Shortened CRISPR-Cas9 arrays enable multiplexed gene targeting in bacteria from a smaller DNA footprint.

CRISPR technologies comprising a Cas nuclease and a guide RNA (gRNA) can utilize multiple gRNAs to enact multi-site editing or regulation in the same cell. Nature devised a highly compact means of encoding gRNAs in the form of CRISPR arrays composed of conserved repeats separated by targeting spacers. However, the capacity to acquire new spacers keeps the arrays longer than necessary for CRISPR technologies. Here, we show that CRISPR arrays utilized by the Cas9 nuclease can be shortened without compromising and sometimes even enhancing targeting activity. Using multiplexed gene repression in E. coli, we found that each region could be systematically shortened to varying degrees before severely compromising targeting activity. Surprisingly, shortening some spacers yielded enhanced targeting activity, which was linked to folding of the transcribed array prior to processing. Overall, shortened CRISPR-Cas9 arrays can facilitate multiplexed editing and gene regulation from a smaller DNA footprint across many bacterial applications of CRISPR technologies.

Optimized metrics for orthogonal combinatorial CRISPR screens.

CRISPR-based gene perturbation enables unbiased investigations of single and combinatorial genotype-to-phenotype associations. In light of efforts to map combinatorial gene dependencies at scale, choosing an efficient and robust CRISPR-associated (Cas) nuclease is of utmost importance. Even though SpCas9 and AsCas12a are widely used for single, combinatorial, and orthogonal screenings, side-by-side comparisons remain sparse. Here, we systematically compared combinatorial SpCas9, AsCas12a, and CHyMErA in hTERT-immortalized retinal pigment epithelial cells and extracted performance-critical parameters for combinatorial and orthogonal CRISPR screens. Our analyses identified SpCas9 to be superior to enhanced and optimized AsCas12a, with CHyMErA being largely inactive in the tested conditions. Since AsCas12a contains RNA processing activity, we used arrayed dual-gRNAs to improve AsCas12a and CHyMErA applications. While this negatively influenced the effect size range of combinatorial AsCas12a applications, it enhanced the performance of CHyMErA. This improved performance, however, was limited to AsCas12a dual-gRNAs, as SpCas9 gRNAs remained largely inactive. To avoid the use of hybrid gRNAs for orthogonal applications, we engineered the multiplex SpCas9-enAsCas12a approach (multiSPAS) that avoids RNA processing for efficient orthogonal gene editing.

Systematically attenuating DNA targeting enables CRISPR-driven editing in bacteria.

Bacterial genome editing commonly relies on chromosomal cleavage with Cas nucleases to counter-select against unedited cells. However, editing normally requires efficient recombination and high transformation efficiencies, which are unavailable in most strains. Here, we show that systematically attenuating DNA targeting activity enables RecA-mediated repair in different bacteria, allowing chromosomal cleavage to drive genome editing. Attenuation can be achieved by altering the format or expression strength of guide (g)RNAs; using nucleases with reduced cleavage activity; or engineering attenuated gRNAs (atgRNAs) with disruptive hairpins, perturbed nuclease-binding scaffolds, non-canonical PAMs, or guide mismatches. These modifications greatly increase cell counts and even improve the efficiency of different types of edits for Cas9 and Cas12a in Escherichia coli and Klebsiella oxytoca. We further apply atgRNAs to restore ampicillin sensitivity in Klebsiella pneumoniae, establishing a resistance marker for genetic studies. Attenuating DNA targeting thus offers a counterintuitive means to achieve CRISPR-driven editing across bacteria.

Differentially Optimized Cell-Free Buffer Enables Robust Expression from Unprotected Linear DNA in Exonuclease-Deficient Extracts.

The use of linear DNA templates in cell-free systems promises to accelerate the prototyping and engineering of synthetic gene circuits. A key challenge is that linear templates are rapidly degraded by exonucleases present in cell extracts. Current approaches tackle the problem by adding exonuclease inhibitors and DNA-binding proteins to protect the linear DNA, requiring additional time- and resource-intensive steps. Here, we delete the recBCD exonuclease gene cluster from the Escherichia coli BL21 genome. We show that the resulting cell-free systems, with buffers optimized specifically for linear DNA, enable near-plasmid levels of expression from σ70 promoters in linear DNA templates without employing additional protection strategies. When using linear or plasmid DNA templates at the buffer calibration step, the optimal potassium glutamate concentrations obtained when using linear DNA were consistently lower than those obtained when using plasmid DNA for the same extract. We demonstrate the robustness of the exonuclease deficient extracts across seven different batches and a wide range of experimental conditions across two different laboratories. Finally, we illustrate the use of the ΔrecBCD extracts for two applications: toehold switch characterization and enzyme screening. Our work provides a simple, efficient, and cost-effective solution for using linear DNA templates in cell-free systems and highlights the importance of specifically tailoring buffer composition for the final experimental setup. Our data also suggest that similar exonuclease deletion strategies can be applied to other species suitable for cell-free synthetic biology.

The Francisella novicida Cas12a is sensitive to the structure downstream of the terminal repeat in CRISPR arrays.

The Class 2 Type V-A CRISPR effector protein Cas12a/Cpf1 has gained widespread attention in part because of the ease in achieving multiplexed genome editing, gene regulation, and DNA detection. Multiplexing derives from the ability of Cas12a alone to generate multiple guide RNAs from a transcribed CRISPR array encoding alternating conserved repeats and targeting spacers. While array design has focused on how to optimize guide-RNA sequences, little attention has been paid to sequences outside of the CRISPR array. Here, we show that a structured hairpin located immediately downstream of the 3' repeat interferes with utilization of the adjacent encoded guide RNA by Francisella novicida (Fn)Cas12a. We first observed that a synthetic Rho-independent terminator immediately downstream of an array impaired DNA cleavage based on plasmid clearance in E. coli and DNA cleavage in a cell-free transcription-translation (TXTL) system. TXTL-based cleavage assays further revealed that inhibition was associated with incomplete processing of the transcribed CRISPR array and could be attributed to the stable hairpin formed by the terminator. We also found that the inhibitory effect partially extended to upstream spacers in a multi-spacer array. Finally, we found that removing the terminal repeat from the array increased the inhibitory effect, while replacing this repeat with an unprocessable terminal repeat from a native FnCas12a array restored cleavage activity directed by the adjacent encoded guide RNA. Our study thus revealed that sequences surrounding a CRISPR array can interfere with the function of a CRISPR nuclease, with implications for the design and evolution of CRISPR arrays.