Background: What is CRISPR? A New Platform for Gene Silencing

Acknowledgements

E. Coli: DNA-Targeting

Design

Future Applications

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are a genomic feature of many bacterial and archael species. CRISPR functions as an adaptive immune system, protecting the host from invasion by plasmids and bacteriophages.

We configured our experimentation with CRISPR systems using the following modular design elements:

CRISPR-Assisted Genetic Engineering Joseph Flay, Madeline Grade, Daniel Garry, Ryan Muller

Arizona State University

ASU’s 2011 iGEM team developed a project which sought to develop a modular platform for gene manipulation. During the execution of our project we:

Its locus consists of a set of CRISPR-associated (Cas) genes, a leader sequence, and an array. This array consists of repeating elements along with “spacers”, which direct the CRISPR machinery to target and destroy a complementary sequence of DNA or RNA in the cell.

Designed a software tool that offers accessibility to others labs who hope to utilize our platform for gene manipulation

Embarked upon a student-led venture at Arizona State University with an undergraduate team of five biomedical engineering students, four life science students, and one computer science student

Explored multiple approaches for testing a system for gene silencing

Expanded our implementation of the BioBrick concept to include using the endogenous genomes of organisms

Spacers (~30bp) can be created to selectively target any sequence of DNA or RNA proto-spacers.

Proto-spacers have short nucleotide tags called proto-spacer adjacent motifs (PAMs). These short sequences are recognized by the Cas machinery.

Spacer Selection:

The sixth lane from the left indicates successful ligation of Leader+RSR

B. halodurans: RNA-targeting

This construct requires the isolation of 6 genes, Cmr1-6, that are all located on a single locus

Endogenous E. Coli Cas Testing Samples containing the Leader+RSR construct showed significantly increased transformation efficiency, contrary to the expected suppression due to the presence of the array.

First row: Test plasmid introduced during final transformation. Second row: Initial plasmid present in competent cells Third row: E. coli Strain (MG1655 Cas +, Cas -)

L. Innocua: DNA-Targeting

Array Construction:

We designed repeat-spacer-repeat (RSR) units to be easily customized and linked together

Leader Sequence:

Promoter sequence that proceeds the RSR array

Cas Genes: The Cas genes can be utilized in two ways: - Amplify gene and insert into testing plasmid - Insert array into strain with functional Cas genes

Advantages of this CRISPR system:

Compactness: A single Cas gene (Cas9) is much smaller and more easily amplifiable than those of E. coli or B. halodurans

Better Regulation: A trans-encoded CRISPR RNA (tracrRNA) is required for function and allows for an extra level of regulation

More Options: The proto-spacer adjacent motif (PAM) is smaller than in other CRISPR hosts, increasing the variety of spacers

Efficient Cloning: Two components are designed to maximize ease of assembly; a part containing a T7 promoter, leader, full RSR, and half of a pseudo-spacer is ligated in one step to subsequent RSRs

A cell-free CRISPR system could be used for: - Verifying successful CRISPR construction and function - Characterizing spacer integration into the RSR array and model spacer targeting - Providing a mechanism to study the evolution of CRISPR in a simplified environment

A functioning RNA-targeting CRISPR system could be used to modulate gene expression in a given production pathway

Diagram: TU Delft iGEM 2010

• Intern Juan Padilla • Deans Mark Jacobs and Margaret Nelson • Dr. William Ditto, Dr. Marco Santello • Deans Paul Johnson and Jim Collofello • ASU President Dr. Michael Crow