Biology subjects

Cooper, R. M.

Publications and source records attributed to Cooper, R. M..

3 recordsLinked to original sources

bioRxiv2021

CRISPR-Cas Inhibits Natural Transformation through Altruistic Group Defense and Self-Sacrifice

CRISPR-Cas systems present an evolutionary tradeoff: does defense against phages and other parasitic DNA also prevent cells from acquiring potentially helpful new genes? Genomic analyses of this conundrum have arrived at often contradictory conclusions. Meanwhile, experimental studies have focused mainly on phages, conjugation, or artificial transformation, but less work has examined natural competence, a major driver of evolution and antibiotic resistance. Here, we use Acinetobacter baylyi, which combines high natural competence with a functional CRISPR-Cas system, to experimentally probe the interactions between CRISPR-Cas and natural competence. In these bacteria, the endogenous CRISPR array largely allows natural transformation by targeted DNA. However, CRISPR-Cas then kills the newly autoimmune cells in a form of programmed cell death. CRISPR-Cas often allows self-targeting cells to form colonies, albeit with fitness costs. Thus CRISPR-Cas appears to block natural transformation in a process more akin to altruistic group defense than an individual immune system.

molecular biology↗

bioRxiv2021

Engineered bacteria detect tumor DNA in vivo

Advances in bacterial engineering have catalysed the development of living cell diagnostics and therapeutics1-3, including microbes that respond to gut inflammation4, intestinal bleeding5, pathogens6 and hypoxic tumors7. Bacteria can access the entire gastrointestinal tract8 to produce outputs measured in stool4 or urine7. Cellular memory, such as bistable switches4,9,10 or genomic rearrangements11, allows bacteria to store information over time. However, living biosensors have not yet been engineered to detect specific DNA sequences or mutations from outside the cell. Here, we engineer naturally competent Acinetobacter baylyi to detect donor DNA from the genomes of colorectal cancer (CRC) cells, organoids and tumors. We characterize the functionality of the biosensors in vitro with co-culture assays and then validate in vivo with sensor bacteria delivered to mice harboring colorectal tumors. We observe horizontal gene transfer from the tumor to the sensor bacteria in our mouse model of CRC. The sensor bacteria achieved 100% discrimination between mice with and without CRC. This Cellular Assay of Targeted, CRISPR-discriminated Horizontal gene transfer (CATCH), establishes a framework for biosensing of mutations or organisms within environments that are difficult to sample, among many other potential applications. Furthermore, the platform could be readily expanded to include production and delivery of antibiotic or antineoplastic therapeutic payloads at the detection site.

synthetic biology↗

bioRxiv2020

One-Day Construction Of Multiplex Arrays to Harness Natural CRISPR Systems

CRISPR-Cas systems are prokaryotic immune systems that have proliferated widely not only in bacteria and archaea, but also much more recently, in human biological research and applications. Much work to date has utilized synthetic sgRNAs along with the CRISPR nuclease Cas9, but the discovery of array-processing nucleases now allows the use of more compact, natural CRISPR arrays in heterologous hosts, in addition to organisms with endogenous systems. Unfortunately, the construction of multiplex natural CRISPR arrays remains technically challenging, expensive, and/or time-consuming. This limitation hampers research involving natural CRISPR arrays in both native and heterologous hosts. To address this problem, we present a method to assemble CRISPR arrays that is simple, rapid, affordable, and highly scalable - we assembled 9-spacer arrays with one days worth of work. We used this method to harness the endogenous CRISPR system of the highly competent bacterium Acinetobacter baylyi, showing that while single spacers are not always completely effective at blocking DNA acquisition through natural competence, multiplex natural CRISPR arrays enable both nearly complete DNA exclusion and genome editing, including with multiple targets for both. In addition to demonstrating a CRISPR array assembly method that will benefit a variety of applications, we also find a potential bet-hedging strategy for balancing CRISPR defense vs. DNA acquisition in naturally competent A. baylyi.

synthetic biology↗