PAM-flexible genome editing with an engineered chimeric Cas9

CRISPR enzymes require a defined protospacer adjacent motif (PAM) flanking a guide RNA-programmed target site, limiting their sequence accessibility for robust genome editing applications. In this study, we recombine the PAM-interacting domain of SpRY, a broad-targeting Cas9 possessing an NRN > NYN (R = A or G, Y = C or T) PAM preference, with the N-terminus of Sc + +, a Cas9 with simultaneously broad, efficient, and accurate NNG editing capabilities, to generate a chimeric enzyme with highly flexible PAM preference: SpRYc. We demonstrate that SpRYc leverages properties of both enzymes to specifically edit diverse PAMs and disease-related loci for potential therapeutic applications. In total, the approaches to generate SpRYc, coupled with its robust flexibility, highlight the power of integrative protein design for Cas9 engineering and motivate downstream editing applications that require precise genomic positioning

Precise and expansive genomic positioning for CRISPR edits

Thesis: Ph. D., Massachusetts Institute of Technology, School of Architecture and Planning, Program in Media Arts and Sciences, 2019Cataloged from PDF version of thesis.Includes bibliographical references (pages 91-105).The recent harnessing of microbial adaptive immune systems, known as CRISPR, has enabled genome-wide engineering across all domains of life. A new generation of gene-editing tools has been fashioned from the natural DNA/RNA-targeting ability of certain CRISPR-associated (Cas) proteins and their guide RNA, which work together to recognize and defend against infectious genetic threats. This straight-forward RNA-programmed sequence recognition by CRISPR has facilitated its rapid global impact on genetic research, diagnostics, therapeutics, and bioproduction. An ideal DNA-editing platform would achieve perfect accuracy on any desired cellular and genomic target. CRISPR systems, however, have limited target fidelity and range, in part due to their evolutionary pressures to defend microbes from fast-mutating viruses without self-targeting their own guide RNA.These natural limitations of CRISPR can especially constrain gene-editing in animals and plants, which are more vulnerable to off-target activity occurring in one of their trillions of cells with genomes that are 1000x larger than those of unicellular microbes that natively harbor CRISPR systems. This thesis overcomes three critical challenges for precise and broad gene-editing of complex organisms: 1) engineering a means of specificity for the type of cells to edit, 2) improving target-matching accuracy, and 3) broadening the editable portion of the genome.This thesis addresses these challenges by integrating custom developed computational design tools and biological validation of the resulting novel CRISPR systems; 1) To target within multicellular heterogeneity, new oligonucleotide-sensing structural motifs are designed and embed into guides that can potentially control CRISPR nuclease activity based on cell-type transcriptome patterns; 2) To discern among increased similarity between a target and non-target sequences in larger genomes, base-pairing thermostability principles are employed to tune the biochemical composition of guides that can evade subtly mismatched off-target sites; 3) To expand the reach of editing techniques with narrow windows of operation, such as base-editing, bioinformatics workflows that discover previously uncharacterized Cas proteins with novel target scope are created.This thesis demonstrates the effectiveness of these strategies in the context of in vitro, bacterial, and human cell culture assays, and contributes advancements in the precision and generality for CRISPR gene-editing.by Noah Michael Jakimo.Ph. D.Ph.D. Massachusetts Institute of Technology, School of Architecture and Planning, Program in Media Arts and Science

Genomic nucleic acid memory storage with directed endonucleases

Thesis: S.M., Massachusetts Institute of Technology, School of Architecture and Planning, Program in Media Arts and Sciences, 2015.Cataloged from PDF version of thesis.Includes bibliographical references (pages 40-41).Technologies for long-term recording of cellular pathway activation are constrained by the difficultly to constantly monitor transient signaling events and expression of target genes. To overcome these limitations we designed a recording tool that uses the transcriptional output of a signaling pathway as the input for an engineered genome encoded memory. The mechanism of recording leverages the programmable nature of the bacterial immune system that consists of Clustered Regularly Interspaced Short Palindromic Repeat Sequences (CRISPR), which can recognize and cleave viral DNA using an RNA-guided directed endonuclease. Cuts left by the endonuclease are repaired by an error-prone DNA damage repair mechanism, namely non-homologous end joining (NHEJ), likely to leave mutations at the cut sites. Defining the cut site with pathway-dependent transcription of guide RNA, this genomic region is sequenced to measure pathway activation by the amount of accumulated mutations. To demonstrate a system to monitor cancer metabolism, guide RNA is expressed in mammalian cell culture with a NF-kappaB promoter. To demonstrate a system that can monitor sugar intake in an environment like the gut, guide RNA is expressed in bacteria with an arabinose promoter.by Noah Jakimo.S.M

Minimal PAM specificity of a highly similar SpCas9 ortholog

RNA-guided DNA endonucleases of the CRISPR-Cas system are widely used for genome engineering and thus have numerous applications in a wide variety of fields. CRISPR endonucleases, however, require a specific protospacer adjacent motif (PAM) flanking the target site, thus constraining their targetable sequence space. In this study, we demonstrate the natural PAM plasticity of a highly similar, yet previously uncharacterized, Cas9 from Streptococcus canis (ScCas9) through rational manipulation of distinguishing motif insertions. To this end, we report affinity to minimal 5′-NNG-3′ PAM sequences and demonstrate the accurate editing capabilities of the ortholog in both bacterial and human cells. Last, we build an automated bioinformatics pipeline, the Search for PAMs by ALignment Of Targets (SPAMALOT), which further explores the microbial PAM diversity of otherwise overlooked Streptococcus Cas9 orthologs. Our results establish that ScCas9 can be used both as an alternative genome editing tool and as a functional platform to discover novel Streptococcus PAM specificities. ©2018 The Authors, some rights reserved

A Cas9 with PAM recognition for adenine dinucleotides

CRISPR-associated (Cas) DNA-endonucleases are remarkably effective tools for genome engineering, but have limited target ranges due to their protospacer adjacent motif (PAM) requirements. We demonstrate a critical expansion of the targetable sequence space for a type II-A CRISPR-associated enzyme through identification of the natural 5′-NAAN-3′ PAM preference of Streptococcus macacae Cas9 (SmacCas9). To achieve efficient editing activity, we graft the PAM-interacting domain of SmacCas9 to its well-established ortholog from Streptococcus pyogenes (SpyCas9), and further engineer an increased efficiency variant (iSpyMac) for robust genome editing activity. We establish that our hybrids can target all adenine dinucleotide PAM sequences and possess robust and accurate editing capabilities in human cells

Evolthon: A community endeavor to evolve lab evolution.

In experimental evolution, scientists evolve organisms in the lab, typically by challenging them to new environmental conditions. How best to evolve a desired trait? Should the challenge be applied abruptly, gradually, periodically, sporadically? Should one apply chemical mutagenesis, and do strains with high innate mutation rate evolve faster? What are ideal population sizes of evolving populations? There are endless strategies, beyond those that can be exposed by individual labs. We therefore arranged a community challenge, Evolthon, in which students and scientists from different labs were asked to evolve Escherichia coli or Saccharomyces cerevisiae for an abiotic stress-low temperature. About 30 participants from around the world explored diverse environmental and genetic regimes of evolution. After a period of evolution in each lab, all strains of each species were competed with one another. In yeast, the most successful strategies were those that used mating, underscoring the importance of sex in evolution. In bacteria, the fittest strain used a strategy based on exploration of different mutation rates. Different strategies displayed variable levels of performance and stability across additional challenges and conditions. This study therefore uncovers principles of effective experimental evolutionary regimens and might prove useful also for biotechnological developments of new strains and for understanding natural strategies in evolutionary arms races between species. Evolthon constitutes a model for community-based scientific exploration that encourages creativity and cooperation