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Detecting Melanocortin 1 Receptor Gene's SNPs by CRISPR/enAsCas12a.
Beyond its powerful genome-editing capabilities, the CRISPR/Cas system has opened up a new era of molecular diagnostics due to its highly specific base recognition and trans-cleavage activity. However, most CRISPR/Cas detection systems are mainly used to detect nucleic acids of bacteria or viruses, while the application of single nucleotide polymorphism (SNP) detection is limited. The MC1R SNPs were investigated by CRISPR/enAsCas12a and are not limited to the protospacer adjacent motif (PAM) sequence in vitro. Specifically, we optimized the reaction conditions, which proved that the enAsCas12a has a preference for divalent magnesium ion (Mg2+) and can effectively distinguish the genes with a single base difference in the presence of Mg2+, and the Melanocortin l receptor (MC1R) gene with three kinds of SNP sites (T305C, T363C, and G727A) was quantitatively detected. Since the enAsCas12a is not limited by PAM sequence in vitro, the method shown here can extend this extraordinary CRISPR/enAsCas12a detection system to other SNP targets, thus providing a general SNP detection toolbox.
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Dual base editor catalyzes both cytosine and adenine base conversions in human cells.
Although base editors are useful tools for precise genome editing, current base editors can only convert either adenines or cytosines. We developed a dual adenine and cytosine base editor (A&C-BEmax) by fusing both deaminases with a Cas9 nickase to achieve C-to-T and A-to-G conversions at the same target site. Compared to single base editors, A&C-BEmax's activity on adenines is slightly reduced, whereas activity on cytosines is higher and RNA off-target activity is substantially decreased.
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Gene Therapy for Glaucoma by Ciliary Body Aquaporin 1 Disruption Using CRISPR-Cas9.
Glaucoma is a common cause of blindness, yet current therapeutic options are imperfect. Clinical trials have invariably shown that reduction in intraocular pressure (IOP) regardless of disease subtype prevents visual loss. Reducing ciliary body aqueous humor production can lower IOP, and the adeno-associated virus ShH10 serotype was identified as able to transduce mouse ciliary body epithelium following intravitreal injection. Using ShH10 to deliver a single vector CRISPR-Cas9 system disrupting Aquaporin 1 resulted in reduced IOP in treated eyes (10.4 ± 2.4 mmHg) compared with control (13.2 ± 2.0 mmHg) or non-injected eyes (13.1 ± 2.8 mmHg; p < 0.001; n = 12). Editing in the aquaporin 1 gene could be detected in ciliary body, and no off-target increases in corneal or retinal thickness were identified. In experimental mouse models of corticosteroid and microbead-induced ocular hypertension, IOP could be reduced to prevent ganglion cell loss (32 ± 4 /mm2) compared with untreated eyes (25 ± 5/mm2; p < 0.01). ShH10 could transduce human ciliary body from post-mortem donor eyes in ex vivo culture with indel formation detectable in the Aquaporin 1 locus. Clinical translation of this approach to patients with glaucoma may permit long-term reduction of IOP following a single injection.
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Gene targeting, genome editing: from Dolly to editors.
One of the most powerful strategies to investigate biology we have as scientists, is the ability to transfer genetic material in a controlled and deliberate manner between organisms. When applied to livestock, applications worthy of commercial venture can be devised. Although initial methods used to generate transgenic livestock resulted in random transgene insertion, the development of SCNT technology enabled homologous recombination gene targeting strategies to be used in livestock. Much has been accomplished using this approach. However, now we have the ability to change a specific base in the genome without leaving any other DNA mark, with no need for a transgene. With the advent of the genome editors this is now possible and like other significant technological leaps, the result is an even greater diversity of possible applications. Indeed, in merely 5 years, these 'molecular scissors' have enabled the production of more than 300 differently edited pigs, cattle, sheep and goats. The advent of genome editors has brought genetic engineering of livestock to a position where industry, the public and politicians are all eager to see real use of genetically engineered livestock to address societal needs. Since the first transgenic livestock reported just over three decades ago the field of livestock biotechnology has come a long way-but the most exciting period is just starting.
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A differentiated and durable allogeneic strategy applicable to cell therapies.
BACKGROUND AIMS: Autologous T-cell therapies have shown profound clinical responses; however, their widespread use has been limited primarily as the result of their individualized manufacturing requirements. METHODS: To develop a persistent "off-the-shelf" allogeneic (Allo) approach, a multiplex Nme2Cas9-based cytosine base editor was deployed to knockout select human leukocyte antigens (HLA) class I and II alleles (HLA-A, HLA-B and the class II transactivator [CIITA]) while retaining HLA-C to protect from natural killer (NK) cell rejection. RESULTS AND CONCLUSION: Matching the residual HLA-C allele from homozygous donors to the host prevented rejection of the donor T cells by allogeneic host T and NK cells. Site-specific integration of a tumor-specific CAR or TCR into the TRAC locus using SpyCas9 nuclease and an adeno-associated virus template allowed for a high localized insertion rate while simultaneously removing the endogenous TCR and preventing graft-versus-host disease. Using an optimized T-cell engineering process involving orthogonal CRISPR/Cas9 cleavage and base editors coupled with lipid nanoparticle delivery, we achieved efficient production of Allo-CAR T cells with high editing rates and cell expansion in a scalable manner. These allogeneic T cells demonstrated comparable functional activity to their autologous counterparts in preclinical assays. Moreover, this gene-editing approach significantly minimized the occurrence of chromosomal aberrations. This promising allogeneic approach also has been applied to induced pluripotent stem cells (iPSCs) with triple edits targeting HLA-A, HLA-B and CIITA (TKO). Pancreatic progenitor cells or cardiomyocytes derived from TKO iPSCs were protected from host peripheral blood mononuclear cell-mediated rejection when matched for HLA-C, suggesting potential applications in regenerative medicine applications.
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Transcription factor competition at the γ-globin promoters controls hemoglobin switching.
BCL11A, the major regulator of fetal hemoglobin (HbF, α2γ2) level, represses γ-globin expression through direct promoter binding in adult erythroid cells in a switch to adult hemoglobin (HbA, α2β2). To uncover how BCL11A initiates repression, we used CRISPR-Cas9, dCas9, dCas9-KRAB and dCas9-VP64 screens to dissect the γ-globin promoters and identified an activator element near the BCL11A-binding site. Using CUT&RUN and base editing, we demonstrate that a proximal CCAAT box is occupied by the activator NF-Y. BCL11A competes with NF-Y binding through steric hindrance to initiate repression. Occupancy of NF-Y is rapidly established following BCL11A depletion, and precedes γ-globin derepression and locus control region (LCR)-globin loop formation. Our findings reveal that the switch from fetal to adult globin gene expression within the >50-kb β-globin gene cluster is initiated by competition between a stage-selective repressor and a ubiquitous activating factor within a remarkably discrete region of the γ-globin promoters.
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An APOBEC3A-Cas9 base editor with minimized bystander and off-target activities.
Base editor technology, which uses CRISPR-Cas9 to direct cytidine deaminase enzymatic activity to specific genomic loci, enables the highly efficient introduction of precise cytidine-to-thymidine DNA alterations. However, existing base editors create unwanted C-to-T alterations when more than one C is present in the enzyme's five-base-pair editing window. Here we describe a strategy for reducing bystander mutations using an engineered human APOBEC3A (eA3A) domain, which preferentially deaminates cytidines in specific motifs according to a TCR>TCY>VCN hierarchy. In direct comparisons with the widely used base editor 3 (BE3) fusion in human cells, our eA3A-BE3 fusion exhibits similar activities on cytidines in TC motifs but greatly reduced editing on cytidines in other sequence contexts. eA3A-BE3 corrects a human β-thalassemia promoter mutation with much higher (>40-fold) precision than BE3. We also demonstrate that eA3A-BE3 shows reduced mutation frequencies on known off-target sites of BE3, even when targeting promiscuous homopolymeric sites.
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CRISPRpred(SEQ): a sequence-based method for sgRNA on target activity prediction using traditional machine learning.
BACKGROUND: The latest works on CRISPR genome editing tools mainly employs deep learning techniques. However, deep learning models lack explainability and they are harder to reproduce. We were motivated to build an accurate genome editing tool using sequence-based features and traditional machine learning that can compete with deep learning models. RESULTS: In this paper, we present CRISPRpred(SEQ), a method for sgRNA on-target activity prediction that leverages only traditional machine learning techniques and hand-crafted features extracted from sgRNA sequences. We compare the results of CRISPRpred(SEQ) with that of DeepCRISPR, the current state-of-the-art, which uses a deep learning pipeline. Despite using only traditional machine learning methods, we have been able to beat DeepCRISPR for the three out of four cell lines in the benchmark dataset convincingly (2.174%, 6.905% and 8.119% improvement for the three cell lines). CONCLUSION: CRISPRpred(SEQ) has been able to convincingly beat DeepCRISPR in 3 out of 4 cell lines. We believe that by exploring further, one can design better features only using the sgRNA sequences and can come up with a better method leveraging only traditional machine learning algorithms that can fully beat the deep learning models.
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Spellchecking for the Story of Life With CRISPR-Cas9 and Base, Prime Editors.
Just four letters-A, G, T, and C-make up the alphabet of the genome. It may seem simple, but a small difference in spelling can create mutations that result in life-threatening diseases. Gene variants that cause genetic diseases come in many varieties. Transition point mutations cause conditions such as progeria, the rapid aging disease. Transversion point mutations cause sickle-cell disease and other major disorders. Small insertions can cause Tay-Sachs, which stops nerves working properly and is usually fatal, and deletions can result in cystic fibrosis.
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Programmable C:G to G:C genome editing with CRISPR-Cas9-directed base excision repair proteins.
Many genetic diseases are caused by single-nucleotide polymorphisms. Base editors can correct these mutations at single-nucleotide resolution, but until recently, only allowed for transition edits, addressing four out of twelve possible DNA base substitutions. Here, we develop a class of C:G to G:C Base Editors to create single-base genomic transversions in human cells. Our C:G to G:C Base Editors consist of a nickase-Cas9 fused to a cytidine deaminase and base excision repair proteins. Characterization of >30 base editor candidates reveal that they predominantly perform C:G to G:C editing (up to 90% purity), with rAPOBEC-nCas9-rXRCC1 being the most efficient (mean 15.4% and up to 37% without selection). C:G to G:C Base Editors target cytidine in WCW, ACC or GCT sequence contexts and within a precise three-nucleotide window of the target protospacer. We further target genes linked to dyslipidemia, hypertrophic cardiomyopathy, and deafness, showing the therapeutic potential of these base editors in interrogating and correcting human genetic diseases.
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