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Inducible in vivo genome editing with CRISPR-Cas9
Abstract
CRISPR-Cas9-based genome editing enables the rapid genetic manipulation of any genomic locus without the need for gene targeting by homologous recombination. Here we describe a conditional transgenic approach that allows temporal control of CRISPR-Cas9 activity for inducible genome editing in adult mice. We show that doxycycline-regulated Cas9 induction enables widespread gene disruption in multiple tissues and that limiting the duration of Cas9 expression or using a Cas9(D10A) (Cas9n) variant can regulate the frequency and size of target gene modifications, respectively. Further, we show that this inducible CRISPR (iCRISPR) system can be used effectively to create biallelic mutation in multiple target loci and, thus, provides a flexible and fast platform to study loss-of-function phenotypes in vivo.
... kb), the overall editing efficiency was low because lentivirus or adeno-associated virus is inefficient to load elements with such a large size [108,109]. Several Cas9-expressing mouse lines were generated to overcome this problem [110][111][112][113][114]. These mouse models enable in vivo genome editing to be conveniently and efficiently performed [102,113,115]. ...
... Mounting the ubiquitously constitutive Cas9 expression system into animal models could dramatically reduce the payload size of viral vectors and the number of components required to be delivered, thus promoting in vivo gene editing efficiency [110][111][112][113]144], but is unable to control the expression of Cas9 protein temporally or spatially. The ubiquitous and sustainable expression of Cas9 could cause genomic damage [145,146], off-target effects [147,148], and immunological clearance responses [149]. ...
Conditional gene regulation systems can control gene expression in predefined tissues or organs at a desired time. Site‐specific recombinase systems and chemically induced gene expression systems are the two most widely used approaches for creating genetically modified (GM) animals with conditional regulation of gene expression. Generation of GM pigs with controllable elements, usually involving multiple gene editing, used to be a major challenge due to a lack of germ line‐competent pluripotent stem cells. With the emergence of artificial endonuclease‐mediated gene editors, a variety of GM pigs with recombinase‐specific recognition elements or chemically induced elements for conditional regulation of gene expression have been generated by the combination of site‐directed knock‐in of somatic cells and somatic cell nuclear transfer technology, allowing conditional deletion of endogenous genes or overexpression of exogenous genes in pigs. These inducible tool pig models will greatly facilitate the production of GM pigs and broaden the applications of transgenic pigs in biomedicine and agriculture fields. In this paper, we review the progress in the construction and application of pigs with controllable elements using gene editing techniques.
... Six paired single guide RNAs (gRNAs) for targeting of the mouse Bap1, Pbrm1, and Setd2 genes were each cloned into the BbsI site of the pX461 plasmid, Addgene #48,140 ( Table 1). The six U6 gRNA cassettes were amplified by PCR and NsiI/ SbfI-cut PCR fragments (390 bp) were cloned sequentially into NsiI-opened, dephosphorylated targeting construct c3GIC9n, Addgene #62,192 17 . See Table 1 for details. ...
... For positive controls we utilized founder mice, which in addition to the BPS allele harbor a R26/rtTA allele 22 . When R26/rtTA mice are provided with dox supplemented drinking water, the R26/rtTA allele, which encodes expression of the reverse tet-transactivator (rtTA, Tet-On), causes a potent activation of the TRE3G promoter in the intestine, skin, and thymus 17 . The intestines from the BPS founder mice consequently served as positive controls for genome editing ( Fig. 2A). ...
Genetically engineered mouse models (GEMMs) are important immunocompetent models for research into the roles of individual genes in cancer and the development of novel therapies. Here we use inducible CRISPR-Cas9 systems to develop two GEMMs which aim to model the extensive chromosome p3 deletion frequently observed in clear cell renal cell carcinoma (ccRCC). We cloned paired guide RNAs targeting early exons of Bap1, Pbrm1, and Setd2 in a construct containing a Cas9D10A (nickase, hSpCsn1n) driven by tetracycline (tet)-responsive elements (TRE3G) to develop our first GEMM. The founder mouse was crossed with two previously established transgenic lines, one carrying the tet-transactivator (tTA, Tet-Off) and one with a triple-mutant stabilized HIF1A-M3 (TRAnsgenic Cancer of the Kidney, TRACK), both driven by a truncated, proximal tubule-specific γ-glutamyltransferase 1 (ggt or γGT) promoter, to create triple-transgenic animals. Our results indicate that this model (BPS-TA) induces low numbers of somatic mutations in Bap1 and Pbrm1 (but not in Setd2), known tumor suppressor genes in human ccRCC. These mutations, largely restricted to kidneys and testis, induced no detectable tissue transformation in a cohort of 13 month old mice (N = 10). To gain insights into the low frequencies of insertions and deletions (indels) in BPS-TA mice we analyzed wild type (WT, N = 7) and BPS-TA (N = 4) kidneys by RNAseq. This showed activation of both DNA damage and immune response, suggesting activation of tumor suppressive mechanisms in response to genome editing. We then modified our approach by generating a second model in which a ggt-driven, cre-regulated Cas9WT(hSpCsn1) was employed to introduce Bap1, Pbrm1, and Setd2 genome edits in the TRACK line (BPS-Cre). The BPS-TA and BPS-Cre lines are both tightly controlled in a spatiotemporal manner with doxycycline (dox) and tamoxifen (tam), respectively. In addition, whereas the BPS-TA line relies on paired guide RNAs (gRNAs), the BPS-Cre line requires only single gRNAs for gene perturbation. In the BPS-Cre we identified increased Pbrm1 gene-editing frequencies compared to the BPS-TA model. Whereas we did not detect Setd2 edits in the BPS-TA kidneys, we found extensive editing of Setd2 in the BPS-Cre model. Bap1 editing efficiencies were comparable between the two models. Although no gross malignancies were observed in our study, this is the first reported GEMM which models the extensive chromosome 3p deletion frequently observed in kidney cancer patients. Further studies are required (1) to model more extensive 3p deletions, e.g. impacting additional genes, and (2) to increase the cellular resolution, e.g. by employing single-cell RNAseq to ascertain the effects of specific combinatorial gene inactivation.
... The Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated system (CRISPR/Cas) is a widely applicable gene-editing tool extensively employed in the study of gene function across various species [1][2][3][4][5][6]. The CRISPR/Cas system consists of two main components: single-guide RNA (sgRNA) and Cas protein. ...
The CRISPR/Cas9 gene-editing system is a standard technique in functional genomics, with widespread applications. However, the establishment of a CRISPR/Cas9 system is challenging. Previous studies have presented numerous methodologies for establishing a CRISPR/Cas9 system, yet detailed descriptions are limited. Additionally, the difficulties in obtaining the necessary plasmids have hindered the replication of CRISPR/Cas9 techniques in other laboratories. In this study, we share a detailed and simple CRISPR/Cas9 knockout system with optimized steps. The results of gene knockout experiments in vitro and in vivo show that this system successfully knocked out the target gene. By sharing detailed information on plasmid sequences, reagent codes, and methods, this study can assist researchers in establishing gene knockout systems.
... Reducing Cas9 activity has been shown to reduce off-target activity [47,48]. Various inducible expression systems have been developed to restrict Cas9 expression [30,49,50]. However, these approaches suffer from decreased targeting activity and the requirement for an induction step. ...
Background
The field of genome editing has been revolutionized by the development of an easily programmable editing tool, the CRISPR–Cas9. Despite its promise, off-target activity of Cas9 posed a great disadvantage for genome editing purposes by causing DNA double strand breaks at off-target locations and causing unwanted editing outcomes. Furthermore, for gene integration applications, which introduce transgene sequences, integration of transgenes to off-target sites could be harmful, hard to detect, and reduce faithful genome editing efficiency.
Method
Here we report the development of a multicolour fluorescence assay for studying CRISPR–Cas9-directed gene integration at an endogenous locus in human cell lines. We examine genetic integration of reporter genes in transiently transfected cells as well as puromycin-selected stable cell lines to determine the fidelity of multiple CRISPR–Cas9 strategies.
Result
We found that there is a high occurrence of unwanted DNA integration which tarnished faithful knock-in efficiency. Integration outcomes are influenced by the type of DNA DSBs, donor design, the use of enhanced specificity Cas9 variants, with S-phase regulated Cas9 activity. Moreover, restricting Cas9 expression with a self-cleaving system greatly improves knock-in outcomes by substantially reducing the percentage of cells with unwanted DNA integration.
Conclusion
Our results highlight the need for a more stringent assessment of CRISPR–Cas9-mediated knock-in outcomes, and the importance of careful strategy design to maximise efficient and faithful transgene integration.
... Additionally, uncontrolled Cas9 expression in animal models can lead to genomic damage (60), off-target effects (61,62), and immunological clearance responses hindering the system's application (63). To test the effect of CAMKV inhibition on NB growth in vivo, we employed a conditional CRISPR/Cas9-mediated gene knockout approach (64)(65)(66)(67). This approach allows for temporal control of CRISPR-Cas9 activity for inducible genome editing in luciferase-expressing CHLA136 cells (CHLA136-Fluc). ...
Neuroblastoma (NB) can be a highly aggressive malignancy in children. However, the precise mechanisms driving NB tumorigenesis remain elusive. This study revealed the critical role of CREB phosphorylation in NB cell proliferation. By employing a CRISPR-Cas9 knockout screen targeting calcium/calmodulin-dependent protein kinase (CaMK) family members, we identified the CaM kinase-like vesicle-associated (CAMKV) protein as a kinase that mediates direct phosphorylation of CREB to promote NB cell proliferation. CAMKV was found to be a transcriptional target of MYCN/MYC in NB cells. CAMKV knockout and knockdown effectively suppressed NB cell proliferation and tumor growth both in vitro and in vivo. Bioinformatic analysis revealed that high CAMKV expression is significantly correlated with poor patient survival. High-risk NB frequently had high CAMKV protein levels by Immunohistochemical staining. Integrated transcriptomic and proteomic analyses of CAMKV knockdown cells unveiled downstream targets involved in CAMKV-regulated phosphorylation and signaling pathways, many of which are linked to neural development and cancer progression. We identified small molecule inhibitors targeting CAMKV and further demonstrated the efficacy of one inhibitor in suppressing NB tumor growth and prolonging the survival of mice bearing xenografted tumors. These findings reveal a critical role for CAMKV kinase signaling in NB growth and identified CAMKV kinase as a potential therapeutic target and prognostic marker for patients with NB.
... This method achieved persistent DNA editing in vivo, while showing fast cargo delivery and rapid peptide turnover. An additional layer of control is provided by regulating CRISPR-Cas expression/activity using split Cas9 (Truong et al., 2015;Zetsche et al., 2015), small chemical molecules (Gonzalez et al., 2014;Davis et al., 2015;Dow et al., 2015), light (Nihongaki et al., 2015), and magnetic nanoparticles . These methods offer precise control over the timing and extent of Cas9 expression, enhancing safety and specificity, primarily within ex vivo editing workflows where high-efficiency payload delivery is feasible. ...
Over the last decade, CRISPR has revolutionized drug development due to its potential to cure genetic diseases that currently do not have any treatment. CRISPR was adapted from bacteria for gene editing in human cells in 2012 and, remarkably, only 11 years later has seen it’s very first approval as a medicine for the treatment of sickle cell disease and transfusion-dependent beta-thalassemia. However, the application of CRISPR systems is associated with unintended off-target and on-target alterations (including small indels, and structural variations such as translocations, inversions and large deletions), which are a source of risk for patients and a vital concern for the development of safe therapies. In recent years, a wide range of methods has been developed to detect unwanted effects of CRISPR-Cas nuclease activity. In this review, we summarize the different methods for off-target assessment, discuss their strengths and limitations, and highlight strategies to improve the safety of CRISPR systems. Finally, we discuss their relevance and application for the pre-clinical risk assessment of CRISPR therapeutics within the current regulatory context.
... Temporal control of gene KO enables analysis of the function of genes at distinct times, evaluation of genes essential for mouse development, and modeling therapeutic inhibition of proteins. Inducible approaches utilizing baseline repression of a gRNA or Cas9 expression that can be relieved with an inducing agent are often leaky, leading to CRISPR-mediated indel formation in the absence of the inducing agent [40][41][42][43] . CRISPR-Switch-On utilizes a gRNA-expression vector that is 'Off' before induction and can be induced via Cre-or Flp-mediated excision of a transcriptional stop in the tracrRNA 14 , thus circumventing leakiness and immunogenicity concerns. ...
Annotation of immunologic gene function in vivo typically requires the generation of knockout mice, which is time consuming and low throughput. We previously developed CHimeric IMmune Editing (CHIME), a CRISPR–Cas9 bone marrow delivery system for constitutive, ubiquitous deletion of single genes. Here we describe X-CHIME, four new CHIME-based systems for modular and rapid interrogation of gene function combinatorially (C-CHIME), inducibly (I-CHIME), lineage-specifically (L-CHIME) or sequentially (S-CHIME). We use C-CHIME and S-CHIME to assess the consequences of combined deletion of Ptpn1 and Ptpn2, an embryonic lethal gene pair, in adult mice. We find that constitutive deletion of both PTPN1 and PTPN2 leads to bone marrow hypoplasia and lethality, while inducible deletion after immune development leads to enteritis and lethality. These findings demonstrate that X-CHIME can be used for rapid mechanistic evaluation of genes in distinct in vivo contexts and that PTPN1 and PTPN2 have some functional redundancy important for viability in adult mice.
... For instance, BEs lead to both genomic and transcriptomic off-target due to long-term expression in vivo in transgenic mice, and mice zygotes injected with ABE7.10 encoded AAV exhibit low birth rates 28 . Although inducible promoters can be used to regulate the expression of ABEs 29,30 , the leaky expressions and the delayed response from transcription to translation are highly undesirable. Post-translational inducible control of Cas proteins [31][32][33][34] can potentially regulate ABE recruitment to the genome but still cannot directly control the deaminase activity of ABE, which does not curtail its off-target effects [25][26][27]35 . ...
DNA base editors use deaminases fused to a programmable DNA-binding protein for targeted nucleotide conversion. However, the most widely used TadA deaminases lack post-translational control in living cells. Here, we present a split adenine base editor (sABE) that utilizes chemically induced dimerization (CID) to control the catalytic activity of the deoxyadenosine deaminase TadA-8e. sABE shows high on-target editing activity comparable to the original ABE with TadA-8e (ABE8e) upon rapamycin induction while maintaining low background activity without induction. Importantly, sABE exhibits a narrower activity window on DNA and higher precision than ABE8e, with an improved single-to-double ratio of adenine editing and reduced genomic and transcriptomic off-target effects. sABE can achieve gene knockout through multiplex splice donor disruption in human cells. Furthermore, when delivered via dual adeno-associated virus vectors, sABE can efficiently convert a single A•T base pair to a G•C base pair on the PCSK9 gene in mouse liver, demonstrating in vivo CID-controlled DNA base editing. Thus, sABE enables precise control of base editing, which will have broad implications for basic research and in vivo therapeutic applications.
... The pPRKRA plasmid, expressing N-terminal 3XFLAG-tagged PRKRA (NCBI Reference Sequence: NM_011871) mouse protein, was generated by Gibson assembly of the following fragments: the EF-1 alpha promoter (from Addgene #2261), the Kozak-3XFLAG fragment (from c3GIC9, Addgene #62191, a gift from Lukas Dow [110]), the PRKRA CDS (amplified from a Mael -/testis cDNA sample) and the P2A-EGFP-ori-AmpR fragment (from Addgene #112101). pTN201, pCEPsmL1 and pCEPsmL1mut plasmids have been described elsewhere [20,76] and were kindly provided by Jeffrey Han (Tulane University); pPAGFP-C1 plasmid (Addgene #11910) was a gift from Svetlana Deryusheva (Carnegie Institution for Science, Embryology). ...
Transposable elements (TE) are mobile DNA sequences whose excessive proliferation endangers the host. Although animals have evolved robust TE-targeting defenses, including Piwi-interacting (pi)RNAs, retrotransposon LINE-1 (L1) still thrives in humans and mice. To gain insights into L1 endurance, we characterized L1 Bodies (LBs) and ORF1p complexes in germ cells of piRNA-deficient Maelstrom null mice. We report that ORF1p interacts with TE RNAs, genic mRNAs, and stress granule proteins, consistent with earlier studies. We also show that ORF1p associates with the CCR4-NOT deadenylation complex and PRKRA, a Protein Kinase R factor. Despite ORF1p interactions with these negative regulators of RNA expression, the stability and translation of LB-localized mRNAs remain unchanged. To scrutinize these findings, we studied the effects of PRKRA on L1 in cultured cells and showed that it elevates ORF1p levels and L1 retrotransposition. These results suggest that ORF1p-driven condensates promote L1 propagation, without affecting the metabolism of endogenous RNAs.
... To establish that MRC dysfunction is consequent to tumor cell genomic and environmental stresses, doxycycline-inducible models have been investigated. While mutationinducible models have been described [311][312][313][314], the investigations by Ying and colleagues [311] assessed the metabolic state of their model post doxycycline induction. In their study, Ying et al. [311] described a doxycycline-KRAS-inducible model wherein activation of a KRAS G12D mutant resulted in development of pancreatic ductal adenocarcinoma in nude mice. ...
Cellular metabolism (or energetics) and epigenetics are tightly coupled cellular processes. It is arguable that of all the described cancer hallmarks, dysregulated cellular energetics and epigenetics are the most tightly coregulated. Cellular metabolic states regulate and drive epigenetic changes while also being capable of influencing, if not driving, epigenetic reprogramming. Conversely, epigenetic changes can drive altered and compensatory metabolic states. Cancer cells meticulously modify and control each of these two linked cellular processes in order to maintain their tumorigenic potential and capacity. This review aims to explore the interplay between these two processes and discuss how each affects the other, driving and enhancing tumorigenic states in certain contexts.
... Double-stranded breaks in DNA effected with nucleases engage a repair pathway called nonhomologous end joining (NHEJ), which leads to indel mutagenesis at target sites. Enzymatic activity can be regulated chemically at some stages, such as enzyme translocation to the nucleus, protein folding, or more generally, protein production stages [45][46][47][48] . For protein function regulation, the time lag between chemical stimulation and activation of protein function should be small. ...
Genome editing had a long history before the appearance of CRISPR. Although a decade has passed since the initial use of CRISPR with mammalian cells, the first attempts at gene editing occurred in the 1980's. Subsequently, many researchers tried to develop methods to edit specific genes. Here, we review the history of genome editing and improvements in genome editing tools. In the last two decades, genome editing tools have been applied in basic sciences, the bio-industry, and therapeutics. We provide examples in which genome editing tools have been applied to various tasks. Recently, new CRISPR-Cas techniques, such as base and prime editing and anti-CRISPR proteins, have attracted considerable interest. Accordingly, these topics are also reviewed.
... Genome editing technologies like CRISPR-Cas9 can be used to rapidly engineer somatic mutations when delivered exogenously or when installed as germline alleles [10][11][12][13][14] . While these models have accelerated the study of putative cancer driver genes, they are most frequently used to induce DNA double-stranded breaks (DSBs), leading to inactivation of tumor suppressor genes via error-prone repair and frameshifting insertion/deletion (indel) formation. ...
Genetically engineered mouse models only capture a small fraction of the genetic lesions that drive human cancer. Current CRISPR–Cas9 models can expand this fraction but are limited by their reliance on error-prone DNA repair. Here we develop a system for in vivo prime editing by encoding a Cre-inducible prime editor in the mouse germline. This model allows rapid, precise engineering of a wide range of mutations in cell lines and organoids derived from primary tissues, including a clinically relevant Kras mutation associated with drug resistance and Trp53 hotspot mutations commonly observed in pancreatic cancer. With this system, we demonstrate somatic prime editing in vivo using lipid nanoparticles, and we model lung and pancreatic cancer through viral delivery of prime editing guide RNAs or orthotopic transplantation of prime-edited organoids. We believe that this approach will accelerate functional studies of cancer-associated mutations and complex genetic combinations that are challenging to construct with traditional models.
... 1,2 However, challenges of spatial and temporal control of CRISPR-Cas need to be addressed, which reduces potential off-target editing and broadens medical applications. Exogenously inducible CRISPR-Cas tools have been rst developed, 3 such as employing blue light 4,5 and small drug molecules [6][7][8] to precisely activate the CRISPR-Cas system to induce interest gene expression. To fully exert the potential of CRISPR in living cells, CRISPR tools need to be manipulated using crucial endogenous biomolecules for studying intracellular biomarkers and controlling the genome of specic cells such as cancer cells and stem cells. ...
Chemical modifications of CRISPR-Cas nucleases help decrease off-target editing and expand the biomedical applications of CRISPR-based gene manipulation tools. Here, we found that epigenetic modifications of guide RNA, such as m6A and m1A methylation, can effectively inhibit both the cis- and trans-DNA cleavage activities of CRISPR-Cas12a. The underlying mechanism is that methylations destabilize the secondary and tertiary structure of gRNA which prevents the assembly of the Cas12a-gRNA nuclease complex, leading to decreased DNA targeting ability. A minimum of three adenine methylated nucleotides are required to completely inhibit the nuclease activity. We also demonstrate that these effects are reversible through the demethylation of gRNA by demethylases. This strategy has been used in the regulation of gene expression, demethylase imaging in living cells and controllable gene editing. The results demonstrate that the methylation-deactivated and demethylase-activated strategy is a promising tool for regulation of the CRISPR-Cas12a system.
... This observation has paved the way for the development of new CRISPR/dCas9 tools coupled to transcriptional effectors (Fig. 4) capable of activating (CRISPRa) or repressing (CRISPRi) gene expression [174] or targeting the epigenetic modifications [175] to modify the gene methylation status (CRISPR DNA methylation) or the acetylation and methylation level of histone proteins (CRISPR Epigenetic modifier). Indeed, histone demethylases (HDM), histone methyltransferases (HTMs), histone acetyltransferases (HATs) and deacetylases (HDACs), and DNA methylation modifying factors such as DNA methyltransferases (DNMTs) and DNA methylases (TET proteins), have been coupled to the CRISPR/dCas9 complex [176][177][178][179]. Inducible CRISPR systems have also been developed [180][181][182][183], based on photoactivated Cas9 and chemically induced CRISPRs [184]. These molecules can selectively target CHRFAM7A gene (Fig. 4) to rescue dysregulation defects in several disorders. ...
The α7 nicotinic receptor (α7 nAChR) is an important entry point for Ca2+ into the cell, which has broad and important effects on gene expression and function. The gene (CHRNA7), mapping to chromosome (15q14), has been genetically linked to a large number of diseases, many of which involve defects in cognition. While numerous mutations in CHRNA7 are associated with mental illness and inflammation, an important control point may be the function of a recently discovered partial duplication CHRNA7, CHRFAM7A, that negatively regulates the function of the α7 receptor, through the formation of heteropentamers; other functions cannot be excluded. The deregulation of this human specific gene (CHRFAM7A) has been linked to neurodevelopmental, neurodegenerative, and inflammatory disorders and has important copy number variations. Much effort is being made to understand its function and regulation both in healthy and pathological conditions. However, many questions remain to be answered regarding its functional role, its regulation, and its role in the etiogenesis of neurological and inflammatory disorders. Missing knowledge on the pharmacology of the heteroreceptor has limited the discovery of new molecules capable of modulating its activity. Here we review the state of the art on the role of CHRFAM7A, highlighting unanswered questions to be addressed. A possible therapeutic approach based on genome editing protocols is also discussed.
... Different inducible promoters in mammalian cells and animal models have been investigated as regulators to control Cas9 activity [98]. A doxycycline-inducible gRNA system has been developed that is responsible for Cas9-mediated genome regulation [99]. ...
The innovative advances in transforming clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) into different variants have taken the art of genome-editing specificity to new heights. Allosteric modulation of Cas9-targeting specificity by sgRNA sequence alterations and protospacer adjacent motif (PAM) modifications have been a good lesson to learn about specificity and activity scores in different Cas9 variants. Some of the high-fidelity Cas9 variants have been ranked as Sniper-Cas9, eSpCas9 (1.1), SpCas9-HF1, HypaCas9, xCas9, and evoCas9. However, the selection of an ideal Cas9 variant for a given target sequence remains a challenging task. A safe and efficient delivery system for the CRISPR/Cas9 complex at tumor target sites faces considerable challenges, and nanotechnology-based stimuli-responsive delivery approaches have significantly contributed to cancer management. Recent innovations in nanoformulation design, such as pH, glutathione (GSH), photo, thermal, and magnetic responsive systems, have modernized the art of CRISPR/Cas9 delivery approaches. These nanoformulations possess enhanced cellular internalization, endosomal membrane disruption/bypass, and controlled release. In this review, we aim to elaborate on different CRISPR/Cas9 variants and advances in stimuli-responsive nanoformulations for the specific delivery of this endonuclease system. Furthermore, the critical constraints of this endonuclease system on clinical translations towards the management of cancer and prospects are described.
... A longer life and a higher concentration of the components of the CRISPR/Cas9 system in the cell promote off-target editing. Use of inducible promoters to express Cas9 (inducible Cas9 (iCas9)) was among the earliest ideas of how to achieve an optimal balance between the efficiency and specificity of the system [112][113][114]. To strictly verify the system specificity in a panel of human cells (293T, HeLa, and SK-BR-3), editing with iCas9 was performed using sgRNAs (both perfectly complementary and mismatch containing) targeted to KDM5C, EMX1, and VEGFA genes. ...
The CRISPR/Cas9 system, which was discovered recently, utilizes nucleases targeted by sequence complementarity and is originally intended to protect bacteria from foreign genetic elements. The system provided a convenient tool for manipulating the genomes of living cells. The CRISPR/Cas9 genomic editing technology moved beyond the laboratory and already found application in biotechnology and agriculture. However, off-target activity of the CRISPR/Cas9 system can cause oncogenic mutations and thus limits its use for genome editing in human cells for medical purposes. Many studies are therefore aimed at developing variants of the CRISPR/Cas9 system with improved accuracy. The review considers the mechanisms of precise and erroneous actions of Cas9 RNA-guided nuclease, natural and artificial variants of RNA-targeted nucleases, possibilities to modulate their specificity through guide RNA modifications, and other approaches to increasing the accuracy of the CRISPR/Cas9 system in genome editing.
... A validated control sgRNA targeting a neutral region in mouse chromosome 8 (ref. 59) was cloned into the pUSEPR backbone and spiked into each of these libraries at a defined fraction to achieve equimolarity between sgRNAs in the library and the control. To assess sgRNA distribution, the sgRNA target region was amplified using primers that append Illumina sequencing adapters on the 5′ and 3′ ends of the amplicon, as well as a random nucleotide stagger and unique demultiplexing barcode on the 5′ end (Supplementary Table 8b). ...
Metastasis frequently develops from disseminated cancer cells that remain dormant after the apparently successful treatment of a primary tumour. These cells fluctuate between an immune-evasive quiescent state and a proliferative state liable to immune-mediated elimination1–6. Little is known about the clearing of reawakened metastatic cells and how this process could be therapeutically activated to eliminate residual disease in patients. Here we use models of indolent lung adenocarcinoma metastasis to identify cancer cell-intrinsic determinants of immune reactivity during exit from dormancy. Genetic screens of tumour-intrinsic immune regulators identified the stimulator of interferon genes (STING) pathway as a suppressor of metastatic outbreak. STING activity increases in metastatic progenitors that re-enter the cell cycle and is dampened by hypermethylation of the STING promoter and enhancer in breakthrough metastases or by chromatin repression in cells re-entering dormancy in response to TGFβ. STING expression in cancer cells derived from spontaneous metastases suppresses their outgrowth. Systemic treatment of mice with STING agonists eliminates dormant metastasis and prevents spontaneous outbreaks in a T cell- and natural killer cell-dependent manner—these effects require cancer cell STING function. Thus, STING provides a checkpoint against the progression of dormant metastasis and a therapeutically actionable strategy for the prevention of disease relapse.
... Engineered inducible Cas9 variants can provide temporal control over targeted DSB generation and subsequent DNA editing [14][15][16][17][18][19][20][21][22][23][24][25][26] . Such temporally controlled Cas9s enable a variety of applications, from studying the kinetics of CRISPR-Cas9 DNA editing and DNA repair to recording biological events in cells [26][27][28][29][30] . ...
CRISPR–Cas9 has yielded a plethora of effectors, including targeted transcriptional activators, base editors and prime editors. Current approaches for inducibly modulating Cas9 activity lack temporal precision and require extensive screening and optimization. We describe a versatile, chemically controlled and rapidly activated single-component DNA-binding Cas9 switch, ciCas9, which we use to confer temporal control over seven Cas9 effectors, including two cytidine base editors, two adenine base editors, a dual base editor, a prime editor and a transcriptional activator. Using these temporally controlled effectors, we analyze base editing kinetics, showing that editing occurs within hours and that rapid early editing of nucleotides predicts eventual editing magnitude. We also reveal that editing at preferred nucleotides within target sites increases the frequency of bystander edits. Thus, the ciCas9 switch offers a simple, versatile approach to generating chemically controlled Cas9 effectors, informing future effector engineering and enabling precise temporal effector control for kinetic studies.
... Cellular immune responses will thus be limited to the period during which CRISPR effector-derived immunogenic epitopes are displayed on the cell surface. Strategies for CRISPR control include the use of inducible promoters (Dow et al., 2015), anti-CRISPR proteins (Harrington et al., 2017;Davidson et al., 2020;Ibraheim et al., 2021), conditional CRISPR effector destabilization (Kleinjan et al., 2017;Senturk et al., 2017), and self-deleting AAV-CRISPR (Li et al., 2019). A self-limiting CRISPR-Cas9 system for LCA10 was developed with sgRNA recognition sites included on the Frontiers in Bioengineering and Biotechnology frontiersin.org ...
CRISPR offers new hope for many patients and promises to transform the way we think of future therapies. Ensuring safety of CRISPR therapeutics is a top priority for clinical translation and specific recommendations have been recently released by the FDA. Rapid progress in the preclinical and clinical development of CRISPR therapeutics leverages years of experience with gene therapy successes and failures. Adverse events due to immunogenicity have been a major setback that has impacted the field of gene therapy. As several in vivo CRISPR clinical trials make progress, the challenge of immunogenicity remains a significant roadblock to the clinical availability and utility of CRISPR therapeutics. In this review, we examine what is currently known about the immunogenicity of CRISPR therapeutics and discuss several considerations to mitigate immunogenicity for the design of safe and clinically translatable CRISPR therapeutics.
... However, the large size of SpCas9 (~4.2kb) is unsuitable for effective in vivo delivery. SpCas9-expressing mouse models have been generated to overcome this hurdle [16,43,44]. For pigs, transgenic models with ubiquitously constitutive Cas9 expression [19,45] and Cre-inducible Cas9 expression [25] have been reported previously. ...
Background
CRISPR-based toolkits have dramatically increased the ease of genome and epigenome editing. SpCas9 is the most widely used nuclease. However, the difficulty of delivering SpCas9 and inability to modulate its expression in vivo hinder its widespread adoption in large animals.
Results
Here, to circumvent these obstacles, a doxycycline-inducible SpCas9-expressing (DIC) pig model was generated by precise knock-in of the binary tetracycline-inducible expression elements into the Rosa26 and Hipp11 loci, respectively. With this pig model, in vivo and/or in vitro genome and epigenome editing could be easily realized. On the basis of the DIC system, a convenient Cas9-based conditional knockout strategy was devised through controlling the expression of rtTA component by tissue-specific promoter, which allows the one-step generation of germline-inherited pigs enabling in vivo spatiotemporal control of gene function under simple chemical induction. To validate the feasibility of in vivo gene mutation with DIC pigs, primary and metastatic pancreatic ductal adenocarcinoma was developed by delivering a single AAV6 vector containing TP53 -sgRNA, LKB1 -sgRNA, and mutant human KRAS gene into the adult pancreases.
Conclusions
Together, these results suggest that DIC pig resources will provide a powerful tool for conditional in vivo genome and epigenome modification for fundamental and applied research.
... Now the targeted modification of Rosa26 in mice has been widely used in continuous and conditional expression of exogenous genes. These modification result in the establishment of several hundred mouse models, which play important roles in the basic study such as gene function, disease models and drug development research [13][14][15]. After the discovery of Rosa26 in mice, the Rosa26 locus of human [16], rat [17], pig [18,19], rabbit [20] and sheep [21] were continually determined through comparative study. ...
The genetic modification of cattle has many agricultural and biomedical applications. However, random integration often leads to the unstable or differentially expression of the exogenous genes, which limit the application and development of transgenic technologies. Finding a safe locus suitable for site-specific insertion and efficient expression of exogenous genes is a good way to overcome these hurdles. In this study, we efficiently integrated three targeted vector into the cattle Rosa26 (cRosa26) by CRISPR/Cas9 technology in which EGFP was driven by CAG , EF1a , PGK and cRosa26 endogenous promoter respectively. The CRISPR/Cas9 knock-in system allows highly efficient gene insertion of different expression units at the cRosa26 locus. We also find that in the four cell lines, EGFP was stable expressed at different times, and the CAG promoter has the highest activity to activate the expression of EGFP, when compared with the cRosa26 , EF1a and PGK promoter. Our results proved that cRosa26 was a locus that could integrate different expression units efficiently, and supported the friendly expression of different expression units. Our findings described here will be useful for a variety of studies using cattle.
... In the mouse, to extend the utility of CRISPR-Cas9 technology, several Cas9 transgenic mice have been generated. 13,[29][30][31] These mouse strains simplify and allow gene editing with high efficiency both in vivo and ex vivo as only sgRNAs are required for a successful gene knockout in Cas9-expressing cells. Previously, Cas9expressing B cells transduced with sgRNA retroviral particles have been used to identify molecules involved in mouse B cell activation and PC differentiation. ...
Human B lymphocytes are attractive targets for immunotherapies in autoantibody-mediated diseases. Gene editing technologies could provide a powerful tool to determine gene regulatory networks regulating B cell differentiation into plasma cells, and identify novel therapeutic targets for prevention and treatment of autoimmune disorders. Here, we describe a new approach that uses CRISPR/Cas9 technology to target genes in primary human B cells in vitro for identifying plasma cell regulators. We found that sgRNA and Cas9 components can be efficiently delivered into primary human B cells through RD114-pseudotyped retroviral vectors. Using this system, we achieved approximate 80% of gene knockout efficiency. We disrupted expression of a triad of transcription factors, IRF4, PRDM1 and XBP1, and showed that human B cell survival and plasma cell differentiation are severely impaired. Specifically, that IRF4, PRDM1 and XBP1 are expressed at different stages during plasma cell differentiation, IRF4, PRDM1 and XBP1-targeted cells fail to progress to the pre-PB, PC state and PC survival, respectively. Our method opens a new avenue to study gene functions in primary human B cells and identify novel plasma cell regulators for therapeutic applications.
... The cells were subsequently cultured with or without Dox before being collected for DNA extraction and deep sequencing ( Figure 4A). Cleavage at all four sites could be detected even in the absence of Dox, an indication of basal SpCas9 expression caused by the leakiness of the tet-on promoter [42][43][44] (Figure 4B-E). As expected, the mutation frequency (indel, %) was low for both on-and off-target cleavage without Dox induction, although considerable variation existed between the four target sites, where EMX1-1 had the highest on-as well as off-targeting levels ( Figure 4B). ...
Genome editing tools based on CRISPR–Cas systems can repair genetic mutations in situ; however, off-target effects and DNA damage lesions that result from genome editing remain major roadblocks to its full clinical implementation. Protein and chemical inhibitors of CRISPR–Cas systems may reduce off-target effects and DNA damage. Here we describe the identification of several lead chemical inhibitors that could specifically inhibit the activity of Streptococcus pyogenes Cas9 (SpCas9). In addition, we obtained derivatives of lead inhibitors that could penetrate the cell membrane and inhibit SpCas9 in cellulo. Two of these compounds, SP2 and SP24, were able to improve the specificity of SpCas9 in cellulo at low-micromolar concentration. Furthermore, microscale thermophoresis (MST) assays showed that SP24 might inhibit SpCas9 activity by interacting with both the SpCas9 protein and the SpCas9–gRNA ribonucleoprotein complex. Taken together, SP24 is a novel chemical inhibitor of SpCas9 which has the potential to enhance therapies that utilize SpCas9.
The recently discovered CRISPR/Cas9 system based on the action of complementary targeted nucleases and originally intended to protect bacteria from foreign genetic elements has become a convenient tool for manipulating the genomes of living cells. The CRISPR/Cas9 genomic editing technology has moved beyond the laboratory and is already finding application in biotechnology and agriculture. However, the use of this method for editing human cells for medical purposes is limited by CRISPR/Cas9 system off-target activity, which can lead to oncogenic mutations. Therefore, many studies aim to develop variants of the CRISPR/Cas9 system with improved accuracy. The review highlights the mechanisms of precise and erroneous action of the RNA-guided nuclease Cas9, natural and artificially created variants of RNA-targeted nucleases, possibilities to modulate their specificity through guide RNA modifications, and other approaches to increase the accuracy of the CRISPR/Cas9 system in genome editing.
Sirtuins (SRTs) are a group of nicotinamide adenine dinucleotide (NAD ⁺ )‐dependent deacetylase that target both histone and nonhistone proteins. The biological function of SRT in horticultural plants has been rarely studied. In this study, FaSRT1‐2 was identified as a key member of the 8 FaSRTs encoded in cultivated strawberry genome. Transient overexpression of FaSRT1‐2 in strawberry fruit accelerated ripening, increased the content of anthocyanins and sugars, enhanced ripening‐related gene expression. Moreover, stable transformation of FaSRT1‐2 in strawberry plants resulted in enhanced vegetative growth, increased sensitivity to heat stress and increased susceptibility to Botrytis cinerea infection. Interestingly, knocking out the homologous gene in woodland strawberry had the opposite effects. Additionally, we found the content of stress‐related hormone abscisic acid (ABA) was decreased, while the growth‐related gibberellin (GA) concentration was increased in FaSRT1‐2 overexpression lines. Gene expression analysis revealed induction of heat shock proteins, transcription factors, stress‐related and antioxidant genes in the FaSRT1‐2 ‐overexpressed plants while knocked‐out of the gene had the opposite impact. In conclusion, our findings demonstrated that FaSRT1‐2 could positively promote strawberry plant vegetative growth and fruit ripening by affecting ABA and GA pathways. However, it negatively regulates the resistance to heat stress and B. cinerea infection by influencing the related gene expression.
Small cell lung cancer (SCLC) remains the most fatal form of lung cancer, with patients in dire need of new and effective therapeutic approaches. Modeling SCLC in an immunocompetent host is essential for understanding SCLC pathogenesis and ultimately discovering and testing new experimental therapeutic strategies. Human SCLC is characterized by near universal genetic loss of the RB1 and TP53 tumor suppressor genes. Twenty years ago, the first genetically-engineered mouse model (GEMM) of SCLC was generated using conditional deletion of both Rb1 and Trp53 in the lungs of adult mice. Since then, several other GEMMs of SCLC have been developed coupling genomic alterations found in human SCLC with Rb1 and Trp53 deletion. Here we summarize how GEMMs of SCLC have contributed significantly to our understanding of the disease in the past two decades. We also review recent advances in modeling SCLC in mice that allow investigators to bypass limitations of the previous generation of GEMMs while studying new genes of interest in SCLC. In particular, CRISPR/Cas9-mediated somatic gene editing can accelerate how new genes of interest are functionally interrogated in SCLC tumorigenesis. Notably, the development of allograft models and precancerous precursor models from SCLC GEMMs provides complementary approaches to GEMMs to study tumor cell-immune microenvironment interactions and test new therapeutic strategies to enhance response to immunotherapy. Ultimately, the new generation of SCLC models can accelerate research and help develop new therapeutic strategies for SCLC.
USAGE OF MOUSE MODELS IN CANCER IMMUNOTHERAPY RESEARCH
DNA targeting Class 2 CRISPR-Cas effector nucleases, including the well-studied Cas9 proteins, evolved protospacer-adjacent motif (PAM) and guide RNA interactions that sequentially license their binding and cleavage activities at protospacer target sites. Both interactions are nucleic acid sequence specific but function constitutively; thus, they provide intrinsic spatial control over DNA targeting activities but naturally lack temporal control. Here we show that engineered Cas9 fusion proteins which bind to nascent RNAs near a protospacer can facilitate spatiotemporal coupling between transcription and DNA targeting at that protospacer: Tr anscription- a ssociated C as9 T argeting (TraCT). Engineered TraCT is enabled when suboptimal PAM interactions limit basal activity in vivo and when one or more nascent RNA substrates are still tethered to the actively transcribing target DNA in cis . We further show that this phenomenon can be exploited for selective editing at one of two identical targets in distinct gene loci, or, in diploid allelic loci that are differentially transcribed. Our work demonstrates that temporal control over Cas9’s targeting activity at specific DNA sites may be engineered without modifying Cas9’s core domains and guide RNA components or their expression levels. More broadly, it establishes RNA binding in cis as a mechanism that can conditionally stimulate CRISPR-Cas DNA targeting in eukaryotes.
Cancers arise through acquisition of mutations in genes that regulate core biological processes like cell proliferation and cell death. Decades of cancer research have led to the identification of genes and mutations causally involved in disease development and evolution, yet defining their precise function across different cancer types and how they influence therapy responses has been challenging. Mouse models have helped define the in vivo function of cancer-associated alterations, and genome-editing approaches using CRISPR have dramatically accelerated the pace at which these models are developed and studied. Here, we highlight how CRISPR technologies have impacted the development and use of mouse models for cancer research and discuss the many ways in which these rapidly evolving platforms will continue to transform our understanding of this disease.
Improved understanding of expression of recombinant immunoglobulin (IgG)-based therapies can decrease manufacturing process costs and bring down costs to patients. Deletion of C-terminal Lysine (C-Lys) from IgG molecules has been shown to greatly impact yield. This study set out to characterise structural components of IgG C-terminal variants which modulate protein expression by examination of the consequences of mutations at the C-terminal of IgG on expression and by the use of fluorescent C-terminal fragment fusion proteins. Cell-based and cell-free experiments were also implemented to characterise how the C-terminal differentially engages with cellular pathways to modulate expression. IgG variants engineered by removal of the C-terminal Lys were expressed at significantly lower rates than control variants by CHO (and HEK) cells. Engineered constructs of mCherry fused with short regions of the C-terminal regions of IgG mimicked the ordering of expressability observed for IgG variants. These fluorescent C-terminal fragment fusions offered the potential to profile how sequences (and point mutations) modified expression. Via combinations of cell and cell-free systems, screening across a range of variants of IgG and mCherry reporter constructs has shown that interactions between specific C-terminal amino acid sequences and the ribosome can regulate the rate and extent of expression. This study highlights the importance of amino acid sequence regulatory events determining the efficiency of production of desirable recombinant proteins, showing that wildtype C-terminal lysine is a necessary capping molecule for IgG1 expression. From a wider perspective, these data are especially significant towards the design of novel entities. The approach has also provided information about novel short C-terminal tags which may be used to provide selective synthesis of specific subunits in the production of multisubunit products. Alternative strategies for removing C-terminal amino acid heterogeneity whilst maintaining efficient rates of expression have been provided.
Genetically engineered mouse models (GEMMs) allow for modeling of spontaneous tumorigenesis within its native microenvironment in mice and have provided invaluable insights into mechanisms of tumorigenesis and therapeutic strategies to treat human disease. However, as their generation requires germline manipulation and extensive animal breeding that is time-, labor-, and cost-intensive, traditional GEMMs are not accessible to most researchers, and fail to model the full breadth of cancer-associated genetic alterations and therapeutic targets. Recent advances in genome-editing technologies and their implementation in somatic tissues of mice have ushered in a new class of mouse models: non-germline GEMMs (nGEMMs). nGEMM approaches can be leveraged to generate somatic tumors de novo harboring virtually any individual or group of genetic alterations found in human cancer in a mouse through simple procedures that do not require breeding, greatly increasing the accessibility and speed and scale on which GEMMs can be produced. Here we describe the technologies and delivery systems used to create nGEMMs and highlight new biological insights derived from these models that have rapidly informed functional cancer genomics, precision medicine, and immune oncology.
Aspergillus is widely distributed in nature and occupies a crucial ecological niche, which has complex and diverse metabolic pathways and can produce a variety of metabolites. With the deepening of genomics exploration, more Aspergillus genomic informations have been elucidated, which not only help us understand the basic mechanism of various life activities, but also further realize the ideal functional transformation. Available genetic engineering tools include homologous recombinant systems, specific nuclease based systems, and RNA techniques, combined with transformation methods, and screening based on selective labeling. Precise editing of target genes can not only prevent and control the production of mycotoxin pollutants, but also realize the construction of economical and efficient fungal cell factories. This paper reviewed the establishment and optimization process of genome technologies, hoping to provide the theoretical basis of experiments, and summarized the recent progress and application in genetic technology, analyzes the challenges and the possibility of future development with regard to Aspergillus .
Here we introduce RNA-CLAMP, a technology which enables site-specific and enzymatic cross-linking (clamping) of two selected stem loops within an RNA of interest. Intramolecular clamping of the RNA can disrupt normal RNA function, whereas subsequent photo-cleavage of the crosslinker restores activity. We applied the RNA-CLAMP technique to the single guide RNA of the CRISPR-Cas9 gene editing system. By clamping two stem loops of the single-guide RNA (sgRNA) with a photo-cleavable cross-linker, gene editing was completely silenced. Visible light irradiation cleaved the crosslinker and restored gene editing with high spatiotemporal resolution. Furthermore, by designing two photo-cleavable linkers which are responsive to different wavelength of lights, we achieved multiplexed photo-activation of gene editing in mammalian cells. Notably, although the Cas9-sgRNA RNP is not capable of DNA cleavage activity upon clamping, it maintained the capability to bind to the target DNA. The RNA-CLAMP enabled photo-activated CRISPR-Cas9 gene editing platform offers clean background, free choice of activation wavelength and multiplexing capability. We believe that this technology to precisely and rapidly control gene editing will serve as a versatile tool in the future development of stimuli responsive gene editing technologies. Beyond gene editing, RNA-CLAMP provides a site-specific tool for manipulating the internal structure of functional RNAs.
CRISPR-Cas systems, adaptive immune systems found in prokaryotes, have undergone tremendous research and development in the past decade, finding a multitude of applications. Beyond the initial genome editing, scientists have used CRISPR-Cas for gene regulation, epigenetic editing, biosensing, and DNA-based data storage. Multiplexing is a key and powerful feature of CRISPR-Cas systems, with CRISPR arrays providing incredibly dense information storage. However, many initial applications appeared to have lost the information density and ease of multiplexing inherent to natural CRISPR arrays, largely due to a move toward synthetic, single guide RNAs (sgRNAs) for the effector nuclease Cas9. Recent developments have restored much of this compactness and multiplexability, both with an effector nuclease that can process its own arrays, Cas12a, and with easier multiplexing methods for Cas9, including some that had long been assumed not to work. This article provides a brief overview of multiplexing in natural CRISPR-Cas systems, multiplexing methods and applications for research and biotechnology, and methods to quickly assemble multiplex CRISPR arrays.
Transformation to aggressive disease histologies generates formidable clinical challenges across cancers, but biological insights remain few. We modeled the genetic heterogeneity of chronic lymphocytic leukemia (CLL) through multiplexed in vivo CRISPR-Cas9 B-cell editing of recurrent CLL loss-of-function drivers in mice and recapitulated the process of transformation from indolent CLL into large cell lymphoma [i.e., Richter syndrome (RS)]. Evolutionary trajectories of 64 mice carrying diverse combinatorial gene assortments revealed coselection of mutations in Trp53, Mga, and Chd2 and the dual impact of clonal Mga/Chd2 mutations on E2F/MYC and interferon signaling dysregulation. Comparative human and murine RS analyses demonstrated tonic PI3K signaling as a key feature of transformed disease, with constitutive activation of the AKT and S6 kinases, downmodulation of the PTEN phosphatase, and convergent activation of MYC/PI3K transcriptional programs underlying enhanced sensitivity to MYC/mTOR/PI3K inhibition. This robust experimental system presents a unique framework to study lymphoid biology and therapy.
Significance
Mouse models reflective of the genetic complexity and heterogeneity of human tumors remain few, including those able to recapitulate transformation to aggressive disease histologies. Herein, we model CLL transformation into RS through multiplexed in vivo gene editing, providing key insight into the pathophysiology and therapeutic vulnerabilities of transformed disease.
The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9 system has been a recent focus of breeders owing to its potential to improve economically significant traits of livestock. The introduction of defined point mutations into the ovine genome via CRISPR/Cas9-mediated homology-directed repair has been reported; however, indel and mosaic events observed in genetically modified animals limit the practical application of this system in sheep breeding. The FecGF mutation (g. G1111A, p. V371M) in the growth differentiation factor 9 (GDF9) gene is strongly associated with litter size in Belclare and Norwegian White Sheep. In the present study, we introduced the FecGF mutation in GDF9 by co-injecting the CRISPR/Cas9 system, single-stranded oligodeoxynucleotide (ssODN), and Scr7 into ovine zygotes. Scr7 at various concentrations (0 μM, 1 μM, and 2 μM) had no adverse effects on embryonic development in vitro. No significant differences in total mutation, point mutation, and indel rates in embryos were observed among groups treated with different concentrations of Scr7. However, the mosaicism rates of embryos from zygotes microinjected with 1 and 2 μM Scr7 were significantly lower than that for 0 μM Scr7 (7.7% and 7.5% vs. 19.7%). We successfully obtained lambs with defined nucleotide substitutions by the coinjection of Cas9 mRNA, sgRNA, ssODN, and 1 μM Scr7 into Altay sheep zygotes. The single nucleotide mutation efficiency was 7.69% (3/39) in newborn lambs, with one mosaic. Our findings provide evidence that Scr7 could improve the specificity of the CRISPR/Cas9 system for the introduction of a defined point mutation in livestock to some extent.
Several advancements have been made to SpCas9, the most widely used CRISPR/Cas genome editing tool, to reduce its unwanted off-target effects. The most promising approach is the development of increased-fidelity nuclease (IFN) variants of SpCas9, however, their fidelity has increased at the cost of reduced activity. SuperFi-Cas9 has been developed recently, and it has been described as a next-generation high-fidelity SpCas9 variant, free from the drawbacks of first-generation IFNs. In this study, we characterize the on-target activity and the off-target propensity of SuperFi-Cas9 in mammalian cells, comparing it to first-generation IFNs. SuperFi-Cas9 demonstrates strongly reduced activity but high fidelity features that are in many aspects similar to those of some first-generation variants, such as evo- and HeFSpCas9. SuperFi-cytosine (CBE3) and -adenine (ABE7.10) base editors, as well as SuperFi-prime editor show no meaningful activity. When combined with ABE8e, SuperFi-Cas9, similarly to HeFSpCas9, executes DNA editing with high activity as well as high specificity reducing both bystander and SpCas9-dependent off-target base editing.
With the aim of expediting drug target discovery and validation for respiratory diseases, we developed an optimized method for in situ somatic gene disruption in murine lung epithelial cells via AAV6-mediated CRISPR/Cas9 delivery. Efficient gene editing was observed in lung type II alveolar epithelial cells and distal airway cells following assessment of single or dual guide AAV vector formats, Cas9 variants, and a unique sequential dosing strategy with combinatorial guide RNA expression cassettes. In particular, we were able to demonstrate population-wide gene disruption within distinct epithelial cell types for separate targets in Cas9 transgenic animals, with minimal to no associated inflammation. We also observed and characterized AAV vector integration events that occurred within directed double-stranded DNA break sites in lung cells, highlighting a complicating factor with AAV-mediated delivery of DNA nucleases. Taken together, we demonstrate a uniquely effective approach for somatic engineering of the murine lung, which will greatly facilitate the modeling of disease and therapeutic intervention.
In 2022, prostate cancer (PCa) is estimated to be the most commonly diagnosed cancer in men in the United States—almost 270,000 American men are estimated to be diagnosed with PCa in 2022. This review compares and contrasts in vivo models of PCa with regards to the altered genes, signaling pathways, and stages of tumor progression associated with each model. The main type of model included in this review are genetically engineered mouse models, which include conditional and constitutive knockout model. 2D cell lines, 3D organoids and spheroids, xenografts and allografts, and patient derived models are also included. The major applications, advantages and disadvantages, and ease of use and cost are unique to each type of model, but they all make it easier to translate the tumor progression that is seen in the mouse prostate to the human prostate. Although both human and mouse prostates are androgen-dependent, the fact that the native, genetically unaltered prostate in mice cannot give rise to carcinoma is an especially critical component of PCa models. Thanks to the similarities between the mouse and human genome, our knowledge of PCa has been expanded, and will continue to do so, through models of PCa.
Chromosomal rearrangements have a central role in the pathogenesis of human cancers and often result in the expression of therapeutically actionable gene fusions. A recently discovered example is a fusion between the genes echinoderm microtubule-associated protein like 4 (EML4) and anaplastic lymphoma kinase (ALK), generated by an inversion on the short arm of chromosome 2: inv(2)(p21p23). The EML4-ALK oncogene is detected in a subset of human non-small cell lung cancers (NSCLC) and is clinically relevant because it confers sensitivity to ALK inhibitors. Despite their importance, modelling such genetic events in mice has proven challenging and requires complex manipulation of the germ line. Here we describe an efficient method to induce specific chromosomal rearrangements in vivo using viral-mediated delivery of the CRISPR/Cas9 system to somatic cells of adult animals. We apply it to generate a mouse model of Eml4-Alk-driven lung cancer. The resulting tumours invariably harbour the Eml4-Alk inversion, express the Eml4-Alk fusion gene, display histopathological and molecular features typical of ALK(+) human NSCLCs, and respond to treatment with ALK inhibitors. The general strategy described here substantially expands our ability to model human cancers in mice and potentially in other organisms.
CRISPR-Cas9 is a versatile genome editing technology for studying the functions of genetic elements. To broadly enable the application of Cas9 in vivo, we established a Cre-dependent Cas9 knockin mouse. We demonstrated in vivo as well as ex vivo genome editing using adeno-associated virus (AAV)-, lentivirus-, or particle-mediated delivery of guide RNA in neurons, immune cells, and endothelial cells. Using these mice, we simultaneously modeled the dynamics of KRAS, p53, and LKB1, the top three significantly mutated genes in lung adenocarcinoma. Delivery of a single AAV vector in the lung generated loss-of-function mutations in p53 and Lkb1, as well as homology-directed repair-mediated Kras(G12D) mutations, leading to macroscopic tumors of adenocarcinoma pathology. Together, these results suggest that Cas9 mice empower a wide range of biological and disease modeling applications.
The study of cancer genes in mouse models has traditionally relied on genetically-engineered strains made via transgenesis or gene targeting in embryonic stem cells. Here we describe a new method of cancer model generation using the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system in vivo in wild-type mice. We used hydrodynamic injection to deliver a CRISPR plasmid DNA expressing Cas9 and single guide RNAs (sgRNAs) to the liver that directly target the tumour suppressor genes Pten (ref. 5) and p53 (also known as TP53 and Trp53) (ref. 6), alone and in combination. CRISPR-mediated Pten mutation led to elevated Akt phosphorylation and lipid accumulation in hepatocytes, phenocopying the effects of deletion of the gene using Cre-LoxP technology. Simultaneous targeting of Pten and p53 induced liver tumours that mimicked those caused by Cre-loxP-mediated deletion of Pten and p53. DNA sequencing of liver and tumour tissue revealed insertion or deletion mutations of the tumour suppressor genes, including bi-allelic mutations of both Pten and p53 in tumours. Furthermore, co-injection of Cas9 plasmids harbouring sgRNAs targeting the β-catenin gene and a single-stranded DNA oligonucleotide donor carrying activating point mutations led to the generation of hepatocytes with nuclear localization of β-catenin. This study demonstrates the feasibility of direct mutation of tumour suppressor genes and oncogenes in the liver using the CRISPR/Cas system, which presents a new avenue for rapid development of liver cancer models and functional genomics.
Bacterial type II CRISPR-Cas9 systems have been widely adapted for RNA-guided genome editing and transcription regulation in eukaryotic cells, yet their in vivo target specificity is poorly understood. Here we mapped genome-wide binding sites of a catalytically inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with single guide RNAs (sgRNAs) in mouse embryonic stem cells (mESCs). Each of the four sgRNAs we tested targets dCas9 to between tens and thousands of genomic sites, frequently characterized by a 5-nucleotide seed region in the sgRNA and an NGG protospacer adjacent motif (PAM). Chromatin inaccessibility decreases dCas9 binding to other sites with matching seed sequences; thus 70% of off-target sites are associated with genes. Targeted sequencing of 295 dCas9 binding sites in mESCs transfected with catalytically active Cas9 identified only one site mutated above background levels. We propose a two-state model for Cas9 binding and cleavage, in which a seed match triggers binding but extensive pairing with target DNA is required for cleavage.
Tetracycline or doxycycline (dox)-regulated control of genetic elements allows inducible, reversible and tissue specific regulation of gene expression in mice. This approach provides a means to investigate protein function in specific cell lineages and at defined periods of development and disease. Efficient and stable regulation of cDNAs or non-coding elements (e.g. shRNAs) downstream of the tetracycline-regulated element (TRE) requires the robust expression of a tet-transactivator protein, commonly the reverse tet-transactivator, rtTA. Most rtTA strains rely on tissue specific promoters that often do not provide sufficient rtTA levels for optimal inducible expression. Here we describe the generation of two mouse strains that enable Cre-dependent, robust expression of rtTA3, providing tissue-restricted and consistent induction of TRE-controlled transgenes. We show that these transgenic strains can be effectively combined with established mouse models of disease, including both Cre/LoxP-based approaches and non Cre-dependent disease models. The integration of these new tools with established mouse models promises the development of more flexible genetic systems to uncover the mechanisms of development and disease pathogenesis.
Targeted gene regulation on a genome-wide scale is a powerful strategy for interrogating, perturbing, and engineering cellular systems. Here, we develop a method for controlling gene expression based on Cas9, an RNA-guided DNA endonuclease from a type II CRISPR system. We show that a catalytically dead Cas9 lacking endonuclease activity, when coexpressed with a guide RNA, generates a DNA recognition complex that can specifically interfere with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This system, which we call CRISPR interference (CRISPRi), can efficiently repress expression of targeted genes in Escherichia coli, with no detectable off-target effects. CRISPRi can be used to repress multiple target genes simultaneously, and its effects are reversible. We also show evidence that the system can be adapted for gene repression in mammalian cells. This RNA-guided DNA recognition platform provides a simple approach for selectively perturbing gene expression on a genome-wide scale.
Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic
CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate
RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases
can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also
be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple
guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian
genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
RNA interference (RNAi) is an extremely effective tool for studying gene function in almost all metazoan and eukaryotic model systems. RNAi in mice, through the expression of short hairpin RNAs (shRNAs), offers something not easily achieved with traditional genetic approaches-inducible and reversible gene silencing. However, technical variability associated with the production of shRNA transgenic strains has so far limited their widespread use. Here we describe a pipeline for the generation of miR30-based shRNA transgenic mice that enables efficient and consistent targeting of doxycycline-regulated, fluorescence-linked shRNAs to the Col1a1 locus. Notably, the protocol details crucial steps in the design and testing of miR30-based shRNAs to maximize the potential for developing effective transgenic strains. In all, this 14-week procedure provides a fast and cost-effective way for any laboratory to investigate gene function in vivo in the mouse.
Transcription activator-like effectors (TALEs) are a class of naturally occurring DNA-binding proteins found in the plant pathogen Xanthomonas sp. The DNA-binding domain of each TALE consists of tandem 34-amino acid repeat modules that can be rearranged according to a simple cipher to target new DNA sequences. Customized TALEs can be used for a wide variety of genome engineering applications, including transcriptional modulation and genome editing. Here we describe a toolbox for rapid construction of custom TALE transcription factors (TALE-TFs) and nucleases (TALENs) using a hierarchical ligation procedure. This toolbox facilitates affordable and rapid construction of custom TALE-TFs and TALENs within 1 week and can be easily scaled up to construct TALEs for multiple targets in parallel. We also provide details for testing the activity in mammalian cells of custom TALE-TFs and TALENs using quantitative reverse-transcription PCR and Surveyor nuclease, respectively. The TALE toolbox described here will enable a broad range of biological applications.
Germ-line mutations of the APC gene are responsible for familial adenomatous polyposis (FAP), an autosomal dominantly inherited disease in humans. Patients with FAP develop multiple benign colorectal tumors. Recently, a mouse lineage that exhibits an autosomal dominantly inherited predisposition to multiple intestinal neoplasia (Min) was described. Linkage analysis showed that the murine homolog of the APC gene (mApc) was tightly linked to the Min locus. Sequence comparison of mApc between normal and Min-affected mice identified a nonsense mutation, which cosegregated with the Min phenotype. This mutation is analogous to those found in FAP kindreds and in sporadic colorectal cancers.
Although Apc is well characterized as a tumor-suppressor gene in the intestine, the precise mechanism of this suppression remains to be defined. Using a novel inducible Ahcre transgenic line in conjunction with a loxP-flanked Apc allele we, show that loss of Apc acutely activates Wnt signaling through the nuclear accumulation of beta-catenin. Coincidentally, it perturbs differentiation, migration, proliferation, and apoptosis, such that Apc-deficient cells maintain a "crypt progenitor-like" phenotype. Critically, for the first time we confirm a series of Wnt target molecules in an in vivo setting and also identify a series of new candidate targets within the same setting.
p53 is an important tumour suppressor with a known role in the later stages of colorectal cancer, but its relevance to the early stages of neoplastic initiation remains somewhat unclear. Although p53-dependent regulation of Wnt signalling activity is known to occur, the importance of these regulatory mechanisms during the early stages of intestinal neoplasia has not been demonstrated.
We have conditionally deleted the Adenomatous Polyposis coli gene (Apc) from the adult murine intestine in wild type and p53 deficient environments and subsequently compared the phenotype and transcriptome profiles in both genotypes.
Expression of p53 was shown to be elevated following the conditional deletion of Apc in the adult small intestine. Furthermore, p53 status was shown to impact on the transcription profile observed following Apc loss. A number of key Wnt pathway components and targets were altered in the p53 deficient environment. However, the aberrant phenotype observed following loss of Apc (rapid nuclear localisation of beta-catenin, increased levels of DNA damage, nuclear atypia, perturbed cell death, proliferation, differentiation and migration) was not significantly altered by the absence of p53.
p53 related feedback mechanisms regulating Wnt signalling activity are present in the intestine, and become activated following loss of Apc. However, the physiological Wnt pathway regulation by p53 appears to be overwhelmed by Apc loss and consequently the activity of these regulatory mechanisms is not sufficient to modulate the immediate phenotypes seen following Apc loss. Thus we are able to provide an explanation to the apparent contradiction that, despite having a Wnt regulatory capacity, p53 loss is not associated with early lesion development.
Cancer is a multistep process that involves mutations and other alterations in oncogenes and tumour suppressor genes. Genome sequencing studies have identified a large collection of genetic alterations that occur in human cancers. However, the determination of which mutations are causally related to tumorigenesis remains a major challenge. Here we describe a novel CRISPR/Cas9-based approach for rapid functional investigation of candidate genes in well-established autochthonous mouse models of cancer. Using a Kras(G12D)-driven lung cancer model, we performed functional characterization of a panel of tumour suppressor genes with known loss-of-function alterations in human lung cancer. Cre-dependent somatic activation of oncogenic Kras(G12D) combined with CRISPR/Cas9-mediated genome editing of tumour suppressor genes resulted in lung adenocarcinomas with distinct histopathological and molecular features. This rapid somatic genome engineering approach enables functional characterization of putative cancer genes in the lung and other tissues using autochthonous mouse models. We anticipate that this approach can be used to systematically dissect the complex catalogue of mutations identified in cancer genome sequencing studies.
Human pluripotent stem cells (hPSCs) offer a unique platform for elucidating the genes and molecular pathways that underlie complex traits and diseases. To realize this promise, methods for rapid and controllable genetic manipulations are urgently needed. By combining two newly developed gene-editing tools, the TALEN and CRISPR/Cas systems, we have developed a genome-engineering platform in hPSCs, which we named iCRISPR. iCRISPR enabled rapid and highly efficient generation of biallelic knockout hPSCs for loss-of-function studies, as well as homozygous knockin hPSCs with specific nucleotide alterations for precise modeling of disease conditions. We further demonstrate efficient one-step generation of double- and triple-gene knockout hPSC lines, as well as stage-specific inducible gene knockout during hPSC differentiation. Thus the iCRISPR platform is uniquely suited for dissection of complex genetic interactions and pleiotropic gene functions in human disease studies and has the potential to support high-throughput genetic analysis in hPSCs.
The type II bacterial CRISPR/Cas system is a novel genome-engineering technology with the ease of multiplexed gene targeting. Here, we created reporter and conditional mutant mice by coinjection of zygotes with Cas9 mRNA and different guide RNAs (sgRNAs) as well as DNA vectors of different sizes. Using this one-step procedure we generated mice carrying a tag or a fluorescent reporter construct in the Nanog, the Sox2, and the Oct4 gene as well as Mecp2 conditional mutant mice. In addition, using sgRNAs targeting two separate sites in the Mecp2 gene, we produced mice harboring the predicted deletions of about 700 bps. Finally, we analyzed potential off-targets of five sgRNAs in gene-modified mice and ESC lines and identified off-target mutations in only rare instances.
Targeted genome editing technologies have enabled a broad range of research and medical applications. The Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a 20 nt guide sequence, which can tolerate certain mismatches to the DNA target and thereby promote undesired off-target mutagenesis. Here, we describe an approach that combines a Cas9 nickase mutant with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity, simultaneous nicking via appropriately offset guide RNAs is required for double-stranded breaks and extends the number of specifically recognized bases for target cleavage. We demonstrate that using paired nicking can reduce off-target activity by 50- to 1,500-fold in cell lines and to facilitate gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency. This versatile strategy enables a wide variety of genome editing applications that require high specificity.
The genetic interrogation and reprogramming of cells requires methods for robust and precise targeting of genes for expression or repression. The CRISPR-associated catalytically inactive dCas9 protein offers a general platform for RNA-guided DNA targeting. Here, we show that fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in human and yeast cells, with the site of delivery determined solely by a coexpressed short guide (sg)RNA. Coupling of dCas9 to a transcriptional repressor domain can robustly silence expression of multiple endogenous genes. RNA-seq analysis indicates that CRISPR interference (CRISPRi)-mediated transcriptional repression is highly specific. Our results establish that the CRISPR system can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence, revealing the potential of CRISPRi as a general tool for the precise regulation of gene expression in eukaryotic cells.
Clustered, regularly interspaced, short palindromic repeat (CRISPR) RNA-guided nucleases (RGNs) have rapidly emerged as a facile and efficient platform for genome editing. Here, we use a human cell-based reporter assay to characterize off-target cleavage of CRISPR-associated (Cas)9-based RGNs. We find that single and double mismatches are tolerated to varying degrees depending on their position along the guide RNA (gRNA)-DNA interface. We also readily detected off-target alterations induced by four out of six RGNs targeted to endogenous loci in human cells by examination of partially mismatched sites. The off-target sites we identified harbored up to five mismatches and many were mutagenized with frequencies comparable to (or higher than) those observed at the intended on-target site. Our work demonstrates that RGNs can be highly active even with imperfectly matched RNA-DNA interfaces in human cells, a finding that might confound their use in research and therapeutic applications.
Mice carrying mutations in multiple genes are traditionally generated by sequential recombination in embryonic stem cells and/or time-consuming intercrossing of mice with a single mutation. The CRISPR/Cas system has been adapted as an efficient gene-targeting technology with the potential for multiplexed genome editing. We demonstrate that CRISPR/Cas-mediated gene editing allows the simultaneous disruption of five genes (Tet1, 2, 3, Sry, Uty - 8 alleles) in mouse embryonic stem (ES) cells with high efficiency. Coinjection of Cas9 mRNA and single-guide RNAs (sgRNAs) targeting Tet1 and Tet2 into zygotes generated mice with biallelic mutations in both genes with an efficiency of 80%. Finally, we show that coinjection of Cas9 mRNA/sgRNAs with mutant oligos generated precise point mutations simultaneously in two target genes. Thus, the CRISPR/Cas system allows the one-step generation of animals carrying mutations in multiple genes, an approach that will greatly accelerate the in vivo study of functionally redundant genes and of epistatic gene interactions.
Bacteria and archaea have evolved adaptive immune defenses, termed clustered regularly interspaced short palindromic repeats
(CRISPR)/CRISPR-associated (Cas) systems, that use short RNA to direct degradation of foreign nucleic acids. Here, we engineer
the type II bacterial CRISPR system to function with custom guide RNA (gRNA) in human cells. For the endogenous AAVS1 locus,
we obtained targeting rates of 10 to 25% in 293T cells, 13 to 8% in K562 cells, and 2 to 4% in induced pluripotent stem cells.
We show that this process relies on CRISPR components; is sequence-specific; and, upon simultaneous introduction of multiple
gRNAs, can effect multiplex editing of target loci. We also compute a genome-wide resource of ~190 K unique gRNAs targeting
~40.5% of human exons. Our results establish an RNA-guided editing tool for facile, robust, and multiplexable human genome
engineering.
We previously established long-term culture conditions under which single crypts or stem cells derived from mouse small intestine expand over long periods. The expanding crypts undergo multiple crypt fission events, simultaneously generating villus-like epithelial domains that contain all differentiated types of cells. We have adapted the culture conditions to grow similar epithelial organoids from mouse colon and human small intestine and colon.
Based on the mouse small intestinal culture system, we optimized the mouse and human colon culture systems.
Addition of Wnt3A to the combination of growth factors applied to mouse colon crypts allowed them to expand indefinitely. Addition of nicotinamide, along with a small molecule inhibitor of Alk and an inhibitor of p38, were required for long-term culture of human small intestine and colon tissues. The culture system also allowed growth of mouse Apc-deficient adenomas, human colorectal cancer cells, and human metaplastic epithelia from regions of Barrett's esophagus.
We developed a technology that can be used to study infected, inflammatory, or neoplastic tissues from the human gastrointestinal tract. These tools might have applications in regenerative biology through ex vivo expansion of the intestinal epithelia. Studies of these cultures indicate that there is no inherent restriction in the replicative potential of adult stem cells (or a Hayflick limit) ex vivo.
RNA interference is a powerful tool for studying gene function, however, the reproducible generation of RNAi transgenic mice remains a significant limitation. By combining optimized fluorescence-coupled miR30-based shRNAs with high efficiency ES cell targeting, we developed a fast, scalable pipeline for the production of shRNA transgenic mice. Using this system, we generated eight tet-regulated shRNA transgenic lines targeting Firefly and Renilla luciferases, Oct4 and tumor suppressors p53, p16(INK4a), p19(ARF) and APC and demonstrate potent gene silencing and GFP-tracked knockdown in a broad range of tissues in vivo. Further, using an shRNA targeting APC, we illustrate how this approach can identify predicted phenotypes and also unknown functions for a well-studied gene. In addition, through regulated gene silencing we validate APC/Wnt and p19(ARF) as potential therapeutic targets in T cell acute lymphoblastic leukemia/lymphoma and lung adenocarcinoma, respectively. This system provides a cost-effective and scalable platform for the production of RNAi transgenic mice targeting any mammalian gene. PAPERCLIP:
Mutation of the APC (adenomatous polyposis coli) gene is an early event in colon tumor development in humans. Mice carrying Min (multiple intestinal neoplasia), a mutant allele of Apc, develop intestinal and mammary tumors as adults. To study the role of the Apc gene in development, we have investigated the phenotype of embryos homozygous for ApcMin (Min). Development of the primitive ectoderm fails prior to gastrulation in homozygous Min embryos. By midgestation, the presumed homozygotes consist of a mass of trophoblast giant cells with an additional cluster of much smaller embryonic cells. These results indicate that functional Apc is required for normal growth of inner cell mass derivatives.
The POU-domain transcription factor Oct-4 is normally expressed in pluripotent cells of the mammalian embryo. In addition, germ-cell tumors and a few somatic tumors show detectable expression of Oct-4. While Oct-4's role during preimplantation development is to maintain embryonic cells in a pluripotent state, little is known about its potential oncogenic properties. Here we investigate the effect of ectopic Oct-4 expression on somatic tissues of adult mice using a doxycycline-dependent expression system. Activation of Oct-4 results in dysplastic growths in epithelial tissues that are dependent on continuous Oct-4 expression. Dysplastic lesions show an expansion of progenitor cells and increased beta-catenin transcriptional activity. In the intestine, Oct-4 expression causes dysplasia by inhibiting cellular differentiation in a manner similar to that in embryonic cells. These data show that certain adult progenitors remain competent to interpret key embryonic signals and support the notion that progenitor cells are a driving force in tumorigenesis.
Author manuscript; available in PMC
Nat Biotechnol. Author manuscript; available in PMC 2015 October 01.
Heritable gene targeting in the mouse and rat using a CRISPR-Cas system
- D Li
Li D, et al. Heritable gene targeting in the mouse and rat using a CRISPR-Cas system. Nat
Biotechnol. 2013; 31:681-683. [PubMed: 23929336]