CRISPR

Summary

CRISPR (ˈkrɪspər) (an acronym for clustered regularly interspaced short palindromic repeats) is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria and archaea. These sequences are derived from DNA fragments of bacteriophages that had previously infected the prokaryote. They are used to detect and destroy DNA from similar bacteriophages during subsequent infections. Hence these sequences play a key role in the antiviral (i.e. anti-phage) defense system of prokaryotes and provide a form of acquired immunity. CRISPR is found in approximately 50% of sequenced bacterial genomes and nearly 90% of sequenced archaea. Cas9 (or "CRISPR-associated protein 9") is an enzyme that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence. Cas9 enzymes together with CRISPR sequences form the basis of a technology known as CRISPR-Cas9 that can be used to edit genes w

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Analysis of long-range gene regulation at the HoxD locus

Ana Rita De Carvalho Amândio Lhopitallier

Hoxd genes are essential for the development of the various body axes in vertebrates and hence the underlying regulatory mechanisms are of paramount importance. Among these various mechanisms are long-range acting enhancers, which are located in the two adjacent regulatory landscapes. Analyses of chromatin architecture at this gene cluster has revealed the existence of two topologically associating domains (TADs) flanking the cluster and encompassing these regulatory landscapes. However, the dynamics of such regulatory regions as well as the stability and functional contribution of specific enhancer-promoter interactions during development remains to be established. In this work, we analysed the 3D chromatin organization and transcription profile at the HoxD locus, at different time points during genital tubercle (GT) development, and observe that the 3D conformation of this regulatory region predates the embryonic emergence of the GT. Along with this tissue development, we observe a reduction in transcript levels correlating with a decrease in enhancer-promoter chromatin loops within the adjacent gene desert. This decrease occurs while maintaining a subset of CTCF/Cohesin associated contacts, which are preserved independently from the transcriptional status of the gene cluster. To further explore the functional contribution of this regulatory landscape, we used CRISPR-Cas9 technology to generate mice carrying partial deletions of this region, as well as targeted deletions of both transient (enhancer associated) and constitutive (CTCF/Cohesin associated) contacts. We observe that single deletions of both transient and constitutive contacts displayed little if any effect on Hoxd genes expression in the GT. On the contrary, the single deletion of a previous characterized Hoxd enhancer, the Prox element, or deletions comprising several enhancers, result in the reduction of Hoxd genes expression levels. Overall our results suggest that not all enhancer elements within a complex regulatory landscape have the same functional strength, and highlight the existence of a dynamic yet robust system to tightly regulate Hoxd genes expression.

EPFL2019

The sheer size of the protein sequence space is massive: a protein of 100 residues can have 20^100 possible sequence combinations; and knowing that this exceeds the number of atoms in the universe, the chance of randomly discovering a stable new sequence with the desired characteristics is infinitesimally small. Therefore, computational methodologies that can search through the sequence space and expand beyond naturally occurring functional protein sequence variants hold enormous potential in biomedicine and nanotechnology.My thesis work leverages machine learning, physics-based, and data-driven techniques to design new protein molecules with distinct shapes (folds) so that they can precisely interact with other molecules to perform biological functions.The first part of my thesis is dedicated to the design of functional proteins. The re-designed of an anti-CRISPR protein that can be controlled via blue light (optogenetic control) to regulate the genome editing activity of the enzyme CRISPRâCas9 is presented. A surface-based design of a broad-spectrum inhibitory Acr towards another natural target SauCas9) exemplifies the re-purposing of existing inhibitory molecules against other related targets. This ultimately led to the development of a general surface-centric design method for generating specific protein-protein interactions from scratch and exemplified by the successful design of novel PD-L1 inhibitors, an immune checkpoint that can halt the immune system from attacking the cancer cells.The successful design of protein-protein interactions heavily relies on the underlying protein fold and structure stabilizing the functional motif in a protein. Because nature has only evolved a small set of protein folds, generated protein-interaction motifs can rarely be incorporated into existing protein structures. To address this problem, the second part of my thesis is dedicated to the development of computational de novo protein design methods for the crafting of proteins with customized folds. To this end, the TopoBuilder framework utilizes a large collection of native proteins to transform a literal description of a protein fold into a physically-realistic protein. Finally, Genesis, a deep neural networks approach for the tailored de novo protein design is presented. Employing both, the TopoBuilder and Genesis, proteins completely absent from the natural repertoire were designed and experimentally validated.My thesis sets the path to explore possibilities of jointly optimizing the protein's shape and its surface geometry to master biological functions. We are now at entering a new era where newly designed protein-based drugs and materials with the potential to solve a vast array of technical challenges and open new avenues for next-generation precision drugs and advanced nanomaterials.

EPFL2022

Engineered anti-CRISPR proteins for optogenetic control of CRISPR-Cas9

Bruno Emanuel Ferreira De Sousa Correia, Zander Harteveld

Anti-CRISPR proteins are powerful tools for CRISPR-Cas9 regulation; the ability to precisely modulate their activity could facilitate spatiotemporally confined genome perturbations and uncover fundamental aspects of CRISPR biology. We engineered optogenetic anti-CRISPR variants comprising hybrids of AcrIIA4, a potent Streptococcus pyogenes Cas9 inhibitor, and the LOV2 photosensor from Avena sativa. Coexpression of these proteins with CRISPR-Cas9 effectors enabled light-mediated genome and epigenome editing, and revealed rapid Cas9 genome targeting in human cells.

NATURE PUBLISHING GROUP2018