9 system and also to know the use of this technique to prevent single genomic defects. In this review discuss the role of the CRISPR/CAS9 system as an active immune system of bacterial cells, classifications, and the CRISPR/CAS9 system (type II) use in the genome-editing mechanism.

New Therapeutics for Extracellular Vesicles: Delivering CRISPR for Cancer Treatment

Cancers are defined by genetic defects, which underlines the prospect of using gene therapy in patient care. During the past decade, CRISPR technology has rapidly evolved into a powerful gene editing tool with high fidelity and precision. However, one of the impediments slowing down the clinical translation of CRISPR-based gene therapy concerns the lack of ideal delivery vectors. Extracellular vesicles (EVs) are nano-sized membrane sacs naturally released from nearly all types of cells. Although EVs are secreted for bio-information conveyance among cells or tissues, they have been recognized as superior vectors for drug or gene delivery. Recently, emerging evidence has spotlighted EVs in CRISPR delivery towards cancer treatment. In this review, we briefly introduce the biology and function of the CRISPR system and follow this with a summary of current delivery methods for CRISPR applications. We emphasize the recent progress in EV-mediated CRISPR editing for various cancer types and target genes. The reported strategies for constructing EV-CRISPR vectors, as well as their limitations, are discussed in detail. The review aims to throw light on the clinical potential of engineered EVs and encourage the expansion of our available toolkit to defeat cancer.

Nonconventional Yeasts Engineered Using the CRISPR-Cas System as Emerging Microbial Cell Factories

Because the petroleum-based chemical synthesis of industrial products causes serious environmental and societal issues, biotechnological production using microorganisms is an alternative approach to achieve a more sustainable economy. In particular, the yeast Saccharomyces cerevisiae is widely used as a microbial cell factory to produce biofuels and valuable biomaterials. However, product profiles are often restricted due to the Crabtree-positive nature of S. cerevisiae, and ethanol production from lignocellulose is possibly enhanced by developing alternative stress-resistant microbial platforms. With desirable metabolic pathways and regulation in addition to strong resistance to diverse stress factors, nonconventional yeasts (NCY) may be considered an alternative microbial platform for industrial uses. Irrespective of their high industrial value, the lack of genetic information and useful gene editing tools makes it challenging to develop metabolic engineering-guided scaled-up applications using yeasts. The recently developed clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system is a powerful gene editing tool for NCYs. This review describes the current status of and recent advances in promising NCYs in terms of industrial and biotechnological applications, highlighting CRISPR-Cas9 system-based metabolic engineering strategies. This will serve as a basis for the development of novel yeast applications.

Plasmid-mediated gene transfer of Cas9 induces vector-related but not SpCas9-related immune responses in human retinal pigment epithelial cells

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) system represents a powerful gene-editing tool and could enable treatment of blinding diseases of the retina. As a peptide of bacterial origin, we investigated the immunogenic potential of Cas9 in models of retinal immunocompetent cells: human microglia (IMhu) and ARPE-19 cells. Transfection with Streptococcus pyogenes-Cas9 expression plasmids (SpCas9 plasmid) induced Cas9 protein expression in both cell lines. However, only ARPE-19 cells, not IMhu cells, responded with pro-inflammatory immune responses as evidenced by the upregulation of IL-8, IL-6, and the cellular activation markers HLA-ABC and CD54 (ICAM). These pro-inflammatory responses were also induced through transfection with equally sized non-coding control plasmids. Moreover, viability rates of ARPE-19 cells were reduced after transfection with both the SpCas9 plasmids and the control plasmids. Although these results demonstrate cell type-specific responses to the DNA plasmid vector, they show no evidence of an immunogenic effect due to the presence of Cas9 in models of human retinal pigment epithelial and microglia cells. These findings add another layer of confidence in the immunological safety of potential future Cas9-mediated retinal gene therapies.