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Gene editing the BmNPV inhibitor of apoptosis protein 2 (iap2) as an antiviral strategy in transgenic silkworm.
Apoptosis is a cellular defense mechanism used for the elimination of host cells infected by viruses. Viruses have evolved corresponding inhibitors of apoptosis genes to promote their replication. Anti-apoptosis-related genes, involved in baculovirus proliferation, have been proposed but it is unclear whether these genes can be manipulated in gene therapy. We constructed a transgenic silkworm, using the CRISPR/Cas9 system to knock out the BmNPV inhibitor of apoptosis 2 (iap2). The sequencing results showed that all the sequences could edit the target site of BmNPV iap2 gene. There were no differences in economic traits and growth tests between the BmNPV iap2 knockout strain transgenic silkworm lines and the control groups. However, the mortality rate was significantly reduced, the median lethal dose (LD50) was about 100 times higher than the control group, and the onset time was prolonged by 1-2 days after knocking out BmNPV iap2. In addition, the expression levels of apoptotic-related genes Bmiap2, BmICE and BmDreed were significantly affected and the activity of caspase 9 was increased after BmNPV iap2 being edited in transgenic silkworm. These results demonstrated that gene editing BmNPV iap2 could significantly inhibit BmNPV replication and proliferation. This approach provides a new strategy for antiviral research.
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Genome Editing of Veterinary Relevant Mycoplasmas Using a CRISPR-Cas Base Editor System.
Mycoplasmas are minimal bacteria that infect humans, wildlife, and most economically relevant livestock species. Mycoplasma infections cause a large range of chronic inflammatory diseases, eventually leading to death in some animals. Due to the lack of efficient recombination and genome engineering tools for most species, the production of mutant strains for the identification of virulence factors and the development of improved vaccine strains is limited. Here, we demonstrate the adaptation of an efficient Cas9-Base Editor system to introduce targeted mutations into three major pathogenic species that span the phylogenetic diversity of these bacteria: the avian pathogen Mycoplasma gallisepticum and the two most important bovine mycoplasmas, Mycoplasma bovis and Mycoplasma mycoides subsp. mycoides. As a proof of concept, we successfully used an inducible SpdCas9-pmcDA1 cytosine deaminase system to disrupt several major virulence factors in these pathogens. Various induction times and inducer concentrations were evaluated to optimize editing efficiency. The optimized system was powerful enough to disrupt 54 of 55 insertion sequence transposases in a single experiment. Whole-genome sequencing of the edited strains showed that off-target mutations were limited, suggesting that most variations detected in the edited genomes are Cas9-independent. This effective, rapid, and easy-to-use genetic tool opens a new avenue for the study of these important animal pathogens and likely the entire class Mollicutes. IMPORTANCE Mycoplasmas are minimal pathogenic bacteria that infect a wide range of hosts, including humans, livestock, and wild animals. Major pathogenic species cause acute to chronic infections involving still poorly characterized virulence factors. The lack of precise genome editing tools has hampered functional studies of many species, leaving multiple questions about the molecular basis of their pathogenicity unanswered. Here, we demonstrate the adaptation of a CRISPR-derived base editor for three major pathogenic species: Mycoplasma gallisepticum, Mycoplasma bovis, and Mycoplasma mycoides subsp. mycoides. Several virulence factors were successfully targeted, and we were able to edit up to 54 target sites in a single step. The availability of this efficient and easy-to-use genetic tool will greatly facilitate functional studies of these economically important bacteria.
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Using CRISPR-Cas9 to Dissect Cancer Mutations in Cell Lines.
The CRISPR-Cas9 technology has revolutionized the scope and pace of biomedical research, enabling the targeting of specific genomic sequences for a wide spectrum of applications. Here we describe assays to functionally interrogate mutations identified in cancer cells utilizing both CRISPR-Cas9 nuclease and base editors. We provide guidelines to interrogate known cancer driver mutations or functionally screen for novel vulnerability mutations with these systems in characterized human cancer cell lines. The proposed platform should be transferable to primary cancer cells, opening up a path for precision oncology on a functional level.
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A-to-G/C/T and C-to-T/G/A dual-function base editor for creating multi-nucleotide variants.
Multi-nucleotide variants (MNVs) are critical genetic variants associated with various genetic diseases. However, tools for precisely installing MNVs are limited. In this study, we present the development of a dual-base editor, BDBE, by integrating TadA-dual and engineered human N-methylpurine DNA glycosylase (eMPG) into nCas9 (D10A). Our results demonstrate that BDBE effectively converts A-to-G/C/T (referred to as A-to-B) and C-to-T/G/A (referred to as C-to-D) simultaneously, yielding nine types of dinucleotides from adjacent CA nucleotides while maintaining minimal off-target effects. Notably, BDBE4 exhibits exceptional performance across multiple human cell lines and successfully simulated all nine dinucleotide MNVs from the gnomAD database. These findings indicate that BDBE significantly expands the product range of base editors and offers a valuable resource for advancing MNV research.
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Genome assembly of two allotetraploid cotton germplasms reveals mechanisms of somatic embryogenesis and enables precise genome editing.
Somatic embryogenesis is crucial for plant genetic engineering, yet the underlying mechanisms in cotton remain poorly understood. Here we present a telomere-to-telomere assembly of Jin668 and a high-quality assembly of YZ1, two highly regenerative allotetraploid cotton germplasms. The completion of the Jin668 genome enables characterization of ~30.1 Mb of centromeric regions invaded by centromeric retrotransposon of maize and Tekay retrotransposons, an ~8.1 Mb 5S rDNA array containing 25,190 copies and a ~75.1 Mb major 45S rDNA array with 8,131 copies. Comparative analyses of regenerative and recalcitrant genotypes reveal dynamic transcriptional patterns and chromatin accessibility during the initial regeneration process. A hierarchical gene regulatory network identifies AGL15 as a contributor to regeneration. Additionally, we demonstrate that genetic variation affects sgRNA target sites, while the Jin668 genome assembly reduces the risk of off-target effects in CRISPR-based genome editing. Together, the complete Jin668 genome reveals the complexity of genomic regions and cotton regeneration, and improves the precision of genome editing.
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Engineering a compact high-fidelity Staphylococcus aureus Cas9 variant with broader targeting range and mechanistic insights into its activation.
Staphylococcus aureus Cas9 (SaCas9) is smaller than the widely used Streptococcus pyogenes Cas9 (SpCas9) and has been harnessed for gene therapy using an adeno-associated virus vector. However, SaCas9 requires a longer NNGRRT (where N is any nucleotide and R is A or G) protospacer adjacent motif (PAM) for target DNA recognition, thereby restricting the targeting range. Although PAM-relaxed Cas9 variants have been developed, expanded targeting is often accompanied by compromised target specificity. Here, we report the rational engineering of eSaCas9-NNG, a SaCas9 variant that recognizes relaxed NNG PAMs while maintaining high target fidelity, thereby overcoming a fundamental trade-off in Cas9-based genome editing. eSaCas9-NNG efficiently induces indels and base conversions at endogenous sites bearing NNG PAMs in human cells and mice, with editing efficiencies comparable to those of other PAM-relaxed nucleases, including SpRY, SpG, and iGeoCas9, but with reduced off-target activity. We further determine the cryo-electron microscopy structures of eSaCas9-NNG in five distinct functional states, revealing the structural basis for its relaxed PAM recognition, improved target specificity, and nuclease activation. Overall, our findings demonstrate that eSaCas9-NNG could be used as a versatile genome editing tool for in vivo gene therapy, and improve our mechanistic understanding of the diverse CRISPR-Cas9 nucleases.
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Design and Generation of Donor Constructs for Genome Engineering in Drosophila.
The generation of precise alterations to the genome using CRISPR requires the combination of CRISPR and a donor construct containing homology to the target site. A double-strand break is first generated at the target locus using CRISPR. It is then repaired using the endogenous homologous recombination (HR) pathway. When a donor construct is provided, it can be used as a template for HR repair and can therefore be exploited to introduce alterations in the genomic sequence with single base-pair precision. Here we describe a protocol for the generation of donor constructs using Golden Gate assembly and discuss some key considerations for donor construct design for use in Drosophila.
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High-throughput PRIME-editing screens identify functional DNA variants in the human genome.
Despite tremendous progress in detecting DNA variants associated with human disease, interpreting their functional impact in a high-throughput and single-base resolution manner remains challenging. Here, we develop a pooled prime-editing screen method, PRIME, that can be applied to characterize thousands of coding and non-coding variants in a single experiment with high reproducibility. To showcase its applications, we first identified essential nucleotides for a 716 bp MYC enhancer via PRIME-mediated single-base resolution analysis. Next, we applied PRIME to functionally characterize 1,304 genome-wide association study (GWAS)-identified non-coding variants associated with breast cancer and 3,699 variants from ClinVar. We discovered that 103 non-coding variants and 156 variants of uncertain significance are functional via affecting cell fitness. Collectively, we demonstrate that PRIME is capable of characterizing genetic variants at single-base resolution and scale, advancing accurate genome annotation for disease risk prediction, diagnosis, and therapeutic target identification.
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Thermodynamics of PAM Recognition by Cas9 of Streptococcus pyogenes.
The CRISPR/Cas9 system from Streptococcus pyogenes (SpCas9) requires a canonical 5'-NGG-3' PAM sequence in target DNA for effective genome editing. Base-specific interactions between the guanines (second and third position) and arginine dyad (R1333 and R1335) ensured specificity. We evaluated the PAM recognition strength of SpCas9 by using alchemical free energy calculations, revealing the energetics that influence genome editing accuracy. SpCas9 does not discriminate at the first position of the NGG sequence, but it penalizes mutations in the second and third positions. SpCas9 imposes a higher penalty for guanine mutation in the third PAM position compared to the second due to the greater conformational rigidity of R1335 in relation to R1333. Conformational rigidity of R1335 prevents side-chain readjustment for new protein-DNA interactions in noncanonical PAMs. A guanine-to-cytosine substitution in either the second or third position of canonical PAM disrupts direct protein-PAM interactions and leads to solvent exposure. This happens due to strong electrostatic repulsion between the arginine dyad's guanidinium groups and the amine group of cytosine. Interestingly, the strength of SpCas9 in disfavoring a single cytosine substitution (by >10 kcal/mol) is comparable to that of disfavoring double base substitutions in the NGG sequence. The ability of SpCas9 to differentiate between noncanonical and canonical PAMs (ΔΔG) is directly related to the number of direct interactions between SpCas9 and the PAM sequence, as well as the degree of solvent exposure. Loss of direct interactions and increased solvent exposure enhance ΔΔG. The calculated ΔΔG adequately explains the observed differences in DNA cleavage activity of SpCas9 across various DNA substrates with different PAM sequences. This study connects thermodynamics, structures, and activity to elucidate PAM selectivity in SpCas9 and may also apply to other CRISPR/Cas systems, offering valuable insights for the rational design of Cas9 variants with modified PAM specificities.
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A Split-Marker System for CRISPR-Cas9 Genome Editing in Methylotrophic Yeasts.
Methylotrophic yeasts such as Ogataea polymorpha and Komagataella phaffii (sin. Hansenula polymorpha and Pichia pastoris, respectively) are commonly used in basic research and biotechnological applications, frequently those requiring genome modifications. However, the CRISPR-Cas9 genome editing approaches reported for these species so far are relatively complex and laborious. In this work we present an improved plasmid vector set for CRISPR-Cas9 genome editing in methylotrophic yeasts. This includes a plasmid encoding Cas9 with a nuclear localization signal and plasmids with a scaffold for the single guide RNA (sgRNA). Construction of a sgRNA gene for a particular target sequence requires only the insertion of a 24 bp oligonucleotide duplex into the scaffold. Prior to yeast transformation, each plasmid is cleaved at two sites, one of which is located within the selectable marker, so that the functional marker can be restored only via recombination of the Cas9-containing fragment with the sgRNA gene-containing fragment. This recombination leads to the formation of an autonomously replicating plasmid, which can be lost from yeast clones after acquisition of the required genome modification. The vector set allows the use of G418-resistance and LEU2 auxotrophic selectable markers. The functionality of this setup has been demonstrated in O. polymorpha, O. parapolymorpha, O. haglerorum and Komagataella phaffii.
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