Recent advances in CRISPR-based functional genomics for the study of disease-associated genetic variants.

Advances in sequencing technology have greatly increased our ability to gather genomic data, yet understanding the impact of genetic mutations, particularly variants of uncertain significance (VUSs), remains a challenge in precision medicine. The CRISPR‒Cas system has emerged as a pivotal tool for genome engineering, enabling the precise incorporation of specific genetic variations, including VUSs, into DNA to facilitate their functional characterization. Additionally, the integration of CRISPR‒Cas technology with sequencing tools allows the high-throughput evaluation of mutations, transforming uncertain genetic data into actionable insights. This allows researchers to comprehensively study the functional consequences of point mutations, paving the way for enhanced understanding and increasing application to precision medicine. This review summarizes the current genome editing tools utilizing CRISPR‒Cas systems and their combination with sequencing tools for functional genomics, with a focus on point mutations.

Magnetically Actuated Trigger Transient Soft Actuators Comprising On-Demand Photo-Initiated and Thermo-Degradable Polypropylene Carbonate-Photo-Acid Generator.

Lifetime-reconfigurable soft robots have emerged as a new class of robots, emphasizing the unmet needs of futuristic sustainability and security. Trigger-transient materials that can both actuate and degrade on-demand are crucial for achieving life-reconfigurable soft robots. Here, we propose the use of transient and magnetically actuating materials that can decompose under ultraviolet light and heat, achieved by adding photo-acid generator (PAG) and magnetic particles (Sr-ferrite) to poly(propylene carbonate) (PPC). Chemical and thermal analyses reveal that the mechanism of PPC-PAG decomposition occurs through PPC backbone cleavage by the photo-induced acid. The self-assembled monolayer (SAM) encapsulation of Sr-ferrite preventing the interaction with the PAG allowed the transience of magnetic soft actuators. We demonstrate remotely controllable and degradable magnetic soft kirigami actuators using blocks with various magnetized directions. This study proposes novel approaches for fabricating lifetime-configurable magnetic soft actuators applicable to diverse environments and applications, such as enclosed/sealed spaces and security/military devices.

Highly efficient genome editing via CRISPR-Cas9 ribonucleoprotein (RNP) delivery in mesenchymal stem cells.

The CRISPR-Cas9 system has significantly advanced regenerative medicine research by enabling genome editing in stem cells. Due to their desirable properties, mesenchymal stem cells (MSCs) have recently emerged as highly promising therapeutic agents, which properties include differentiation ability and cytokine production. While CRISPR-Cas9 technology is applied to develop MSC-based therapeutics, MSCs exhibit inefficient genome editing, and susceptibility to plasmid DNA. In this study, we compared and optimized plasmid DNA and RNP approaches for efficient genome engineering in MSCs. The RNP-mediated approach enabled genome editing with high indel frequency and low cytotoxicity in MSCs. By utilizing Cas9 RNPs, we successfully generated B2M-knockout MSCs, which reduced T-cell differentiation, and improved MSC survival. Furthermore, this approach enhanced the immunomodulatory effect of IFN-r priming. These findings indicate that the RNP-mediated engineering of MSC genomes can achieve high efficiency, and engineered MSCs offer potential as a promising therapeutic strategy. [BMB Reports 2024; 57(1): 60-65].

CRISPR prime editing for unconstrained correction of oncogenic KRAS variants.

KRAS is the most commonly mutated RAS family gene and is a primary cause of the occurrence of several types of cancer. However, KRAS mutations have several unique and diverse molecular identities, making it difficult to find specific treatments. Here, we developed universal pegRNAs which can correct all types of G12 and G13 oncogenic KRAS mutations with CRISPR-mediated prime editors (PEs). The universal pegRNA successfully corrected 12 types of KRAS mutations, accounting for 94% of all known KRAS mutations, by up to 54.8% correction frequency in HEK293T/17 cells. We also applied the universal pegRNA to correct endogenous KRAS mutations in human cancer cells and found that G13D KRAS mutation was successfully corrected to wild-type KRAS sequences with up to 40.6% correction frequency without indel mutations. We propose prime editing with the universal pegRNA as a 'one-to-many' potential therapeutic strategy for KRAS oncogene variants.

Dysfunction in parkin aggravates inflammatory bone erosion by reinforcing osteoclast activity.

BACKGROUND: Parkin dysfunction associated with the progression of parkinsonism contributes to a progressive systemic skeletal disease characterized by low bone mineral density. However, the role of parkin in bone remodeling has not yet been elucidated in detail. RESULT: We observed that decreased parkin in monocytes is linked to osteoclastic bone-resorbing activity. siRNA-mediated knockdown of parkin significantly enhanced the bone-resorbing activity of osteoclasts (OCs) on dentin without any changes in osteoblast differentiation. Moreover, Parkin-deficient mice exhibited an osteoporotic phenotype with a lower bone volume accompanied by increased OC-mediated bone-resorbing capacity displaying increased acetylation of α-tubulin compared to wild-type (WT) mice. Notably, compared to WT mice, the Parkin-deficient mice displayed increased susceptibility to inflammatory arthritis, reflected by a higher arthritis score and a marked bone loss after arthritis induction using K/BxN serum transfer, but not ovariectomy-induced bone loss. Intriguingly, parkin colocalized with microtubules and parkin-depleted-osteoclast precursor cells (Parkin-/- OCPs) displayed augmented ERK-dependent acetylation of α-tubulin due to failure of interaction with histone deacetylase 6 (HDAC6), which was promoted by IL-1ß signaling. The ectopic expression of parkin in Parkin-/- OCPs limited the increase in dentin resorption induced by IL-1ß, accompanied by the reduced acetylation of α-tubulin and diminished cathepsin K activity. CONCLUSION: These results indicate that a deficiency in the function of parkin caused by a decrease in parkin expression in OCPs under the inflammatory condition may enhance inflammatory bone erosion by altering microtubule dynamics to maintain OC activity.

Therapeutic gene correction for Lesch-Nyhan syndrome using CRISPR-mediated base and prime editing.

Lesch-Nyhan syndrome (LNS) is inherited as an X-linked recessive genetic disorder caused by mutations in hypoxanthine-guanine phosphoribosyl transferase 1 (HPRT1). Patients with LNS show various clinical phenotypes, including hyperuricemia, gout, devastating behavioral abnormality, intellectual disability, and self-harm. Although uric acid overproduction can be modulated with the xanthine oxidase inhibitor allopurinol, there exists no treatment for behavioral and neurological manifestations of LNS. In the current study, CRISPR-mediated base editors (BEs) and prime editors (PEs) were utilized to generate LNS-associated disease models and correct the disease models for therapeutic approach. Cytosine BEs (CBEs) were used to induce c.430C>T and c.508C>T mutations in HAP1 cells, and then adenine BEs (ABEs) were used to correct these mutations without DNA cleavage. PEs induced a c.333_334ins(A) mutation, identified in a Korean patient with LNS, in HAP1 cells, which was corrected in turn by PEs. Furthermore, improved PEs corrected the same mutation in LNS patient-derived fibroblasts by up to 14% without any unwanted mutations. These results suggest that CRISPR-mediated BEs and PEs would be suggested as a potential therapeutic strategy of this extremely rare, devastating genetic disease.

Functional Analysis of Variants in BRCA1 Using CRISPR Base Editors.

To date, methods such as fluorescent reporter assays, embryonic stem cell viability assays, and therapeutic drug-based sensitivity assays have been used to evaluate the function of the variants of uncertain significance (VUS) of the BRCA genes. However, these methods have limitations as they are associated with overexpression and do not apply to post-transcriptional regulation. Therefore, there are several VUS whose functions are unclear. Recently, we devised a new way to assess the functionality of variants in BRCA1 via a CRISPR-mediated base editor to overcome these limitations. We precisely introduced the target nucleotide substitution in living cells and identified variants whose functions were not defined. Here, we describe the methods for the functional appraisal of BRCA1 variants using CRISPR-based base editors.

Identification of Genes Involved in EGF-induced Apoptosis Using CRISPR/Cas9 Knockout Screening: Implications for Novel Therapeutic Targets in EGFR-Overexpressing Cancers.

PURPOSE: Exogenous epidermal growth factor (EGF) causes apoptosis in EGF receptor (EGFR)-overexpressing cell lines. The apoptosis-inducing factors could be a therapeutic target. We aimed to determine the mechanism of EGF-induced apoptosis using a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-based knockout screen. Materials and Methods: Two-vector system of the human genome-scale CRISPR knockout library v2 was used to target 19,050 genes using 123,411 single guide RNAs (sgRNAs). Recombinant human EGF (100 nM) or distilled water four times was administered to the experimental and control groups, respectively. The read counts of each sgRNA obtained from next-generation sequencing were analyzed using the edgeR algorithm. We used another EGFR-overexpressing cell line (A549) and short hairpin RNAs (shRNAs) targeting five EGF-resistance genes for validation. DUSP1 expression in A431, A549, and HEK293FT cells was calculated using reverse transcription-quantitative polymerase chain reaction. RESULTS: We found 77 enriched and 189 depleted genes in the experimental group using the CRISPR-based knockout screen and identified the top five EGF-resistance genes: DDX20, LHFP, REPS1, DUSP1,<.i> and KRTAP10-12. Transfecting shRNAs targeting these genes into A549 cells significantly increased the surviving fractions after EGF treatment, compared with those observed in the control shRNA-transfected cells. The expression ratio of DUSP1 (inhibits ERK signaling) increased in A431 and A549 cells after EGF treatment. However, DUSP1 expression remained unchanged in HEK293FT cells after EGF treatment. CONCLUSION: The CRISPR-based knockout screen revealed 266 genes possibly responsible for EGF-induced apoptosis. DUSP1 might be a critical component of EGF-induced apoptosis and a novel target for EGFR-overexpressing cancers.

Targeted dual base editing with Campylobacter jejuni Cas9 by single AAV-mediated delivery.

Various CRISPR‒Cas9 orthologs are used in genome engineering. One of the smallest Cas9 orthologs is cjCas9 derived from Campylobacter jejuni, which is a highly specific genome editing tool. Here, we developed cjCas9-based base editors including a cytosine base editor (cjCBEmax) and an adenine base editor (cjABE8e) that can successfully induce endogenous base substitutions by up to 91.2% at the HPD gene in HEK293T cells. Analysis of the base editing efficiency of 13 endogenous target sites showed that the active windows of cjCBEmax and cjABE8e are wider than those of spCas9-based base editors and that their specificities are slightly lower than that of cjCas9. Importantly, engineered cjCas9 and gRNA scaffolds can improve the base editing efficiency of cjABE8e by up to 6.4-fold at the HIF1A gene in HEK293T cells. Due to its small size, cjABE8e can be packaged in a single adeno-associated virus vector with two tandem arrays of gRNAs, and the delivery of the resulting AAV could introduce base substitutions at endogenous ANGPT2 and HPD target sites. Overall, our findings have expanded the potential of the use of base editors for in vivo or ex vivo therapeutic approaches.

Targeted genomic translocations and inversions generated using a paired prime editing strategy.

A variety of cancers have been found to have chromosomal rearrangements, and the genomic abnormalities often induced expression of fusion oncogenes. To date, a pair of engineered nucleases including ZFNs, TALENs, and CRISPR-Cas9 nucleases have been used to generate chromosomal rearrangement in living cells and organisms for disease modeling. However, these methods induce unwanted indel mutations at the DNA break junctions, resulting in incomplete disease modeling. Here, we developed prime editor nuclease-mediated translocation and inversion (PETI), a method for programmable chromosomal translocation and inversion using prime editor 2 nuclease (PE2 nuclease) and paired pegRNA. Using PETI method, we successfully introduced DNA recombination in episomal fluorescence reporters as well as precise chromosomal translocations in human cells. We applied PETI to create cancer-associated translocations and inversions such as NPM1-ALK and EML4-ALK in human cells. Our findings show that PETI generated chromosomal translocation and inversion in a programmable manner with efficiencies comparable of Cas9. PETI methods, we believe, could be used to create disease models or for gene therapy.

Antibody-mediated blockade for galectin-3 binding protein in tumor secretome abrogates PDAC metastasis.

The major challenges in pancreatic ductal adenocarcinoma (PDAC) management are local or distant metastasis and limited targeted therapeutics to prevent it. To identify a druggable target in tumor secretome and to explore its therapeutic intervention, we performed a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic analysis of tumors obtained from a patient-derived xenograft model of PDAC. Galectin-3 binding protein (Gal-3BP) is identified as a highly secreted protein, and its overexpression is further validated in multiple PDAC tumors and primary cells. Knockdown and exogenous treatment of Gal-3BP showed that it is required for PDAC cell proliferation, migration, and invasion. Mechanistically, we revealed that Gal-3BP enhances galectin-3-mediated epidermal growth factor receptor signaling, leading to increased cMyc and epithelial-mesenchymal transition. To explore the clinical impact of these findings, two antibody clones were developed, and they profoundly abrogated the metastasis of PDAC cells in vivo. Altogether, our data demonstrate that Gal-3BP is an important therapeutic target in PDAC, and we propose its blockade by antibody as a therapeutic option for suppressing PDAC metastasis.

Highly efficient heat-dissipation power driven by ferromagnetic resonance in MFe2O4 (M = Fe, Mn, Ni) ferrite nanoparticles.

We experimentally demonstrated that heat-dissipation power driven by ferromagnetic resonance (FMR) in superparamagnetic nanoparticles of ferrimagnetic MFe2O4 (M = Fe, Mn, Ni) gives rise to highly localized incrementation of targeted temperatures. The power generated thereby is extremely high: two orders of magnitude higher than that of the conventional Néel-Brownian model. From micromagnetic simulation and analytical derivation, we found robust correlations between the temperature increment and the intrinsic material parameters of the damping constant as well as the saturation magnetizations of the nanoparticles' constituent materials. Furthermore, the magnetization-dissipation-driven temperature increments were reliably manipulated by extremely low strengths of applied AC magnetic fields under resonance field conditions. Our experimental results and theoretical formulations provide for a better understanding of the effect of FMR on the efficiency of heat generation as well as straightforward guidance for the design of advanced materials for control of highly localized incrementation of targeted temperatures using magnetic particles in, for example, magnetic hyperthermia bio-applications.

AWP1 Restrains the Aggressive Behavior of Breast Cancer Cells Induced by TNF-α.

TNF-α plays a crucial role in cancer initiation and progression by enhancing cancer cell proliferation, survival, and migration. Even though the known functional role of AWP1 (zinc finger AN1 type-6, ZFAND6) is as a key mediator of TNF-α signaling, its potential role in the TNF-α-dependent responses of cancer cells remains unclear. In our current study, we found that an AWP1 knockdown using short hairpin RNAs increases the migratory potential of non-aggressive MCF-7 breast cancer cells with no significant alteration of their proliferation in response to TNF-α. A CRISPR/Cas9-mediated AWP1 knockout in MCF-7 cells led to mesenchymal cell type morphological changes and an accelerated motility. TNF-α administration further increased this migratory capacity of these AWP1-depleted cells through the activation of NF-κB accompanied by increased epithelial-mesenchymal transition-related gene expression. In particular, an AWP1 depletion augmented the expression of Nox1, reactive oxygen species (ROS) generating enzymes, and ROS levels and subsequently promoted the migratory potential of MCF-7 cells mediated by TNF-α. These TNF-α-mediated increases in the chemotactic migration of AWP1 knockout cells were completely abrogated by an NF-κB inhibitor and a ROS scavenger. Our results suggest that a loss-of-function of AWP1 alters the TNF-α response of non-aggressive breast cancer cells by potentiating ROS-dependent NF-κB activation.

Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors.

Recent studies have investigated the risks associated with BRCA1 gene mutations using various functional assessment methods such as fluorescent reporter assays, embryonic stem cell viability assays, and therapeutic drug-based sensitivity assays. Although they have clarified a lot of BRCA1 variants, these assays involving the use of exogenously expressed BRCA1 variants are associated with overexpression issues and cannot be applied to post-transcriptional regulation. To resolve these limitations, we previously reported a method for functional analysis of BRCA1 variants via CRISPR-mediated cytosine base editor that induce targeted nucleotide substitution in living cells. Using this method, we identified variants whose functions remain ambiguous, including c.-97C>T, c.154C>T, c.3847C>T, c.5056C>T, and c.4986+5G>A, and confirmed that CRISPR-mediated base editors are useful tools for reclassifying the variants of uncertain significance in BRCA1. Here, we describe a protocol for functional analysis of BRCA1 variants using CRISPR-based cytosine base editor. This protocol provides guidelines for the selection of target sites, functional analysis and evaluation of BRCA1 variants.

Ultra-high rate of temperature increment from superparamagnetic nanoparticles for highly efficient hyperthermia.

The magneto-thermal effect, which represents the conversion of magnetostatic energy to heat from magnetic materials, has been spotlighted for potential therapeutic usage in hyperthermia treatments. However, the realization of its potential has been challenged owing to the limited heating from the magnetic nanoparticles. Here, we explored a new-concept of magneto-thermal modality marked by low-power-driven, fast resonant spin-excitation followed by consequent energy dissipation, which concept has yet to be realized for current hyperthermia applications. We investigated the effect of spin resonance-mediated heat dissipation using superparamagnetic Fe3O4 nanoparticles and achieved an extraordinary initial temperature increment rate of more than 150 K/s, which is a significant increase in comparison to that for the conventional magnetic heat induction of nanoparticles. This work would offer highly efficient heat generation and precision wireless controllability for realization of magnetic-hyperthermia-based medical treatment.

Small-molecule inhibitors of histone deacetylase improve CRISPR-based adenine base editing.

CRISPR-based base editors (BEs) are widely used to induce nucleotide substitutions in living cells and organisms without causing the damaging DNA double-strand breaks and DNA donor templates. Cytosine BEs that induce C:G to T:A conversion and adenine BEs that induce A:T to G:C conversion have been developed. Various attempts have been made to increase the efficiency of both BEs; however, their activities need to be improved for further applications. Here, we describe a fluorescent reporter-based drug screening platform to identify novel chemicals with the goal of improving adenine base editing efficiency. The reporter system revealed that histone deacetylase inhibitors, particularly romidepsin, enhanced base editing efficiencies by up to 4.9-fold by increasing the expression levels of proteins and target accessibility. The results support the use of romidepsin as a viable option to improve base editing efficiency in biomedical research and therapeutic genome engineering.

Engineered prime editors with PAM flexibility.

Although prime editors are a powerful tool for genome editing, which can generate various types of mutations such as nucleotide substitutions, insertions, and deletions in the genome without double-strand breaks or donor DNA, the conventional prime editors are still limited to their target scopes because of the PAM preference of the Streptococcus pyogenes Cas9 (spCas9) protein. Here, we describe the engineered prime editors to expand the range of their target sites using various PAM-flexible Cas9 variants. Using the engineered prime editors, we could successfully generate more than 50 types of mutations with up to 51.7% prime-editing activity in HEK293T cells. In addition, we successfully introduced the BRAF V600E mutation, which could not be induced by conventional prime editors. These variants of prime editors will broaden the applicability of CRISPR-based prime editing technologies in biological research.

Gene Manipulation Using Fusion Guide RNAs for Cas9 and Cas12a.

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) protein has emerged as a genome engineering tool for various organisms. Known as the CRISPR-Cas system, Cas endonucleases such as Cas9 and Cas12a (also known as Cpf1) and guide RNA (gRNA) complexes recognize and cleave the target DNA, allowing for targeted gene manipulation. Along with the Cas protein engineering, gRNA engineering has broadened the applications of the CRISPR-Cas system. Recently, we have developed fusion guide RNAs (fgRNAs) for orthogonal gene manipulation using Cas9 and Cas12a. Here, we describe the methods for designing and generating fgRNAs-expression constructs to achieve multiplex genome editing and gene manipulation in human cells.

Protein Translocation Acquires Substrate Selectivity Through ER Stress-Induced Reassembly of Translocon Auxiliary Components.

Protein import across the endoplasmic reticulum membrane is physiologically regulated in a substrate-selective manner to ensure the protection of stressed ER from the overload of misfolded proteins. However, it is poorly understood how different types of substrates are accurately distinguished and disqualified during translocational regulation. In this study, we found poorly assembled translocon-associated protein (TRAP) complexes in stressed ER. Immunoaffinity purification identified calnexin in the TRAP complex in which poor assembly inhibited membrane insertion of the prion protein (PrP) in a transmembrane sequence-selective manner, through translocational regulation. This reaction was induced selectively by redox perturbation, rather than calcium depletion, in the ER. The liberation of ERp57 from calnexin appeared to be the reason for the redox sensitivity. Stress-independent disruption of the TRAP complex prevented a pathogenic transmembrane form of PrP (ctmPrP) from accumulating in the ER. This study uncovered a previously unappreciated role for calnexin in assisting the redox-sensitive function of the TRAP complex and provided insights into the ER stress-induced reassembly of translocon auxiliary components as a key mechanism by which protein translocation acquires substrate selectivity.

Impact of proactive high-throughput functional assay data on BRCA1 variant interpretation in 3684 patients with breast or ovarian cancer.

The clinical utility of BRCA1/2 genotyping was recently extended from the selection of subjects at high risk for hereditary breast and ovary cancer to the identification of candidates for poly (ADP-ribose) polymerase (PARP) inhibitor treatment. This underscores the importance of accurate interpretation of BRCA1/2 genetic variants and of reducing the number of variants of uncertain significance (VUSs). Two recent studies by Findlay et al. and Starita et al. introduced high-throughput functional assays, and proactively analyzed variants in specific regions regardless of whether they had been previously observed. We retrospectively reviewed all BRCA1 and BRCA2 germline genetic test reports from patients with breast or ovarian cancer examined at Asan Medical Center (Seoul, Korea) between September 2011 and December 2018. Variants were assigned pathogenic or benign strong evidence codes according to the functional classification and were reclassified according to the ACMG/AMP 2015 guidelines. Among 3684 patients with available BRCA1 and BRCA2 germline genetic test reports, 429 unique variants (181 from BRCA1) were identified. Of 34 BRCA1 variants intersecting with the data reported by Findlay et al., three missense single-nucleotide variants from four patients (0.11%, 4/3684) were reclassified from VUSs to likely pathogenic variants. Four variants scored as functional were reclassified into benign or likely benign variants. Three variants that overlapped with the data reported by Starita et al. could not be reclassified. In conclusion, proactive high-throughput functional study data are useful for the reclassification of clinically observed VUSs. Integrating additional evidence, including functional assay results, may help reduce the number of VUSs.