Introduction:
CRISPR-ology, the study of genome editing, has advanced our understanding of biology and its applications. Focus on the use of CRISPR in microbes at the Innovative Genomics Institute (IGI).

CRISPR Background:
CRISPR-Cas9, a revolutionary RNA-guided DNA cleaver, was discovered in 2012. CRISPR systems are adaptive immune mechanisms in bacteria and archaea against foreign genetic material. CRISPR-Cas proteins use RNA guides to identify and cleave matching DNA sequences.

Initial Research:
Early funding from LBNL enabled biochemical studies on CRISPR systems. Blake Widenheft conducted groundbreaking research on the biochemistry and structural biology of CRISPR-Cas systems. Collaboration with Emmanuel Charpentier revealed the programmability of CRISPR-associated RNA guides.

The Power of a Simple Question:
Martin Yinek’s experiment demonstrated the targeted DNA cleavage capability of CRISPR-Cas9 with a simplified single guide RNA format. This experiment showcased the potential of CRISPR as a powerful tool for genome manipulation.

CRISPR Applications:
CRISPR-Cas9 platform has been utilized for various applications, including gene disruption/replacement, transcription control, molecular diagnostics, and cell-based imaging.

Experimental Systems for CRISPR Research:
Investigating the fundamental mechanisms of CRISPR genome editing. Developing CRISPR-based tools for microbiome research, such as targeted gene disruption and gene regulation. Applying CRISPR to study microbial interactions, community dynamics, and metabolic pathways.

Cas9 Structure and Function:
Cas9 is an enzyme that recognizes a specific sequence in double-stranded DNA, guided by a complementary RNA molecule. This recognition requires a protospacer adjacent motif (PAM) sequence adjacent to the target DNA. Cas9 unwinds DNA through interaction with the PAM motif, allowing the RNA guide to interrogate the adjacent sequence. Extensive complementarity between the RNA guide and DNA leads to the formation of an R-loop and DNA cleavage.

Cas9 Dynamics and DNA Interrogation:
Cas9 undergoes a large rearrangement between closed and open forms, enabling DNA bending and unwinding near PAM sequences. Initial complementarity between the RNA guide and DNA prolongs the interaction, allowing further interrogation. The search speed of Cas9, influenced by intrinsic protein dynamics, is distinct from DNA binding and cutting speeds.

Applications in Microbiome Biology:
CRISPR technology can be used to study the fundamental biology of microbiomes. Applications range from basic research to medicine and agriculture. Exploring CRISPR in the native organisms that encode CRISPR systems, such as microbes, offers exciting new directions. CRISPR can potentially unlock microbiome biology, enabling targeted manipulation and understanding of microbial communities.

BioForge Team at IGI:
BioForge is a group of young investigators at the Innovative Genomics Institute focused on uncovering new biology using microbial community editing. Their goal is to develop a set of genome editing tools optimized for microbes, allowing fundamental biological studies and manipulation of symbiotic relationships. Members include Ben Rubin, Brady Kress, and Spencer Diamond, who have expertise in microbiome editing, tool development, and bioinformatics.

Importance of Microbiomes:
Microbiomes play crucial roles in human health, neurological function, and agriculture (e.g., livestock emissions). Microbiome manipulation is challenging, with antibiotics having limited effectiveness and undesirable side effects.

Development of CRISPR for Microbiome Manipulation:
Ben Rubin led efforts to develop ways to use CRISPR in natural microbial communities, initially focusing on synthetic communities. The strategy involved determining which microbes in a community can take up foreign DNA and developing targeted CRISPR editing approaches for specific organisms.

Environmental Transformation Sequencing (ETSeq):
Ben Rubin developed ETSeq, a method to quantitatively measure genetic accessibility in microbial communities. ETSeq uses non-targeted transposons to make genomic modifications that can be mapped using sequencing, providing a measure of insertion efficiency.

DNA Uptake Preferences:
ETSeq revealed that different microbes have distinct preferences for DNA delivery methods, such as natural transformation, conjugation, and electroporation. This information is critical for manipulating specific organisms within complex communities.

Future Directions:
Ongoing work aims to extend the sensitivity of methods like ETSeq to reach deeper into microbial communities and manipulate even inabundant organisms.

Genetic Accessibility Measurements:
Researchers developed a method to measure the genetic accessibility of abundant community members. This approach involves manipulating microbes genetically in the context of their microbial neighbors. The method can be used to optimize DNA delivery and other aspects of genome manipulation.

CRISPR-Cas Transposase Editing:
Researchers used a targeted CRISPR-Cas editing system to introduce genes into microbes. The system couples a transposase to an RNA-guided CRISPR-associated protein, allowing targeted integration of DNA. This strategy avoids the double-stranded DNA break intermediate, which is detrimental to many microbes.

VC-DART System:
The Sternberg Lab developed a system called VC-DART, which combines all the necessary components for CRISPR-Cas transposase editing into a single plasmid. VC-DART includes the guide RNA, the transposon cargo, and the Cas genes required for assembling a functional RNA-guided transposase complex.

Specificity of VC-DART:
VC-DART exhibited high specificity in integration events. Integration events were only detected at sequences in the genome that were targeted by the RNA guide. This specificity is advantageous compared to alternative CRISPR transposases, which are less selective and less dependent on the RNA guide.

Editing a Microbial Community:
Researchers used the CRISPR-Cas-guided transposase to edit a synthetic microbial community. By changing the guide RNA, they could direct integration events to different organisms in the population without affecting others. This approach was successful in introducing a desired sequence into specific community members.

CRISPR Transposases for Selective Targeting:
Cascaded transposases can selectively target different organisms within a microbial community by integrating a selectable marker, allowing the targeted organism to outgrow others.

Experiments with Infant Gut Microbiome:
E. coli strains with different virulence genes were present in infant gut microbiome samples. Guide RNAs were designed to selectively target one E. coli strain over the other. Selective outgrowth of the targeted strain was observed after transposase treatment and selection, demonstrating the feasibility of precise targeting in a natural microbial population.

Challenges and Future Directions:
Increasing the accessibility of different organisms for editing. Deep understanding of metagenomic sequences to identify editable targets. Developing methods for outgrowth measurement without relying on selection. Establishing a pipeline for strain-specific editing, enrichment, and genome assembly.

Generalizable Tool Set for Community Editing:
Using sequencing-based strategies to assess genetic accessibility of organisms in a community. Delivering DNA effectively to introduce it into organisms of interest. Utilizing species and locus-specific editing through CRISPR transposases.

Advancements in CRISPR Transposase Research:
Rapid discovery and experimental testing of new CRISPR transposases. Potential for inserting DNA sequences into plasmids in eukaryotic cells.

Demonstrated Editing Pipeline in Natural Communities:
Successful editing in infant gut microbiome samples. Potential for manipulating individual organisms in natural microbiomes.

Current CRISPR Technology:
CRISPR programmable transposons can edit complex microbiomes. They have high specificity but low efficiency. Future goals include increasing efficiency.

Expanding CRISPR Applications:
Increased efficiency will allow access to more species. New transformation methods may be discovered through studying natural mechanisms.

Technology Development and Fundamental Biology:
Technology advancements drive fundamental biology questions. Fundamental biology discoveries enhance technology development. Synergy between technology and biology drives the field forward.

Team Recognition:
Doudna Lab retreat brought together undergraduate, summer, and joint students, technicians, and others. Shoutout to Josh Kofsky, Kasia Soksyk, Brady, Ben, and Spencer for their contributions. Appreciation for the Banfield Group, Adam Deutschbauer, Rodolf Barrango, and Trent Northern’s collaboration.

Funding Sources:
NIH, MCAFES, Department of Energy, and Jay Bae and the Sheryl and Kay Kersey Foundation provided funding.

Overcoming Antibiotic Selection:
Introduction of beneficial genes that provide an environmental advantage is one approach. Future goal is to manipulate bugs for environmental advantage without antibiotics.