CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering

Vo, Phuc Leo H.; Ronda, Carlotta; Klompe, Sanne E.; Chen, Ethan E.; Acree, Christopher; Wang, Harris H.; Sternberg, Samuel H.

doi:10.1038/s41587-020-00745-y

Cited by 229 publications

(275 citation statements)

References 76 publications

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“…Interestingly, whereas >98% of SMRT-seq reads for the V. cholerae CRISPR-Tn represented on-target DNA integration, only 65-70% of SMRT-seq reads for the S. hofmannii CRISPR-Tn were on-target ( Fig. 1e ), consistent with our prior analysis of genome-wide integration specificity (18). These results conclusively demonstrate that CRISPR RNA-guided transposons lacking functional TnsA are unable to fully excise from their donor sites as a linear species, and instead mobilize through a copy-and-paste pathway that relies on extensive DNA replication and vector backbone insertion.…”

Section: Resultssupporting

confidence: 87%

“…Using a two-plasmid expression system ( Methods ), we transformed E. coli cells and targeted the genome for integration using crRNA-13. Unlike our previous SMRT-seq experiments, in which individual clones were analyzed (18), here we pooled a large population of transformants, isolated gDNA, performed WGS, and carried out downstream analyses using circular consensus sequence (CCS) reads. Given the >11 kb average CCS read lengths ( Supplementary Table 1 ) and unbiased library preparation, we reasoned that transposon-containing reads would unambiguously report the type and genetic structure of integration products sampled across the entire population.…”

Section: Resultsmentioning

confidence: 99%

“…Indeed, the observation biases imposed by previous analyses of CRISPR-Cas9 and CRISPR-transposons have obscured key unanticipated byproducts such as large deletions or vector insertions (6, 16, 22, 23). By applying SMRT-WGS, in conjunction with prior transposon-insertion sequencing approaches (5, 18), we have now exhaustively characterized the types and sites of genome-wide transposition products afforded by Tn 7- and Tn 5053 -like CRISPR-transposons. Our data confirms the decisive role of TnsA in mediating non-replicative, cut-and-paste transposition across diverse systems.…”

Section: Resultsmentioning

confidence: 99%

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Unbiased profiling of CRISPR RNA-guided transposition products by long-read sequencing

Acree

Smith

et al. 2021

Preprint

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Bacterial transposons propagate through either non-replicative (cut-and-paste) or replicative (copy-and-paste) pathways, depending on how the mobile element is excised from its donor source. In the well-characterized E. coli transposon Tn7, a heteromeric TnsA-TnsB transposase directs cut-and-paste transposition by cleaving both strands at each transposon end during the excision step. Whether a similar pathway is involved for RNA-guided transposons, in which CRISPR-Cas systems confer DNA target specificity, has not been determined. Here, we apply long-read, population-based whole-genome sequencing (WGS) to unambiguously resolve transposition products for two evolutionarily distinct transposon classes that employ either Cascade or Cas12k for RNA-guided DNA integration. Our results reveal that RNA-guided transposon systems lacking functional TnsA primarily generate cointegrate products containing genomically inserted vector backbones. Finally, we report natural and engineered transposon variants encoding a TnsAB fusion protein, revealing a novel strategy for achieving RNA-guided transposition with fewer molecular components.

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Section: Resultssupporting

confidence: 87%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Unbiased profiling of CRISPR RNA-guided transposition products by long-read sequencing

Acree

Smith

et al. 2021

Preprint

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“…Intriguingly, multiple examples exist where CRISPR-Cas defense systems have been coopted by mobile elements to support their own maintenance and dispersal 2 . Of interest are multiple examples where CRISPR-Cas systems associate with a specialized type of transposons called Tn7-like elements 12, 13 . These natural systems of guide RNA-directed transposition show promise as tools for precision integration of genetic payloads, but fundamental questions remain with how they normally function and how they interact with canonical active CRISPR-Cas systems 4-7 .…”

Section: Introductionmentioning

confidence: 99%

“…All of the known associations between CRISPR-Cas systems and transposons occurred with Tn7-like elements, which have a dedicated system of target site selection 12, 13 . In addition to the transposase that is responsible for recognizing the cis -acting ends of the element and joining them to a new target DNA, Tn7-like elements have a conserved transposition regulator protein, TnsC, and a target site identification protein, TniQ/TnsD.…”

Section: Introductionmentioning

confidence: 99%

Tn7-CRISPR-Cas12K elements manage pathway choice using truncated repeat-spacer units to target tRNA attachment sites

Hsieh

Peters

2021

Preprint

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CRISPR-Cas systems provide a defense against mobile elements. These defense systems have been naturally coopted multiple times for guide RNA-directed transposition by Tn7-like transposons. Elements associated with a type I-F CRISPR-Cas system categorize guide RNAs, maintaining a standard CRISPR array capable of acquiring new spacers targeting other mobile elements while maintaining a special guide RNA allowing integration into a conserved site in the chromosome called an attachment site. We show here that Tn7-like elements associated with a type V-K (Cas12K-based) system use a similar strategy to target diverse tRNA genes as attachment sites. These guides are encoded as truncated minimal repeat-spacer units and are found in distinct locations. Multiple pieces of information support that V-K guide RNAs are acquired using a type I-D adaptation system, but remain private to the V-K transposition process. This catalog of Cas12K elements and naturally occurring insertions will help future work engineering precision integration systems.

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DNA on the move: mechanisms, functions and applications of transposable elements

Schmitz,

Querques

2023

FEBS Open Bio

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Transposons are mobile genetic elements that have invaded all domains of life by moving between and within their host genomes. Due to their mobility (or transposition), transposons facilitate horizontal gene transfer in bacteria and foster the evolution of new molecular functions in prokaryotes and eukaryotes. As transposition can lead to detrimental genomic rearrangements, organisms have evolved a multitude of molecular strategies to control transposons, including genome defense mechanisms provided by CRISPR‐Cas systems. Apart from their biological impacts on genomes, DNA transposons have been leveraged as efficient gene insertion vectors in basic research, transgenesis and gene therapy. However, the close to random insertion profile of transposon‐based tools limits their programmability and safety. Despite recent advances brought by the development of CRISPR‐associated genome editing nucleases, a strategy for efficient insertion of large, multi‐kilobase transgenes at user‐defined genomic sites is currently challenging. The discovery and experimental characterization of bacterial CRISPR‐associated transposons (CASTs) led to the attractive hypothesis that these systems could be repurposed as programmable, site‐specific gene integration technologies. Here, we provide a broad overview of the molecular mechanisms underpinning DNA transposition and of its biological and technological impact. The second focus of the article is to describe recent mechanistic and functional analyses of CAST transposition. Finally, current challenges and desired future advances of CAST‐based genome engineering applications are briefly discussed.

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CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering

Cited by 229 publications

References 76 publications

Unbiased profiling of CRISPR RNA-guided transposition products by long-read sequencing

Unbiased profiling of CRISPR RNA-guided transposition products by long-read sequencing

Tn7-CRISPR-Cas12K elements manage pathway choice using truncated repeat-spacer units to target tRNA attachment sites

DNA on the move: mechanisms, functions and applications of transposable elements

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