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Modeling pediatric AML FLT3 mutations using CRISPR/Cas12a- mediated gene editing.
Clustered regularly interspaced palindromic repeats (CRISPR) with the associated (Cas) nuclease complexes have democratized genetic engineering through their precision and ease-of-use. We have applied a variation of this technology, known as CRISPR-directed mutagenesis (CDM), to reconstruct genetic profiles within the FLT3 gene of AML patients. We took advantage of the versatility of CDM and built expression vectors that, in combination with a specifically designed donor DNA fragment, recapitulate simple and complex mutations within the FLT3 gene. We generate insertions and point mutations including combinations of these mutations originating from individual patient samples. We then analyze how these complex genetic profiles modulate transformation of Ba/F3 cells. Our results show that FLT3 expression plasmids bearing patient-specific single or multiple mutations recapitulate cellular transformation properties induced by FLT3 ITDs and modify their sensitivity or resistance in response to established AML drugs as a function of these complex mutations.
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Applications of CRISPR in Parasitology.
Clustered Regions of Interspersed Palindromic Repeat (CRISPR)-based techniques have been utilized in various research areas, including agriculture, biotechnology, and medicine. With the use of a short sequence guide RNA and CRISPR-associated (Cas) protein, this technique allows for robust, site-specific manipulation of the genome, aiding researchers in making important biomedical discoveries and scientific advancements. In this review, we explored the applications of CRISPR/Cas systems in the field of parasitology for the identification and validation of novel functional genes, diagnosis of parasitic infections, reduction of parasite virulence, and the disruption of disease transmission. We also discussed how CRISPR can be used for the development of therapeutics, vaccines, and drug discovery. Furthermore, the challenges and future perspectives of this technology are also highlighted.
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CRISPR-Cas3 induces broad and unidirectional genome editing in human cells.
Although single-component Class 2 CRISPR systems, such as type II Cas9 or type V Cas12a (Cpf1), are widely used for genome editing in eukaryotic cells, the application of multi-component Class 1 CRISPR has been less developed. Here we demonstrate that type I-E CRISPR mediates distinct DNA cleavage activity in human cells. Notably, Cas3, which possesses helicase and nuclease activity, predominantly triggered several thousand base pair deletions upstream of the 5'-ARG protospacer adjacent motif (PAM), without prominent off-target activity. This Cas3-mediated directional and broad DNA degradation can be used to introduce functional gene knockouts and knock-ins. As an example of potential therapeutic applications, we show Cas3-mediated exon-skipping of the Duchenne muscular dystrophy (DMD) gene in patient-induced pluripotent stem cells (iPSCs). These findings broaden our understanding of the Class 1 CRISPR system, which may serve as a unique genome editing tool in eukaryotic cells distinct from the Class 2 CRISPR system.
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Give CRISPR a Chance: the GeneSprout Initiative.
Did you know that a group of early-career researchers launched an initiative enabling open dialog on new plant breeding techniques, such as genome editing? We developed a wide-ranging initiative that aims to facilitate public engagement and provide a platform for young plant scientists to encourage participation in science communication.
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From Evolution to Revolution: Accelerating Crop Domestication through Genome Editing.
Crop domestication has a tremendous impact on socioeconomic conditions and human civilization. Modern cultivars were domesticated from their wild progenitors thousands of years ago by the selection of natural variation by humans. New cultivars are being developed by crossing two or more compatible individuals. But the limited genetic diversity in the cultivars severely affects the yield and renders the crop susceptible to many biotic and abiotic stresses. Crop wild relatives (CWRs) are the rich reservoir for many valuable agronomic traits. The incorporation of useful genes from CWR is one of the sustainable approaches for enriching the gene pool of cultivated crops. However, CWRs are not suited for urban and intensive cultivation because of several undesirable traits. Researchers have begun to study the domestication traits in the CWRs and modify them using genome-editing tools to make them suitable for extensive cultivation. Growing evidence has shown that modification in these genes is not sufficient to bring the desired change in the neodomesticated crop. However, the other dynamic genetic factors such as microRNAs (miRNAs), transposable elements, cis-regulatory elements and epigenetic changes have reshaped the domesticated crops. The creation of allelic series for many valuable domestication traits through genome editing holds great potential for the accelerated development of neodomesticated crops. The present review describes the current understanding of the genetics of domestication traits that are responsible for the agricultural revolution. The targeted mutagenesis in these domestication genes via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 could be used for the rapid domestication of CWRs.
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CRISPR-TSKO: A Tool for Tissue-Specific Genome Editing in Plants.
Functional genomics is at the core of studying the exact function of genes. However, homozygous knockouts of essential and pleiotropic effectors (almost 10% of the genome) are not always possible, thus, functions of these genes remain obscured. The tissue-specific genome editing tool (CRISPR-TSKO) recently described by Decaestecker et al., can characterize these indispensable genes and has wide applications in plants.
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Engineered extracellular vesicles as versatile ribonucleoprotein delivery vehicles for efficient and safe CRISPR genome editing.
Transient delivery of CRISPR-based genome editing effectors is important to reduce off-target effects and immune responses. Recently extracellular vesicles (EVs) have been explored for Cas9 ribonucleoprotein (RNP) delivery. However, lack of mechanisms to enrich RNPs into EVs limited the efficiency of EVs as a RNP delivery vehicle. Here we describe a mechanism to actively enrich RNPs into EVs. We used the specific interaction between RNA aptamer and aptamer-binding protein (ABP) to enrich RNPs into EVs. We inserted RNA aptamer com into single guide RNA (sgRNA), and fused com-binding ABP Com to both termini of tetraspan protein CD63 that is abundant in exosomes. We found that the Com/com interaction enriched Cas9 and adenine base editor (ABE) RNPs into EVs, via forming a three-component complex including CD63-Com fusion protein, com-modified sgRNA and Cas9 or ABE. The RNP enriched EVs are efficient in genome editing and transiently expressed. The system is capable of delivering RNPs targeting multiple loci for multiplex genome editing. In addition, Cas9 from different species can be used together. The EV-delivered RNPs are active in vivo. The data show that the aptamer and ABP interactions can be utilized to actively enrich RNPs into EVs for improved genome editing efficiency and safety.
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Functional interrogation of non-coding DNA through CRISPR genome editing.
Methodologies to interrogate non-coding regions have lagged behind coding regions despite comprising the vast majority of the genome. However, the rapid evolution of clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing has provided a multitude of novel techniques for laboratory investigation including significant contributions to the toolbox for studying non-coding DNA. CRISPR-mediated loss-of-function strategies rely on direct disruption of the underlying sequence or repression of transcription without modifying the targeted DNA sequence. CRISPR-mediated gain-of-function approaches similarly benefit from methods to alter the targeted sequence through integration of customized sequence into the genome as well as methods to activate transcription. Here we review CRISPR-based loss- and gain-of-function techniques for the interrogation of non-coding DNA.
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He Jiankui´s gene-editing experiment and the non-identity problem.
Genetic engineering has been a topic of discussion for over 50 years, but it is only recently that gene editing has become a reality. CRISPR biotechnologies have made gene editing much safer, precise and feasible. We have witnessed the first cases of human germline genetic modification resulting in live births, conducted by He Jiankui. In this paper, we will analyse He Jiankui's case in relation to one of the most difficult problems in procreative ethics (or the ethics of future generations): the non-identity problem. We believe that this analysis will help us to understand the ethics involved in gene editing and hopefully allow for a better, more philosophically grounded legislation on CRISPR and other gene-editing technologies.
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Tumor immunology CRISPR screening: present, past, and future.
Recent advances in immunotherapy have fundamentally changed the landscape of cancer treatment by leveraging the specificity and selectivity of the adaptive immune system to kill cancer cells. These successes have ushered in a new wave of research aimed at understanding immune recognition with the hope of developing newer immunotherapies. The advent of clustered regularly interspaced short palindromic repeats (CRISPR) technologies and advancement of multiomics modalities have greatly accelerated the discovery process. Here, we review the current literature surrounding CRISPR screens within the context of tumor immunology, provide essential components needed to conduct immune-specific CRISPR screens, and present avenues for future research.
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