Protospacer Adjacent Motif Research Articles

CRISPR-based screening of small RNA modulators of bile susceptibility in Bacteroides thetaiotaomicron

Microbiota-centric interventions are limited by our incomplete understanding of the gene functions of many of its constituent species. This applies in particular to small RNAs (sRNAs), which are emerging as important regulators in microbiota species yet tend to be missed by traditional functional genomics approaches. Here, we establish CRISPR interference (CRISPRi) in the abundant microbiota member Bacteroides thetaiotaomicron for genome-wide sRNA screens. By assessing the abundance of different protospacer-adjacent motifs, we identify the Prevotella bryantii B14 Cas12a as a suitable nuclease for CRISPR screens in these bacteria and generate an inducible Cas12a expression system. Using a luciferase reporter strain, we infer guide design rules and use this knowledge to assemble a computational pipeline for automated gRNA design. By subjecting the resulting guide library to a phenotypic screen, we uncover the sRNA BatR to increase susceptibility to bile salts through the regulation of genes involved in Bacteroides cell surface structure. Our study lays the groundwork for unlocking the genetic potential of these major human gut mutualists and, more generally, for identifying hidden functions of bacterial sRNAs.

Targeted mutagenesis in mice via an engineered AsCas12f1 system

SpCas9 and AsCas12a are widely utilized as genome editing tools in human cells, but their applications are largely limited by their bulky size. Recently, AsCas12f1 protein, with a small size (422 amino acids), has been demonstrated to be capable of cleaving double-stranded DNA protospacer adjacent motif (PAM). However, low editing efficiency and large differences in activity against different genomic loci have been a limitation in its application. Here, we show that engineered AsCas12f1 sgRNA has significantly improved the editing efficiency in human cells and mouse embryos. Moreover, we successfully generated three stable mouse mutant disease models using the engineered CRISPR-AsCas12f1 system in this study. Collectively, our work uncovers the engineered AsCas12f1 system expands mini CRISPR toolbox, providing a remarkable promise for therapeutic applications.

CRISPR-Cas12a for Highly Efficient and Marker-Free Targeted Integration in Human Pluripotent Stem Cells.

The CRISPR-Cas12a platform has attracted interest in the genome editing community because the prototypical Acidaminococcus Cas12a generates a staggered DNA double-strand break upon binding to an AT-rich protospacer-adjacent motif (PAM, 5'-TTTV). The broad application of the platform in primary human cells was enabled by the development of an engineered version of the natural Cas12a protein, called Cas12a Ultra. In this study, we confirmed that CRISPR-Cas12a Ultra ribonucleoprotein complexes enabled allelic gene disruption frequencies of over 90% at multiple target sites in human T cells, hematopoietic stem and progenitor cells (HSPCs), and induced pluripotent stem cells (iPSCs). In addition, we demonstrated, for the first time, the efficient knock-in potential of the platform in human iPSCs and achieved targeted integration of a GFP marker gene into the AAVS1 safe harbor site and a CSF2RA super-exon into CSF2RA in up to 90% of alleles without selection. Clonal analysis revealed bi-allelic integration in >50% of the screened iPSC clones without compromising their pluripotency and genomic integrity. Thus, in combination with the adeno-associated virus vector system, CRISPR-Cas12a Ultra provides a highly efficient genome editing platform for performing targeted knock-ins in human iPSCs.

Prime editing using CRISPR-Cas12a and circular RNAs in human cells.

Genome editing with prime editors based on CRISPR-Cas9 is limited by the large size of the system and the requirement for a G/C-rich protospacer-adjacent motif (PAM) sequence. Here, we use the smaller Cas12a protein to develop four circular RNA-mediated prime editor (CPE) systems: nickase-dependent CPE (niCPE), nuclease-dependent CPE (nuCPE), split nickase-dependent CPE (sniCPE) and split nuclease-dependent CPE (snuCPE). CPE systems preferentially recognize T-rich genomic regions and possess a potential multiplexing capacity in comparison to corresponding Cas9-based systems. The efficiencies of the nuclease-based systems are up to 10.42%, whereas niCPE and sniCPE reach editing frequencies of up to 24.89% and 40.75% without positive selection in human cells, respectively. A derivative system, called one-sniCPE, combines all three RNA editing components under a single promoter. By arraying CRISPR RNAs for different targets in one circular RNA, we also demonstrate low-efficiency editing of up to four genes simultaneously with the nickase prime editors niCPE and sniCPE.

The pAblo·pCasso self-curing vector toolset for unconstrained cytidine and adenine base-editing in Gram-negative bacteria.

A synthetic biology toolkit, exploiting clustered regularly interspaced short palindromic repeats (CRISPR) and modified CRISPR-associated protein (Cas) base-editors, was developed for genome engineering in Gram-negative bacteria. Both a cytidine base-editor (CBE) and an adenine base-editor (ABE) have been optimized for precise single-nucleotide modification of plasmid and genome targets. CBE comprises a cytidine deaminase conjugated to a Cas9 nickase from Streptococcus pyogenes (SpnCas9), resulting in C→T (or G→A) substitutions. Conversely, ABE consists of an adenine deaminase fused to SpnCas9 for A→G (or T→C) editing. Several nucleotide substitutions were achieved using these plasmid-borne base-editing systems and a novel protospacer adjacent motif (PAM)-relaxed SpnCas9 (SpRY) variant. Base-editing was validated in Pseudomonas putida and other Gram-negative bacteria by inserting premature STOP codons into target genes, thereby inactivating both fluorescent proteins and metabolic (antibiotic-resistance) functions. The functional knockouts obtained by engineering STOP codons via CBE were reverted to the wild-type genotype using ABE. Additionally, a series of induction-responsive vectors was developed to facilitate the curing of the base-editing platform in a single cultivation step, simplifying complex strain engineering programs without relying on homologous recombination and yielding plasmid-free, modified bacterial cells.

The Development of a CRISPR-FnCpf1 System for Large-Fragment Deletion and Multiplex Gene Editing in Acinetobacter baumannii.

Acinetobacter baumannii is a low-GC-content Gram-negative opportunistic pathogen that poses a serious global public health threat. Convenient and rapid genetic manipulation is beneficial for elucidating its pathogenic mechanisms and developing novel therapeutic methods. In this study, we report a new CRISPR-FnCpf1-based two-plasmid system for versatile and precise genome editing in A. baumannii. After identification, this new system prefers to recognize the 5'-TTN-3' (N = A, T, C or G) and the 5'-CTV-3' (V = A, C or G) protospacer-adjacent motif (PAM) sequence and utilize the spacer with lengths ranging from 19 to 25 nt. In direct comparison with the existing CRISPR-Cas9 system, it exhibits approximately four times the targetable range in A. baumannii. Moreover, by employing a tandem dual crRNA expression cassette, the new system can perform large-fragment deletion and simultaneous multiple gene editing, which is difficult to achieve via CRISPR-Cas9. Therefore, the new system is valuable and can greatly expand the genome editing toolbox of A. baumannii.

Computational analysis of cas proteins unlocks new potential in HIV-1 targeted gene therapy.

Introduction: The human immunodeficiency virus type 1 (HIV-1) pandemic has been slowed with the advent of anti-retroviral therapy (ART). However, ART is not a cure and as such has pushed the disease into a chronic infection. One potential cure strategy that has shown promise is the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas gene editing system. It has recently been shown to successfully edit and/or excise the integrated provirus from infected cells and inhibit HIV-1 in vitro, ex vivo, and in vivo. These studies have primarily been conducted with SpCas9 or SaCas9. However, additional Cas proteins are discovered regularly and modifications to these known proteins are being engineered. The alternative Cas molecules have different requirements for protospacer adjacent motifs (PAMs) which impact the possible targetable regions of HIV-1. Other modifications to the Cas protein or gRNA handle impact the tolerance for mismatches between gRNA and the target. While reducing off-target risk, this impacts the ability to fully account for HIV-1 genetic variability. Methods: This manuscript strives to examine these parameter choices using a computational approach for surveying the suitability of a Cas editor for HIV-1 gene editing. The Nominate, Diversify, Narrow, Filter (NDNF) pipeline measures the safety, broadness, and effectiveness of a pool of potential gRNAs for any PAM. This technique was used to evaluate 46 different potential Cas editors for their HIV therapeutic potential. Results: Our examination revealed that broader PAMs that improve the targeting potential of editors like SaCas9 and LbCas12a have larger pools of useful gRNAs, while broader PAMs reduced the pool of useful SpCas9 gRNAs yet increased the breadth of targetable locations. Investigation of the mismatch tolerance of Cas editors indicates a 2-missmatch tolerance is an ideal balance between on-target sensitivity and off-target specificity. Of all of the Cas editors examined, SpCas-NG and SPRY-Cas9 had the highest number of overall safe, broad, and effective gRNAs against HIV. Discussion: Currently, larger proteins and wider PAMs lead to better targeting capacity. This implies that research should either be targeted towards delivering longer payloads or towards increasing the breadth of currently available small Cas editors. With the discovery and adoption of additional Cas editors, it is important for researchers in the HIV-1 gene editing field to explore the wider world of Cas editors.

MeHOLMES: A CRISPR-cas12a-based method for rapid detection of DNA methylation in a sequence-independent manner

Aberrant DNA methylation is closely associated with various diseases, particularly cancer, and its precise detection plays an essential role in disease diagnosis and monitoring. In this study, we present a novel DNA methylation detection method (namely meHOLMES), which integrates both the TET2/APOBEC-mediated cytosine deamination step and the CRISPR-Cas12a-based signal readout step. TET2/APOBEC efficiently converts unmethylated cytosine to uracil, which is subsequently changed to thymine after PCR amplification. Utilizing a rationally designed crRNA, Cas12a specifically identifies unconverted methylated cytosines and generates detectable signals using either fluorescent reporters or lateral flow test strips. meHOLMES quantitatively detects methylated CpG sites with or without Protospacer Adjacent Motif (PAM) sequences in both artificial and real biological samples. In addition, meHOLMES can complete the whole detection process within 6 h, which is much faster than traditional bisulfite-based sample pre-treatment method. Above all, meHOLMES provides a simpler, faster, more accurate, and cost-effective approach for quantitation of DNA methylation levels in a sequence-independent manner.

Occurrence and diversity pattern of CRISPR-Cas systems in Acetobacter genus provides insights on adaptive defense mechanisms against to invasive DNAs.

The Acetobacter genus is primarily known for its significance in acetic acid production and its application in various industrial processes. This study aimed to shed light on the prevalence, diversity, and functional implications of CRISPR-Cas systems in the Acetobacter genus using a genome mining approach. The investigation analyzed the CRISPR-Cas architectures and components of 34 Acetobacter species, as well as the evolutionary strategies employed by these bacteria in response to phage invasion and foreign DNA. Furthermore, phylogenetic analysis based on CAS1 protein sequences was performed to gain insights into the evolutionary relationships among Acetobacter strains, with an emphasis on the potential of this protein for genotyping purposes. The results showed that 15 species had orphan, while20 species had complete CRISPR-Cas systems, resulting in an occurrence rate of 38% for complete systems in Acetobacter strains. The predicted complete CRISPR-Cas systems were categorized into I-C, I-F, I-E, and II-C subtypes, with subtype I-E being the most prevalent in Acetobacter. Additionally, spacer homology analysis revealed against such the dynamic interaction between Acetobacter strains and foreign invasive DNAs, emphasizing the pivotal role of CRISPR-Cas systems in defending against such invasions. Furthermore, the investigation of the secondary structures of CRISPR arrays revealed the conserved patterns within subtypes despite variations in repeat sequences. The exploration of protospacer adjacent motifs (PAMs) identified distinct recognition motifs in the flanking regions of protospacers. In conclusion, this research not only contributes to the growing body of knowledge on CRISPR-Cas systems but also establishes a foundation for future studies on the adaptive defense mechanisms of Acetobacter. The findings provide valuable insights into the intricate interplay between bacteria and phages, with implications for industrial applications and potential biotechnological advancements.

A one-pot CRISPR-RCA strategy for ultrasensitive and specific detection of circRNA.

Accurate and precise detection of circular RNA (circRNA) is imperative for its clinical use. However, the inherent challenges in circRNA detection, arising from its low abundance and potential interference from linear isomers, necessitate innovative solutions. In this study, we introduce, for the first time, the application of the CRISPR/Cas12a system to establish a one-pot, rapid (30 minutes to 2 hours), specific and ultrasensitive circRNA detection strategy, termed RETA-CRISPR (reverse transcription-rolling circle amplification (RT-RCA) with the CRISPR/Cas12a). This method comprises two steps: (1) the RT-RCA process of circRNA amplification, generating repeat units containing the back-splicing junction (BSJ) sequences; and (2) leveraging the protospacer adjacent motif (PAM)-independent Cas12a/crRNA complex to precisely recognize target sequences with BSJ, thereby initiating the collateral cleavage activity of Cas12a to generate a robust fluorescence signal. Remarkably, this approach exhibits the capability to detect circRNAs at a concentration as low as 300 aM. The sensor has been successfully employed for accurate detection of a potential hepatocellular carcinoma biomarker hsa_circ_0001445 (circRNA1445) in various cell lines. In conclusion, RETA-CRISPR seamlessly integrates the advantages of exponential amplification reaction and the robust collateral cleavage activity of Cas12a, positioning it as a compelling tool for practical CRISPR-based diagnostics.

Development and design of CRISPR-based diagnostic for Acinetobacter baumannii by employing off-target gene editing of sgRNA

Acinetobacter baumannii is widely recognized as a human opportunistic pathogen in nosocomial infections. The proliferation of multidrug-resistant strains of A. baumannii has presented an array of difficulties for clinical anti-infective therapies and diagnostic procedures, owing to the existence of numerous variations. The development of therapy utilizing CRISPR/Cas9 for treatment and diagnosis necessitates an in-depth study of potential off-target consequences. The objective of this work is to assess potential off-target effects associated with a single guide RNA (sgRNA) designed to identify several variants present in A. baumannii. The current investigation involved the identification of Cas12 nuclease-specific protospacer adjacent motif (PAM) and downstream target sequences. This was achieved by utilizing computational tools and software to analyze conserved sections of the A. baumannii siderophore protein gene. Further, the in-silico expression vector was created with the SnapGene software. A total of 24 potential off-target sequences were identified in these sequences with 100% query identity with 96 different A. baumannii strains. In addition, a target-specific oligonucleotide single-guide RNA (sgRNA) template was synthesized by appending an additional nucleotide 'G' to the 5′ end. This research uses A. baumannii as an example of a problem that affects all treatments and diagnosis procedures to illustrate the significance of screening off-targets in different variants of a pathogen. Our findings may impact the safety and effectiveness of CRISPR/Cas9, which may have wider implications for additional targets that are currently being used therapeutically.

Cas-based bacterial detection: recent advances and perspectives.

Persistent bacterial infections pose a formidable threat to global health, contributing to widespread challenges in areas such as food safety, medical hygiene, and animal husbandry. Addressing this peril demands the urgent implementation of swift and highly sensitive detection methodologies suitable for point-of-care testing and large-scale screening. These methodologies play a pivotal role in the identification of pathogenic bacteria, discerning drug-resistant strains, and managing and treating diseases. Fortunately, new technology, the CRISPR/Cas system, has emerged. The clustered regularly interspaced short joint repeats (CRISPR) system, which is part of bacterial adaptive immunity, has already played a huge role in the field of gene editing. It has been employed as a diagnostic tool for virus detection, featuring high sensitivity, specificity, and single-nucleotide resolution. When applied to bacterial detection, it also surpasses expectations. In this review, we summarise recent advances in the detection of bacteria such as Mycobacterium tuberculosis (MTB), methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli (E. coli), Salmonella and Acinetobacter baumannii (A. baumannii) using the CRISPR/Cas system. We emphasize the significance and benefits of this methodology, showcasing the capability of diverse effector proteins to swiftly and precisely recognize bacterial pathogens. Furthermore, the CRISPR/Cas system exhibits promise in the identification of antibiotic-resistant strains. Nevertheless, this technology is not without challenges that need to be resolved. For example, CRISPR/Cas systems must overcome natural off-target effects and require high-quality nucleic acid samples to improve sensitivity and specificity. In addition, limited applicability due to the protospacer adjacent motif (PAM) needs to be addressed to increase its versatility. Despite the challenges, we are optimistic about the future of bacterial detection using CRISPR/Cas. We have already highlighted its potential in medical microbiology. As research progresses, this technology will revolutionize the detection of bacterial infections.

Recent Advancements in Reducing the Off-Target Effect of CRISPR-Cas9 Genome Editing.

The CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)) and the associated protein (Cas9) system, a young but well-studied genome-editing tool, holds plausible solutions to a wide range of genetic disorders. The single-guide RNA (sgRNA) with a 20-base user-defined spacer sequence and the Cas9 endonuclease form the core of the CRISPR-Cas9 system. This sgRNA can direct the Cas9 nuclease to any genomic region that includes a protospacer adjacent motif (PAM) just downstream and matches the spacer sequence. The current challenge in the clinical applications of CRISPR-Cas9 genome-editing technology is the potential off-target effects that can cause DNA cleavage at the incorrect sites. Off-target genome editing confuses and diminishes the therapeutic potential of CRISPR-Cas9 in addition to potentially casting doubt on scientific findings regarding the activities of genes. In this review, we summarize the recent technological advancements in reducing the off-target effect of CRISPR-Cas9 genome editing.

Enhancement and inactivation effect of CRISPR/Cas12a via extending hairpin activators for detection of transcription factors.

An enhancement effect for the activation of CRISPR/Cas12a (CRISPR = clustered regularly interspaced short palindromic repeats; Cas = CRISPR-associated) was discovered. That was, a hairpin model with dangling 5' end complementary to crRNA (CRISPR RNA) greatly improved the activity of CRISPR/Cas12a after extention of two random sequences. But, the corresponding intact hairpin without PAM (protospacer adjacent motif) or suboptimal PAM sequences was completely inactive to CRISPR/Cas12a because of the superhigh stability of intact hairpin. According to the finding, a CRISPR/Cas12a-based strategy coupled with a signal reported system was designed for transcription factors detection. By using mono-labeled ssDNA (single-stranded DNA) as reporter and two newly synthesized N-C (nitrogen-doped carbon) nanosheets as scavenger to eliminate the fluorescent background, the strategy realized the detection of NF-ĸB p50 (p50 subunit of nuclear factor kappa-B) with a linear detection range of 0.8 - 2000.0pM and a LOD of 0.5pM. The discovery of "enhancement and inactivation effect" not only deepened insight into CRISPR/Cas12a but also broadened the practical application of CRISPR/Cas systems for the molecular detection and disease diagnostics.

Base-editing mutagenesis maps alleles to tune human T cell functions.

CRISPR-enabled screening is a powerful tool for the discovery of genes that control T cell function and has nominated candidate targets for immunotherapies1-6. However, new approaches are required to probe specific nucleotide sequences within key genes. Systematic mutagenesis in primary human T cells could reveal alleles that tune specific phenotypes. DNA base editors are powerful tools for introducing targeted mutations with high efficiency7,8. Here we develop a large-scale base-editing mutagenesis platform with the goal of pinpointing nucleotides that encode amino acid residues that tune primary human T cell activation responses. We generated a library of around 117,000 single guide RNA molecules targeting base editors to protein-coding sites across 385 genes implicated in T cell function and systematically identified protein domains and specific amino acid residues that regulate T cell activation and cytokine production. We found a broad spectrum of alleles with variants encoding critical residues in proteins including PIK3CD, VAV1, LCP2, PLCG1 and DGKZ, including both gain-of-function and loss-of-function mutations. We validated the functional effects of many alleles and further demonstrated that base-editing hits could positively and negatively tune T cell cytotoxic function. Finally, higher-resolution screening using a base editor with relaxed protospacer-adjacent motif requirements9 (NG versus NGG) revealed specific structural domains and protein-protein interaction sites that can be targeted to tune T cell functions. Base-editing screens in primary immune cells thus provide biochemical insights with thepotential to accelerate immunotherapy design.

Unraveling Exogenous DNA Processing in Cas4-Lacking Crispr Systems: A Novel Bypass Pathway Explored.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems are widely distributed adaptive immune systems found in prokaryotes. The process involves three main stages: adaptation, expression, and interference. While the adaptation stage has been extensively studied, there is still an incomplete understanding of the mechanisms underlying the capture, trimming, and integration of exogenous DNA. For instance, Cas4, a CRISPR-Cas protein with endonuclease activity, is responsible for selecting and processing protospacer adjacent motif (PAM) sequences. However, some CRISPR isoforms lack Cas4 activity, relying on other enzymes for adaptive immunity. Recently, Wang etal. presented a novel model of exogenous DNA processing in a type I-E CRISPR system lacking Cas4 in a Nature article. This model integrates protospacer processing into CRISPR arrays through fine-tuned synthases formed by DnaQ-like exonuclease (DEDDh) and Cas1-Cas2 complexes. Their study introduces a novel model, shedding new light on the evolution of CRISPR adaptive immunity. This perspective comprehensively examines the fundamental process of CRISPR adaptive immunity, detailing both the classical pathway mediated by Cas4 and the alternative pathway mediated by DEDDh. Furthermore, a thorough evaluation of Wang etal.'s work is conducted, highlighting its strengths, weaknesses, and existing research challenges.

Optimization of base editors for the functional correction of SMN2 as a treatment for spinal muscular atrophy.

Spinal muscular atrophy (SMA) is caused by mutations in SMN1. SMN2 is a paralogous gene with a C•G-to-T•A transition in exon 7, which causes this exon to be skipped in most SMN2 transcripts, and results in low levels of the protein survival motor neuron (SMN). Here we show, in fibroblasts derived from patients with SMA and in a mouse model of SMA that, irrespective of the mutations in SMN1, adenosine base editors can be optimized to target the SMN2 exon-7 mutation or nearby regulatory elements to restore the normal expression of SMN. After optimizing and testing more than 100 guide RNAs and base editors, and leveraging Cas9 variants with high editing fidelity that are tolerant of different protospacer-adjacent motifs, we achieved the reversion of the exon-7 mutation via an A•T-to-G•C edit in up to 99% of fibroblasts, with concomitant increases in the levels of the SMN2 exon-7 transcript and of SMN. Targeting the SMN2 exon-7 mutation via base editing or other CRISPR-based methods may provide long-lasting outcomes to patients with SMA.

Phage-assisted evolution of compact Cas9 variants targeting a simple NNG PAM.

Compact Cas9 nucleases hold great promise for therapeutic applications. Although several compact Cas9 nucleases have been developed, many genomic loci still could not be edited due to a lack of protospacer adjacent motifs (PAMs). We previously developed a compact SlugCas9 recognizing an NNGG PAM. Here we demonstrate that SlugCas9 displays comparable activity to SpCas9. We developed a simple phage-assisted evolution to engineer SlugCas9 for unique PAM requirements. Interestingly, we generated a SlugCas9 variant (SlugCas9-NNG) that could recognize an NNG PAM, expanding the targeting scope. We further developed a SlugCas9-NNG-based adenine base editor and demonstrated that it could be delivered by a single adeno-associated virus to disrupt PCSK9 splice donor and splice acceptor. These genome editors greatly enhance our ability for in vivo genome editing.

Development of the Cas12a-based microdeletion and microinsertion detection system

CRISPR/Cas-based systems are widely used as genome editing systems, nucleic acid detection systems and molecular visualization instruments [1]. In our laboratory using CRISPR/Cas9 technology we have obtained several KO mouse lines harboring deletions ranging from 2 to 20 base pairs. While 20 bp deletions are easily PCR-detected, when it comes to 2 bp deletions it is essential to genotype numerous mice using time-consuming Sanger sequencing. We propose a microdeletion/microinsertion detection system based on Cas12a nuclease fromLachnospiraceaebacterium (LbCas12a). Its active complex consists of the Cas12a enzyme and one crisprRNA [2]. A special sequence called protospacer adjacent motif (PAM) is required for target recognition by LbCas12a. In our laboratory we have discovered new PAM TTAA recognized by LbCas12a [3]. Via agarose electrophoresis and fluorescent analysis using FAM-labeled probes we have shown that new PAM allowed detection of 1 bp substitutions in target DNAin vitro. Also we have tested different FAM-labeled probes and have shown that AT-rich probes longer than 10 bp are cleaved most effectively. Finally we have used our system for detecting 2 bp deletions in Pde6b-KO mice and Grin3A-KO mice and successfully distinguished these mice from wild type mice.
 In conclusion, new PAM TTAA greatly increases specificity of DNA cleavage allowing to use this system as an instrument for rapid detection if microdeletions in mice.
 This work was supported by a Saint Petersburg State University grant for the development of scientific research (ID 94030690) and the Genome Research Centre development program “Kurchatov Genome Centre–PNPI” (agreement No. 075-15-2019-1663)

Evaluation of non-specific CRISPR/Cas9 activity in a yeast model

CRISPR/Cas9-based genome editing systems are widely used for genetic modification of different organisms. One of the drawbacks of CRISPR/Cas9 methods is the non-specific activity of Cas9, which can lead to accumulation of unwanted mutations in the edited genome [1]. Therefore, the development ofin vivomodels for high-throughput analysis of factors influencing the frequency of mutagenesis associated with the use of CRISPR/Cas9 technologies is a relevant task. YeastSaccharomycescerevisiaeis a convenient object for developing such models [2].
 Here we represent a yeastin vivomodel that allows us to evaluate the effects of nucleotide sequence of the protospacer adjacent motif (PAM) and the guide RNA (gRNA) on the efficiency of binding between the gRNA/Cas9 complex and the target sequence in the genome. Since the Cas9 activity is lethal in cells lacking a donor sequence for homologous repair of double-strand breaks caused by this endonuclease, in the proposed test-system, the reduced efficiency of transformation by a plasmid encoding Cas9 and various gRNA variants reflects the efficiency of recognition of the target gene by the gRNA/Cas9 complex.
 To study the influence of different PAM variants, with a consensus of NGG, on CRISPR/Cas9 activity, we obtained four isogenic strains that differ in their PAM sequence (AGG, TGG, CGG, GGG) in the codon 202 of the chromosomal copy of the reporter geneURA3. To evaluate the effect of incomplete matching between gRNA and the target site sequences, we propose using a series of plasmids based on the pML107 vector, encoding Cas9 and one of the 20 possible gRNA variants with single nucleotide substitutions at each of the 20 positions. The results obtained so far indicate the potential of the proposed approach.