Functionally diverse type V CRISPR-Cas systems

Science ◽  
2018 ◽  
Vol 363 (6422) ◽  
pp. 88-91 ◽  
Author(s):  
Winston X. Yan ◽  
Pratyusha Hunnewell ◽  
Lauren E. Alfonse ◽  
Jason M. Carte ◽  
Elise Keston-Smith ◽  
...  
Keyword(s):  
Functional Diversity ◽  
Single Strand ◽  
Double Stranded Dna ◽  
Type V ◽  
Functionally Diverse

Type V CRISPR-Cas systems are distinguished by a single RNA-guided RuvC domain-containing effector, Cas12. Although effectors of subtypes V-A (Cas12a) and V-B (Cas12b) have been studied in detail, the distinct domain architectures and diverged RuvC sequences of uncharacterized Cas12 proteins suggest unexplored functional diversity. Here, we identify and characterize Cas12c, -g, -h, and -i. Cas12c, -h, and -i demonstrate RNA-guided double-stranded DNA (dsDNA) interference activity. Cas12i exhibits markedly different efficiencies of CRISPR RNA spacer complementary and noncomplementary strand cleavage resulting in predominant dsDNA nicking. Cas12g is an RNA-guided ribonuclease (RNase) with collateral RNase and single-strand DNase activities. Our study reveals the functional diversity emerging along different routes of type V CRISPR-Cas evolution and expands the CRISPR toolbox.

scholarly journals Mechanistic insights into the R-loop formation and cleavage in CRISPR-Cas12i1

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Bo Zhang ◽  
Diyin Luo ◽  
Yu Li ◽  
Vanja Perčulija ◽  
Jing Chen ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Cleavage ◽  
Binary Complex ◽  
Loop Formation ◽  
Dna Duplex ◽  
Target Dna ◽  
Before And After ◽  
Dna Strand ◽  
Type V ◽  
Functionally Diverse

AbstractCas12i is a newly identified member of the functionally diverse type V CRISPR-Cas effectors. Although Cas12i has the potential to serve as genome-editing tool, its structural and functional characteristics need to be investigated in more detail before effective application. Here we report the crystal structures of the Cas12i1 R-loop complexes before and after target DNA cleavage to elucidate the mechanisms underlying target DNA duplex unwinding, R-loop formation and cis cleavage. The structure of the R-loop complex after target DNA cleavage also provides information regarding trans cleavage. Besides, we report a crystal structure of the Cas12i1 binary complex interacting with a pseudo target oligonucleotide, which mimics target interrogation. Upon target DNA duplex binding, the Cas12i1 PAM-interacting cleft undergoes a remarkable open-to-closed adjustment. Notably, a zipper motif in the Helical-I domain facilitates unzipping of the target DNA duplex. Formation of the 19-bp crRNA-target DNA strand heteroduplex in the R-loop complexes triggers a conformational rearrangement and unleashes the DNase activity. This study provides valuable insights for developing Cas12i1 into a reliable genome-editing tool.

scholarly journals Functional diversity increases the efficacy of phage combinations

2021 ◽  
Author(s):  
Rosanna C. T. Wright ◽  
Ville Friman ◽  
Margaret C. M. Smith ◽  
Michael Brockhurst
Keyword(s):  
Functional Diversity ◽  
Functional Traits ◽  
Bacterial Infections ◽  
Phage Therapy ◽  
Promising Alternative ◽  
Functionally Diverse

Phage therapy is a promising alternative to traditional antibiotics for treating bacterial infections. Such phage-based therapeutics typically contain multiple phages, but how the efficacy of phage combinations scales with phage richness, identity and functional traits is unclear. Here, we experimentally tested the efficacy of 827 unique phage combinations ranging in phage richness from 1 to 12 phages. The efficacy of phage combinations increased with phage richness. However, complementarity between functionally diverse phages allowed efficacy to be maximised at lower levels of phage richness in functionally diverse combinations. These findings suggest that phage functional diversity is the key property of effective phage combinations, enabling the design of simple but effective phage therapies that overcome the practical and regulatory hurdles that limit development of more diverse phage therapy cocktails.

scholarly journals CRISPR-Cas12a exploits R-loop asymmetry to form double-strand breaks

2020 ◽  
Author(s):  
Joshua C. Cofsky ◽  
Deepti Karandur ◽  
Carolyn J. Huang ◽  
Isaac P. Witte ◽  
John Kuriyan ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Strand Break ◽  
Loop Structure ◽  
Loop Formation ◽  
Strand Breaks ◽  
Guide Rna ◽  
Double Stranded Dna ◽  
Dna Strands ◽  
Type V

ABSTRACTMost type V CRISPR-Cas interference proteins use a single RuvC active site to make RNA-guided breaks in double-stranded DNA substrates, an activity essential for both bacterial immunity and genome editing applications. The best-studied of these enzymes, Cas12a, initiates DNA cutting by forming a 20-nucleotide R-loop in which the guide RNA displaces one of the DNA strands of a double-helical substrate, positioning the DNase active site for first-strand cleavage. However, crystal structures and biochemical data have not explained how the second strand is cut to complete the double-strand break. Here, we show that Cas12a-mediated R-loop formation destabilizes DNA at the second-strand cleavage site, which is located outside of the R-loop structure and beyond the 3′ end of the guide RNA. Chemical and fluorescent DNA probes reveal that this destabilization is an intrinsic feature of DNA flanking the RNA-3′ side of R-loops and does not require direct protein interactions. Interestingly, DNA flanking the RNA-5′ side of R-loops is not intrinsically unstable. This asymmetry in R-loop structure may explain the uniformity of guide RNA architecture and the single-active-site cleavage mechanism that are fundamental features of all type V CRISPR-Cas systems.

Promotion of Single-Strand Invasion of PNA to Double-Stranded DNA by Pseudo-Complementary Base Pairing

2019 ◽  
Vol 92 (2) ◽  
pp. 330-335 ◽  
Author(s):  
Narumi Shigi ◽  
Yuki Mizuno ◽  
Hiroko Kunifuda ◽  
Kazunari Matsumura ◽  
Makoto Komiyama
Keyword(s):  
Single Strand ◽  
Base Pairing ◽  
Double Stranded Dna ◽  
Strand Invasion

scholarly journals Reaping the Rewards of Functional Diversity in Healthcare Teams: Why Team Processes Improve Performance

2018 ◽  
Vol 43 (3) ◽  
pp. 440-474 ◽  
Author(s):  
Anya Johnson ◽  
Helena Nguyen ◽  
Markus Groth ◽  
Les White
Keyword(s):  
Conflict Management ◽  
Role Conflict ◽  
Functional Diversity ◽  
Team Performance ◽  
Interpersonal Conflict ◽  
Relative Importance ◽  
Team Process ◽  
Team Processes ◽  
Healthcare Teams ◽  
Functionally Diverse

Functional diversity in healthcare teams—where members from different healthcare professions work together—is often advocated as the key to achieving quality patient outcomes. However, although functionally diverse teams promise much, they do not always deliver on that promise. Based on the informational diversity perspective, we argue that functional diversity increases role conflict and is an important factor that can help to explain why functional diversity does not always lead to effective team performance. We also argue that team processes play an important role in moderating the effect of functional diversity on role conflict and that depending on how functionally diverse healthcare teams are, certain team processes are more important than others for reducing role conflict thereby leading to improved team performance. We contrast two specific team processes (interpersonal conflict management and back up and helping) and argue for their relative importance depending on the level of functional diversity in healthcare teams. Data from 75 hospital teams support our differential predictions that interpersonal conflict management is a particularly important team process for reducing role conflict and improving team performance for teams with high functional diversity, whereas for teams with low functional diversity, back up and helping is a more important team process. These results have important implications for the management of functionally diverse healthcare teams. By identifying the relative importance of team processes, these results provide evidence for investing in team processes that enable healthcare teams to reap the rewards of functional diversity.

scholarly journals Human Aquaporins: Functional Diversity and Potential Roles in Infectious and Non-infectious Diseases

2021 ◽  
Vol 12 ◽  
Author(s):  
Abul Kalam Azad ◽  
Topu Raihan ◽  
Jahed Ahmed ◽  
Al Hakim ◽  
Tanvir Hossain Emon ◽  
...  
Keyword(s):  
Infectious Diseases ◽  
Functional Diversity ◽  
Parasitic Infections ◽  
Microbial Pathogenesis ◽  
Potential Therapeutic Target ◽  
Physiological Relevance ◽  
Living Organisms ◽  
Functionally Diverse ◽  
Transmembrane Diffusion ◽  
Shed Light

Aquaporins (AQPs) are integral membrane proteins and found in all living organisms from bacteria to human. AQPs mainly involved in the transmembrane diffusion of water as well as various small solutes in a bidirectional manner are widely distributed in various human tissues. Human contains 13 AQPs (AQP0–AQP12) which are divided into three sub-classes namely orthodox aquaporin (AQP0, 1, 2, 4, 5, 6, and 8), aquaglyceroporin (AQP3, 7, 9, and 10) and super or unorthodox aquaporin (AQP11 and 12) based on their pore selectivity. Human AQPs are functionally diverse, which are involved in wide variety of non-infectious diseases including cancer, renal dysfunction, neurological disorder, epilepsy, skin disease, metabolic syndrome, and even cardiac diseases. However, the association of AQPs with infectious diseases has not been fully evaluated. Several studies have unveiled that AQPs can be regulated by microbial and parasitic infections that suggest their involvement in microbial pathogenesis, inflammation-associated responses and AQP-mediated cell water homeostasis. This review mainly aims to shed light on the involvement of AQPs in infectious and non-infectious diseases and potential AQPs-target modulators. Furthermore, AQP structures, tissue-specific distributions and their physiological relevance, functional diversity and regulations have been discussed. Altogether, this review would be useful for further investigation of AQPs as a potential therapeutic target for treatment of infectious as well as non-infectious diseases.

scholarly journals Functional diversity increases the efficacy of phage combinations

Microbiology ◽  
2021 ◽  
Vol 167 (12) ◽  
Author(s):  
Rosanna C. T. Wright ◽  
Ville-Petri Friman ◽  
Margaret C. M. Smith ◽  
Michael A. Brockhurst
Keyword(s):  
Functional Diversity ◽  
Functional Traits ◽  
Bacterial Infections ◽  
Phage Therapy ◽  
Promising Alternative ◽  
Functionally Diverse

Phage therapy is a promising alternative to traditional antibiotics for treating bacterial infections. Such phage-based therapeutics typically contain multiple phages, but how the efficacy of phage combinations scales with phage richness, identity and functional traits is unclear. Here, we experimentally tested the efficacy of 827 unique phage combinations ranging in phage richness from one to 12 phages. The efficacy of phage combinations increased with phage richness. However, complementarity between functionally diverse phages allowed efficacy to be maximized at lower levels of phage richness in functionally diverse combinations. These findings suggest that phage functional diversity is the key property of effective phage combinations, enabling the design of simple but effective phage therapies that overcome the practical and regulatory hurdles that limit development of more diverse phage therapy cocktails.

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