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A promoterless AAV6.2FF-based lung gene editing platform for the correction of surfactant protein B deficiency.
Surfactant protein B (SP-B) deficiency is a rare genetic disease that causes fatal respiratory failure within the first year of life. Currently, the only corrective treatment is lung transplantation. Here, we co-transduced the murine lung with adeno-associated virus 6.2FF (AAV6.2FF) vectors encoding a SaCas9-guide RNA nuclease or donor template to mediate insertion of promoterless reporter genes or the (murine) Sftpb gene in frame with the endogenous surfactant protein C (SP-C) gene, without disrupting SP-C expression. Intranasal administration of 3 × 1011 vg donor template and 1 × 1011 vg nuclease consistently edited approximately 6% of lung epithelial cells. Frequency of gene insertion increased in a dose-dependent manner, reaching 20%-25% editing efficiency with the highest donor template and nuclease doses tested. We next evaluated whether this promoterless gene editing platform could extend survival in the conditional SP-B knockout mouse model. Administration of 1 × 1012 vg SP-B-donor template and 5 × 1011 vg nuclease significantly extended median survival (p = 0.0034) from 5 days in the untreated off doxycycline group to 16 days in the donor AAV and nuclease group, with one gene-edited mouse living 243 days off doxycycline. This AAV6.2FF-based gene editing platform has the potential to correct SP-B deficiency, as well as other disorders of alveolar type II cells.
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CRISPR based therapeutics: a new paradigm in cancer precision medicine.
BACKGROUND: Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) systems are the latest addition to the plethora of gene-editing tools. These systems have been repurposed from their natural counterparts by means of both guide RNA and Cas nuclease engineering. These RNA-guided systems offer greater programmability and multiplexing capacity than previous generation gene editing tools based on zinc finger nucleases and transcription activator like effector nucleases. CRISPR-Cas systems show great promise for individualization of cancer precision medicine. MAIN BODY: The biology of Cas nucleases and dead Cas based systems relevant for in vivo gene therapy applications has been discussed. The CRISPR knockout, CRISPR activation and CRISPR interference based genetic screens which offer opportunity to assess functions of thousands of genes in massively parallel assays have been also highlighted. Single and combinatorial gene knockout screens lead to identification of drug targets and synthetic lethal genetic interactions across different cancer phenotypes. There are different viral and non-viral (nanoformulation based) modalities that can carry CRISPR-Cas components to different target organs in vivo. CONCLUSION: The latest developments in the field in terms of optimization of performance of the CRISPR-Cas elements should fuel greater application of the latter in the realm of precision medicine. Lastly, how the already available knowledge can help in furtherance of use of CRISPR based tools in personalized medicine has been discussed.
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Establishing CRISPR-Cas9 in the sexually dimorphic moss, Ceratodon purpureus.
The development of CRISPR technologies provides a powerful tool for understanding the evolution and functionality of essential biological processes. Here we demonstrate successful CRISPR-Cas9 genome editing in the dioecious moss species, Ceratodon purpureus. Using an existing selection system from the distantly related hermaphroditic moss, Physcomitrium patens, we generated knock-outs of the APT reporter gene by employing CRISPR-targeted mutagenesis under expression of native U6 snRNA promoters. Next, we used the native homology-directed repair (HDR) pathway, combined with CRISPR-Cas9, to knock in two reporter genes under expression of an endogenous RPS5A promoter in a newly developed landing site in C. purpureus. Our results show that the molecular tools developed in P. patens can be extended to other mosses across this ecologically important and developmentally variable group. These findings pave the way for precise and powerful experiments aimed at identifying the genetic basis of key functional variation within the bryophytes and between the bryophytes and other land plants.
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CRISPR/Cas 9 system for the treatment of dilated cardiomyopathy: A hypothesis related to function of a MAP kinase.
Dilated cardiomyopathy (DCM) is a disease with high incidence and mortality rates. Its therapies have one primary goal, which is to minimize symptoms and it has only one effective approach to healing, the heart transplantation. As it is widely associated with genetic causes, the use of gene therapies, such as the CRISPR/Cas9 system, is a promising alternative to treat DCM. For this purpose, it is necessary to analyze possible target genes for this approach and what would be the implications of their use. Here, we hypothesized that cardiac troponin I type 3 interacting kinase (TNI3K), involved with superoxide production in DCM patients, besides other factors, could be a good target for the use of gene editing.
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Stimuli-responsive delivery strategies for controllable gene editing in tumor therapeutics.
The CRISPR system has attracted significant attention due to its great potential in tumor therapy. Developing effective, precise and safe delivery vectors is a prerequisite for CRISPR applications. Some disease-related biological signals provide a rationale for the development of precise delivery vehicles for stimuli-response delivery. Therefore, combining the linker of endogenous signal and exogenous signal stimulus responses with the nanocarrier is the key to designing and synthesizing the controllable release vector in stimuli-response delivery. This review summarizes the synthesis of stimuli-responsive nanocarriers by designing key chemical structures to achieve controllable release. Nanocarriers controlled by biochemical or physical signals are then discussed. Finally, potential challenges and prospects for existing nanocarriers are discussed.
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Sharing mutations: are biobanks still required in the post-CRISPR/Cas9 era?
Cryopreservation is seen as a key aspect of good colony management which supports the drive towards improvements in animal care and the implementation of the 3Rs. However, following the advent of gene editing technologies, the generation of new mouse models is quicker and cheaper than ever before. This has led some to question the future value of biobanks around the world. In the following commentary, we argue that the need to cryopreserve mouse strains and distribute them from well-funded repositories is as strong as it has ever been. Repositories are not simply archives for unwanted mouse strains. Biobanks distribute identical QC verified mouse strains to the community and eliminate the need to recreate mice. They provide a check point in the development of mouse strains that minimises genetic drift and breeding failures. What is more, cryopreservation makes resource sharing easier, cheaper and improves animal care by eliminating the need for live animal shipments.
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Research Progress and Prospects of Molecular Breeding in Bermudagrass (Cynodon dactylon).
Bermudagrass (Cynodon dactylon L.) is a warm-season grass species of significant ecological and economic importance. It is widely utilized in turf management and forage production due to its resilience to drought, salt, and other environmental stresses. Recent advancements in molecular breeding, particularly through genomics technology and gene editing, have enabled the efficient identification of key genes associated with stress tolerance and turf quality. The use of techniques such as overexpression and CRISPR/Cas has enhanced resistance to drought, salt, cold, and heat, while the application of molecular markers has accelerated the development of superior varieties. The integration of multi-omics, such as genomics, transcriptomics, and proteomics, provides deeper insights into the molecular mechanisms of bermudagrass, thereby improving breeding efficiency and precision. Additionally, artificial intelligence is emerging as a powerful tool for analyzing genomic data, predicting optimal trait combinations, and accelerating breeding processes. These technologies, when combined with traditional breeding methods, hold great potential for optimizing bermudagrass varieties for both turf and forage use. Future research will focus on further integrating these tools to address the challenges of breeding posed by climate change to breeding climate-resilient turf and forage crops.
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CRISPR/Cas9 Methods for Identification and Validation of Genes Regulating BCR-Mediated Antigen Uptake.
Genome-wide CRISPR screens are a powerful tool to interrogate and identify gene function in a wide variety of applications and cell types. CRISPR-Cas9 technology using pooled CRISPR single guide RNA (sgRNA) libraries enables genetic editing in bulk in a large population of cells of interest. After selection of gene-edited cells, phenotyping effects can be evaluated by quantifying abundance (over- or under-representation) of individual sgRNAs using DNA sequencing. In addition to cell survival, these assays can be applied to investigations of drug sensitivity, as well as almost any cellular process with a clear phenotypic read out, for example, receptor internalization, migration, autophagy, and differentiation.Here, taking as an example the identification of molecular components governing B-cell antigen uptake through the B-cell receptor, we describe whole-genome, small-scale, and in vivo validation methods to identify and validate genes regulating BCR-mediated antigen uptake.
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An anti-CRISPR targets the sgRNA to block Cas9 and guides the design of enhanced genome editors.
Bacteriophages have evolved anti-CRISPR (Acr) proteins to combat the adaptive immunity provided by bacterial CRISPR-Cas systems. Here, we report the cryo-electron microscopy structure of an anti-Cas9 protein AcrIIA27 bound to SpyCas9-sgRNA (single guide RNA) complex. Our structure reveals that AcrIIA27 binds the solvent-exposed phosphate backbone of the sgRNA, acting as a potent inhibitor of diverse Cas9 orthologs. AcrIIA27 in the structure is positioned near the protospacer-adjacent motif DNA-binding pocket on SpyCas9, causing steric hindrance that prevents substrate DNA recognition. This mechanism suggests solvent-exposed regions of sgRNAs (PTP RNAs), prone to nonspecific binding of positively charged components, may compromise CRISPR-Cas genome-editing efficiency. Indeed, truncations of the PTP RNAs in different editing systems significantly enhance genome-editing efficiency in human cells. Overall, our findings reveal a previously uncharacterized inhibition mechanism of an anti-Cas protein and offers a general strategy for developing more efficient genome-editing tools.
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OTX2 represses sister cell fate choices in the developing retina to promote photoreceptor specification.
During vertebrate retinal development, subsets of progenitor cells generate progeny in a non-stochastic manner, suggesting that these decisions are tightly regulated. However, the gene-regulatory network components that are functionally important in these progenitor cells are largely unknown. Here we identify a functional role for the OTX2 transcription factor in this process. CRISPR/Cas9 gene editing was used to produce somatic mutations of OTX2 in the chick retina and identified similar phenotypes to those observed in human patients. Single cell RNA sequencing was used to determine the functional consequences OTX2 gene editing on the population of cells derived from OTX2-expressing retinal progenitor cells. This confirmed that OTX2 is required for the generation of photoreceptors, but also for repression of specific retinal fates and alternative gene regulatory networks. These include specific subtypes of retinal ganglion and horizontal cells, suggesting that in this context, OTX2 functions to repress sister cell fate choices.
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