CRISPR Journal最新文献

The advent of clustered regularly interspaced short palindromic repeats (CRISPR)-based technologies has revolutionized genome editing, with continued interest in expanding the CRISPR-associated proteins (Cas) toolbox with diverse, efficient, and specific effectors. CRISPR-Cas12a is a potent, programmable RNA-guided dual nickase, broadly used for genome editing. Here, we mined dairy cow microbial metagenomes for CRISPR-Cas systems, unraveling novel Cas12a enzymes. Using in silico pipelines, we characterized and predicted key drivers of CRISPR-Cas12a activity, encompassing guides and protospacer adjacent motifs for five systems. We next assessed their functional potential in cell-free transcription-translation assays with GFP-based fluorescence readouts. Lastly, we determined their genome editing potential in vivo in Escherichia coli by generating 1 kb knockouts. Unexpectedly, we observed natural sequence variation in the bridge-helix domain of the best-performing candidate and used mutagenesis to alter the activity of Cas12a orthologs, resulting in increased gene editing capabilities of a relatively inefficient candidate. This study illustrates the potential of underexplored metagenomic sequence diversity for the development and refinement of genome editing effectors.

Custom CRISPR screens are powerful tools for rapid, hypothesis-driven discovery, but their design is often complex and time-consuming. Green Listed v2.0 simplifies this process with an intuitive workflow for designing custom CRISPR spacer libraries and supports downstream analysis for all users, irrespective of their computational experience. The web application features a user-friendly graphical interface freely accessible at https://greenlisted.cmm.se. Version 2.0 includes significant upgrades to the original 2016 version that were implemented based on user feedback. This includes a new gene synonym tool, expanded library options, optimized output lists, performance improvements, and linked scripts for the rational design of custom CRISPR screen gene sets.

CRISPR-Cas9 genome editing holds tremendous potential for accelerating genetic improvements in aquaculture. The success of the CRISPR-Cas9 system relies on the specificity and efficiency of engineered single-guide RNAs (sgRNAs). In this study, we optimized an in vitro validation protocol for sgRNAs to streamline the gene editing process, capitalizing on the limited breeding season of the Pacific oyster (Crassostrea gigas). We evaluated the efficiency of 11 sgRNAs targeting four genes both in vitro and in vivo in C. gigas. In addition, we found that Cas9 protein differs from Cas9 mRNA in gene editing efficiency at various stages of early development. Cas9 protein proved particular efficacy in achieving early and efficient gene knockout, functioning effectively during the first cell division and facilitating biallelic gene knockouts. Statistical analysis showed that in the protein group, the biallelic editing frequency ranged from 12.5% to 57.8%, and the overall editing frequency reached as high as 75-90.6%. The mRNA group exhibited a biallelic editing frequency of 3.1-14.0% and the overall editing frequency spanning 65.6-78.1%. Contrary to expectations, low-temperature incubation (20°C) of oyster embryos prolonged the time window for the first cell division but did not improve gene editing efficiency, likely due to the high temperature sensitivity of Cas9 enzyme activity. Together, this study provides a comprehensive analysis of factors affecting the efficiency of CRISPR-Cas9 gene editing in C. gigas, providing a robust framework for future gene editing endeavors in mollusks and other marine invertebrates.

Bacteria and archaea acquire resistance to genetic parasites by preferentially integrating short fragments of foreign DNA at one end of a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR). "Leader" DNA upstream of CRISPR loci regulates transcription and foreign DNA integration into the CRISPR. Here, we analyze 37,477 CRISPRs from 39,277 bacterial and 556 archaeal genomes to identify conserved sequence motifs in CRISPR leaders. A global analysis of all leader sequences fails to identify universally conserved motifs. However, an analysis of leader sequences that have been grouped by 16S rRNA-based taxonomy and CRISPR subtype reveals 87 specific motifs in type I, II, III, and V CRISPR leaders. Fourteen of these leader motifs have biochemically demonstrated roles in CRISPR biology including integration, transcription, and CRISPR RNA processing. Another 28 motifs are related to DNA binding sites for proteins with functions that are consistent with regulating CRISPR activity. In addition, we show that these leader motifs can be used to improve existing CRISPR detection methods and enhance the accuracy of CRISPR classification.

Type V CRISPR systems are highly diverse in sequence, mechanism, and function. Although recent efforts have greatly expanded our understanding of their evolution, the diversity of type V systems remains to be completely explored, and many clades have not been experimentally characterized. In this work, we mined metagenomic databases to identify three new subtypes and nine new variants of Cas12, the effector of Type V systems, and provide experimental and computational characterization of their Protospacer-Adjacent Motif (PAM), interference activity, loci architecture, and tracrRNA dependence. Half of the new Cas12s are found in phages or prophages. New subtypes Cas12o and Cas12p lack the canonical RuvC catalytic residues, suggesting they interfere with the target without cleavage, possibly by blocking transcription or replication. One variant, Cas12f10, displays substantial activity on PAM-less targets. Our work expands the diversity of the functionally characterized Cas12 effectors and provides some promising candidates for genome engineering tools.

CRISPR gene editing is a cutting-edge technology that has advanced tremendously in recent years. The first clinical CRISPR applications have been approved, and more gene editing therapies are to be expected in human medicine. Consequently, continuous basic research is needed to assess possibilities and prime future clinical applications. Because this technology not only offers new possibilities for treating diseases but also raises important ethical and societal questions, collaboration between human, life, biomedical, and medical sciences is needed. In this article, we discuss the practical challenges of such interdisciplinary projects and present strategies for addressing them based on our experience of conducting an interdisciplinary project on CRISPR. This work aims to help and encourage interdisciplinary collaborations and discussions on modern scientific endeavors that, such as gene editing, tend to blur the lines between traditional disciplines. The strategies suggested include realistic expectations, shared goals, space setting, and expert and lay dialogue.

The incorporation of nuclear localization signal (NLS) sequences at one or both termini of CRISPR enzymes is a widely adopted strategy to facilitate genome editing. Engineered variants of CRISPR enzymes with diverse NLS sequences have demonstrated superior performance, promoting nuclear localization and efficient DNA editing. However, limiting NLS fusion to the CRISPR protein's termini can negatively impact protein yield via recombinant expression. Here we present a distinct strategy involving the installation of hairpin internal NLS sequences (hiNLS) at rationally selected sites within the backbone of CRISPR-Cas9. We evaluated the performance of these hiNLS Cas9 variants by editing genes in human primary T cells following the delivery of ribonucleoprotein enzymes via either electroporation or co-incubation with amphiphilic peptides. We show that hiNLS Cas9 variants can improve editing efficiency in T cells compared with constructs with terminally fused NLS sequences. Furthermore, many hiNLS Cas9 constructs can be produced with high purity and yield, even when these constructs contain as many as nine NLS. These hiNLS Cas9 constructs represent a key advance in optimizing CRISPR effector design and may contribute to improved editing outcomes in research and therapeutic applications.