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 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 NNN PAMs and disease-related loci for potential therapeutic applications. In total, the unique 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.

The CRISPR-Cas type V-I is a family of Cas12i-containing programmable nuclease systems guided by a short crRNA without requirement for a tracrRNA. Here we present an engineered Type V-I CRISPR system (Cas12i), ABR-001, which utilizes a tracr-less guide RNA. The compact Cas12i effector is capable of self-processing pre-crRNA and cleaving dsDNA targets, which facilitates versatile delivery options and multiplexing, respectively. We apply an unbiased mutational scanning approach to enhance initially low editing activity of Cas12i2. The engineered variant, ABR-001, exhibits broad genome editing capability in human cell lines, primary T cells, and CD34+ hematopoietic stem and progenitor cells, with both robust efficiency and high specificity. In addition, ABR-001 achieves a high level of genome editing when delivered via AAV vector to HEK293T cells. This work establishes ABR-001 as a versatile, specific, and high-performance platform for ex vivo and in vivo gene therapy.

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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-IIA CRISPR-associated enzyme through identification of the natural 5’-NAA-3’ PAM specificity of a Streptococcus macacae Cas9 (Smac Cas9). We further recombine protein domains between Smac Cas9 and its well-established ortholog from Streptococcus pyogenes (Spy Cas9), as well as an “increased” nucleolytic variant (iSpy Cas9), to achieve consistent mediation of gene modification and base editing. In a comparison to previously reported Cas9 and Cas12a enzymes, we show that our hybrids recognize all adenine dinucleotide PAM sequences and possess robust editing efficiency in human cells.

CRISPR enzymes require a protospacer-adjacent motif (PAM) near the target cleavage site, constraining the sequences accessible for editing. In the present study, we combine protein motifs from several orthologs to engineer two variants of Streptococcus canis Cas9—Sc ++ and a higher-fidelity mutant HiFi-Sc ++ —that have simultaneously broad 5′-NNG-3′ PAM compatibility, robust DNA-cleavage activity and minimal off-target activity. Sc ++ and HiFi-Sc ++ extend the use of CRISPR editing for diverse applications.

Programmable CRISPR enzymes are powerful and versatile tools for genome editing. They, however, require a specific protospacer adjacent motif (PAM) flanking the target site, which constrains the accessible sequence space for position-specific genome editing applications, such as base editing and homology-directed repair. For example, the standard Cas9 from Streptococcus pyogenes requires a PAM sequence of 5’-NGG-3’ downstream of its RNA-programmed target. Recently, three separate Cas9 enzymes (xCas9-3.7, SpCas9-NG, and ScCas9) have been independently engineered or discovered to reduce the PAM specificity to a single guanine (G) nucleotide, thus greatly expanding the number of targetable sequences. In this study, we have employed motifs from closely-related orthologs to engineer and optimize ScCas9 to exhibit enhanced genome editing and higher fidelity. Our engineered variants demonstrate superior activity within gene repression and nucleolytic contexts and possess effective base editing capabilities.

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.

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.

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. The range of sequences that CRISPR endonucleases can recognize, however, is constrained by the need for a specific protospacer adjacent motif (PAM) flanking the target site. 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 a divergent affinity to 5’-NNGT-3’ PAM sequences and demonstrate the editing capabilities of the ortholog in both bacterial and human cells. Finally, 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 utilized both as an alternative genome editing tool and as a functional platform to discover novel Streptococcus PAM specificities.

RNA and DNA profiles can help classify a variety of biological states, including disease, metabolism and cell type. In a proof-of-concept study on novel genetically encodable components for detecting single-stranded oligonucleotides, we engineered switchable CRISPR guide RNA (swigRNA) with Cas9 affinity that is conditional on sensing an oligo trigger molecule (trigRNA or trigDNA). RNA- and DNA-sensing swigRNAs that serve as off-to-on and on-to-off switches for Cas9 cleavage were achieved by computational design of hybridization dependencies in the guide. This study highlights functional swigRNA structures that implement toehold-gated strand-displacement with their triggers, and should merit further engineering and kinetic characterization.

Oligonucleotide-guided nucleases (OGNs) have enabled rapid advances in precision genome engineering. Though much effort has gone into characterizing and mitigating mismatch tolerance for the most widely adopted OGN, Streptococcus pyogenes Cas9 (SpCas9), potential off-target interactions may still limit applications where on-target specificity is critical. Here we present a new axis to control mismatch sensitivity along the recognition-conferring spacer sequence of SpCas99s guide RNA (gRNA). We introduce mismatch-evading lowered-thermostability guides (melt-guides) and exhibit how nucleotide-type substitutions in the spacer can reduce cleavage of sequences mismatched by as few as a single base pair. Co-transfecting melt-guides into human cell culture with an exonuclease involved in DNA repair, we observe indel formation on a standard genomic target at approximately 70% the rate of canonical gRNA and undetectable on off-target data.