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Advances in CRISPR-Cas systems for human bacterial disease.
Prokaryotic adaptive immune systems called CRISPR-Cas systems have transformed genome editing by allowing for precise genetic alterations through targeted DNA cleavage. This system comprises CRISPR-associated genes and repeat-spacer arrays, which generate RNA molecules that guide the cleavage of invading genetic material. CRISPR-Cas is classified into Class 1 (multi-subunit effectors) and Class 2 (single multi-domain effectors). Its applications span combating antimicrobial resistance (AMR), targeting antibiotic resistance genes (ARGs), resensitizing bacteria to antibiotics, and preventing horizontal gene transfer (HGT). CRISPR-Cas3, for example, effectively degrades plasmids carrying resistance genes, providing a precise method to disarm bacteria. In the context of ESKAPE pathogens, CRISPR technology can resensitize bacteria to antibiotics by targeting specific resistance genes. Furthermore, in tuberculosis (TB) research, CRISPR-based tools enhance diagnostic accuracy and facilitate precise genetic modifications for studying Mycobacterium tuberculosis. CRISPR-based diagnostics, leveraging Cas endonucleases' collateral cleavage activity, offer highly sensitive pathogen detection. These advancements underscore CRISPR's transformative potential in addressing AMR and enhancing infectious disease management.
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Putting CRISPR-Cas system in action: a golden window for efficient and precise genome editing for crop improvement.
The daunting task of feeding an ever-growing population is an immense challenge for the contemporary scientific community, especially in view of the rapidly changing climate throughout the world. Amidst these threatening crises, we witness rapid development in genome editing (GE) technologies, revolutionizing the field of applied genomics and molecular breeding. Various GE tools have been developed during the last two decades, but the CRISPR/Cas system has most recently made a significant impact on crop improvement. The major breakthroughs of this versatile toolbox are genomic modifications like single base-substitutions, multiplex GE, gene regulation, screening mutagenesis, and enhancing the breeding of wild crop plants. Previously, this toolbox was used to modify genes related to significant traits such as biotic/abiotic resistance/tolerance, post-harvest traits, nutritional regulation, and to address self-incompatibility analysis-related challenges. In the present review, we have demonstrated the functional dynamics of CRISPR-based GE and its applicability in targeting genes to accomplish novel editing of crops. The compiled knowledge will provide a solid foundation for highlighting the primary source for applying CRISPR/Cas as a toolbox for enhancing crops, to achieve food and nutritional security.
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The initiation, propagation and dynamics of CRISPR-SpyCas9 R-loop complex.
CRISPR-Cas9 system has been widely used for efficient genome editing. Although the structures of Cas9 protein in complex with single-guided RNA (sgRNA) and target DNA have been resolved, the molecular details about the formation of Cas9 endonuclease R-loop structure remain elusive. Here we examine the DNA cleavage activities of Streptococcus pyogenes Cas9 (SpyCas9) and its mutants using various target sequences and study the conformational dynamics of R-loop structure during target binding using single-molecule fluorescence energy transfer (smFRET) technique. Our results show that Cas9-sgRNA complex divides the target DNA into several distinct domains: protospacer adjacent motif, linker, Seed, Middle and Tail. After seed pairing, the Cas9 transiently retains a semi-active conformation and induces the cleavage of either target or non-target strand. smFRET studies demonstrate that an intermediate state exists in prior to the formation of the fully stable R-loop complex. Kinetics analysis of this new intermediate state indicates that the lifetime of this state increases when the base-pairing length of guide-DNA hybrid duplex increases and reaches the maximum at the size of 18 bp. These data provide new insights into the process of R-loop formation and reveal the source of off-targeting in CRISPR/Cas9 system.
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A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells.
Translation of the CRISPR-Cas9 system to human therapeutics holds high promise. However, specificity remains a concern especially when modifying stem cell populations. We show that existing rationally engineered Cas9 high-fidelity variants have reduced on-target activity when using the therapeutically relevant ribonucleoprotein (RNP) delivery method. Therefore, we devised an unbiased bacterial screen to isolate variants that retain activity in the RNP format. Introduction of a single point mutation, p.R691A, in Cas9 (high-fidelity (HiFi) Cas9) retained the high on-target activity of Cas9 while reducing off-target editing. HiFi Cas9 induces robust AAV6-mediated gene targeting at five therapeutically relevant loci (HBB, IL2RG, CCR5, HEXB, and TRAC) in human CD34+ hematopoietic stem and progenitor cells (HSPCs) as well as primary T cells. We also show that HiFi Cas9 mediates high-level correction of the sickle cell disease (SCD)-causing p.E6V mutation in HSPCs derived from patients with SCD. We anticipate that HiFi Cas9 will have wide utility for both basic science and therapeutic genome-editing applications.
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Protein-nucleic acid language model-assisted design of precise and compact adenine base editor.
Adenine base editors (ABEs) are powerful tools for gene therapy. However, efficient version of ABEs (e.g. ABE8e) always induce excessive bystander and off-target editing events and are large in size, hindering their potential in clinical disease treatment. Here, we develop a pre-trained Protein-Nucleic Acid Constrained Language Model to design ABE8e with high activity, reduced editing window and decreased size. By further engineering, the smallest ABE8e- PNLM-pcABE- with a 27% size reduction, exhibits high activity, precise 3-nt editing window, and reduced off-target events near background level in HEK293T cells. Compared to ABE8e, PNLM-pcABE has up to 133.5-fold precision improvement in pathogenic mutation correction. By PNLM-pcABE, the albino mouse model carrying desired base mutation is nearly 100% obtained via zygotes microinjection and the expression of PCSK9 substantially decreases in mice receiving in vivo delivery with lipid nanoparticle (LNP), indicating their great potential in gene therapy and disease modeling.
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Biallelic mutations in the gene encoding eEF1A2 cause seizures and sudden death in F0 mice.
De novo heterozygous missense mutations in the gene encoding translation elongation factor eEF1A2 have recently been found to give rise to neurodevelopmental disorders. Children with mutations in this gene have developmental delay, epilepsy, intellectual disability and often autism; the most frequently occurring mutation is G70S. It has been known for many years that complete loss of eEF1A2 in mice causes motor neuron degeneration and early death; on the other hand heterozygous null mice are apparently normal. We have used CRISPR/Cas9 gene editing in the mouse to mutate the gene encoding eEF1A2, obtaining a high frequency of biallelic mutations. Whilst many of the resulting founder (F0) mice developed motor neuron degeneration, others displayed phenotypes consistent with a severe neurodevelopmental disorder, including sudden unexplained deaths and audiogenic seizures. The presence of G70S protein was not sufficient to protect mice from neurodegeneration in G70S/- mice, showing that the mutant protein is essentially non-functional.
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Programmed genome editing of the omega-1 ribonuclease of the blood fluke, Schistosoma mansoni.
CRISPR/Cas9-based genome editing has yet to be reported in species of the Platyhelminthes. We tested this approach by targeting omega-1 (ω1) of Schistosoma mansoni as proof of principle. This secreted ribonuclease is crucial for Th2 polarization and granuloma formation. Schistosome eggs were exposed to Cas9 complexed with guide RNA complementary to ω1 by electroporation or by transduction with lentiviral particles. Some eggs were also transfected with a single stranded donor template. Sequences of amplicons from gene-edited parasites exhibited Cas9-catalyzed mutations including homology directed repaired alleles, and other analyses revealed depletion of ω1 transcripts and the ribonuclease. Gene-edited eggs failed to polarize Th2 cytokine responses in macrophage/T-cell co-cultures, while the volume of pulmonary granulomas surrounding ω1-mutated eggs following tail-vein injection into mice was vastly reduced. Knock-out of ω1 and the diminished levels of these cytokines following exposure showcase the novel application of programmed gene editing for functional genomics in schistosomes.
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Homology-directed repair in mouse cells increased by CasRx-mediated knockdown or co-expressing Kaposi's sarcoma-associated herpesvirus ORF52.
Precise genome editing with directed base insertion or targeted point mutations can be achieved by CRISPR/Cas9-mediated homology-directed repair (HDR) and is of great significance in clinical disease therapy. However, HDR efficiency, compared with non-homologous end-joining (NHEJ), is inherently low. To enhance HDR, enabling the insertion of precise genetic modifications, we compared two strategies during surrogate reporter assays in mouse N2A cells: the suppression of DNA ligase IV, a key molecule in NHEJ, using the CasRx (Ruminococcus flavefaciens Cas13d) system, and co-expression of Kaposi's sarcoma-associated herpesvirus (KSHV) ORF52 proteins. We found that suppression of DNA ligase IV promotes HDR efficiency by 1.4-fold. When co-expressed with the Cas9 system, ORF52 improved HDR efficiency by up to 2.1-fold. In addition, we used ORF52 co-expression to modify the ACTB and Tubb3 genes of mouse N2A and E14 cells, which further increased HDR efficiency by approximately two- to four-fold. In conclusion, our data suggest that ORF52 co-expression is effective for enhancing CRISPR/Cas9-mediated HDR, which may be useful for future studies involving precise genome editing.
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CYP6AE gene cluster knockout in Helicoverpa armigera reveals role in detoxification of phytochemicals and insecticides.
The cotton bollworm Helicoverpa armigera, is one of the world's major pest of agriculture, feeding on over 300 hosts in 68 plant families. Resistance cases to most insecticide classes have been reported for this insect. Management of this pest in agroecosystems relies on a better understanding of how it copes with phytochemical or synthetic toxins. We have used genome editing to knock out a cluster of nine P450 genes and show that this significantly reduces the survival rate of the insect when exposed to two classes of host plant chemicals and two classes of insecticides. Functional expression of all members of this gene cluster identified the P450 enzymes capable of metabolism of these xenobiotics. The CRISPR-Cas9-based reverse genetics approach in conjunction with in vitro metabolism can rapidly identify the contributions of insect P450s in xenobiotic detoxification and serve to identify candidate genes for insecticide resistance.
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Targeted Forward Genetics: Population-Scale Analyses of Allele Replacements Spanning Thousands of Base Pairs in Fission Yeast.
Precise allele replacement (genome editing), without unwanted changes to the genome, provides a powerful tool to define the functions of DNA elements and encoded factors in their normal biological context. While CRISPR is now used extensively for gene targeting, its utility for precise allele replacement at population scale is limited because: (A) there is a strict requirement for a correctly positioned PAM motif to introduce recombinogenic dsDNA breaks (DSBs); (B) efficient replacements only occur very close to the DSBs; and (C) indels and off-target changes are frequently generated. Here we show, using a saturated mutation library with about 15,000 alleles of the ade6 gene of Schizosaccharomyces pombe, that pop-in, pop-out allele replacement circumvents these problems. Two rounds of selection ensure that clones arise by homologous recombination with the target locus. Moreover, the exceptionally high efficiency allows one to carry out the process in bulk, then screen individual clones for phenotypes and genotypes. Alleles were introduced successfully throughout the region targeted, up to 1,956 base pairs from the DSB. About 11% of mutant alleles were hypomorphic, demonstrating utility for analyses of essential genes and genetic elements. This process of "targeted forward genetics" can be used to analyze comprehensively, across thousands of base pairs within a specific target region, a variety of allelic changes, such as scanning amino acid substitutions, deletions, and epitope tags. The overall approach and optimized workflow are extensible to other organisms that support gene targeting.
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