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1.
CRISPR J ; 6(3): 261-277, 2023 06.
Article in English | MEDLINE | ID: mdl-37272861

ABSTRACT

Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 nucleases have been extensively used in biotechnology and therapeutics. However, many applications are not possible owing to the size, targetability, and potential off-target effects associated with currently known systems. In this study, we identified thousands of CRISPR type II effectors by mining an extensive, genome-resolved metagenomics database encompassing hundreds of thousands of microbial genomes. We developed a high-throughput pipeline that enabled us to predict tracrRNA sequences, to design single guide RNAs, and to demonstrate nuclease activity in vitro for 41 newly described subgroups. Active systems represent an extensive diversity of protein sequences and guide RNA structures and require diverse protospacer adjacent motifs (PAMs) that collectively expand the known targeting capability of current systems. Several nucleases showed activity levels comparable to or significantly higher than SpCas9, despite being smaller in size. In addition, top systems exhibited low levels of off-target editing in mammalian cells, and PAM-interacting domain engineered chimeras further expanded their targetability. These newly discovered nucleases are attractive enzymes for translation into many applications, including therapeutics.


Subject(s)
CRISPR-Cas Systems , Gene Editing , Animals , CRISPR-Cas Systems/genetics , CRISPR-Associated Protein 9/genetics , CRISPR-Associated Protein 9/metabolism , Biotechnology , RNA, Guide, CRISPR-Cas Systems , Mammals/genetics , Mammals/metabolism
2.
Nat Commun ; 13(1): 7602, 2022 12 15.
Article in English | MEDLINE | ID: mdl-36522342

ABSTRACT

Programmable, RNA-guided nucleases are diverse enzymes that have been repurposed for biotechnological applications. However, to further expand the therapeutic application of these tools there is a need for targetable systems that are small enough to be delivered efficiently. Here, we mined an extensive genome-resolved metagenomics database and identified families of uncharacterized RNA-guided, compact nucleases (between 450 and 1,050 aa). We report that Cas9d, a new CRISPR type II subtype, contains Zinc-finger motifs and high arginine content, features that we also found in nucleases related to HEARO effectors. These enzymes exhibit diverse biochemical characteristics and are broadly targetable. We show that natural Cas9d enzymes are capable of genome editing in mammalian cells with >90% efficiency, and further engineered nickase variants into the smallest base editors active in E. coli and human cells. Their small size, broad targeting potential, and translatability suggest that Cas9d and HEARO systems will enable a variety of genome editing applications.


Subject(s)
Escherichia coli , Gene Editing , Animals , Humans , Escherichia coli/genetics , Escherichia coli/metabolism , Endonucleases/genetics , Endonucleases/metabolism , Clustered Regularly Interspaced Short Palindromic Repeats , Ribonucleases/genetics , RNA , CRISPR-Cas Systems/genetics , Mammals/genetics
3.
iScience ; 24(8): 102875, 2021 Aug 20.
Article in English | MEDLINE | ID: mdl-34386733

ABSTRACT

Lak phages with alternatively coded ∼540 kbp genomes were recently reported to replicate in Prevotella in microbiomes of humans that consume a non-Western diet, baboons, and pigs. Here, we explore Lak phage diversity and broader distribution using diagnostic polymerase chain reaction and genome-resolved metagenomics. Lak phages were detected in 13 animal types, including reptiles, and are particularly prevalent in pigs. Tracking Lak through the pig gastrointestinal tract revealed significant enrichment in the hindgut compared to the foregut. We reconstructed 34 new Lak genomes, including six curated complete genomes, all of which are alternatively coded. An anomalously large (∼660 kbp) complete genome reconstructed for the most deeply branched Lak from a horse microbiome is also alternatively coded. From the Lak genomes, we identified proteins associated with specific animal species; notably, most have no functional predictions. The presence of closely related Lak phages in diverse animals indicates facile distribution coupled to host-specific adaptation.

4.
CRISPR J ; 3(6): 454-461, 2020 12.
Article in English | MEDLINE | ID: mdl-33146573

ABSTRACT

Cas12a enzymes are quickly being adopted for use in a variety of genome-editing applications. These programmable nucleases are part of adaptive microbial immune systems, the natural diversity of which has been largely unexplored. Here, we identified novel families of Type V-A CRISPR nucleases through a large-scale analysis of metagenomes collected from a variety of complex environments, and developed representatives of these systems into gene-editing platforms. The nucleases display extensive protein variation and can be programmed by a single-guide RNA with specific motifs. The majority of these enzymes are part of systems recovered from uncultivated organisms, some of which also encode a divergent Type V effector. Biochemical analysis uncovered unexpected protospacer adjacent motif diversity, indicating that these systems will facilitate a variety of genome-engineering applications. The simplicity of guide sequences and activity in human cell lines suggest utility in gene and cell therapies.


Subject(s)
Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , CRISPR-Associated Proteins/isolation & purification , CRISPR-Associated Proteins/metabolism , Endodeoxyribonucleases/isolation & purification , Endodeoxyribonucleases/metabolism , Gene Editing/methods , Bacteria/genetics , Bacterial Proteins/genetics , CRISPR-Associated Protein 9/genetics , CRISPR-Associated Proteins/genetics , CRISPR-Cas Systems/genetics , Clustered Regularly Interspaced Short Palindromic Repeats , Endodeoxyribonucleases/genetics , Endonucleases/genetics , Gene Editing/trends , Humans , Metagenomics/methods , Phylogeny , RNA, Guide, Kinetoplastida/genetics
5.
Nat Microbiol ; 4(4): 693-700, 2019 04.
Article in English | MEDLINE | ID: mdl-30692672

ABSTRACT

Bacteriophages (phages) dramatically shape microbial community composition, redistribute nutrients via host lysis and drive evolution through horizontal gene transfer. Despite their importance, much remains to be learned about phages in the human microbiome. We investigated the gut microbiomes of humans from Bangladesh and Tanzania, two African baboon social groups and Danish pigs; many of these microbiomes contain phages belonging to a clade with genomes >540 kilobases in length, the largest yet reported in the human microbiome and close to the maximum size ever reported for phages. We refer to these as Lak phages. CRISPR spacer targeting indicates that Lak phages infect bacteria of the genus Prevotella. We manually curated to completion 15 distinct Lak phage genomes recovered from metagenomes. The genomes display several interesting features, including use of an alternative genetic code, large intergenic regions that are highly expressed and up to 35 putative transfer RNAs, some of which contain enigmatic introns. Different individuals have distinct phage genotypes, and shifts in variant frequencies over consecutive sampling days reflect changes in the relative abundance of phage subpopulations. Recent homologous recombination has resulted in extensive genome admixture of nine baboon Lak phage populations. We infer that Lak phages are widespread in gut communities that contain the Prevotella species, and conclude that megaphages, with fascinating and underexplored biology, may be common but largely overlooked components of human and animal gut microbiomes.


Subject(s)
Bacteria/virology , Bacteriophages/isolation & purification , Gastrointestinal Microbiome , Microbiota , Papio/microbiology , Prevotella/virology , Swine/microbiology , Adult , Animals , Bacteriophages/classification , Bacteriophages/genetics , Female , Genome, Viral , Humans , Male , Metagenome , Middle Aged , Phylogeny , Prevotella/classification , Prevotella/genetics
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