cell-free-protein-synthesis-from-exonuclease-deficient-cellular

Cell-Free Protein Synthesis from Exonuclease-Deficient Cellular Extracts Utilizing Linear DNA Templates

Cell-free protein synthesis (CFPS) has recently become very popular in the field of synthetic biology due to its numerous advantages. Using linear DNA ...

CRISPR-Cas systems must enact robust immunity against foreign genetic material without inducing cytotoxic autoimmunity. For type VI systems that use Cas13 nucleases and recognize RNA targets, immune activation requires extensive CRISPR RNA (crRNA) guide-target complementarity and a target-flanking motif. Here, we report a third requirement shaping the immune response: the expression of the target transcript exceeding a threshold. We found that endogenous non-essential transcripts targeted by crRNAs rarely elicited autoimmunity. Instead, autoimmune induction required over-expressing the targeted transcripts above a threshold. A genome-wide screen confirmed target expression levels as a global determinant of cytotoxic autoimmunity and revealed that this threshold shifts with each guide-target pair. This threshold further ensured defense against a lytic bacteriophage yet allowed the tolerance of a targeted beneficial gene expressed from an invading plasmid. These findings establish target expression levels as an additional criterion for immune defense by RNA-targeting CRISPR-Cas systems, preventing autoimmunity and distinguishing pathogenic and benign invaders.

A target expression threshold dictates invader defense and prevents autoimmunity by CRISPR-Cas13.

The characterization of CRISPR-Cas immune systems in bacteria was quickly followed by the discovery of anti-CRISPR proteins (Acrs) in bacteriophages. These proteins block different steps of CRISPR-based immunity and, as some inhibit Cas nucleases, can offer tight control over CRISPR technologies. While Acrs have been identified against a few CRISPR-Cas systems, likely many more await discovery and application. Here, we report a rapid and scalable method for characterizing putative Acrs against Cas nucleases using an E. coli-derived cell-free transcription-translation system. Using known Acrs against type II Cas9 nucleases as models, we demonstrate how the method can be used to measure the inhibitory activity of individual Acrs in under two days. We also show how the method can overcome non-specific inhibition of gene expression observed for some Acrs. In total, the method should accelerate the interrogation and application of Acrs as CRISPR-Cas inhibitors.

An enhanced assay to characterize anti-CRISPR proteins using a cell-free transcription-translation system.

Genome editing is essential for probing genotype-phenotype relationships and for enhancing chemical production and phenotypic robustness in industrial bacteria. Currently, the most popular tools for genome editing couple recombineering with DNA cleavage by the CRISPR nuclease Cas9 from Streptococcus pyogenes. Although successful in some model strains, CRISPR-based genome editing has been slow to extend to the multitude of industrially relevant bacteria. In this review, we analyze existing barriers to implementing CRISPR-based editing across diverse bacterial species. We first compare the efficacy of current CRISPR-based editing strategies. Next, we discuss alternatives when the S. pyogenes Cas9 does not yield colonies. Finally, we describe different ways bacteria can evade editing and how elucidating these failure modes can improve CRISPR-based genome editing across strains. Together, this review highlights existing obstacles to CRISPR-based editing in bacteria and offers guidelines to help achieve and enhance editing in a wider range of bacterial species, including non-model strains.

Barriers to genome editing with CRISPR in bacteria.

CRISPR-Cas systems inherently multiplex through CRISPR arrays-whether to defend against different invaders or mediate multi-target editing, regulation, imaging, or sensing. However, arrays remain difficult to generate due to their reoccurring repeat sequences. Here, we report a modular, one-pot scheme called CRATES to construct CRISPR arrays and array libraries. CRATES allows assembly of repeat-spacer subunits using defined assembly junctions within the trimmed portion of spacers. Using CRATES, we construct arrays for the single-effector nucleases Cas9, Cas12a, and Cas13a that mediated multiplexed DNA/RNA cleavage and gene regulation in cell-free systems, bacteria, and yeast. CRATES further allows the one-pot construction of array libraries and composite arrays utilized by multiple Cas nucleases. Finally, array characterization reveals processing of extraneous CRISPR RNAs from Cas12a terminal repeats and sequence- and context-dependent loss of RNA-directed nuclease activity via global RNA structure formation. CRATES thus can facilitate diverse multiplexing applications and help identify factors impacting crRNA biogenesis.

Modular one-pot assembly of CRISPR arrays enables library generation and reveals factors influencing crRNA biogenesis.

CRISPR-Cas systems naturally rely on CRISPR arrays to achieve immunity against multiple foreign invaders, where these arrays are also being utilized for multiplexed targeting as part of CRISPR technologies. However, CRISPR arrays have proven difficult to synthesize or assemble to-date due to the repetitive DNA repeats in each array. To overcome this barrier, we recently reported a cloning method we term CRATES (CRISPR Assembly through Trimmed Ends of Spacers) for the single-step, efficient generation of large Class 2 CRISPR arrays. CRATES generates CRISPR arrays through assembly of multiple repeat-spacer subunits using defined junction sequences within the trimmed portion of the CRISPR spacers. These arrays can be utilized by single-effector nucleases associated with Class 2 CRISPR-Cas systems, such as Cas9, Cas12a/Cpf1, or Cas13a/C2c2. Here, we describe in detail the steps for generating arrays utilized by Cas9 and Cas12a as well as composite arrays co-utilized by both nucleases. We also generate a representative three-spacer array and demonstrate multiplexed DNA cleavage through plasmid-clearance assays in Escherichia coli. This method is expected to simplify the study of natural CRISPR arrays and facilitate multiplexed targeting with programmable nucleases from Class 2 Cas nucleases across the myriad applications of CRISPR technologies.

CRATES: A one-step assembly method for Class 2 CRISPR arrays.

Methods for characterizing, applying, and teaching CRISPR-Cas systems.

Jin et al. (2020) engineered new variants of CRISPR base editors that make precise genomic edits in rice protoplasts while minimizing untargeted mutagenesis.

Your Base Editor Might Be Flirting with Single (Stranded) DNA: Faithful On-Target CRISPR Base Editing without Promiscuous Deamination.

The ever-expanding set of CRISPR technologies and their programmable RNA-guided nucleases exhibit remarkable flexibility in DNA targeting. However, this flexibility comes with an ever-present constraint: the requirement for a protospacer adjacent motif (PAM) flanking each target. While PAMs play an essential role in self/nonself discrimination by CRISPR-Cas immune systems, this constraint has launched a far-reaching expedition for nucleases with relaxed PAM requirements. Here, we review ongoing efforts toward realizing PAM-free nucleases through natural ortholog mining and protein engineering. We also address potential consequences of fully eliminating PAM recognition and instead propose an alternative nuclease repertoire covering all possible PAM sequences.

CRISPR technologies and the search for the PAM-free nuclease.

CRISPR-Cas systems recognize foreign genetic material using CRISPR RNAs (crRNAs). In type II systems, a trans-activating crRNA (tracrRNA) hybridizes to crRNAs to drive their processing and utilization by Cas9. While analyzing Cas9-RNA complexes from , we discovered tracrRNA hybridizing to cellular RNAs, leading to formation of "noncanonical" crRNAs capable of guiding DNA targeting by Cas9. Our discovery inspired the engineering of reprogrammed tracrRNAs that link the presence of any RNA of interest to DNA targeting with different Cas9 orthologs. This capability became the basis for a multiplexable diagnostic platform termed LEOPARD (leveraging engineered tracrRNAs and on-target DNAs for parallel RNA detection). LEOPARD allowed simultaneous detection of RNAs from different viruses in one test and distinguished severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its D614G (Asp→Gly) variant with single-base resolution in patient samples.

Noncanonical crRNAs derived from host transcripts enable multiplexable RNA detection by Cas9.

CRISPR transposons (CASTs) represent unique mobile genetic elements that co-opted CRISPR-Cas immune systems for RNA-guided DNA transposition. However, CAST-encoded CRISPR arrays rarely match the CAST's chromosomal location. A recent publication in Cell helps resolve this paradox by revealing CRISPR-array-independent mechanisms of chromosomal homing unique to different CAST types.

CRISPR transposons on the move.

CRISPR-Cas adaptive immune systems in bacteria and archaea utilize short CRISPR RNAs (crRNAs) to guide sequence-specific recognition and clearance of foreign genetic material. Multiple crRNAs are stored together in a compact format called a CRISPR array that is transcribed and processed into the individual crRNAs. While the exact processing mechanisms vary widely, some CRISPR-Cas systems, including those encoding the Cas9 nuclease, rely on a -activating crRNA (tracrRNA). The tracrRNA was discovered in 2011 and was quickly co-opted to create single-guide RNAs as core components of CRISPR-Cas9 technologies. Since then, further studies have uncovered processes extending beyond the traditional role of tracrRNA in crRNA biogenesis, revealed Cas nucleases besides Cas9 that are dependent on tracrRNAs, and established new applications based on tracrRNA engineering. In this review, we describe the biology of the tracrRNA and how its ongoing characterization has garnered new insights into prokaryotic immune defense and enabled key technological advances.

The tracrRNA in CRISPR Biology and Technologies.

Cell-free transcription-translation (TXTL) systems produce RNAs and proteins from added DNA. By coupling their production to a biochemical assay, these biomolecules can be rapidly and scalably characterized without the need for purification or cell culturing. Here, we describe how TXTL can be applied to characterize Cas13 nucleases from Type VI CRISPR-Cas systems. These nucleases employ guide RNAs to recognize complementary RNA targets, leading to the nonspecific collateral cleavage of nearby RNAs. In turn, RNA targeting by Cas13 has been exploited for numerous applications, including in vitro diagnostics, programmable gene silencing in eukaryotes, and sequence-specific antimicrobials. As part of the described method, we detail how to set up TXTL assays to measure on-target and collateral RNA cleavage by Cas13 as well as how to assay for putative anti-CRISPR proteins. Overall, the method should be useful for the characterization of Type VI CRISPR-Cas systems and their use in ranging applications.

Rapidly Characterizing CRISPR-Cas13 Nucleases Using Cell-Free Transcription-Translation Systems.

Diverse DNA repair pathways correct ranging types of damage and play central roles in genome editing. In this issue of Cell, two publications leverage a new high-throughput screen that links pathway genes with the outcomes of repair, yielding mechanistic insights into the repair process as well as means to shape editing outcomes.

Illuminating the path to DNA repair.

Type I CRISPR-Cas systems represent the most common and diverse type of these prokaryotic defense systems and are being harnessed for a growing set of applications. As these systems rely on multi-protein effector complexes, their characterization remains challenging. Here, we report a rapid and straightforward method to characterize these systems in a cell-free transcription-translation (TXTL) system. A ribonucleoprotein complex is produced and binds to its target next to a recognized PAM, thereby preventing the targeted sequence from being cleaved by a restriction enzyme. Selection for uncleaved targeted plasmids leads to an enrichment of recognized sequences within a PAM library. This assay will aid the exploration of CRISPR-Cas diversity and evolution and help contribute new systems for CRISPR technologies and applications.

A TXTL-Based Assay to Rapidly Identify PAMs for CRISPR-Cas Systems with Multi-Protein Effector Complexes.

CRISPR-Cas biology and technologies have been largely shaped to date by the characterization and use of single-effector nucleases. By contrast, multi-subunit effectors dominate natural systems, represent emerging technologies, and were recently associated with RNA-guided DNA transposition. This disconnect stems from the challenge of working with multiple protein subunits in vitro and in vivo. Here, we apply cell-free transcription-translation (TXTL) systems to radically accelerate the characterization of multi-subunit CRISPR effectors and transposons. Numerous DNA constructs can be combined in one TXTL reaction, yielding defined biomolecular readouts in hours. Using TXTL, we mined phylogenetically diverse I-E effectors, interrogated extensively self-targeting I-C and I-F systems, and elucidated targeting rules for I-B and I-F CRISPR transposons using only DNA-binding components. We further recapitulated DNA transposition in TXTL, which helped reveal a distinct branch of I-B CRISPR transposons. These capabilities will facilitate the study and exploitation of the broad yet underexplored diversity of CRISPR-Cas systems and transposons.

Rapid cell-free characterization of multi-subunit CRISPR effectors and transposons.

CRISPR-Cas systems store fragments of foreign DNA, called spacers, as immunological recordings used to combat future infections. Of the many spacers stored in a CRISPR array, the most recent are known to be prioritized for immune defence. However, the underlying mechanism remains unclear. Here we show that the leader region upstream of CRISPR arrays in CRISPR-Cas9 systems enhances CRISPR RNA (crRNA) processing from the newest spacer, prioritizing defence against the matching invader. Using the CRISPR-Cas9 system from Streptococcus pyogenes as a model, we found that the transcribed leader interacts with the conserved repeats bordering the newest spacer. The resulting interaction promotes transactivating crRNA (tracrRNA) hybridization with the second of the two repeats, accelerating crRNA processing. Accordingly, disruption of this structure reduces the abundance of the associated crRNA and immune defence against targeted plasmids and bacteriophages. Beyond the S. pyogenes system, bioinformatics analyses revealed that leader-repeat structures appear across CRISPR-Cas9 systems. CRISPR-Cas systems thus possess an RNA-based mechanism to prioritize defence against the most recently encountered invaders.

Spacer prioritization in CRISPR-Cas9 immunity is enabled by the leader RNA.

The bacterial genus Lactobacillus comprises a vast range of strains with varying metabolic and probiotic traits, with genome editing representing an essential tool to probe genotype-phenotype relationships and enhance their beneficial properties. Currently, one of the most effective means of genome editing in bacteria couples low-efficiency recombineering with high-efficiency counterselection by nucleases from CRISPR-Cas systems. In lactobacilli, several CRISPR-based genome editing methods exist that have shown varying success in different strains. Here, we detail a fast and simple approach using two shuttle vectors encoding a recombineering template as well as the Streptococcus pyogenes Cas9, a trans-activating RNA, and a CRISPR array. We provide a step-by-step procedure for cloning the shuttle vectors, sequentially transforming the vectors into lactobacilli, screening for the desired edit, and finally clearing the shuttle vectors from the mutant strain. As CRISPR-based genome editing in bacteria can fail for various reasons, we also lay out instructions for probing mechanisms of escape. Finally, we include practical notes along the way to facilitate each stage of genome editing, and we illustrate the technique using a representative edit in a strain of Lactobacillus plantarum. Overall, this method should serve as a complete guide to performing genome editing in lactobacilli.

Genome Editing with Cas9 in Lactobacilli.

As part of the ongoing bacterial-phage arms race, CRISPR-Cas systems in bacteria clear invading phages whereas anti-CRISPR proteins (Acrs) in phages inhibit CRISPR defenses. Known Acrs have proven extremely diverse, complicating their identification. Here, we report a deep learning algorithm for Acr identification that revealed an Acr against type VI-B CRISPR-Cas systems. The algorithm predicted numerous putative Acrs spanning almost all CRISPR-Cas types and subtypes, including over 7,000 putative type IV and VI Acrs not predicted by other algorithms. By performing a cell-free screen for Acr hits against type VI-B systems, we identified a potent inhibitor of Cas13b nucleases we named AcrVIB1. AcrVIB1 blocks Cas13b-mediated defense against a targeted plasmid and lytic phage, and its inhibitory function principally occurs upstream of ribonucleoprotein complex formation. Overall, our work helps expand the known Acr universe, aiding our understanding of the bacteria-phage arms race and the use of Acrs to control CRISPR technologies.

Anti-CRISPR prediction using deep learning reveals an inhibitor of Cas13b nucleases.

CRISPR-based detection and recording technologies are gaining increasing attention in disease surveillance and prevention. In this chapter, we describe how our recent discovery of noncanonical crRNAs inspired the engineering of reprogrammed tracrRNAs and led to a powerful platform for multiplexed RNA detection. We provide detailed protocols regarding how to design reprogrammed tracrRNA and carry out assays in vitro and in vivo.

Article	Year
A target expression threshold dictates invader defense and prevents autoimmunity by CRISPR-Cas13. Cell host & microbe\| PubMed ID: 35690065	2022
An enhanced assay to characterize anti-CRISPR proteins using a cell-free transcription-translation system. Methods (San Diego, Calif.)\| PubMed ID: 31121300	2020
Barriers to genome editing with CRISPR in bacteria. Journal of industrial microbiology & biotechnology\| PubMed ID: 31165970	2019
Modular one-pot assembly of CRISPR arrays enables library generation and reveals factors influencing crRNA biogenesis. Nature communications\| PubMed ID: 31270316	2019
CRATES: A one-step assembly method for Class 2 CRISPR arrays. Methods in enzymology\| PubMed ID: 31727255	2019
Methods for characterizing, applying, and teaching CRISPR-Cas systems. Methods (San Diego, Calif.)\| PubMed ID: 31954772	2020
Your Base Editor Might Be Flirting with Single (Stranded) DNA: Faithful On-Target CRISPR Base Editing without Promiscuous Deamination. Molecular cell\| PubMed ID: 32888434	2020
CRISPR technologies and the search for the PAM-free nuclease. Nature communications\| PubMed ID: 33483498	2021
Noncanonical crRNAs derived from host transcripts enable multiplexable RNA detection by Cas9. Science (New York, N.Y.)\| PubMed ID: 33906967	2021
CRISPR transposons on the move. Cell host & microbe\| PubMed ID: 33984272	2021
The tracrRNA in CRISPR Biology and Technologies. Annual review of genetics\| PubMed ID: 34416117	2021
Rapidly Characterizing CRISPR-Cas13 Nucleases Using Cell-Free Transcription-Translation Systems. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 34694607	2022
Illuminating the path to DNA repair. Cell\| PubMed ID: 34715020	2021
A TXTL-Based Assay to Rapidly Identify PAMs for CRISPR-Cas Systems with Multi-Protein Effector Complexes. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 34985758	2022
Rapid cell-free characterization of multi-subunit CRISPR effectors and transposons. Molecular cell\| PubMed ID: 35216669	2022
Spacer prioritization in CRISPR-Cas9 immunity is enabled by the leader RNA. Nature microbiology\| PubMed ID: 35314780	2022
Genome Editing with Cas9 in Lactobacilli. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 35583743	2022
Anti-CRISPR prediction using deep learning reveals an inhibitor of Cas13b nucleases. Molecular cell\| PubMed ID: 35649413	2022
Reprogramming TracrRNAs for Multiplexed RNA Detection. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 35666448	2022

Chase L. Beisel

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