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Found: 26 108
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Engineered prime editors with PAM flexibility.
Although prime editors are a powerful tool for genome editing, which can generate various types of mutations such as nucleotide substitutions, insertions, and deletions in the genome without double-strand breaks or donor DNA, the conventional prime editors are still limited to their target scopes because of the PAM preference of the Streptococcus pyogenes Cas9 (spCas9) protein. Here, we describe the engineered prime editors to expand the range of their target sites using various PAM-flexible Cas9 variants. Using the engineered prime editors, we could successfully generate more than 50 types of mutations with up to 51.7% prime-editing activity in HEK293T cells. In addition, we successfully introduced the BRAF V600E mutation, which could not be induced by conventional prime editors. These variants of prime editors will broaden the applicability of CRISPR-based prime editing technologies in biological research.
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CRISPR-Cas9 therapies in experimental mouse models of cancer.
The CRISPR-Cas9, a part of the defence mechanism from bacteria, has rapidly become the simplest, fastest and the most precise genome-editing tool available. The therapeutic applications of CRISPR are boundless: correction of mutations in several disorders, inactivation of oncogenes and viral oncoproteins, and activation of tumor suppressor genes. In this review, we expose recent advances concerning animal models of cancer that use CRISPR-Cas9, addressing also the current efforts to develop CRISPR-Cas9-based therapies, focusing on proof-of-concept studies. Finally, the review exposes some of the main challenges that this genome-editing tool faces. The key issue remains: does CRISPR-Cas9 have real potential for therapeutic application or will it just remain a wonderful research tool?
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Targeted RNA Knockdown by a Type III CRISPR-Cas Complex in Zebrafish.
RNA interference is a powerful experimental tool for RNA knockdown, but not all organisms are amenable. Here, we provide a proof of principle demonstration that a type III Csm effector complex can be used for programmable mRNA transcript degradation in eukaryotes. In zebrafish, Streptococcus thermophilus Csm complex (StCsm) proved effective for knockdown of maternally expressed EGFP in germ cells of Tg(ddx4:ddx4-EGFP) fish. It also led to significant, albeit less drastic, fluorescence reduction at one day postfertilization in Tg(myl7:GFP) and Tg(fli1:EGFP) fish that express EGFP zygotically. StCsm targeted against the endogenous tdgf1 elicited the characteristic one-eyed phenotype with greater than 50% penetrance, and hence with similar efficiency to morpholino-mediated knockdown. We conclude that Csm-mediated knockdown is very efficient for maternal transcripts and can also be used for mixed maternal/early zygotic and early zygotic transcripts, in some cases reaching comparable efficiency to morpholino-based knockdown without significant off-target effects.
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Genome-Wide CRISPRi-Based Identification of Targets for Decoupling Growth from Production.
Growth decoupling can be used to optimize microbial production of biobased compounds by inhibiting excess biomass formation and redirect carbon flux from growth to product formation. However, identifying suitable genetic targets through rational design is challenging. Here, we conduct a genome-wide CRISPRi screen to discover growth switches suitable for decoupling growth and production. Using an sgRNA library covering 12 238 loci in the Escherichia coli genome, we screen for targets that inhibit growth while allowing for continued protein production. In total, we identify 1332 sgRNAs that simultaneously decrease growth and maintain or increase accumulation of GFP. The top target sibB/ibsB shows more than 5-fold increase in GFP accumulation and 45% decrease in biomass formation. Overall, our genome-wide CRISPRi screen provides key targets for growth decoupling, and the approach can be applied to improve biobased production in other microorganisms.
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A CRISPR-Cpf1-Assisted Non-Homologous End Joining Genome Editing System of Mycobacterium smegmatis.
Mycobacterium smegmatis is an important model strain of Mycobacterium for scientific study because it is non-pathogenic and grows rapidly. However, research is limited by the low efficiency and time-consuming nature of existing genome editing tools. Although the Streptococcus pyogenes CRISPR-Cas9 system is widely used in bacterial genome editing, it cannot be introduced into M. smegmatis because of its toxicity. The authors test 14 different Cas effector proteins in M. smegmatis. Cas9 (TdCas9_m) from Treponema denticola, Cas9 (NmCas9) from Neisseria meningitidis, and Corynebacterium glutamicum codon-optimized Cpf1 (FnCpf1_cg) from Francisella tularensis do not affect cell growth. The numbers of transformant plasmids expressing TdCas9_m, NmCas9, or FnCpf1_cg, and guide RNAs (gRNA) targeting ku(MSMEG_5580), ligD(MSMEG_6301), pta(MSMEG_0783), or ackA(MSMEG_0784) decreases by about 10-, 10-, or 100-fold, respectively, compared with plasmids expressing only the Cas effector proteins. Non-homologous end joining (NHEJ) is detected only in the CRISPR-FnCpf1_cg system. The one-plasmid-based, CRISPR-FnCpf1-assisted NHEJ system enables N iterative rounds of genome editing in 7N + 2 days, with an editing efficiency up to 70%; thus, this system should greatly reduce the necessary genome manipulation time for M. smegmatis.
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Small Molecule SOS1 Agonists Modulate MAPK and PI3K Signaling via Independent Cellular Responses.
Activating mutations in RAS can lead to oncogenesis by enhancing downstream signaling, such as through the MAPK and PI3K pathways. Therefore, therapeutically targeting RAS may perturb multiple signaling pathways simultaneously. One method for modulating RAS signaling is to target the activity of the guanine nucleotide exchange factor SOS1. Our laboratory has discovered compounds that bind to SOS1 and activate RAS. Interestingly, these SOS1 agonist compounds elicit biphasic modulation of ERK phosphorylation and simultaneous inhibition of AKT phosphorylation levels. Here, we utilized multiple chemically distinct compounds to elucidate whether these effects on MAPK and PI3K signaling by SOS1 agonists were mechanistically linked. In addition, we used CRISPR/Cas9 gene-editing to generate clonally derived SOS1 knockout cells and identified a potent SOS1 agonist that rapidly elicited on-target molecular effects at substantially lower concentrations than those causing off-target effects. Our findings will allow us to further define the on-target utility of SOS1 agonists.
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Hierarchical self-uncloaking CRISPR-Cas13a-customized RNA nanococoons for spatial-controlled genome editing and precise cancer therapy.
CRISPR-Cas13a holds enormous potential for developing precise RNA editing. However, spatial manipulation of CRISPR-Cas13a activity remains a daunting challenge for elaborately regulating localized RNase function. Here, we designed hierarchical self-uncloaking CRISPR-Cas13a-customized RNA nanococoons (RNCOs-D), featuring tumor-specific recognition and spatial-controlled activation of Cas13a, for precise cancer synergistic therapy. RNCOs-D consists of programmable RNA nanosponges (RNSs) capable of targeted delivery and caging chemotherapeutic drug, and nanocapsules (NCs) anchored on RNSs for cloaking Cas13a/crRNA ribonucleoprotein (Cas13a RNP) activity. The acidic endo/lysosomal microenvironment stimulates the outer decomposition of NCs with concomitant Cas13a RNP activity revitalization, while the inner disassembly through trans-cleavage of RNSs initiated by cis-recognition and cleavage of EGFR variant III (EGFRvIII) mRNA. RNCOs-D demonstrates the effective EGFRvIII mRNA silencing for synergistic therapy of glioblastoma cancer cells in vitro and in vivo. The engineering of RNSs, together with efficient Cas13a activity regulation, holds immense prospect for multimodal and synergistic cancer therapy.
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A Comprehensive Review on CRISPR-Based Screening and Its Applications.
CRISPR-based tools have quickly moved from specialist techniques to routine instruments in biology and medicine, and they are now central to large-scale loss-of-function and perturbation screens. In this review, we focus on how pooled CRISPR screens are used to interrogate gene function in living cells, most often through cell fitness or simple selectable markers, and contrast this with arrayed formats that trade throughput for richer molecular readouts, such as transcriptome-wide changes. We bring together current strategies for library design, delivery, and selection and show how different Cas nucleases, including Cas9, Cas12, and Cas13, broaden the range of genome and transcriptome perturbations that can be assayed. We then discuss recent applications in drug response, viral infection, and cancer biology and consider how improvements in high-content technologies, data analysis, and emerging diagnostic uses are likely to shape the next generation of CRISPR-based screening studies.
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Pooled library screening with multiplexed Cpf1 library.
Capitalizing on the inherent multiplexing capability of AsCpf1, we developed a multiplexed, high-throughput screening strategy that minimizes library size without sacrificing gene targeting efficiency. We demonstrated that AsCpf1 can be used for functional genomics screenings and that an AsCpf1-based multiplexed library performs similarly as compared to currently available monocistronic CRISPR/Cas9 libraries, with only one vector required for each gene. We construct the smallest whole-genome CRISPR knock-out library, Mini-human, for the human genome (n = 17,032 constructs targeting 16,977 protein-coding genes), which performs favorably compared to conventional Cas9 libraries.
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A toolkit for rapid CRISPR-SpCas9 assisted construction of hexose-transport-deficient Saccharomyces cerevisiae strains.
Hexose transporter-deficient yeast strains are valuable testbeds for the study of sugar transport by native and heterologous transporters. In the popular Saccharomyces cerevisiae strain EBY.VW4000, deletion of 21 transporters completely abolished hexose transport. However, repeated use of the LoxP/Cre system in successive deletion rounds also resulted in major chromosomal rearrangements, gene loss and phenotypic changes. In the present study, CRISPR/SpCas9 was used to delete the 21 hexose transporters in an S. cerevisiae strain from the CEN.PK family in only three deletion rounds, using 11 unique guide RNAs. Even upon prolonged cultivation, the resulting strain IMX1812 (CRISPR-Hxt0) was unable to consume glucose, while its growth rate on maltose was the same as that of a strain equipped with a full set of hexose transporters. Karyotyping and whole-genome sequencing of the CRISPR-Hxt0 strain with Illumina and Oxford Nanopore technologies did not reveal chromosomal rearrangements or other unintended mutations besides a few SNPs. This study provides a new, 'genetically unaltered' hexose transporter-deficient strain and supplies a CRISPR toolkit for removing all hexose transporter genes from most S. cerevisiae laboratory strains in only three transformation rounds.
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