Extra (embryonic) dialogues: Keys to improved stem cell-based embryo models.

Despite advances in stem cell cultures, modeling early human development with stem cells in a dish remains challenging. Research by Hislop et al.,1 Okubo et al.,2 and Wei et al.3 paves the way for improved in vitro embryo models and culture conditions, offering valuable insights for regenerative medicine.

ßTrCP1 promotes SLC35F2 protein ubiquitination and inhibits cancer progression in HeLa cells.

The solute carrier family 35 F2 (SLC35F2) belongs to membrane-bound carrier proteins that are associated with multiple cancers. The main factor that determines cancer progression is the expression level of SLC35F2. Thus, identifying the E3 ligase that controls SLC35F2 protein abundance in cancer cells is critical. Here, we identified ßTrCP1 interacting with and reducing the SLC35F2 protein level. ßTrCP1 signals SLC35F2 protein ubiquitination and reduces SLC35F2 protein half-life. The mRNA expression pattern between ßTrCP1 and SLC35F2 across a panel of cancer cell lines showed a negative correlation. Additionally, the depletion of ßTrCP1 accumulated SLC35F2 protein and promoted SLC35F2-mediated cell growth, migration, invasion, and colony formation ability in HeLa cells. Overall, we demonstrate that ßTrCP1 acts as a tumor suppressor by controlling SLC35F2 protein abundance in cancer cells. The depletion of ßTrCP1 promotes SLC35F2-mediated carcinogenesis. Thus, we envision that ßTrCP1 may be a potential target for cancer therapeutics.

E3 ubiquitin ligase APC/CCdh1 regulates SLC35F2 protein turnover and inhibits cancer progression in HeLa cells.

BACKGROUND: The solute carrier family 35 F2 (SLC35F2), belongs to membrane-bound carrier proteins that control various physiological functions and are activated in several cancers. However, the molecular mechanism regulating SLC35F2 protein turnover and its implication in cancer progression remains unexplored. Therefore, screening for E3 ligases that promote SLC35F2 protein degradation is essential during cancer progression. METHODS: The immunoprecipitation and Duolink proximity ligation assays (PLA) were used to determine the interaction between APC/CCdh1 and SLC35F2 proteins. A CRISPR/Cas9-mediated knockdown and rescue experiment were used to validate the functional significance of APC/CCdh1 on SLC35F2 protein stabilization. The ubiquitination function of APC/CCdh1 on SLC35F2 protein was validated using in vitro ubiquitination assay and half-life analysis. The role of APC/CCdh1 regulating SLC35F2-mediated tumorigenesis was confirmed by in vitro oncogenic experiments in HeLa cells. RESULTS: Based on the E3 ligase screen and in vitro biochemical experiments, we identified that APC/CCdh1 interacts with and reduces SLC35F2 protein level. APC/CCdh1 promotes SLC35F2 ubiquitination and decreases the half-life of SLC35F2 protein. On the other hand, the CRISPR/Cas9-mediated depletion of APC/CCdh1 increased SLC35F2 protein levels. The mRNA expression analysis revealed a negative correlation between APC/CCdh1 and SLC35F2 across a panel of cancer cell lines tested. Additionally, we demonstrated that depletion in APC/CCdh1 promotes SLC35F2-mediated cell proliferation, colony formation, migration, and invasion in HeLa cells. CONCLUSION: Our study highlights that APC/CCdh1 is a critical regulator of SLC35F2 protein turnover and depletion of APC/CCdh1 promotes SLC35F2-mediated tumorigenesis. Thus, we envision that APC/CCdh1-SLC35F2 axis might be a therapeutic target in cancer.

Quantification of Genetic Heterogeneity Using Long-Read Targeted Individual DNA Molecule Sequencing.

Understanding genetic heterogeneity is of paramount importance in unraveling the intricate functioning of biological systems, as it contributes to the diversity of phenotypes of gene-environment interactions. We have developed a method termed targeted Individual DNA Molecule Sequencing (IDMseq) to accurately quantify genetic heterogeneity within cell populations, even those with rare variants present at low frequencies. IDMseq ensures that each original DNA molecule is distinctively represented by one unique molecule identifier (UMI) group, preventing false UMI groups and enabling precise quantification of allele frequency within the original population. IDMseq is a versatile sequencing technique that combines error correction and long-read sequencing, enabling sensitive detection of various genetic variants, including single nucleotide variants and large structural variants in both basic and clinical research settings. This protocol provides a comprehensive, step-by-step guide to preparing samples and performing IDMseq to determine genetic variations. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: UMI labeling and amplification of DNA Support Protocol 1: AMPure XP beads cleanup Support Protocol 2: Suggested data analysis pipeline.

USP19 Negatively Regulates p53 and Promotes Cervical Cancer Progression.

p53 is a tumor suppressor gene activated in response to cellular stressors that inhibits cell cycle progression and induces pro-apoptotic signaling. The protein level of p53 is well balanced by the action of several E3 ligases and deubiquitinating enzymes (DUBs). Several DUBs have been reported to negatively regulate and promote p53 degradation in tumors. In this study, we identified USP19 as a negative regulator of p53 protein level. We demonstrate a direct interaction between USP19 and p53 by pull down assay. The overexpression of USP19 promoted ubiquitination of p53 and reduced its protein half-life. We also demonstrate that CRISPR/Cas9-mediated knockout of USP19 in cervical cancer cells elevates p53 protein levels, resulting in reduced colony formation, cell migration, and cell invasion. Overall, our results indicate that USP19 negatively regulates p53 protein levels in cervical cancer progression.

Dual role of deubiquitinating enzyme USP19 regulates mitotic progression and tumorigenesis by stabilizing survivin.

Survivin is a component of the chromosomal passenger complex, which includes Aurora B, INCENP, and Borealin, and is required for chromosome segregation and cytokinesis. We performed a genome-wide screen of deubiquitinating enzymes for survivin. For the first time, we report that USP19 has a dual role in the modulation of mitosis and tumorigenesis by regulating survivin expression. Our results found that USP19 stabilizes and interacts with survivin in HCT116 cells. USP19 deubiquitinates survivin protein and extends its half-life. We also found that USP19 functions as a mitotic regulator by controlling the downstream signaling of survivin protein. Targeted genome knockout verified that USP19 depletion leads to several mitotic defects, including cytokinesis failure. In addition, USP19 depletion results in significant enrichment of apoptosis and reduces the growth of tumors in the mouse xenograft. We envision that simultaneous targeting of USP19 and survivin in oncologic drug development would increase therapeutic value and minimize redundancy.

CRISPR/Cas9-based genome-wide screening for deubiquitinase subfamily identifies USP1 regulating MAST1-driven cisplatin-resistance in cancer cells.

Background: Cisplatin is one of the frontline anticancer agents. However, development of cisplatin-resistance limits the therapeutic efficacy of cisplatin-based treatment. The expression of microtubule-associated serine/threonine kinase 1 (MAST1) is a primary factor driving cisplatin-resistance in cancers by rewiring the MEK pathway. However, the mechanisms responsible for MAST1 regulation in conferring drug resistance is unknown. Methods: We implemented a CRISPR/Cas9-based, genome-wide, dual screening system to identify deubiquitinating enzymes (DUBs) that govern cisplatin resistance and regulate MAST1 protein level. We analyzed K48- and K63-linked polyubiquitination of MAST1 protein and mapped the interacting domain between USP1 and MAST1 by immunoprecipitation assay. The deubiquitinating effect of USP1 on MAST1 protein was validated using rescue experiments, in vitro deubiquitination assay, immunoprecipitation assays, and half-life analysis. Furthermore, USP1-knockout A549 lung cancer cells were generated to validate the deubiquitinating activity of USP1 on MAST1 abundance. The USP1-MAST1 correlation was evaluated using bioinformatics tool and in different human clinical tissues. The potential role of USP1 in regulating MAST1-mediated cisplatin resistance was confirmed using a series of in vitro and in vivo experiments. Finally, the clinical relevance of the USP1-MAST1 axis was validated by application of small-molecule inhibitors in a lung cancer xenograft model in NSG mice. Results: The CRISPR/Cas9-based dual screening system identified USP1 as a novel deubiquitinase that interacts, stabilizes, and extends the half-life of MAST1 by preventing its K48-linked polyubiquitination. The expression analysis across human clinical tissues revealed a positive correlation between USP1 and MAST1. USP1 promotes MAST1-mediated MEK1 activation as an underlying mechanism that contributes to cisplatin-resistance in cancers. Loss of USP1 led to attenuation of MAST1-mediated cisplatin-resistance both in vitro and in vivo. The combined pharmacological inhibition of USP1 and MAST1 using small-molecule inhibitors further abrogated MAST1 level and synergistically enhanced cisplatin efficacy in a mouse xenograft model. Conclusions: Overall, our study highlights the role of USP1 in the development of cisplatin resistance and uncovers the regulatory mechanism of MAST1-mediated cisplatin resistance in cancers. Co-treatment with USP1 and MAST1 inhibitors abrogated tumor growth and synergistically enhanced cisplatin efficacy, suggesting a novel alternative combinatorial therapeutic strategy that could further improve MAST1-based therapy in patients with cisplatin-resistant tumors.

The role of the CRISPR-Cas system in cancer drug development: Mechanisms of action and therapy.

BACKGROUND: The recent emergence of gene editing using Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated system (Cas) tools and advances in genomics and proteomics has revolutionized drug discovery and personalized medicine. PURPOSE AND SCOPE: The CRISPR-Cas system has enabled gene and cell-based therapies, screening for novel drug targets, a new generation of disease models, elucidation of drug resistance mechanisms, and drug efficacy testing. Here, we summarized recent investigations and strategies involved in cancer-related drug discovery using the CRISPR-Cas system. CONCLUSION: CRISPR-Cas-mediated gene editing has shown great potential in the development of next generation drugs for treatment of Mendelian disorders and various cancer types. In this review, we focused on the impact of the CRISPR-Cas system in drug discovery and its application to biomarker identification and validation, high-end target genes, and breakthrough anticancer cell therapies. We also highlighted the role of CRISPR-Cas in precision disease modeling and functional drug screening.

USP32 confers cancer cell resistance to YM155 via promoting ER-associated degradation of solute carrier protein SLC35F2.

Background: The most commonly preferred chemotherapeutic agents to treat cancers are small-molecule drugs. However, the differential sensitivity of various cancer cells to small molecules and untargeted delivery narrow the range of potential therapeutic applications. The mechanisms responsible for drug resistance in a variety of cancer cells are also largely unknown. Several deubiquitinating enzymes (DUBs) are the main determinants of drug resistance in cancer cells. Methods: We used CRISPR-Cas9 to perform genome-scale knockout of the entire set of genes encoding ubiquitin-specific proteases (USPs) and systematically screened for DUBs resistant to the clinically evaluated anticancer compound YM155. A series of in vitro and in vivo experiments were conducted to reveal the relationship between USP32 and SLC35F2 on YM155-mediated DNA damage in cancer cells. Results: CRISPR-based dual-screening method identified USP32 as a novel DUB that governs resistance for uptake of YM155 by destabilizing protein levels of SLC35F2, a solute-carrier protein essential for the uptake of YM155. The expression of USP32 and SLC35F2 was negatively correlated across a panel of tested cancer cell lines. YM155-resistant cancer cells in particular exhibited elevated expression of USP32 and low expression of SLC35F2. Conclusion: Collectively, our DUB-screening strategy revealed a resistance mechanism governed by USP32 associated with YM155 resistance in breast cancers, one that presents an attractive molecular target for anti-cancer therapies. Targeted genome knockout verified that USP32 is the main determinant of SLC35F2 protein stability in vitro and in vivo, suggesting a novel way to treat tumors resistant to small-molecule drugs.

Smart Vitamin Micelles as Cancer Nanomedicines for Enhanced Intracellular Delivery of Doxorubicin.

Chemotherapy is one of the most effective treatments for cancer. However, intracellular delivery of many anticancer drugs is hindered by their hydrophobicity and low molecular weight. Here, we describe highly biocompatible and biodegradable amphiphilic vitamin conjugates comprising hydrophobic vitamin E and hydrophilic vitamin B labeled with dual pH and glutathione-responsive degradable linkages. Vitamin-based micelles (vitamicelles), formed by self-assembly in aqueous solutions, were optimized based on their stability after encapsulation of doxorubicin (DOX). The resulting vitamicelles have great potential as vehicles for anticancer drugs because they show excellent biocompatibility (>94% after 48 h of incubation) and rapid biodegradability (>90% after 2.5 h). Compared with free DOX, DOX-loaded vitamicelles showed a markedly enhanced anticancer effect as they released the drug rapidly and inhibited drug efflux out of cells efficiently. By exploiting these advantages, this study not only provides a promising strategy for circumventing existing challenges regarding the delivery of anticancer drugs but also extends the utility of current DOX-induced chemotherapy.

Ubiquitin Specific Protease 29 Functions as an Oncogene Promoting Tumorigenesis in Colorectal Carcinoma.

Colorectal carcinoma is the third foremost cause of cancer-related deaths and accounts for 5.8% of all deaths globally. The molecular mechanisms of colon cancer progression and metastasis control are not well studied. Ubiquitin-specific protease 29 (USP29), a deubiquitinating enzyme, is involved in the occurrence and development of wide variety of cancers. However, its clinical significance and biological roles in colorectal carcinoma (CRC) remain unexplored. In this research, we observed that the rate of USP29 overexpression was higher in colon cancer patient tissues relative to its corresponding normal tissues. CRISPR-Cas9-mediated depletion of USP29 triggered DNA double strand breaks and delayed cell-cycle progression in HCT116 cells. We also demonstrated that USP29 depletion hampers the colony formation and increases apoptosis of HCT116 cells. USP29 knockdown significantly decreased CRC cell proliferation in vitro. Depletion of USP29 in HCT116 cells substantially reduced the tumor volume of mouse xenografts. In conclusion, our study shows that elevated expression of USP29 promotes malignancy in CRC, suggesting that USP29 could be a promising target for colon cancer therapy.

Ubiquitin-Specific Protease 29 Regulates Cdc25A-Mediated Tumorigenesis.

Cell division cycle 25A (Cdc25A) is a dual-specificity phosphatase that is overexpressed in several cancer cells and promotes tumorigenesis. In normal cells, Cdc25A expression is regulated tightly, but the changes in expression patterns in cancer cells that lead to tumorigenesis are unknown. In this study, we showed that ubiquitin-specific protease 29 (USP29) stabilized Cdc25A protein expression in cancer cell lines by protecting it from ubiquitin-mediated proteasomal degradation. The presence of USP29 effectively blocked polyubiquitination of Cdc25A and extended its half-life. CRISPR-Cas9-mediated knockdown of USP29 in HeLa cells resulted in cell cycle arrest at the G0/G1 phase. We also showed that USP29 knockdown hampered Cdc25A-mediated cell proliferation, migration, and invasion of cancer cells in vitro. Moreover, NSG nude mice transplanted with USP29-depleted cells significantly reduced the size of the tumors, whereas the reconstitution of Cdc25A in USP29-depleted cells significantly increased the tumor size. Altogether, our results implied that USP29 promoted cell cycle progression and oncogenic transformation by regulating protein turnover of Cdc25A.

E3 Ubiquitin Ligase APC/CCdh1 Regulation of Phenylalanine Hydroxylase Stability and Function.

Phenylketonuria (PKU) is an autosomal recessive metabolic disorder caused by the dysfunction of the enzyme phenylalanine hydroxylase (PAH). Alterations in the level of PAH leads to the toxic accumulation of phenylalanine in the blood and brain. Protein degradation mediated by ubiquitination is a principal cellular process for maintaining protein homeostasis. Therefore, it is important to identify the E3 ligases responsible for PAH turnover and proteostasis. Here, we report that anaphase-promoting complex/cyclosome-Cdh1 (APC/C)Cdh1 is an E3 ubiquitin ligase complex that interacts and promotes the polyubiquitination of PAH through the 26S proteasomal pathway. Cdh1 destabilizes and declines the half-life of PAH. In contrast, the CRISPR/Cas9-mediated knockout of Cdh1 stabilizes PAH expression and enhances phenylalanine metabolism. Additionally, our current study demonstrates the clinical relevance of PAH and Cdh1 correlation in hepatocellular carcinoma (HCC). Overall, we show that PAH is a prognostic marker for HCC and Cdh1 could be a potential therapeutic target to regulate PAH-mediated physiological and metabolic disorders.

YM155 sensitizes HeLa cells to TRAIL-mediated apoptosis via cFLIP and survivin downregulation.

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis is a safe method for the treatment of various types of cancer. However, TRAIL therapy is less effective in certain types of cancer, including cervical cancer. To address this problem, a combinatorial approach was employed to sensitize cervical cancer at low dosages. YM155, a survivin inhibitor, was used at low dosages along with TRAIL to induce apoptosis in HeLa cells. The effects of the individual treatment with TRAIL and YM155 on apoptosis were assessed by propidium iodide assay. In addition, to validate the DNA damage exhibited by the combination treatment, the phosphorylation status of γH2A histone family member X was investigated by immunofluorescence and western blot analysis. TRAIL or YM155 alone had no significant effect on DNA damage and apoptosis. However, the TRAIL/YM155 combination triggered a synergistic pro-apoptotic stimulus in HeLa cells. The mRNA and protein levels of CASP8- and FADD-like apoptosis regulator (cFLIP), death receptor 5 (DR5) and survivin were monitored using RT-PCR and western blot analysis, respectively. This combinatorial approach downregulated both mRNA and protein expression levels of cFLIP and survivin. Further experimental results suggested that the combination treatment significantly reduced cell viability, invasion and migration of HeLa cells. Overall, the present findings indicated that the low dosage of YM155 sensitized HeLa cells to TRAIL-induced apoptosis via a mechanism involving downregulation of cFLIP and survivin. The results indicated the importance of combination drug treatment and reveal an effective therapeutic alternative for TRAIL therapy in human cervical cancer.

HAUSP stabilizes Cdc25A and protects cervical cancer cells from DNA damage response.

Resistance to DNA-damaging agents is one of the main reasons for the low survival of cervical cancer patients. Previous reports have suggested that the Cdc25A oncoprotein significantly affects the level of susceptibility to DNA-damaging agents, but the molecular mechanism remains unclear. In this study, we used Western blot and flow cytometry analyses to demonstrate that the deubiquitinating enzyme HAUSP stabilizes Cdc25A protein level. Furthermore, in a co-immunoprecipitation assay, we found that HAUSP interacts with and deubiquitinates Cdc25A both exogenously and endogenously. HAUSP extends the half-life of the Cdc25A protein by circumventing turnover. HAUSP knockout in HeLa cells using the CRISPR/Cas9 system caused a significant delay in Cdc25A-mediated cell cycle progression, cell migration, and colony formation and attenuated tumor progression in a mouse xenograft model. Furthermore, HAUSP-mediated stabilization of the Cdc25A protein produced enhanced resistance to DNA-damaging agents. Overall, our study suggests that targeting Cdc25A and HAUSP could be a promising combinatorial approach to halt progression and minimize antineoplastic resistance in cervical cancer.

Genome-scale screening of deubiquitinase subfamily identifies USP3 as a stabilizer of Cdc25A regulating cell cycle in cancer.

Conventional screening methods for deubiquitinating enzymes (DUBs) have important limitations. A loss-of-function study based on the knockout of DUB genes in mammalian cells can provide an excellent model for exploring DUB function. Here, we used CRISPR-Cas9 to perform genome-scale knockout of the entire set of genes encoding ubiquitin-specific proteases (USPs), a DUB subfamily, and then systematically screened for DUBs that stabilize the Cdc25A oncoprotein. USP3 was identified as a deubiquitinase of Cdc25A. USP3 depletion reduces the Cdc25A protein level, resulting in a significant delay in cell-cycle progression, and reduces the growth of cervical tumor xenografts in nude mice. Clinically, USP3 expression is positively correlated with Cdc25A protein expression and the poorest survival in breast cancer. We envision that our DUB knockout library kit will facilitate genome-scale screening of functional DUBs for target proteins of interest in a wide range of biomedical fields.

CRISPR-mediated upregulation of DR5 and downregulation of cFLIP synergistically sensitize HeLa cells to TRAIL-mediated apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has received attention as an anticancer therapy because it mediates apoptosis of several cancer cell types but not normal human cell types. In this study, we implemented genome editing techniques to upregulate DR5 and downregulate cFLIP in HeLa cells to stimulate TRAIL-induced apoptosis. We designed and validated sgRNAs to enrich the endogenous level of DR5 by dead Cas9 (dCas9). Similarly, we designed two sgRNAs to disrupt the cFLIP gene by CRISPR/Cas9. We analyzed the effect of TRAIL on tumor cells by co-transfecting HeLa cells with the best combinations of sgRNAs regulating DR5 and cFLIP genes. TRAIL-induced apoptosis in HeLa cells was evaluated by the γH2AX foci formation assay to check for double-strand break and propidium iodide and Annexin V staining to quantify apoptotic cells. Viable cells were identified by CCK-8 assay, and cleaved-PARP level was evaluated by Western blot. This is the first study to demonstrate that genome editing techniques can be used as an effective combinatorial treatment strategy to induce apoptosis of cancer cells. In particular, enhancement of DR5 expression and inhibition of cFLIP expression by genome editing had a synergistic effect of inhibiting proliferation and inducing apoptosis in TRAIL-resistant HeLa cells. These results suggest that combinatorial treatment strategies mediated by the CRISPR/Cas9 system may be effective for design of other human TRAIL-resistant cell types.

Deubiquitinating enzymes as cancer biomarkers: new therapeutic opportunities?

Cancer remains a life-threatening disease and accounts for the major mortality rates worldwide. The practice of using biomarkers for early detection, staging, and customized therapy may increase cancer patients' survival. Deubiquitinating enzymes (DUBs) are a family of proteases that remove ubiquitin tags from proteins of interest undergoing proteasomal degradation. DUBs play several functional roles other than deubiquitination. One of the important roles of DUBs is regulation of tumor progression. Several reports have suggested that the DUB family members were highly-elevated in various cancer cells and tissues in different stages of cancer. These findings suggest that the DUBs could be used as drug targets in cancer therapeutics. In this review, we recapitulate the role of the DUB family members, including ubiquitinspecific protease, otubain protease, and important candidates from other family members. Our aim was to better understand the connection between DUB expression profiles and cancers to allow researchers to design inhibitors or gene therapies to improve diagnosis and prognosis of cancers. [BMB Reports 2019; 52(3): 181-189].

The stability and oncogenic function of LIN28A are regulated by USP28.

RNA-binding protein LIN28A is often highly expressed in human malignant tumors and is involved in tumor metastasis and poor prognosis. Knowledge about post-translational regulatory mechanisms governing LIN28A protein stability and function is scarce. Here, we investigated the role of ubiquitination and deubiquitination on LIN28A protein stability and report that LIN28A protein undergoes ubiquitination. Ubiquitin-specific protease 28 (USP28), a deubiquitinating enzyme, interacts with and stabilizes LIN28A protein to extend its half-life. USP28, through its deubiquitinating activity, antagonizes LIN28A protein turnover by reversing its proteasomal degradation. Our study describes the consequential impacts of USP28-mediated stabilization of LIN28A protein on enhancing cancer cell viability, migration and ultimately augmenting LIN28A-mediated tumor progression. Overall, our data suggest that a synergistic, combinatorial approach of targeting LIN28A with USP28 would contribute to effective cancer therapeutics.

Different Methods of Delivering CRISPR/Cas9 Into Cells.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (Cas) is comprised of repetitive bases followed by short fragments of DNA from a previously invading organism that provide immunity to the most prokaryotic organisms. An RNA-dependent spacer is required for CRISPR/Cas9 to recognize the target DNA. Delivery of the CRISPR/Cas9-guide RNA (gRNA) complex to any cell results in modification of the target sequence. The CRISPR/Cas9-mediated genome editing technique is currently in the spotlight and has several research interests, including molecular medicine and agriculture. There are several factors that hinder the delivery of this complex, such as the large size of the plasmid or high dosage of the chemical agent. There are several methods available to deliver CRISPR/Cas9 and its components to the target cells. It includes viral, non-viral and physical methods to deliver plasmid or ribonucleoprotein (RNP) of CRISPR components. But in vivo CRISPR/Cas9 delivery remains challenging to the researchers due to insertional mutagenesis, targeted delivery, immunogenicity, and off-targets. However, studies suggesting that the CRISPR/Cas9-RNP delivery can overcome these hurdles. Here, we review the various methods for delivery of CRISPR/Cas9 and gRNA to several cell lines, highlighting the limitations of each approach, and suggest possible alternative methods.