Guide RNA Categorization Enables Target Site Choice in Tn7-CRISPR-Cas Transposons

Petassi, Michael T.; Hsieh, Shan-Chi; Peters, Joseph E.

doi:10.1016/j.cell.2020.11.005

Cited by 78 publications

(80 citation statements)

References 49 publications

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“…MUCICAT is simple to operate and machine friendly, showing great potential to be applied by laboratory automation systems to revolutionize cell programming. As more and more types of CRISPR-Cas transposase systems are discovered, 34 MUCI-CAT can introduce multiple orthogonal systems to produce two-dimensional, three-dimensional, or even multidimensional diversity changes in organisms, such as (1) system A produces m kinds of gene disruption diversity and system B produces n copies of gene integration diversity; (2) system A generates diversity of m copies for pathway 1 and system B generates diversity of n copies for pathway 2, and so on; and (3) combining ( 1) and (2) to create more possibilities. Furthermore, this technology has the potential to be applied to bacteria other than E. coli.…”

Section: Discussionmentioning

confidence: 99%

Programming Cells by Multicopy Chromosomal Integration Using CRISPR-Associated Transposases

Zhang

Yang

et al. 2021

The CRISPR Journal

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Directed evolution and targeted genome editing have been deployed to create genetic variants with usefully altered phenotypes. However, these methods are limited to high-throughput screening methods or serial manipulation of single genes. In this study, we implemented multicopy chromosomal integration using CRISPRassociated transposases (MUCICAT) to simultaneously target up to 11 sites on the Escherichia coli chromosome for multiplex gene interruption and/or insertion, generating combinatorial genomic diversity. The MUCICAT system was improved by replacing the isopropyl-beta-D-thiogalactoside (IPTG)-dependent promoter to decouple gene editing and product synthesis and truncating the right end to reduce the leakage expression of cargo. We applied MUCICAT to engineer and optimize the N-acetylglucosamine (GlcNAc) biosynthesis pathway in E. coli to overproduce the industrially important GlcNAc in only 8 days. Two rounds of transformation, the first round for disruption of two degradation pathways related gene clusters and the second round for multiplex integration of the GlcNAc gene cassette, would generate a library with 1-11 copies of the GlcNAc cassette. We isolated a best variant with five copies of GlcNAc cassettes, producing 11.59 g/L GlcNAc, which was more than sixfold than that of the strain containing the pET-GNAc plasmid. Our multiplex approach MUCICAT has potential to become a powerful tool of cell programing and can be widely applied in many fields such as synthetic biology.

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Section: Discussionmentioning

confidence: 99%

Programming Cells by Multicopy Chromosomal Integration Using CRISPR-Associated Transposases

Zhang

Yang

et al. 2021

The CRISPR Journal

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“…For VchCAST, pDonorVerA(Vch), pTns(Vch) and pQCascade(Vch)-crRNA4 with the tetracycline (Tet) promoter were used. For AsaCAST, pMTP170 ( 7 )(Addgene #164263) was reconstructed to target the lacZ gene (spacer #6 as reported) and pDonorAsa derived from pMTP112 ( 7 ) (Addgene #164260) was reconstructed as well. They were used together with pMTP130 ( 7 ) (Addgene #162461) and pMTP140 ( 7 ) (Addgene #164262).…”

Section: Methodsmentioning

confidence: 99%

“…reported that VchCAST and type V-K CAST from Scytonema hofmannii PCC 7110 (ShoCAST) were orthogonal in that they recognize different transposon ends ( 12 ), but type V-K CASTs tend to cointegrate its donor plasmid ( 13–16 ) due to lack of protein TnsA activity, and have a relatively low integration efficiency on multiple targets ( 2 ). Type I-F3 CAST from Aeromonas salmonicida S44 (AsaCAST) exhibits RNA-guided DNA transposition activity when introduced to E. coli , but the integration efficiency of AsaCAST seems much lower than VchCAST ( 7 ). Highly active CASTs capable of MUCICAT need to be identified for the implementation of a parallel and multiplexed genome editing system with VchCAST.…”

Section: Introductionmentioning

confidence: 99%

Orthogonal CRISPR-associated transposases for parallel and multiplexed chromosomal integration

Yang

Zhang

et al. 2021

Nucleic Acids Research

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Cell engineering is commonly limited to the serial manipulation of a single gene or locus. The recently discovered CRISPR-associated transposases (CASTs) could manipulate multiple sets of genes to achieve predetermined cell diversity, with orthogonal CASTs being able to manipulate them in parallel. Here, a novel CAST from Pseudoalteromonas translucida KMM520 (PtrCAST) was characterized without a protospacer adjacent motif (PAM) preference which can achieve a high insertion efficiency for larger cargo and multiplexed transposition and tolerate mismatches out of 4-nucleotide seed sequence. More importantly, PtrCAST operates orthogonally with CAST from Vibrio cholerae Tn6677 (VchCAST), though both belonging to type I-F3. The two CASTs were exclusively active on their respective mini-Tn substrate with their respective crRNAs that target the corresponding 5 and 2 loci in one Escherichia coli cell. The multiplexed orthogonal MUCICAT (MUlticopy Chromosomal Integration using CRISPR-Associated Transposases) is a powerful tool for cell programming and appears promising with applications in synthetic biology.

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“…Prokaryotes have evolved genome defense mechanisms to restrict parasitic transposition, including adaptive immunity provided by CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated) systems that rely on Cas proteins and CRISPR RNA (crRNA) guides to target invasive genetic elements for nucleolytic degradation 1,10 . In contrast to the defensive role of canonical CRISPR-Cas systems, several nucleasedeficient type I-F, I-B and V-K systems have been instead co-opted by a distinct group of Tn7-like transposons to direct RNA-guided transposon DNA insertion into specific target sites [2][3][4][5][6][7] . In these systems, DNA targeting relies on transposon-encoded Cas-RNPs, involving a Cas3-less multisubunit effector complex (termed Cascade) in type I systems 3 or a single catalytically inactive Cas12k protein in type V-K systems 7 .…”

Section: Introductionmentioning

confidence: 99%

“…

Although the canonical function of CRISPR-Cas systems is to provide adaptive immunity against mobile genetic elements 1 , type I-F, I-B and V-K systems have been adopted by Tn7-like transposons to direct RNA-guided transposon insertion [2][3][4][5][6][7] . Type V-K CRISPR-associated transposons rely on the activities of the pseudonuclease Cas12k, the transposase TnsB, the AAA+ ATPase TnsC and the zinc-finger protein TniQ 7 .

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mentioning

confidence: 99%

Molecular mechanism of target site selection and remodeling by type V CRISPR-associated transposons

Querques

Schmitz

Oberli

et al. 2021

Preprint

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Although the canonical function of CRISPR-Cas systems is to provide adaptive immunity against mobile genetic elements, type I-F, I-B and V-K systems have been adopted by Tn7-like transposons to direct RNA-guided transposon insertion. Type V-K CRISPR-associated transposons rely on the activities of the pseudonuclease Cas12k, the transposase TnsB, the AAA+ ATPase TnsC and the zinc-finger protein TniQ. However, the molecular and structural details of RNA-directed DNA transposition have remained elusive. Here we report cryo-electron microscopic structures of a Cas12k-guide RNA-target DNA complex and a DNA-bound, polymeric TnsC filament. The Cas12k complex structure reveals an intricate guide RNA architecture and critical interactions mediating RNA-guided target DNA recognition. The assembly of the TnsC helical filament is ATP-dependent and accompanied by structural remodeling of the bound DNA duplex. In vivo transposition assays corroborate key features of the structures, and biochemical experiments further show that TniQ restricts TnsC polymerization, while the TnsB transposase interacts directly with TnsC filaments to trigger their disassembly upon ATP hydrolysis. Together, these results suggest a mechanistic model whereby RNA-directed target selection by Cas12k primes TnsC polymerization and DNA remodeling, generating a recruitment platform for TnsB to catalyze site-specific transposon insertion. The present work advances our mechanistic understanding of the cross-talk between CRISPR effectors and the transposition machinery and will inform design efforts to harness CRISPR-associated transposons as programmable site-specific gene insertion tools for genome engineering applications.

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Guide RNA Categorization Enables Target Site Choice in Tn7-CRISPR-Cas Transposons

Cited by 78 publications

References 49 publications

Programming Cells by Multicopy Chromosomal Integration Using CRISPR-Associated Transposases

Programming Cells by Multicopy Chromosomal Integration Using CRISPR-Associated Transposases

Orthogonal CRISPR-associated transposases for parallel and multiplexed chromosomal integration

Molecular mechanism of target site selection and remodeling by type V CRISPR-associated transposons

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