Recent Advances in Improving Gene-Editing Specificity through CRISPR–Cas9 Nuclease Engineering
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ABSTRACT:
SUBMITTER: Huang X
PROVIDER: S-EPMC9319960 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:CRISPR (Clustered Regularly-Interspaced Short Palindromic Repeats)-Cas9 (CRISPR associated protein 9) has rapidly become the most promising genome editing tool with great potential to revolutionize medicine. Through guidance of a 20 nucleotide RNA (gRNA), CRISPR-Cas9 finds and cuts target protospacer DNA precisely 3 base pairs upstream of a PAM (Protospacer Adjacent Motif). The broken DNA ends are repaired by either NHEJ (Non-Homologous End Joining) resulting in small indels, or by HDR (Homology Directed Repair) for precise gene or nucleotide replacement. Theoretically, CRISPR-Cas9 could be used to modify any genomic sequences, thereby providing a simple, easy, and cost effective means of genome wide gene editing. However, the off-target activity of CRISPR-Cas9 that cuts DNA sites with imperfect matches with gRNA have been of significant concern because clinical applications require 100% accuracy. Additionally, CRISPR-Cas9 has unpredictable efficiency among different DNA target sites and the PAM requirements greatly restrict its genome editing frequency. A large number of efforts have been made to address these impeding issues, but much more is needed to fully realize the medical potential of CRISPR-Cas9. In this article, we summarize the existing problems and current advances of the CRISPR-Cas9 technology and provide perspectives for the ultimate perfection of Cas9-mediated genome editing.
| S-EPMC4927198 | biostudies-literature
Project description:The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome editing system has been a major technological breakthrough that has brought revolutionary changes to genome editing for therapeutic and diagnostic purposes and precision medicine. With the advent of the CRISPR/Cas9 system, one of the critical limiting factors has been the safe and efficient delivery of this system to cells or tissues of interest. Several approaches have been investigated to find delivery systems that can attain tissue-targeted delivery, lowering the chances of off-target editing. While viral vectors have shown promise for in vitro, in vivo and ex vivo delivery of CRISPR/Cas9, their further clinical applications have been restricted due to shortcomings including limited cargo packaging capacity, difficulties with large-scale production, immunogenicity and insertional mutagenesis. Rapid progress in nonviral delivery vectors, including the use of lipid, polymer, peptides, and inorganic nanoparticle-based delivery systems, has established nonviral delivery approaches as a viable alternative to viral vectors. This review will introduce the molecular mechanisms of the CRISPR/Cas9 gene editing system, current strategies for delivering CRISPR/Cas9-based tools, an overview of strategies for overcoming off-target genome editing, and approaches for improving genome targeting and tissue targeting. We will also highlight current developments and recent clinical trials for the delivery of CRISPR/Cas9. Finally, future directions for overcoming the limitations and adaptation of this technology for clinical trials will be discussed.
| S-EPMC10052255 | biostudies-literature
Project description:Rapid advancement in genome editing technologies has provided new promises for treating neoplasia, cardiovascular, neurodegenerative, and monogenic disorders. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has emerged as a powerful gene editing tool offering advantages, including high editing efficiency and low cost over the conventional approaches. Human pluripotent stem cells (hPSCs), with their great proliferation and differentiation potential into different cell types, have been exploited in stem cell-based therapy. The potential of hPSCs and the capabilities of CRISPR/Cas9 genome editing has been paradigm-shifting in medical genetics for over two decades. Since hPSCs are categorized as hard-to-transfect cells, there is a critical demand to develop an appropriate and effective approach for CRISPR/Cas9 delivery into these cells. This review focuses on various strategies for CRISPR/Cas9 delivery in stem cells.
| S-EPMC10661828 | biostudies-literature
Project description:The cephalochordate amphioxus is a promising animal model for studying the origin of vertebrates due to its key phylogenetic position among chordates. Although transcription activator-like effector nucleases (TALENs) have been adopted in amphioxus genome editing, its labor-intensive construction of TALEN proteins limits its usage in many laboratories. Here we reported an application of the CRISPR/Cas9 system, a more amenable genome editing method, in this group of animals. Our data showed that while co-injection of Cas9 mRNAs and sgRNAs into amphioxus unfertilized eggs caused no detectable mutations at targeted loci, injections of Cas9 mRNAs and sgRNAs at the two-cell stage, or of Cas9 protein and sgRNAs before fertilization, can execute efficient disruptions of targeted genes. Among the nine tested sgRNAs (targeting five genes) co-injected with Cas9 protein, seven introduced mutations with efficiency ranging from 18.4% to 90% and four caused specific phenotypes in the injected embryos. We also demonstrated that monomerization of sgRNAs via thermal treatment or modifying the sgRNA structure could increase mutation efficacies. Our study will not only promote application of genome editing method in amphioxus research, but also provide valuable experiences for other organisms in which the CRISPR/Cas9 system has not been successfully applied.
| S-EPMC7694359 | biostudies-literature
Project description:We have adapted a bacterial CRISPR RNA/Cas9 system to precisely engineer the Drosophila genome and report that Cas9-mediated genomic modifications are efficiently transmitted through the germline. This RNA-guided Cas9 system can be rapidly programmed to generate targeted alleles for probing gene function in Drosophila.
| S-EPMC3730909 | biostudies-other
Project description:Clustered, regularly interspaced, short palindromic repeat (CRISPR) RNA-guided nucleases (RGNs) are highly efficient genome editing tools. CRISPR-associated 9 (Cas9) RGNs are directed to genomic loci by guide RNAs (gRNAs) containing 20 nucleotides that are complementary to a target DNA sequence. However, RGNs can induce mutations at sites that differ by as many as five nucleotides from the intended target. Here we report that truncated gRNAs, with shorter regions of target complementarity <20 nucleotides in length, can decrease undesired mutagenesis at some off-target sites by 5,000-fold or more without sacrificing on-target genome editing efficiencies. In addition, use of truncated gRNAs can further reduce off-target effects induced by pairs of Cas9 variants that nick DNA (paired nickases). Our results delineate a simple, effective strategy to improve the specificities of Cas9 nucleases or paired nickases.
| S-EPMC3988262 | biostudies-literature
Project description:The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a system is a powerful tool in gene editing; however, crRNA-DNA mismatches might induce unwanted cleavage events, especially at the distal end of the PAM. To minimize this limitation, we engineered a hyper fidelity AsCas12a variant carrying the mutations S186A/R301A/T315A/Q1014A/K414A (termed HyperFi-As) by modifying amino acid residues interacting with the target DNA and crRNA strand. HyperFi-As retains on-target activities comparable to wild-type AsCas12a (AsCas12aWT) in human cells. We demonstrated that HyperFi-As has dramatically reduced off-target effects in human cells, and HyperFi-As possessed notably a lower tolerance to mismatch at the position of the PAM-distal region compared with the wild type. Further, a modified single-molecule DNA unzipping assay at proper constant force was applied to evaluate the stability and transient stages of the CRISPR/Cas ribonucleoprotein (RNP) complex. Multiple states were sensitively detected during the disassembly of the DNA-Cas12a-crRNA complexes. On off-target DNA substrates, the HyperFi-As-crRNA was harder to maintain the R-loop complex state compared to the AsCas12aWT, which could explain exactly why the HyperFi-As has low off-targeting effects in human cells. Our findings provide a novel version of AsCas12a variant with low off-target effects, especially capable of dealing with the high off-targeting in the distal region from the PAM. An insight into how the AsCas12a variant behaves at off-target sites was also revealed at the single-molecule level and the unzipping assay to evaluate multiple states of CRISPR/Cas RNP complexes might be greatly helpful for a deep understanding of how CRISPR/Cas behaves and how to engineer it in future.
| S-EPMC10965058 | biostudies-literature
Project description:Due to the development of CRISPR technology, the era of effective editing of target genes has arrived. However, the offtarget problem that occurs when recognizing target DNA due to the inherent nature of CRISPR components remains the biggest task to be overcome in the future. In this review, the principle of inducing such unintended off-target editing is analyzed from the structural aspect of CRISPR, and the methodology that has been developed to reduce off-target editing until now is summarized. [BMB Reports 2024; 57(1): 12-18].
| S-EPMC10828434 | biostudies-literature
Project description:The ability to rationally target disease-causing mutations has been made possible with programmable nucleases with the CRISPR/Cas9 system representing a facile platform for individualized gene-based medicine. In this study we employed footprint free reprogramming of fibroblasts from a patient with mutations to the Fanconi anemia I (FANCI) gene to generate induced pluripotent stem cells (iPSC). This process was accomplished without gene complementation and the resultant iPSC were able to be gene corrected in a robust manner using the Cas9 nickase. The self-renewing iPSC that were maintained under feeder free conditions were differentiated into cells with characteristics of definitive hematopoiesis. This defined and highly efficient procedure employed small molecule modulation of the hematopoietic differentiation pathway and a vascular induction technique to generate hematopoietic progenitors. In sum, our results demonstrate the ability to induce patient derived FA cells to pluripotency for patient specific therapeutic cell derivation.
| S-EPMC5035838 | biostudies-other
Project description:B cells offer unique opportunities for gene therapy because of their ability to secrete large amounts of protein in the form of antibody and persist for the life of the organism as plasma cells. Here, we report optimized CRISPR/Cas9 based genome engineering of primary human B cells. Our procedure involves enrichment of CD19+ B cells from PBMCs followed by activation, expansion, and electroporation of CRISPR/Cas9 reagents. We are able expand total B cells in culture 10-fold and outgrow the IgD+?IgM+?CD27- naïve subset from 35% to over 80% of the culture. B cells are receptive to nucleic acid delivery via electroporation 3 days after stimulation, peaking at Day 7 post stimulation. We tested chemically modified sgRNAs and Alt-R gRNAs targeting CD19 with Cas9 mRNA or Cas9 protein. Using this system, we achieved genetic and protein knockout of CD19 at rates over 70%. Finally, we tested sgRNAs targeting the AAVS1 safe harbor site using Cas9 protein in combination with AAV6 to deliver donor template encoding a splice acceptor-EGFP cassette, which yielded site-specific integration frequencies up to 25%. The development of methods for genetically engineered B cells opens the door to a myriad of applications in basic research, antibody production, and cellular therapeutics.
| S-EPMC6092381 | biostudies-literature