Cas9 Protein Research Articles

The morphogen Hedgehog is essential for proper adult morphogenesis in Bombyx mori

The well-known morphogen Hedgehog (Hh) is indispensable for embryo patterning and organ development from invertebrates to vertebrates. The role of Hh signaling pathway has been extensively investigated in the model organism Drosophila melanogaster, whereas its biological functions are still poorly understood in non-drosophilid insects. In the current study, we describe comprehensive investigation of Hh biological roles in the model lepidopteran insect Bombyx mori by using both CRISPR/Cas9-mediated gene ablation and Gal4/UAS-mediated ectopic expression. Direct injection of Cas9 protein and Hh-specific sgRNAs into preblastoderm embryos induced complete lethality. In contrast, Hh mutants obtained by the binary transgenic CRISPR/Cas9 system showed no deleterious phenotypes during embryonic and larval stages. However, mutants showed abnormalities from the pupal stage and most of adult body appendages exhibited severe developmental defects. Molecular analysis focused on wing development reveal that Hh signaling, Imd signaling and Wnt signaling pathways were distorted in Hh mutant wings. Ectopic expression by using the binary Gal4/UAS system induce early larval lethality. On contrary, moderate overexpression of Hh by using a unitary transgenic system resulted in severe defects in adult leg and antenna development. Our data directly provide genetic evidence that Hh plays vital roles in imaginal discs development and proper adult morphogenesis in B. mori.

Single nucleotide polymorphism in the genomic target affects the recombination efficiency of CRISPR/Cas9-mediated gene editing in zebrafish

The CRISPR/Cas9 gene editing tool is becoming increasingly popular in the scientific community as one of the most efficient gene editing tools because of its high specificity and precision in targeted mutagenesis. A typical CRISPR system includes two components: a single guide RNA (sgRNA or gRNA) and a CRISPR associated endonuclease (Cas9). In CRISPR, the specificity of the Cas9 protein in cleaving the target DNA is decided by the homology of the spacer sequence, particularly the seed sequence (8–10 bases at the 3′ end of the spacer sequence) and any mismatch in the seed sequence abolishes the target cleavage and the subsequent generation of indels. Here, using Poly (A)specific ribonuclease (PARN) as an example in zebrafish, we show that the presence of single nucleotide polymorphism (SNP) in the sgRNA target, particularly in the seed sequence, affects the Cas9 activity resulting in a reduced recombination efficiency of CRISPR. Thus, while selecting target sites, prior screening for SNPs in the chosen targets across different wild-type lines/strains could be one of the easiest and effective ways of avoiding reduced recombination efficiency of CRISPR-mediated gene editing in zebrafish.

Deficiency of Adipose Triglyceride Lipase Induces Metabolic Syndrome and Cardiomyopathy in Zebrafish

Lipid metabolism dysfunction is related to clinical disorders including obesity, cancer, liver steatosis, and cardiomyopathy. Impaired lipolytic enzymes result in altered release of free fatty acids. The dramatic change in dyslipidemia is important in lipotoxic cardiomyopathy. Adipose triglyceride lipase (ATGL) catalyzes the lipolysis of triacylglycerol to reduce intramyocardial triglyceride levels in the heart and improve myocardial function. We examined the role of ATGL in metabolic cardiomyopathy by developing an Atgl knockout (ALKO) zebrafish model of metabolic cardiomyopathy disease by continuously expressing CRISPR/Cas9 protein and atgl gene guide RNAs (gRNAs). The expressed Cas9 protein bound to four gRNAs targeting the atgl gene locus, facilitating systemic gene KO. Ablation of Atgl interfered with lipid metabolism, which induced hyperlipidemia and hyperglycemia. ALKO adults and embryos displayed hypertrophic hearts. ALKO presented a typical dilated cardiomyopathy profile with a remarkable reduction in four sarcomere genes (myosin heavy chain 7-like, actin alpha cardiac muscle 1b, myosin binding protein C3, and troponin T type 2a) and two Ca2+ handling regulator genes (tropomyosin 4b and ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2b). Immune cell infiltration in cardiac tissue of ALKO provided direct evidence of advanced metabolic cardiomyopathy. The presently described model could become a powerful tool to clarify the underlying mechanism between metabolic disorders and cardiomyopathies.

CaExTun: Mitigating Cas9-Related Toxicity in Streptomyces through Species-Specific Expression Tuning with Randomized Constitutive Promoters.

The CRISPR/Cas9 system provides an efficient tool for engineering genomes. However, its application to Streptomyces genome engineering has been hampered by excessive toxicity associated with overexpression of Cas9 protein. As the level of Cas9 toxicity varies significantly between Streptomyces species, species-specific optimization of Cas9 expression is a strategy to mitigate its toxicity while maintaining sufficient double-strand break (DSB) activity for genome engineering. Using a pool of randomized constitutive promoters and a blue pigment indigoidine biosynthetic gene (IndC) as a reporter, we developed the CaExTun (Cas9 Expression Tuning) platform, which enables rapid screening of a large pool of promoter-Cas9 constructs to quickly recover the one with high DSB activity and no apparent toxicity. We demonstrate the utility of CaExTun using four model Streptomyces species. We also show that CaExTun can be applied to the CRISPRi system by allowing the construction of a library of promoter-dCas9 constructs that confer a wide range of gene repression levels. As demonstrated here, CaExTun is a versatile tool for the rapid optimization of the CRISPR/Cas9 system in a species-specific manner and thus will facilitate CRISPR/Cas9-based genome engineering efforts in Streptomyces.

Novel configurations of type I-E CRISPR-Cas system in Corynebacterium striatum clinical isolates

Clustered regularly interspaced short palindromic repeats (CRISPR) are a prokaryotic adaptive immune system that, through Cas proteins, promote the degradation of foreign nucleic acids such as phages and plasmids. We analyzed 10 genomes of Corynebacterium striatum clinical isolates from a public hospital in Rio de Janeiro, Brazil, the most emergent multidrug-resistant Corynebacterium species. All isolates were submitted to antimicrobial susceptibility testing. The occurrence and diversity of the CRISPR system were investigated by bioinformatics tools. Our analysis revealed that the isolates exhibited type I-E gene arrangements, and 3 more multidrug-resistant isolates, alternative type I-E gene arrangements, showing a divergent gene arrangement within the cas operon. Phylogenetic analysis of the cas1 gene of this type I-E CRISPR-Cas system alternative arrangement, termed here type I-E’, showed a cluster in a distinct clade of the type I-E CRISPR-Cas system. The systems’ guanine-cytosine (GC) content is lower than the genomic DNA’s GC content, and mobile genetic elements were found in some isolates near the CRISPR-Cas system. Most CRISPR spacers are unknown indicating that there is a reservoir of unexplored corynebacteriophages and plasmids. Some spacers showed perfect homologies with phage and plasmid sequences. Intact phage regions were found in 3 of our isolates, ranging from 9.1 to 43.8 kb, with regions showing similarity to Rhodococcus and Corynebacterium phages. Our results may contribute to research about the CRISPR-Cas system diversity in C. striatum, where there are no published data to date.

Multi-locus gene editing effectively knocked out cyp19a1a and foxl2 in Monopterus albus, a hermaphroditic fish

Homozygous mutant populations are typically obtained when studying gene function using gene editing. However, because of asynchronous testicular and ovarian development, it is difficult to obtain homozygous mutant offspring of hermaphrodite fish by mating males and females. The rice field eel (Monopterus albus) was the first hermaphrodite teleost to be identified. Natural sex change characteristics render M. albus a good model for studying vertebrate sex differentiation. Rice field eels are also an economically important aquaculture species in East and Southeast Asia. In this study, we established a highly efficient multi-locus gene editing method for rice field eels. We selected cyp19a1a and foxl2 for editing and targeted seven and 11 sites in the coding regions of these genes, respectively. Fertilized eggs were co-injected with the Cas9 protein and the guide RNAs of each gene. The efficiency of gene editing–induced mutations in cyp19a1a and foxl2 was approximately 100% in the microinjected embryos and adult fish tissues. In cyp19a1a mutants, serum E2 levels were decreased, and ovarian development was blocked. Only germ stem cells or oogonia were present in the gonads of the 12-month-old cyp19a1a mutant fish. These results suggest that cyp19a1a plays a key role in regulating primary gonadal development in the ovaries of rice field eels. In foxl2 mutants, cyp19a1a transcription and serum E2 levels were minimally decreased, whereas the transcription of foxl2-l, foxl3, and dmrt1 was increased. Nevertheless, the gonads of foxl2 mutant fish differentiated into ovaries, thereby indicating that foxl2 does not directly affect ovarian development in rice field eels. The multi-locus gene editing method established in this study provides an effective approach for studying gene function study and improving the genetic traits in rice field eels.

Research status of CRISPR/Cas9 delivery system

Deriving from the adaptive immune system of prokaryotes, the emerging gene-editing technology CRISPR/Cas9, has become the most popular and powerful tool for the manipulation of genes at the molecular level. It has the advantages of easy construction, high efficiency along with good specificity. CRISPR/Cas9 system has been extensively applied to model constructions, clinical treatment and mechanisms of tumorigenesis. As a number of strategies for CRISPR/Cas9 system have been developed, this review systematically summarizes the commonly applied methods along with discussions on their advantages and limitations and different scenarios for specific applications. The basic delivery forms include plasmid DNA with sgRNA, mRNA with sgRNA and a complex of Cas9 protein and sgRNA, ribonucleoprotein (RNP). For the methods of delivery into cells, there are viral vectors and non-viral vectors, which further consist of physical and chemical methods. While the efficiency and rate of successful transfection vary in different strategies, they commonly suffer from off-target effects, mutagenesis, cytotoxicity and immunogenicity to different extents.

Global phylogenomic novelty of the Cas1 gene from hot spring microbial communities.

The Cas1 protein is essential for the functioning of CRISPR-Cas adaptive systems. However, despite the high prevalence of CRISPR-Cas systems in thermophilic microorganisms, few studies have investigated the occurrence and diversity of Cas1 across hot spring microbial communities. Phylogenomic analysis of 2,150 Cas1 sequences recovered from 48 metagenomes representing hot springs (42-80°C, pH 6-9) from three continents, revealed similar ecological diversity of Cas1 and 16S rRNA associated with geographic location. Furthermore, phylogenetic analysis of the Cas1 sequences exposed a broad taxonomic distribution in thermophilic bacteria, with new clades of Cas1 homologs branching at the root of the tree or at the root of known clades harboring reference Cas1 types. Additionally, a new family of casposases was identified from hot springs, which further completes the evolutionary landscape of the Cas1 superfamily. This ecological study contributes new Cas1 sequences from known and novel locations worldwide, mainly focusing on under-sampled hot spring microbial mat taxa. Results herein show that circumneutral hot springs are environments harboring high diversity and novelty related to adaptive immunity systems.

Antiviral Activity of CRISPR/Cas9 Ribonucleoprotein Complexes on a Hepatitis B Virus Model In Vivo

Chronic hepatitis B (CHB) is caused by hepatitis B virus (HBV) infection. This disease is a key issue for global health. Modern methods of therapy do not completely eliminate HBV from infected cells and do not cure chronic infection. The CRISPR/Cas9 systems of site-specific nucleases can effectively cleave do not target DNA including viral genomes. The cleavage of the major form of the HBV genome, i.e., covalently closed circular DNA (cccDNA), leads to a robust reduction in viral replication and degradation or mutational inactivation of cccDNA. CRISPR/Cas9-based approaches are one of the most promising ways to achieve a 'sterilizing' cure of CHB, i.e., complete elimination of the virus from the body. Here, the HBV mouse model in vivo has been used to analyze the antiviral activity of the high-specific Cas9 protein and sgRNA targeting HBV genome. We have found that a single injection of short-lived ribonucleoprotein complexes of CRISPR/Cas9 results in a ~10-fold reduction in HBV DNA levels in the serum and liver of mice as early as 48 h after the start of the experiment. The remaining HBV DNAs have been found to harbor rare indel mutations. Developing new antivirals for treating CHB based on CRISPR/Cas9 ribonucleoprotein complexes could substantially reduce the duration of CHB therapy and, potentially, achieve complete elimination of viral infection.

A CRISPR view on autophagy.

Autophagy is a fundamental pathway for the degradation of cytoplasmic content in response to pleiotropic extracellular and intracellular stimuli. Recent advances in the autophagy field have demonstrated that different organelles can also be specifically targeted for autophagy with broad implications on cellular and organismal health. This opens new dimensions in the autophagy field and more unanswered questions on the rationale and underlying mechanisms to degrade different organelles. Functional genomics via clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-based screening has gained popularity in the autophagy field to understand the common and unique factors that are implicated in the signaling, recognition, and execution of different cargo-specific autophagies. We focus on recent applications of CRISPR-based screens in the autophagy field, their discoveries, and the future directions of autophagy screens.

Thou shalt not cleave DNA—only repress transcription: A compact Cas protein representing a new CRISPR-Cas subtype

Wu etal.1 characterize Cas12m, a compact Cas protein that silences transcription without cleaving DNA and is a prototype protein of the novel CRISPR-Cas subtype V-M.

A Test System for Assessment of the Activity of Mutant Cas9 Variants in Saccharomyces cerevisiae

The key component of the revolutionary Streptococcus pyogenes CRISPR/Cas genome editing technology is the multidomain protein Cas9. However, the specificity of wild type Cas9 is not sufficiently high for editing large genomes of higher eukaryotes, which limits the realization of the potential of genomic editing both in fundamental investigations and in the therapy of genetic diseases. The main way to obtain more specific variants of Cas9 is through mutagenesis followed by characterization of mutant proteins in in vitro or in vivo test systems. The in vitro and some in vivo test systems described in the literature are often labor-intensive and have scaling limitations, which makes it challenging to screen SpCas9 mutant variant libraries. In order to develop a simple method for high-throughput screening of Cas9 mutants in vivo, we characterized three test systems using CRISPR/Cas9-mediated inactivation of the reporter genes, tsPurple, ADE2, and URA3, in the Saccharomyces cerevisiae yeast as a model subject. We measured the activities of high-precision forms of Cas9, evoCas9, and HiFiCas9, and compared them with the wild-type form. ADE2 gene inactivation was found to be the most valid method for the evaluation of Cas9 activity. In the test-system developed, the sensitivity to chromatin structure was demonstrated for the high-fidelity variant of Cas9, HiFiCas9. The proposed test-system can be used for the development of new generation genome editors.

Using CRISPR-Cas9 In Human Fetuses to Prevent Trisomy 16 and Trisomy 22 Induced Miscarriages

About 50% of abortions have been found to be caused by aneuploidy, roughly 60% of which are trisomy. The most common trisomy occurs on chromosome 16, 21, and 22. However, trisomy 21 (also called Down Syndrome) is viable in about 57% of cases, while trisomy 16 and 22 result in miscarriage in nearly every pregnancy. Thus, additional therapies and treatments must be explored, especially through quickly advancing techniques such as gene editing. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) combined with the cleaving endonuclease CRISPR-associated protein (Cas9) harbors potential for targeted chromosome deletion, possibly increasing the chance of survival in fetuses with trisomy 16 or 22. However, the risks and safety benefits during genetic diagnosing and potential treatment of the fetus should also be considered. Successfully demonstrated approaches of editing or eliminating extraneous chromosomes in studies of both animal models and lab-cultured human embryos will be discussed. In particular, this paper will examine possible gene editing approaches such as elimination of entire chromosomes, large-scale deletions, and chromosomal truncations to target trisomy. In order to determine the efficacy of these approaches in trisomy, the use of CRISPR-Cas9 to specifically treat trisomy via autosomal deletion and counter-selection markers will be deliberated. Delivery methods for the in-utero therapy includes viral techniques such as retroviruses and adeno-associated vectors (AAVs) as well as non-viral techniques like electroporation and nanoparticles. This paper will propose hypothetical treatments for CRISPR-Cas9 in human embryos to target trisomy 16 and 22 while also examining the bioethical implications of doing so.

Characterization of homozygous Foxn1 mutations induced in rat embryos by different delivery forms of Cas9 nuclease.

The Cas9 nuclease is delivered in the form of either Cas9 protein or mRNA along with CRISPR guide RNA (gRNA: dual-crRNA:tracrRNA or chimeric single-guide RNA) or in a plasmid package encoding both Cas9 and the CRISPR gRNA. We directly compared the efficiency of producing rat blastocysts with homozygous mutations of the Foxn1 locus by pronuclear injection of Cas9 in the form of protein, mRNA, or plasmid DNA. For highly efficient production of rat blastocysts with homozygous Foxn1 mutations, pronuclear injection of Cas9 protein at 60ng/µl was likely optimal. While blastocyst harvest in the mRNA groups was higher than those in the protein and plasmid DNA groups, genotype analysis showed that 63.6%, 8.7-20.0%, and 25.0% of the analyzed blastocysts were homozygous mutants in the protein, mRNA, and plasmid DNA groups, respectively. The high efficiency of producing homozygous mutant blastocysts in the 60ng/µl protein group may be associated with primary genome editing being initiated before the first cleavage. In most cases, homozygous mutations at the target Foxn1 locus are triggered by deletion and repair via nonhomologous end joining or microhomology-mediated end joining. Deletion downstream of the Cas9 break site was more likely than deletion in the upstream direction. The Cas9 nuclease in protein form, when coinjected with the CRISPR gRNA (ribonucleoprotein) into a rat zygote pronucleus, can access the target genome site and induce double-strand breaks promptly, resulting in the efficient production of homozygous mutants.

Complete and Prolonged Inhibition of Herpes Simplex Virus Type 1 Infection In Vitro by CRISPR/Cas9 and CRISPR/CasX Systems

Almost all people become infected with herpes viruses, including herpes simplex virus type 1 (HSV-1), during their lifetime. Typically, these viruses persist in a latent form that is resistant to all available antiviral medications. Under certain conditions, such as immunosuppression, the latent forms reactivate and cause disease. Moreover, strains of herpesviruses that are drug-resistant have rapidly emerged. Therefore, it is important to develop alternative methods capable of eradicating herpesvirus infections. One promising direction is the development of CRISPR/Cas systems for the therapy of herpesvirus infections. We aimed to design a CRISPR/Cas system for relatively effective long-term and safe control of HSV-1 infection. Here, we show that plasmids encoding the CRISPR/Cas9 system from Streptococcus pyogenes with a single sgRNA targeting the UL30 gene can completely suppress HSV-1 infection of the Vero cell line within 6 days and provide substantial protection within 9 days. For the first time, we show that CRISPR/CasX from Deltaproteobacteria with a single guide RNA against UL30 almost completely suppresses HSV-1 infection of the Vero cell line for 3 days and provides substantial protection for 6 days. We also found that the Cas9 protein without sgRNAs attenuates HSV-1 infection. Our results show that the developed CRISPR/Cas systems are promising therapeutic approaches to control HSV-1 infections.

Dissecting Locus-Specific Chromatin Interactions by CRISPR CAPTURE.

The spatiotemporal control of tissue-specific gene expression is coordinated by cis-regulatory elements (CREs) and associated trans-acting factors. Despite major advances in genome-wide annotation of candidate CREs, the in situ regulatory composition of the vast majority of CREs remain unknown. To address this challenge, we developed the CRISPR affinity purification in situ of regulatory elements (CAPTURE) toolbox that employs an in vivo biotinylated nuclease-deficient Cas9 (dCas9) protein and programmable single-guide RNAs (sgRNAs) to identify CRE-associated macromolecular complexes and chromatin looping. In this chapter, we provide a detailed protocol for implementing the latest iteration of the CRISPR-based CAPTURE methods to interrogate the molecular composition of locus-specific chromatin complexes and configuration in a mammalian genome.

CRISPR/Cas systems: Delivery and application in gene therapy.

The CRISPR/Cas systems in prokaryotes such as bacteria and archaea are the adaptive immune system to prevent infection from viruses, phages, or other foreign substances. When viruses or phages first invade the bacteria, Cas proteins recognize and cut the DNA from viruses or phages into short fragments that will be integrated into the CRISPR array. Once bacteria are invaded again, the modified CRISPR and Cas proteins react quickly to cut DNA at the specified target location, protecting the host. Due to its high efficiency, versatility, and simplicity, the CRISPR/Cas system has become one of the most popular gene editing technologies. In this review, we briefly introduce the CRISPR/Cas systems, focus on several delivery methods including physical delivery, viral vector delivery, and non-viral vector delivery, and the applications of disease therapy. Finally, some problems in CRISPR/Cas9 technology have been proposed, such as the off-target effects, the efficiency of DNA repair mechanisms, and delivery of CRISPR/Cas system safely and efficiently to the target location.

Preparation of NanoMEDIC Extracellular Vesicles to Deliver CRISPR-Cas9 Ribonucleoproteins for Genomic Exon Skipping.

The CRISPR-Cas9 system has quickly become the standard tool for genome editing. To deliver this system to target cells, adeno-associated virus (AAV) vectors are commonly used. In fact, AAV vectors have been utilized to deliver the CRISPR-Cas9 system to induce genomic exon skipping and restore the dystrophin protein in various Duchenne muscular dystrophy model animals. Despite the high transduction efficiency, AAV vector-mediated delivery has several limitations, such as the packaging size, prolonged overexpression of Cas9, immunogenicity against the AAV capsid, and the risk of integrating a part of the AAV genomic sequence into the host cell. To overcome these issues, we have recently engineered a transient delivery system utilizing VSV-G pseudotyped extracellular vesicles (EVs) termed NanoMEDIC (nanomembrane-derived extracellular vesicles for the delivery of macromolecular cargo). NanoMEDIC utilizes an HIV-derived Gag protein to package Cas9 protein and gRNA into EVs. The Cas9 and Gag proteins are fused to a heterodimerizer and conditionally dimerized by the addition of an inducible chemical ligand to recruit Cas9 protein into EVs. sgRNA is packaged into EVs through an HIV-derived RNA packaging signal and is subsequently released by two self-cleaving ribozymes. Utilizing these features, NanoMEDIC can achieve highly efficient packaging of the Cas9 protein and gRNA for genome editing into a variety of target cells and in vivo. Here, we describe a step-by-step protocol, including the gRNA-expressing vector construction and large-scale NanoMEDIC production, for in vivo genome editing.

CRISPR-Cas-Integrated LAMP.

Pathogen-specific point-of-care (PoC) diagnostic tests have become an important need in the fight against infectious diseases and epidemics in recent years. PoC diagnostic tests are designed with the following parameters in mind: rapidity, accuracy, sensitivity, specificity, and ease of use. Molecular techniques are the gold standard for pathogen detection due to their accuracy and specificity. There are various limitations in adapting molecular diagnostic methods to PoC diagnostic tests. Efforts to overcome limitations are focused on the development of integrated molecular diagnostics by utilizing the latest technologies available to create the most successful PoC diagnostic platforms. With this point of view, a new generation technology was developed by combining loop-mediated isothermal amplification (LAMP) technology with clustered regularly interspaced short palindromic repeat (CRISPR)-associated (CRISPR-Cas) technology. This integrated approach benefits from the properties of LAMP technology, namely its high efficiency, short turnaround time, and the lack of need for a complex device. It also makes use of the programmable function of CRISPR-Cas technology and the collateral cleavage activity of certain Cas proteins that allow for convenient reporter detection. Thus, this combined technology enables the development of PoC diagnostic tests with high sensitivity, specificity, and ease of use without the need for complicated devices. In this review, we discuss the advantages and limitations of the CRISPR/Cas combined LAMP technology. We review current limitations to convert CRISPR combined LAMP into pathogen-specific PoC platforms. Furthermore, we point out the need to design more useful PoC platforms using microfabrication technologies by developing strategies that overcome the limitations of this new technology, reduce its complexity, and reduce the risk of contamination.

CD33 rs12459419 SNP Regulated the Total CD33 Expression Level

Background: Gemutuzumab ozogamicin (GO) is a molecularly targeted drug which combines a humanized anti-CD33 antibody (p67.6) with calicheamicin. GO has differed therapeutic efficacy in cases of acute myeloid leukemia (AML). Single nucleotide polymorphisms (SNPs) in the CD33 gene have been investigated as one of the reasons for this difference. SNPs (CC, CT, and TT types) in rs12459419 have been reported to be associated with clinical outcomes in pediatric AML patients treated with GO-containing chemotherapy. On the other hand, there are reports of poor association in adult AML patients, and the significance of rs12459419 SNPs in remains to be clarified. CD33 is composed of two extracellular domains, IgV and IgC. p67.6, anti-CD33 antibodies, recognize IgV, while HIM3-4 antibodies recognize IgC. rs12459419 SNP is located in the splicing acceptor region of exon 2 of the CD33 gene, when the SNP is T, transcripts that skip exon 2 which encodes the IgV domain, are more likely to occur. The increasing expression of this exon skipping product may be accompanied by a decreasing amount of wild-type CD33 (CD33FL) and an increasing amount of IgV domain-deficient CD33 (CD33ΔE2). GO only recognizes CD33FL, but not CD33ΔE2. We used CRISPR/Cas9 genome editing system to generate cell lines with specifically modified CD33 SNPs to elucidate the expression of CD33FL and CD33ΔE2 by CD33 SNPs and its effect on sensitivity to GO in a uniform genetic background. Methods and Results: First, we analyzed CD33 SNPs in 23 AML patients treated with GO (CC type: 13; CT type; 9: TT type: 1). The outcome of 17 patients, excluding a history of hematopoietic stem cell transplantation before treated with GO, was evaluated. Three-year overall survival rate for the type of CC vs. CT was 70.7% vs. 83.3% (p value = 0.609), and 3-year disease-free survival rate for the type of CC vs. CT was 80.8% vs. 83.3% (p value = 0.895). There was no significant difference in CC and CT. Next, using p67.6 recognizes only CD33FL and HIM3-4 recognizes both CD33FL and CD33ΔE2, we evaluated the expression ratio of CD33FL and CD33ΔE2 in relation with CD33 SNP. We assumed that the expression levels of MFI(median fluorescence intensity) were roughly similar in HIM3-4, but the MFI evaluated by both antibodies tended to be higher in the CT and CC types than in the TT type (MFI for p67.6 of CC, CT and TT was 2675, 1752 and 245, respectively; MFI for HIM3-4 of CC, CT and TT was 699, 629 and 53, respectively). The patient samples differ in many genetic backgrounds besides CD33 SNPs, making accurate evaluation difficult. Genome editing was performed by co-transfection of guide RNAs targeting CD33 SNPs, Cas9 protein and with single-stranded oligonucleotides to the target leukemia cell line. Single cell cloning was performed by limiting dilution to generate single nucleotide altered cells for the CD33 SNP. OCI-AML3 wild-type (TT type) and the obtained CD33 SNP-modified clones (CC type, 4 clones and CT type, 4 clones) were evaluated by flow cytometry with p67.6 and HIM3-4.The results showed that the MFI of both antibodies was higher in the CT and CC types than in the TT, the wild type (MFI for p67.6 of CC, CT and TT was 10760, 13686 and 4801, respectively; MFI for HIM3-4 of CC, CT and TT was 3115, 4067 and 887, respectively). These results showed similar trends to the clinical samples implying that differences in CD33 SNPs affect the amount of total CD33 expression on the cell surface itself. Conclusion: The CD33 rs12459419 SNP regulated the expression level of total CD33 in clinical samples and gene-edited cell lines. There was no obvious difference in the expression levels of total CD33 and CD33FL at the cell surface between the CC and CT types, but it was higher than that of the TT type. The difference in CD33 expression may influence the therapeutic efficacy of GO.