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Application of CRISPR-Cas systems in neuroscience.
CRISPR-Cas systems have, over the years, emerged as indispensable tools for Genetic interrogation in contexts of clinical interventions, elucidation of genetic pathways and metabolic engineering and have pervaded almost every aspect of modern biology. Within this repertoire, the nervous system comes with its own set of perplexities and mysteries. Scientists have, over the years, tried to draw up a clearer genetic picture of the neuron and how it functions in a network, mainly in an endeavor to mitigate diseases of the human nervous system like Alzheimer's, Parkinson's, Huntington's, Autism Spectrum Disorder (ASD), etc. With most being progressive in nature, these diseases have plagued mankind for centuries. In spite of our immense progress in modern biology, we are yet to get a grasp over these diseases and unraveling their mechanisms is of utmost importance. Before CRISPR-Cas systems came along, the elucidation of the complex interactome of the mammalian nervous system was attempted with erstwhile existing electrophysiological, histological and pharmacological techniques coupled with Next Generation Sequencing and cell-specific targeting technologies. Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), imparted excellent sequence specific DNA targeting capabilities but came with their huge baggage of extensive protein engineering requirements, which practically rendered them unsuitable for high throughput exercises. With the discovery of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and CRISPR Associated proteins(CAS) systems by Ishino (1987)1, the era of extensive custom made endonuclease targeting was ushered in. For the first time in 2012, Jinek et al. (2012)2 repurposed the CRISPR-Cas mediated bacterial immune system for customizable mammalian gene editing. The CRISPR-Cas technology made it possible to easily customize Cas9 endonucleases to cleave near specifically targeted sequences, thereby facilitating knock-ins or knock-outs, silencing or activating or editing any gene, at any locus of the genome, both at the base-pair level or at the epigenetic level. With this enhanced degree of freedom, decrypting the nervous system and therapeutic interventions for neuropathies became significantly less cumbersome an exercise. Here we take a brisk walk through the several endeavors of research that show how the humble bacteria's CRISPR-Cas system gave us the "nerves" to "talk" to our nerves with ease.
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Prime editing-mediated correction of the leptin receptor in muscle cells of db/db mice.
Genetic diseases can be caused by monogenic diseases, which result from a single gene mutation in the DNA sequence. Many innovative approaches have been developed to cure monogenic genetic diseases, namely by genome editing. A specific type of genomic editing, prime editing, has the potential advantage to edit the human genome without requiring double-strand breaks or donor DNA templates for editing. Additionally, prime editing does not require a precisely positioned protospacer adjacent motif (PAM) sequence, which offers flexible target and more precise genomic editing. Here we detail a novel construction of a prime editing extended guide RNA (pegRNA) to target mutated leptin receptors in B6.BKS(D)-Leprdb/J mice (db/db mice). The pegRNA was then injected into the flexor digitorum brevis (FDB) muscle of db/db mice to demonstrate in vivo efficacy, which resulted in pegRNA mediated base transversion at endogenous base transversion. Genomic DNA sequencing confirmed that prime editing could correct the mutation of leptin receptor gene in db/db mice. Furthermore, prime editing treated skeletal muscle exhibited enhanced leptin receptor signals. Thus, the current study showed in vivo efficacy of prime editing to correct mutant protein and rescue the physiology associated with functional protein.
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Increased lateral root formation by CRISPR/Cas9-mediated editing of arginase genes in cotton.
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An SpG-Cas9-based cytosine base editor expands the scope of genome editing in carrot plants.
SpG Cas9 significantly expands the genome editing scope in carrot with NGN PAM recognition.
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Decrypting the mechanistic basis of CRISPR/Cas9 protein.
CRISPR/Cas system, a newly but extensively investigated genome-editing method, harbors practical solutions for various genetic problems. It relies on short guide RNAs (gRNAs) to recruit the Cas9 protein, a DNA cleaving enzyme, to its genomic target DNAs. The Cas9 enzyme exhibits some unique properties, like the ability to differentiate self vs. non-self - DNA strands using the base-pairing potential of crRNA, i.e., only CRISPR DNA is entirely complementary to the CRISPR repeat sequences at the crRNA whereas the presence of mismatches in the upstream region of the spacer permit CRISPR interference which is inhibited in case of CRISPR-DNA, allosteric regulation in its domains, and domain reorientation on sgRNA binding. Several groups have contributed their efforts in understanding the functioning of the CRISPR/Cas system, but even then, there is a lot more to explore in this area. The structural and sequence-based understanding of the whole CRISPR-associated bacterial ortholog family landscape is still ambiguous. A better understanding of the underlying energetics of the CRISPR/Cas9 system should reveal critical parameters to design better CRISPR/Cas9s.
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Efficient production of transgene-free, gene-edited carrot plants via protoplast transformation.
We have developed and validated an efficient protocol for producing gene-edited carrot plants that do not result in the stable incorporation of foreign DNA in the edited plant's genome. We report here a method for producing transgene-free, gene-edited carrot (Daucus carota subs. sativus) plants. With this approach, PEG-mediated transformation is used to transiently express a cytosine base editor and a guide RNA in protoplasts to induce targeted mutations in the carrot genome. These protoplasts are then cultured under conditions that lead to the production of somatic embryos which subsequently develop into carrot plants. For this study, we used the Centromere-Specific Histone H3 (CENH3) gene as a target for evaluating the efficiency with which regenerated, edited plants could be produced. After validating sgRNA performance and protoplast transformation efficiency using transient assays, we performed two independent editing experiments using sgRNAs targeting different locations within CENH3. In the first experiment, we analyzed 184 regenerated plants and found that 22 of them (11.9%) carried targeted mutations within CENH3, while in the second experiment, 28 out of 190 (14.7%) plants had mutations in CENH3. Of the 50 edited carrot lines that we analyzed, 43 were homozygous or bi-allelic for mutations in CENH3. No evidence of the base editor expression plasmid was found in the edited lines tested, indicating that this approach is able to produce transgene-free, gene-edited lines. The protocol that we describe provides an efficient method for easily generating large numbers of transgene-free, gene-edited carrot plants.
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Highly efficient and genotype-independent barley gene editing based on anther culture.
Recalcitrance to tissue culture and genetic transformation is the major bottleneck for gene manipulation in crops. In barley, immature embryos of Golden Promise have typically been used as explants for transformation. However, the genotype dependence of this approach limits the genetic modification of commercial varieties. Here, we developed an anther culture-based system that permits the effective creation of transgenic and gene-edited plants from commercial barley varieties. The protocol was tested in Golden Promise and four Australian varieties, which differed in phenology, callus induction, and green plant regeneration responses. Agrobacterium-mediated transformation was performed on microspore-derived callus to target the HvPDS gene, and T0 albinos with targeted mutations were successfully obtained from commercial varieties. Further editing of three targets was achieved with an average mutation rate of 53% in the five varieties. In 51 analyzed T0 individuals, Cas9 induced a large proportion (69%) of single-base indels and two-base deletions in the target sites, with variable mutation rates among targets and varieties. Both on-target and off-target activities were detected in T1 progenies. Compared with immature embryo protocols, this genotype-independent platform can deliver a high editing efficiency and more regenerant plants within a similar time frame. It shows promise for functional genomics and the application of CRISPR technologies for the precise improvement of commercial varieties.
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Prime editing via precise sequence insertion restores function of the recessive rc allele in rice.
An improved prime editing system precisely corrected a 14-bp deletion in the rc gene of white rice, restoring the production of brown pigments.
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Viral and nonviral nanocarriers for in vivo CRISPR-based gene editing.
The continued development of clustered regularly interspaced short palindromic repeats (CRISPR) technology has the potential to greatly impact clinical medicine, particularly for disease diagnosis and treatment. Despite high demand for the in vivo delivery of CRISPR-based therapies, significant challenges persist. These include rapid degradation by enzymes, inefficient disease site targeting, and the risk of undesired off-target outcomes. Nanoparticulate platforms, with their tailorable properties, have been engineered to efficiently package CRISPR payloads in various formats, including as plasmid DNA, mRNA, and ribonucleoprotein complexes, for in vivo delivery. Among them, recombinant adeno-associated viruses, virus-like particles, and lipid nanoparticles have displayed exceptional promise. This review will discuss the development of these and other nanocarriers for in vivo CRISPR-based genome editing.
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Toward optimizing diversifying base editors for high-throughput mutational scanning studies.
Base editors, including diversifying base editors that create C>N mutations, are potent tools for systematically installing point mutations in mammalian genomes and studying their effect on cellular function. Numerous base editor options are available for such studies, but little information exists on how the composition of the editor (deaminase, recruitment method, and fusion architecture) affects editing. To address this knowledge gap, the effect of various design features, such as deaminase recruitment and delivery method (electroporation or lentiviral transduction), on editing was assessed across ∼200 synthetic target sites. The direct fusion of a hyperactive variant of activation-induced cytidine deaminase to the N-terminus of dCas9 (DivA-BE) produced the highest editing efficiency, ∼4-fold better than the previous CRISPR-X method. Additionally, DivA-BE mutagenized the DNA strand that anneals to the targeting sgRNA (target strand) to create complementary C>N mutations, which were absent when the deaminase was fused to the C-terminus of dCas9. Based on these studies that comprehensively analyze the editing patterns of several popular base editors, DivA-BE editors efficiently diversified their target sites, albeit with increased indel frequencies. Overall, the improved editing efficiency makes the DivA-BE editors ideal for discovering functional variants in mutational scanning assays.
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