CRISPR-based engineering of phages for in situ bacterial base editing

Abstract

Investigation of microbial gene function is essential to the elucidation of ecological roles and complex genetic interactions that take place in microbial communities. While microbiome studies have increased in prevalence, the lack of viable in situ editing strategies impedes experimental progress, rendering genetic knowledge and manipulation of microbial communities largely inaccessible. Here, we demonstrate the utility of phage-delivered CRISPR-Cas payloads to perform targeted genetic manipulation within a community context, deploying a fabricated ecosystem (EcoFAB) as an analog for the soil microbiome. First, we detail the engineering of two classical phages for community editing using recombination to replace nonessential genes through Cas9-based selection. We show efficient engineering of T7, then demonstrate the expression of antibiotic resistance and fluorescent genes from an engineered λ prophage within an Escherichia coli host. Next, we modify λ to express an APOBEC-1-based cytosine base editor (CBE), which we leverage to perform C-to-T point mutations guided by a modified Cas9 containing only a single active nucleolytic domain (nCas9). We strategically introduce these base substitutions to create premature stop codons in-frame, inactivating both chromosomal ( lacZ ) and plasmid-encoded genes (mCherry and ampicillin resistance) without perturbation of the surrounding genomic regions. Furthermore, using a multigenera synthetic soil community, we employ phage-assisted base editing to induce host-specific phenotypic alterations in a community context both in vitro and within the EcoFAB, observing editing efficiencies from 10 to 28% across the bacterial population. The concurrent use of a synthetic microbial community, soil matrix, and EcoFAB device provides a controlled and reproducible model to more closely approximate in situ editing of the soil microbiome.

Genome-wide Characterization of Diverse Bacteriophages Enabled by RNA-Binding CRISPRi

“…Beyond basic understanding of phages, resolution of gene essentiality can allow for rapid inference of deletable gene content. In tandem with wild-type Cas13 phage gene editing strategies (23), CRISPRi-ART can guide phage genome reduction for phage therapy or microbiome editing applications (4,(47)(48)(49). Furthermore, given the broad vulnerability of phages to CRISPRi-ART targeting independent of genome protection strategies (15, 17-19, 36, 39), we anticipate CRISPRi-ART to enable phage functional genomics across phage diversity.…”

PHEIGES, all-cell-free phage synthesis and selection from engineered genomes

“…These conditions eliminate many of the DNA assembly methods that have been reported recently. Editing procedures based on CRISPR 42 13 15 14 or living chassis 16 43 are time-consuming and relatively costly. The Gibson and Golden Gate DNA assembly methods are achieved in buffers that do not interface well with TXTL, which requires additional purification steps after assembly 21 .…”

“…The yeast cloning approach to phage genome engineering takes on the order of one week to achieve 64 65 . CRISPR approaches take at least one week to achieve as plasmids must be prepared for homologous recombination first 13 14 . The Golden Gate Assembly method (GGA) takes about two to three days to carry out without taking into account the step of whole genome synthesis to remove unwanted GGA sites 63 .…”

“…Bacteriophages (phages) comprise an immense reservoir of biotechnologically-relevant bioactive materials such as DNA engineering tools 1 and phage display 2 with broad applications in phage therapy 3 4 5 6 7 8 , nanotechnology 9 10 , and vaccine scaffolds engineering 11 12 . Despite this, current in vivo phage engineering approaches, based on CRISPR 13 14 15 , yeast assembly 16 17 or integrases 18 limit phage’s usage due to time consuming cloning steps and complex selection processes 19 , especially challenging for obligated lytic phages 20 . On the other hand, in vitro genome engineering is emerging as a complementary approach to assemble and edit bacteriophages with novel properties.…”

PHEIGES: all-cell-free phage synthesis and selection from engineered genomes

“…Bacteriophages (phages) comprise an immense reservoir of biotechnologically-relevant bioactive materials such as DNA engineering tools 1 and phage display 2 with broad applications in phage therapy 3 – 8 , nanotechnology 9 , 10 , and vaccine scaffolds engineering 11 , 12 . Despite this, current in vivo phage engineering approaches, based on CRISPR 13 – 15 , yeast assembly 16 , 17 or integrases 18 limit phage’s usage due to time consuming cloning steps and complex selection processes 19 , especially challenging for obligated lytic phages 20 . On the other hand, in vitro genome engineering is emerging as a complementary approach to assemble and edit bacteriophages with novel properties.…”