A tool for more specific DNA integration.

The efficiency of targeted DNA insertion by CRISPR transposons is improved.

Cas4/1 dual nuclease activities enable prespacer maturation and directional integration in a type I-G CRISPR-Cas system.

CRISPR-Cas adaptive immune systems uptake short "spacer" sequences from foreign DNA and incorporate them into the host genome to serve as templates for CRISPR RNAs that guide interference against future infections. Adaptation in CRISPR systems is mediated by Cas1-Cas2 complexes that catalyze integration of prespacer substrates into the CRISPR array. Many DNA targeting systems also require Cas4 endonucleases for functional spacer acquisition. Cas4 selects prespacers containing a protospacer adjacent motif (PAM) and removes the PAM prior to integration, both of which are required to ensure host immunization. Cas1 has also been shown to function as a nuclease in some systems, but a role for this nuclease activity in adaptation has not been demonstrated. We identified a type I-G Cas4/1 fusion with a nucleolytically active Cas1 domain that can directly participate in prespacer processing. The Cas1 domain is both an integrase and a sequence-independent nuclease that cleaves the non-PAM end of a prespacer, generating optimal overhang lengths that enable integration at the leader side. The Cas4 domain sequence specifically cleaves the PAM end of the prespacer, ensuring integration of the PAM end at the spacer side. The two domains have varying metal ion requirements. While Cas4 activity is Mn2+ dependent, Cas1 preferentially uses Mg2+ over Mn2+. The dual nuclease activity of Cas4/1 eliminates the need for additional factors in prespacer processing making the adaptation module self-reliant for prespacer maturation and directional integration.

Cas4/1 dual nuclease activities enable prespacer maturation and directional integration in a type I-G CRISPR-Cas system.

CRISPR-Cas adaptive immune systems uptake short 'spacer' sequences from foreign DNA and incorporate them into the host genome to serve as templates for crRNAs that guide interference against future infections. Adaptation in CRISPR systems is mediated by Cas1-Cas2 complexes that catalyze integration of prespacer substrates into the CRISPR array. Many DNA targeting systems also require Cas4 endonucleases for functional spacer acquisition. Cas4 selects prespacers containing a protospacer adjacent motif (PAM) and removes the PAM prior to integration, both of which are required to ensure host immunization. Cas1 has also been shown to function as a nuclease in some systems, but a role for this nuclease activity in adaptation has not been demonstrated. We identified a type I-G Cas4/1 fusion with a nucleolytically active Cas1 domain that can directly participate in prespacer processing. The Cas1 domain is both an integrase and a sequence-independent nuclease that cleaves the non-PAM end of a prespacer, generating optimal overhang lengths that enable integration at the leader side. The Cas4 domain sequence-specifically cleaves the PAM end of the prespacer, ensuring integration of the PAM end at the spacer side. The two domains have varying metal ion requirements. While Cas4 activity is Mn 2+ dependent, Cas1 preferentially uses Mg 2+ over Mn 2+ . The dual nuclease activity of Cas4/1 eliminates the need for additional factors in prespacer processing, making the adaptation module self-reliant for prespacer maturation and directional integration.

PAM binding ensures orientational integration during Cas4-Cas1-Cas2-mediated CRISPR adaptation.

Adaptation in CRISPR-Cas systems immunizes bacteria and archaea against mobile genetic elements. In many DNA-targeting systems, the Cas4-Cas1-Cas2 complex is required for selection and processing of DNA segments containing PAM sequences prior to integration of these "prespacer" substrates as spacers in the CRISPR array. We determined cryo-EM structures of the Cas4-Cas1-Cas2 adaptation complex from the type I-C system that encodes standalone Cas1 and Cas4 proteins. The structures reveal how Cas4 specifically reads out bases within the PAM sequence and how interactions with both Cas1 and Cas2 activate Cas4 endonuclease activity. The Cas4-PAM interaction ensures tight binding between the adaptation complex and the prespacer, significantly enhancing integration of the non-PAM end into the CRISPR array and ensuring correct spacer orientation. Corroborated with our biochemical results, Cas4-Cas1-Cas2 structures with substrates representing various stages of CRISPR adaptation reveal a temporally resolved mechanism for maturation and integration of functional spacers into the CRISPR array.

Miniature CRISPR-Cas12 endonucleases - Programmed DNA targeting in a smaller package.

CRISPR-associated (Cas) endonucleases specifically target and cleave RNA or DNA based on complementarity to a guide RNA. Cas endonucleases - including Cas9, Cas12a, and Cas13 - have been adopted for a wide array of biotechnological tools, including gene editing, transcriptional modulation, and diagnostics. These tools are facilitated by ready reprogramming of guide RNA sequences and the varied nucleic acid binding and cleavage activities observed across diverse Cas endonucleases. However, the large size of most Cas endonucleases (950-1400 amino acids) can restrict applications. The recent discovery of miniature Cas endonucleases (400-800 amino acids) provides the potential to overcome this limitation. Here, we review recent advances in understanding the structural mechanisms of two miniature Cas endonucleases, Cas12f and Cas12j.

Arabidopsis thaliana possesses two novel ELL associated factor homologs.

Transcription elongation is one of the key steps at which RNA polymerase II-directed expression of protein-coding genes is regulated in eukaryotic cells. Different proteins have been shown to control this process, including the ELL/EAF family. ELL Associated Factors (EAFs) were first discovered in a yeast two-hybrid screen as interaction partners of the human ELL (Eleven nineteen Lysine-rich Leukemia) transcription elongation factor. Subsequently, they have been identified in different organisms, including Schizosaccharomyces pombe. However, no homolog(s) of EAF has as yet been characterized from plants. In the present work, we identified EAF orthologous sequences in different plants and have characterized two novel Arabidopsis thaliana EAF homologs, AtEAF-1 (At1g71080) and AtEAF-2 (At5g38050). Sequence analysis showed that both AtEAF-1 and AtEAF-2 exhibit similarity with its S. pombe EAF counterpart. Moreover, both Arabidopsis thaliana and S. pombe EAF orthologs share conserved sequence characteristic features. Computational tools also predicted a high degree of disorder in regions towards the carboxyl terminus of these EAF proteins. We demonstrate that AtEAF-2, but not AtEAF-1 functionally complements growth deficiencies of Schizosaccharomyces pombe eaf mutant. We also show that only AtEAF-1 displays transactivation potential resembling the S. pombe EAF ortholog. Subsequent expression analysis in A. thaliana showed that both homologs were expressed at varying levels during different developmental stages and in different tissues tested in the study. Individual null-mutants of either AtEAF-1 or AtEAF-2 are developmentally normal implying their functional redundancy. Taken together, our results provide first evidence that A. thaliana also possesses functional EAF proteins, suggesting an evolutionary conservation of these proteins across organisms.

The Cas4-Cas1-Cas2 complex mediates precise prespacer processing during CRISPR adaptation.

CRISPR adaptation immunizes bacteria and archaea against viruses. During adaptation, the Cas1-Cas2 complex integrates fragments of invader DNA as spacers in the CRISPR array. Recently, an additional protein Cas4 has been implicated in selection and processing of prespacer substrates for Cas1-Cas2, although this mechanism remains unclear. We show that Cas4 interacts directly with Cas1-Cas2 forming a Cas4-Cas1-Cas2 complex that captures and processes prespacers prior to integration. Structural analysis of the Cas4-Cas1-Cas2 complex reveals two copies of Cas4 that closely interact with the two integrase active sites of Cas1, suggesting a mechanism for substrate handoff following processing. We also find that the Cas4-Cas1-Cas2 complex processes single-stranded DNA provided in cis or in trans with a double-stranded DNA duplex. Cas4 cleaves precisely upstream of PAM sequences, ensuring the acquisition of functional spacers. Our results explain how Cas4 cleavage coordinates with Cas1-Cas2 integration and defines the exact cleavage sites and specificity of Cas4.