RNA-based gene writing in human cells

Introduction

CRISPR development has enormously accelerated genetic engineering principles, and precise methods to modify small alleles (such as base or prime editing) are now available. However, generating large genomic changes still presents enormous challenges.

Challenges in Gene Transfer

Large modifications, such as gene transfers, are performed generally with viral vectors, which have been associated with toxicities in the clinic, and often lack the versatility needed for basic science experimentation. Newer CRISPR-based techniques for gene transfer suffer from significant efficacy and safety problems when used for large message writing.

Goals of SCRIBE

The overall goal of SCRIBE is to create new strategies for gene writing and define their molecular principles. These new writers will use RNA to both encode and transfer the message.

Methodology

The SCRIBE strategies will take advantage of the retrotransposon capacity for writing genes from RNA, and the precision of CRISPR in addressing specific sites of the genome. Thus, the “find” function will be dominated by CRISPR components, and “copy-paste” activity will be executed by retroelement components.

1. To develop and optimize such a technology, we will use evolutionary analysis to select those retroelements with the highest activity and orthogonality.
2. We will modulate their message writing capacity by engineering their components.
3. We will test various CRISPR and retrotransposon combinations, and adapt both of them to converge into a unified molecular machine.
4. We will use artificial intelligence applied to protein design and a novel concept of synthetically oriented evolution to accelerate the emergence of the new function.

Application

Finally, we will deploy new gene writing principles for RNA-based in vivo gene delivery.

Conclusion

In sum, we will develop a new family of tools for engineering life. The real breakthrough will be the establishment of gene writing as a simple and general method for both research advancement and applied purposes.