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A simple protocol to isolate a single human cell PRDX1 knockout generated by CRISPR-Cas9 system.
Here, we describe a protocol for human PRDX1 gene knockout cells using the CRISPR-Cas9 system. The protocol describes all the steps sequentially: (1) single-guide RNA design, cloning, and transfection; (2) gene editing evaluation by T7EI assay; (3) single-cell isolation; and (4) knockout verification to determine indels in one or both alleles by Sanger sequencing. This strategy is based on the efficiency of DNA editing, avoids antibiotic selection, and bypasses the need for cell sorting.
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A mechanistic study on the tolerance of PAM distal end mismatch by SpCas9.
The therapeutic application of CRISPR-Cas9 is limited due to its off-target activity. To have a better understanding of this off-target effect, we focused on its mismatch-prone PAM distal end. The off-target activity of SpCas9 depends directly on the nature of mismatches, which in turn results in deviation of the active site of SpCas9 due to structural instability in the RNA-DNA duplex strand. In order to test the hypothesis, we designed an array of mismatched target sites at the PAM distal end and performed in vitro and cell line-based experiments, which showed a strong correlation for Cas9 activity. We found that target sites having multiple mismatches in the 18th to 15th position upstream of the PAM showed no to little activity. For further mechanistic validation, Molecular Dynamics simulations were performed, which revealed that certain mismatches showed elevated root mean square deviation values that can be attributed to conformational instability within the RNA-DNA duplex. Therefore, for successful prediction of the off-target effect of SpCas9, along with complementation-derived energy, the RNA-DNA duplex stability should be taken into account.
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Machine learning-based prediction models to guide the selection of Cas9 variants for efficient gene editing.
The increasing emergence of Cas9 variants has attracted broad interest, as these variants were designed to expand CRISPR applications. New Cas9 variants typically feature higher editing efficiency, improved editing specificity, or alternative PAM sequences. To select Cas9 variants and gRNAs for high-fidelity and efficient genome editing, it is crucial to systematically quantify the editing performances of gRNAs and develop prediction models based on high-quality datasets. Using synthetic gRNA-target paired libraries and next-generation sequencing, we compared the activity and specificity of gRNAs of four SpCas9 variants. The nucleotide composition in the PAM-distal region had more influence on the editing efficiency of HiFi Cas9 and LZ3 Cas9. We further developed machine learning models to predict the gRNA efficiency and specificity for the four Cas9 variants. To aid users from broad research areas, the machine learning models for the predictions of gRNA editing efficiency within human genome sites are available on our website.
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Genome editing and cancer: How far has research moved forward on CRISPR/Cas9?
Cancer accounted for almost ten million deaths worldwide in 2020. Metastasis, characterized by cancer cell invasion to other parts of the body, is the main cause of cancer morbidity and mortality. Therefore, understanding the molecular mechanisms of tumor formation and discovery of potential drug targets are of great importance. Gene editing techniques can be used to find novel drug targets and study molecular mechanisms. In this review, we describe how popular gene-editing methods such as CRISPR/Cas9, TALEN and ZFNs work, and, by comparing them, we demonstrate that CRISPR/Cas9 has superior efficiency and precision. We further provide an overview of the recent applications of CRISPR/Cas9 to cancer research, focusing on the most common cancers such as breast cancer, lung cancer, colorectal cancer, and prostate cancer. We describe how these applications will shape future research and treatment of cancer, and propose new ways to overcome current challenges.
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[Recombinase-Based Engineering of Plant Genomes in the Era of Genome Editing].
The rapidly evolving CRISPR/Cas-based genome editing technologies, which have dominated nearly all areas of molecular biology over the past decade, still face several unresolved challenges. One of the major limitations of current genome editing tools is the low efficiency of targeted long-sequence insertions. This issue is particularly critical in plant systems, where genome editing efficiency is hindered by specific cellular characteristics. Site-specific recombinases (SSRs), which have long been employed in genetic engineering to mediate various genomic rearrangements-including deletions, duplications, insertions, and inversions-are limited in their application by the requirement for preexisting recombination recognition sites in the genome. However, CRISPR/Cas and recombinase tools complement each other, and their combined use offers a powerful strategy to overcome key limitations of genome editing. The discovery of CRISPR-associated transposons such as CAST and OMEGA, which naturally utilize their own recombinases, marks a significant advance in genome engineering, providing an elegant example of the natural convergence between CRISPR and recombinase technologies.
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New Gene Editing Research Hopes to Eliminate Genetically Related Male Infertility.
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Disrupting Protein Expression with Double-Clicked sgRNA-Cas9 Complexes: A Modular Approach to CRISPR Gene Editing.
CRISPR-Cas9 is currently the most versatile technique to perform gene editing in living organisms. In this approach, the Cas9 endonuclease is guided toward its DNA target sequence by the guide RNA (gRNA). Chemical synthesis of a functional single gRNA (sgRNA) is nontrivial because of the length of the RNA strand. Recently we demonstrated that a sgRNA can be stitched together from three smaller fragments through a copper-catalyzed azide-alkyne cycloaddition, making the process highly modular. Here we further advance this approach by leveraging this modulator platform by incorporating chemically modified nucleotides at both ends of the modular sgRNA to increase resistance against ribonucleases. Modified nucleotides consisted of a 2'-O-Me group and a phosphorothioate backbone in varying number at both the 5'- and 3'-ends of the sgRNA. It was observed that three modified nucleotides at both ends of the sgRNA significantly increased the success of Cas9 in knocking out a gene of interest. Using these chemically stabilized sgRNAs facilitates multigene editing at the protein level, as demonstrated by successful knockout of both Siglec-3 and Siglec-7 using two fluorophores in conjunction with fluorescence-activated cell sorting. These results demonstrate the versatility of this modular platform for assembling sgRNAs from small, chemically modified strands to simultaneously disrupt the gene expression of two proteins.
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Use of CRISPR/Cas ribonucleoproteins for high throughput gene editing of induced pluripotent stem cells.
Induced pluripotent stem cells (iPSCs) have become widely used for disease modelling, particularly with regard to predisposing genetic risk factors and causal gene variants. Alongside this, technologies such as the CRISPR/Cas system have been adapted to enable programmable gene editing in human cells. When combined, CRISPR/Cas gene editing of donor-specific iPSC to generate isogenic cell lines that differ only at specific gene variants provides a powerful model with which to investigate genetic variants associated with diseases affecting many organs, including the brain and eye. Here we describe our optimized protocol for using CRISPR/Cas ribonucleoproteins to edit disease causing gene variants in human iPSCs. We discuss design of crRNAs and homology-directed repair templates, assembly of CRISPR/Cas ribonucleoproteins, optimization of delivery via nucleofection, and strategies for single cell cloning, efficient clone cryopreservation and genotyping for identifying iPSC clones for further characterization.
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[Advances of using CRISPR-Cas13a system for tumor diagnosis and treatment].
CRISPR-Cas systems are well known gene editing tools, among which CRISPR-Cas9 system targeting DNA is the most well developed. Compared with CRISPR-Cas9 system, CRISPR-C2c2/ Cas13a system derived from TYPE VI of CRISPR family that can target RNA has attracted increasingly intense investigations in recent years. The CRISPR-Cas13a system is featured by specific recognition and binding of single stranded RNA sequences, thus playing a role in non-specific cleavage of RNA. This feature could be potentially applied to detect free nucleic acid in tumors or peripheral blood as a diagnostic approach. Since Cas13a specifically targets RNA, it can directly edit mRNA transcripts of genomic DNA to achieve the downregulation of target proteins without involving DNA editing. Therefore, Cas13a system could be used in tumor treatment. This review summarized the advances of using CRISPR-Cas13a for RNA targeting in tumor diagnosis and treatment, and prospected future applications.
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Clinical progress in genome-editing technology and in vivo delivery techniques.
There is wide interest in applying genome-editing tools to prevent, treat, and cure a variety of diseases. Since the discovery of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems, these techniques have been used in combination with different delivery systems to create highly efficacious treatment options. Each delivery system has its own advantages and disadvantages and is being used for various applications. With the large number of gene-editing applications being studied but very few being brought into the clinic, we review current progress in the field, specifically where genome editing has been applied in vivo and in the clinic, and identify current challenges and areas of future growth.
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