The RNA-guided DNA endonuclease Cas9 cleaves double-stranded DNA targets complementary to an RNA guide, and is widely used as a powerful genome-editing tool. Here, we report the crystal structure of Brevibacillus laterosporus Cas9 (BlCas9, also known as BlatCas9), in complex with a guide RNA and its target DNA at 2.4-Å resolution. The structure reveals that the BlCas9 guide RNA adopts an unexpected architecture containing a triple-helix, which is specifically recognized by BlCas9, and that BlCas9 recognizes a unique N4CNDN protospacer adjacent motif through base-specific interactions on both the target and non-target DNA strands. Based on the structure, we rationally engineered a BlCas9 variant that exhibits enhanced genome- and base-editing activities with an expanded target scope in human cells. This approach may further improve the performance of the enhanced BlCas9 variant to generate useful genome-editing tools that require only a single C PAM nucleotide and can be packaged into a single AAV vector for in vivo gene therapy.

Streptococcus pyogenes Cas9 (SpCas9) is the most popular tool in gene editing; however, off-target mutagenesis is one of the biggest impediments in its application. In our previous study, we proposed the HH theory, which states that sgRNA/DNA hybrid (hybrid) extrusion-induced enhancement of hydrophobic interactions between the hybrid and REC3/HNH is a key factor in cleavage initiation. Based on the HH theory, we analyzed the interactions between the REC3 domain and hybrid and obtained 8 mutant sites. We designed 8 SpCas9 variants (V1-V8), used digital droplet PCR to assess SpCas9-induced DNA indels in human cells, and developed high-fidelity variants. Thus, the HH theory may be employed to further optimize SpCas9-mediated genome editing systems, and the resultant V3, V6, V7, and V8 SpCas9 variants may be valuable for applications requiring high-precision genome editing.

It has been 10 years since CRISPR/Cas technology was applied to edit the genomes of various organisms. Its ability to produce a double-strand break in a DNA region specified by the researcher started a revolution in bioengineering. Later, the Base Editing (BE) method was developed. BE is performed via the formation of single-strand breaks by the mutant form of Cas nuclease (nickase), fused with deaminases and other enzymes. It can be used to promote A ↔ G and C ↔ T transitions, and a C → G transversion. Just over 3 years ago, a new Prime Editing (PE) variant of CRISPR/Cas was invented. Unlike BE, in PE the nickase is fused with reverse transcriptase, capable of building a new DNA chain using the pegRNA template. The pegRNA consists of an elongated guide RNA with an extra sequence at the 3\'-end. Prime editing makes it possible to insert the desired mutations into this extra sequence and to carry out any substitutions and indels of bases without the use of special donor DNA. To date, a number of PE variants have been proposed; they are briefly considered in this review with an emphasis on prime editing of plant genomes. Some attention is also paid to pegRNA design programs, as well as evaluation of the efficiency of the editing. Such a variety of PE techniques is due to the opportunities of high-precision introduction of desired changes with a rather low frequency of off-target mutations in the genomes of various organisms. The relatively low efficiency of prime editing inspires researchers to offer new approaches. There is hope that further development of the technology will improve PE enough to take its rightful place among the genome targeting methods that are suitable for any organisms, and will have a positive impact on the agricultural sector, industrial biotechnologies, and medicine.

The clinical success of CRISPR therapies hinges on the safety and efficacy of Cas proteins. The Cas9 from Francisella novicida (FnCas9) is highly precise, with a negligible affinity for mismatched substrates, but its low cellular targeting efficiency limits therapeutic use. Here, we rationally engineer the protein to develop enhanced FnCas9 (enFnCas9) variants and broaden their accessibility across human genomic sites by ~3.5-fold. The enFnCas9 proteins with single mismatch specificity expanded the target range of FnCas9-based CRISPR diagnostics to detect the pathogenic DNA signatures. They outperform Streptococcus pyogenes Cas9 (SpCas9) and its engineered derivatives in on-target editing efficiency, knock-in rates, and off-target specificity. enFnCas9 can be combined with extended gRNAs for robust base editing at sites which are inaccessible to PAM-constrained canonical base editors. Finally, we demonstrate an RPE65 mutation correction in a Leber congenital amaurosis 2 (LCA2) patient-specific iPSC line using enFnCas9 adenine base editor, highlighting its therapeutic utility.

Milk is a good source of nutrition but is also a source of allergenic proteins such as α-lactalbumin, β-lactoglobulin (BLG), casein, and immunoglobulins. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas technology has the potential to edit any gene, including milk allergens. Previously, CRISPR/Cas has been successfully employed in dairy cows and goats, but buffaloes remain unexplored for any milk trait. In this study, we utilized the CRISPR/Cas9 system to edit the major milk allergen BLG gene in buffaloes. First, the editing efficiency of designed sgRNAs was tested in fibroblast cells using the T7E assay and Sanger sequencing. The most effective sgRNA was selected to generate clonal lines of BLG-edited cells. Analysis of 15 single-cell clones, through TA cloning and Sanger sequencing, revealed that 7 clones exhibited bi-allelic (-/-) heterozygous, bi-allelic (-/-) homozygous, and mono-allelic (-/+) disruptions in BLG. Bioinformatics prediction analysis confirmed that non-multiple-of-3 edited nucleotide cell clones have frame shifts and early truncation of BLG protein, while multiple-of-3 edited nucleotides resulted in slightly disoriented protein structures. Somatic cell nuclear transfer (SCNT) method was used to produce blastocyst-stage embryos that have similar developmental rates and quality with wild-type embryos. This study demonstrated the successful bi-allelic editing (-/-) of BLG in buffalo cells through CRISPR/Cas, followed by the production of BLG-edited blastocyst stage embryos using SCNT. With CRISPR and SCNT methods described herein, our long-term goal is to generate gene-edited buffaloes with BLG-free milk.

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BACKGROUND: In recent years, a gene-editing technology known as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has been developed and is progressively advancing into clinical trials. While current antiviral therapies are unable to eliminate the Hepatitis B virus (HBV), it stands as a prime target for the CRISPR/Cas9 technology. The objective of this study was to enhance the efficacy of CRISPR/Cas9 in suppressing HBV replication, lowering HBsAg and HBeAg levels, and eliminating covalently closed circular DNA (cccDNA).
METHODS: To enhance the anti-HBV effectiveness of CRISPR/Cas9, our study delved into a dual-guide RNA (gRNA) strategy. After evaluating the antiviral activities of multiple gRNAs that effectively impeded HBV replication, we identified three specific gRNAs-namely 10, 4, and 21. These gRNAs were selected for their targeting of distinct yet conserved regions within the HBV genome.
RESULTS: In HBV-stable cell lines, namely HepAD38, and HBV infection models of HepG2-NTCP cells, our investigation revealed that the co-application of gRNA-10 with either gRNA-4 or gRNA-21 within the CRISPR/Cas9 system demonstrated heightened efficacy in impeding HBV replication, reducing the levels of HBsAg, HBeAg, and cccDNA levels, along with a more pronounced promotion of HBsAg clearance when compared to the use of a single gRNA.
CONCLUSIONS: The CRISPR/Cas9 system employing dual gRNAs has proven highly effective in both suppressing HBV replication and facilitating HBsAg clearance. This promising outcome suggests that it holds potential to emerge as a novel approach for achieving the functional cure of patients with HBV infection.

CRISPR-Cas technology is a widely utilized gene-editing tool that involves gRNA-guided sequence recognition and Cas nuclease-mediated cleavage. The design and evaluation of gRNA are essential for enhancing CRISPR/Cas editing efficiency. Various assays such as single-strand annealing, in vitro cleavage, and T7 endonuclease I (T7EI) are commonly used to assess gRNA-mediated Cas protein cleavage activity. In this study, a firefly luciferase and Renilla luciferase co-expressed and a cleavage-based single-plasmid dual-luciferase surrogate reporter was built to evaluate the gRNA-mediated Cas12a cleavage efficiency. The cleavage activities of CRISPR-Cas12a can be quantitatively determined by the recovery degree of firefly luciferase activity. The cleavage efficiency of CRISPR-Cas12a can be quantitatively measured by the recovery of firefly luciferase activity. By using this system, the cleavage efficiency of CRISPR-Cas12a on hepatitis B virus (HBV)/D expression plasmid was evaluated, revealing a negative correlation between gRNA cleavage efficiency and HBV gene expression measured using an enzyme-linked immunosorbent assay. This simple, efficient, and quantifiable system only requires the dual-luciferase vector and CRISPR-Cas12a vector, making it a valuable tool for selecting effective gRNAs for gene editing.

The revolutionary CRISPR-Cas9 technology has revolutionized genetic engineering, and it holds immense potential for therapeutic interventions. However, the presence of off-target mutations and mismatch capacity poses significant challenges to its safe and precise implementation. In this study, we explore the implications of off-target effects on critical gene regions, including exons, introns, and intergenic regions. Leveraging a benchmark dataset and using innovative data preprocessing techniques, we have put forth the advantages of categorical encoding over one-hot encoding in training machine learning classifiers. Crucially, we use latent class analysis (LCA) to uncover subclasses within the off-target range, revealing distinct patterns of gene region disruption. Our comprehensive approach not only highlights the critical role of model complexity in CRISPR applications but also offers a transformative off-target scoring procedure based on ML classifiers and LCA. By bridging the gap between traditional target-off scoring and comprehensive model analysis, our study advances the understanding of off-target effects and opens new avenues for precision genome editing in diverse biological contexts. This work represents a crucial step toward ensuring the safety and efficacy of CRISPR-based therapies, underscoring the importance of responsible genetic manipulation for future therapeutic applications.

Although CRISPR-dCas13, the RNA-guided RNA-binding protein, was recently exploited as a translation-level gene expression modulator, it has still been difficult to precisely control the level due to the lack of detailed characterization. Here, we develop a synthetic tunable translation-level CRISPR interference (Tl-CRISPRi) system based on the engineered guide RNAs that enable precise and predictable down-regulation of mRNA translation. First, we optimize the Tl-CRISPRi system for specific and multiplexed repression of genes at the translation level. We also show that the Tl-CRISPRi system is more suitable for independently regulating each gene in a polycistronic operon than the transcription-level CRISPRi (Tx-CRISPRi) system. We further engineer the handle structure of guide RNA for tunable and predictable repression of various genes in Escherichia coli and Vibrio natriegens. This tunable Tl-CRISPRi system is applied to increase the production of 3-hydroxypropionic acid (3-HP) by 14.2-fold via redirecting the metabolic flux, indicating the usefulness of this system for the flux optimization in the microbial cell factories based on the RNA-targeting machinery.