Good guide, bad guide:spacer sequence-dependent cleavage efficiency of Cas12a

Genome editing has recently made a revolutionary development with the introduction of the CRISPR–Cas technology. The programmable CRISPR-associated Cas9 and Cas12a nucleases generate specific dsDNA breaks in the genome, after which host DNA-repair mechanisms can be manipulated to implement the desired editing. Despite this spectacular progress, the efficiency of Cas9/Cas12a-based engineering can still be improved. Here, we address the variation in guide-dependent efficiency of Cas12a, and set out to reveal the molecular basis of this phenomenon. We established a sensitive and robust in vivo targeting assay based on loss of a target plasmid encoding the red fluorescent protein (mRFP). Our results suggest that folding of both the precursor guide (pre-crRNA) and the mature guide (crRNA) have a major influence on Cas12a activity. Especially, base pairing of the direct repeat, other than with itself, was found to be detrimental to the activity of Cas12a. Furthermore, we describe different approaches to minimize base-pairing interactions between the direct repeat and the variable part of the guide. We show that design of the 3′ end of the guide, which is not involved in target strand base pairing, may result in substantial improvement of the guide's targeting potential and hence of its genome editing efficiency

Characterizing a thermostable Cas9 for bacterial genome editing and silencing

CRISPR-Cas9-based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including engineering at elevated temperatures. Here we identify and characterize ThermoCas9 from the thermophilic bacterium Geobacillus thermodenitrificans T12. We show that in vitro ThermoCas9 is active between 20 and 70 °C, has stringent PAM-preference at lower temperatures, tolerates fewer spacer-protospacer mismatches than SpCas9 and its activity at elevated temperatures depends on the sgRNA-structure. We develop ThermoCas9-based engineering tools for gene deletion and transcriptional silencing at 55 °C in Bacillus smithii and for gene deletion at 37 °C in Pseudomonas putida. Altogether, our findings provide fundamental insights into a thermophilic CRISPR-Cas family member and establish a Cas9-based bacterial genome editing and silencing tool with a broad temperature range

CRISPR-Cas ribonucleoprotein mediated homology-directed repair for efficient targeted genome editing in microalgae Nannochloropsis oceanica IMET1

Background: Microalgae are considered as a sustainable feedstock for the production of biofuels and other value-added compounds. In particular, Nannochloropsis spp. stand out from other microalgal species due to their capabilities to accumulate both triacylglycerol (TAG) and polyunsaturated fatty acids (PUFAs). However, the commercialization of microalgae-derived products is primarily hindered by the high production costs compared to less sustainable alternatives. Efficient genome editing techniques leading to effective metabolic engineering could result in strains with enhanced productivities of interesting metabolites and thereby reduce the production costs. Competent CRISPR-based genome editing techniques have been reported in several microalgal species, and only very recently in Nannochloropsis spp. (2017). All the reported CRISPR-Cas-based systems in Nannochloropsis spp. rely on plasmid-borne constitutive expression of Cas9 and a specific guide, combined with repair of double-stranded breaks (DSB) by non-homologous end joining (NHEJ) for the target gene knockout. Results: In this study, we report for the first time an alternative approach for CRISPR-Cas-mediated genome editing in Nannochloropsis sp.; the Cas ribonucleoproteins (RNP) and an editing template were directly delivered into microalgal cells via electroporation, making Cas expression dispensable and homology-directed repair (HDR) possible with high efficiency. Apart from widely used SpCas9, Cas12a variants from three different bacterium were used for this approach. We observed that FnCas12a from Francisella novicida generated HDR-based targeted mutants with highest efficiency (up to 93% mutants among transformants) while AsCas12a from Acidaminococcus sp. resulted in the lowest efficiency. We initially show that the native homologous recombination (HR) system in N. oceanica IMET1 is not efficient for easy isolation of targeted mutants by HR. Cas9/sgRNA RNP delivery greatly enhanced HR at the target site, generating around 70% of positive mutant lines. Conclusion: We show that the delivery of Cas RNP by electroporation can be an alternative approach to the presently reported plasmid-based Cas9 method for generating mutants of N. oceanica. The co-delivery of Cas-RNPs along with a dsDNA repair template efficiently enhanced HR at the target site, resulting in a remarkable higher percentage of positive mutant lines. Therefore, this approach can be used for efficient generation of targeted mutants in Nannochloropsis sp. In addition, we here report the activity of several Cas12a homologs in N. oceanica IMET1, identifying FnCas12a as the best performer for high efficiency targeted genome editing.</p

Pathogen-induced activation of disease-suppressive functions in the endophytic root microbiome

Microorganisms living inside plants can promote plant growth and health, but their genomic and functional diversity remain largely elusive. Here, metagenomics and network inference show that fungal infection of plant roots enriched for Chitinophagaceae and Flavobacteriaceae in the root endosphere and for chitinase genes and various unknown biosynthetic gene clusters encoding the production of nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). After strain-level genome reconstruction, a consortium of Chitinophaga and Flavobacterium was designed that consistently suppressed fungal root disease. Site-directed mutagenesis then revealed that a previously unidentified NRPS-PKS gene cluster from Flavobacterium was essential for disease suppression by the endophytic consortium. Our results highlight that endophytic root microbiomes harbor a wealth of as yet unknown functional traits that, in concert, can protect the plant inside out.</p

Guide-free Cas9 from pathogenic Campylobacter jejuni bacteria causes severe damage to DNA

CRISPR-Cas9 systems are enriched in human pathogenic bacteria and have been linked to cytotoxicity by an unknown mechanism. Here, we show that upon infection of human cells, Campylobacter jejuni secretes its Cas9 (CjeCas9) nuclease into their cytoplasm. Next, a native nuclear localization signal enables CjeCas9 nuclear entry, where it catalyzes metal-dependent nonspecific DNA cleavage leading to cell death. Compared to CjeCas9, native Cas9 of Streptococcus pyogenes (SpyCas9) is more suitable for guide-dependent editing. However, in human cells, native SpyCas9 may still cause some DNA damage, most likely because of its ssDNA cleavage activity. This side effect can be completely prevented by saturation of SpyCas9 with an appropriate guide RNA, which is only partially effective for CjeCas9. We conclude that CjeCas9 plays an active role in attacking human cells rather than in viral defense. Moreover, these unique catalytic features may therefore make CjeCas9 less suitable for genome editing applications

Microbial treasure trove : Unravelling the potential of class 2 CRISPR–Cas systems

Animals are known to possess a large repertoire of immune systems with a high degree of sophistication. On the other hand, the immune systems found in bacteria and archaea appeared to be much more rudimentary. However, the ground-breaking discovery of a novel immune system, CRISPR-Cas, proved otherwise. CRISPR-Cas, is unique in being both adaptive and heritable, and it relies on small RNA molecules that specifically guide the defence system to matching invader DNA sequences. This natural defence system has been successfully repurposed into a valuable molecular technology which is a robust, efficient, easy-to-use method to precisely alter DNA sequences of living organisms. The current CRISPR-based technologies, mostly employ the Cas9 protein, for diverse biotechnological applications. Nevertheless, the natural diversity of CRISPR-Cas systems is remarkably extensive, including systems that target DNA, systems that target RNA, and systems that target both DNA and RNA. The diverse class 2 CRISPR nucleases have unique molecular features that contribute to an expansive toolbox for genome and transcriptome engineering. These nucleases differ greatly in their structure and mechanisms. These differences could be exploited as complementary applications creating numerous CRISPR-based technologies possibly with favourable specificity, efficiency and/or delivery. This thesis explores the diversity of Class 2 CRISPR–Cas systems and provides mechanistic insights into different class 2 nucleases. In addition, it describes potential applications to expand the current repertoire of CRISPR-based technologies. Due to the everlasting arms race between prokaryotes and their viruses, the rapid evolution of CRISPR–Cas systems has resulted in extreme structural and functional diversity. As a result, a plethora of distinct CRISPR–Cas systems are represented in genomes of most archaea and almost half of the bacteria. The key players of this system are the crRNA binding effector complexes, and the associated nuclease domains. CRISPR–Cas systems are currently grouped into two classes each of which is subdivided into three types. Class 1 systems (consisting of types I, III, and IV) use a multi-subunit protein complex to achieve interference, and class 2 systems (consisting of types II, V, and VI) utilize a single multi-domain protein, that have been repurposed for genome editing applications in a wide range of organisms. The mechanism of crRNA maturation in CRISPR–Cas12a systems was unravelled during this thesis. Unlike the type II nuclease Cas9, which utilizes a tracrRNA as well as endogenous RNaseIII for maturation of its dual crRNA/tracrRNA guides, pre-crRNA processing in the Cas12a system proceeds in the absence of tracrRNA or other Cas proteins. It was demonstrated that Cas12a nucleases possess a previously unknown RNase domain that is responsible for cleaving the pre-crRNA to generate the mature crRNAs. The typical cleavage pattern revealed that Cas12a recognizes specific secondary structures and/or motifs on its direct repeats. Furthermore, the ability to autonomously process crRNA has significant implications from a genome editing standpoint, as it provides a simple route to editing multiple genomic loci at a time (multiplex editing). Using a single customized CRISPR array up to four genes in mammalian cells ex vivo and up to three genes in mouse brain cells in vivo were shown to be edited simultaneously. The characterisation of a novel, diminutive type V-U1 Cas protein from Mycolicibacterium mucogenicum (MmuC2c4) was described in this thesis. Type V-U proteins are highly similar to the typical transposon-encoded TnpB-like proteins and each of them (type VU-1 to type VU-5) appear to have originated independently from distinct TnpB families. Akin to most type V proteins, MmuC2c4 was shown to recognize a 5’-TTN-3’ PAM on a double-stranded target DNA. The characterisation of a type II-C Cas9 orthologue of the thermophilic bacterium Geobacillus thermodenitrificans T12, ThermoCas9 is described. This is one of the first reports that provides fundamental insights into a thermophilic CRISPR–Cas9 family member. It was demonstrated that ThermoCas9 is active in vitro between 20 and 70 ℃, that the structure of its sgRNA influences its activity at elevated temperatures, it has a more stringent PAM-preference at lower temperatures, it does not tolerate extensive spacer-protospacer mismatches, and it preferentially cleaves plasmid DNA compared to linear DNA. Furthermore, ThermoCas9 was employed for pyrF gene deletion and transcriptional silencing of ldhL gene at 55 ℃ in Bacillus smithii ET 138 and for pyrF gene deletion at 37 ℃ in Pseudomonas putida. This is the first time Cas9-based bacterial genome editing and silencing tools were used at temperatures above 42 ℃. Four Cas12a orthologues were assessed for their salt tolerance as well as pH- and temperature stability using biochemical assays as described. Subsequently, Francisella tularensis subsp. novicida (FnCas12a) and Eubacterium eligens (EeCas12a) were applied for genome editing in a moderate thermophilic bacterium, Bacillus smithii. It is demonstrated that FnCas12a and EeCas12a are sub-optimally active in vivo at temperatures above 45 ℃. The wide growth temperature range of B. smithii ET 138 was employed for the controllable induction of Cas12a expression at temperatures below the 45 ℃ threshold. It was demonstrated that a mutant can be generated within a short span of 2-3 days. This process can be easily adapted for gene editing applications in a wide variety of both mesophilic and moderate thermophilic organisms. potential to harness the activity of anti-CRISPR (Acr) proteins for controllable bacterial genome engineering was also investigated. The Acr protein from Neisseria meningitidis (AcrIIC1Nme) was employed as an “on/off-switch” to control the activity of thermostable Cas9 orthologues from Geobacillus thermodenitrificans T12 (ThermoCas9) and Geobacillus stearothermophilus (GeoCas9). Initially, it was proven that both ThermoCas9 and GeoCas9 can introduce lethal dsDNA breaks in E. coli at 37 ℃ in a tuneable manner. Next, it was demonstrated that AcrIIC1Nme traps both tested Cas9 orthologues in a DNA-bound, catalytically inactive state. The Cas9/AcrIIC1Nme complexes can promote a transcriptional silencing effect with efficiency comparable to the catalytically “dead” ThermodCas9 and GeodCas9 variants. Finally, a single-vector, tightly controllable and highly efficient Cas9/AcrIIC1Nme-based tool for coupled silencing and targeting in E. coli was developed. This tool may serve as a basis for further developing a controllable genome editing and transcriptional regulation in model as well as non-model microorganisms. Furthermore, a novel biological role and mechanism for the CRISPR–Cas9 system of the pathogen Campylobacter jejuni (CjeCas9) was uncovered. It was demonstrated that upon C. jejuni infection of human cells, CjeCas9 is secreted into the cytoplasm of the infected cells and it can autonomously enter the nucleus. Inside the nucleus, it catalyses metal-dependent and sequence-independent nicking of double stranded DNA, eventually leading to cell death. Genome editing using CjeCas9 was compared with the commonly used Cas9 from Streptococcus pyogenes (SpyCas9), and the latter was shown to be superior in creating indels. It was concluded that the unique catalytic features make CjeCas9 nuclease less suitable for genome editing applications. In conclusion, the research described in this PhD thesis has uncovered novel molecular requirements and mechanisms of several unique Class 2 CRISPR–Cas nucleases. Besides gaining insights into their biochemical mechanism, the potential of Class 2 nucleases has been harnessed for biotechnological applications. Additionally, a unique role and mechanism of CRISPR–Cas in virulence has been elucidated. The characterisation of nucleases such as FnCas12a, EeCas12a, MmuC2c4 and ThermoCas9 opens up exciting possibilities of utilizing them as genome and transcriptome engineering tools.</p

Heterologous Expression and Purification of the CRISPR-Cas12a/Cpf1 Protein

This protocol provides step by step instructions (Figure 1) for heterologous expression of Francisella novicida Cas12a (previously known as Cpf1) in Escherichia coli. It additionally includes a protocol for high-purity purification and briefly describes how activity assays can be performed. These protocols can also be used for purification of other Cas12a homologs and the purified proteins can be used for subsequent genome editing experiments

CRISPR type V-U1 system from Mycobacterium mucogenicum and uses thereof

The type V-U1 system from the bacterium Mycobacterium mucogenicum CCH10-A2 (Mmu) has a nuclease which binds dsDNA but it does not cleave it. Additionally, after dsDNA binding by the nuclease an RuvC-dependent interference of nascent transcript (mRNA) takes place and this mechanism has not been described before for any CRISPR system. CRISPR based gene manipulation can therefore use CRISPR endonucleases from the Type V-U1 system from Mycobacterium mucogenicum, including variant and modified endonucleases, so as to provide for methods of expression control and gene editing in cells of any living organism or of any nucleic acid in vitro.</p

Alternative functions of CRISPR–Cas systems in the evolutionary arms race

CRISPR–Cas systems of bacteria and archaea comprise chromosomal loci with typical repetitive clusters and associated genes encoding a range of Cas proteins. Adaptation of CRISPR arrays occurs when virus-derived and plasmid-derived sequences are integrated as new CRISPR spacers. Cas proteins use CRISPR-derived RNA guides to specifically recognize and cleave nucleic acids of invading mobile genetic elements. Apart from this role as an adaptive immune system, some CRISPR-associated nucleases are hijacked by mobile genetic elements: viruses use them to attack their prokaryotic hosts, and transposons have adopted CRISPR systems for guided transposition. In addition, some CRISPR–Cas systems control the expression of genes involved in bacterial physiology and virulence. Moreover, pathogenic bacteria may use their Cas nuclease activity indirectly to evade the human immune system or directly to invade the nucleus and damage the chromosomal DNA of infected human cells. Thus, the evolutionary arms race has led to the expansion of exciting variations in CRISPR mechanisms and functionalities. In this Review, we explore the latest insights into the diverse functions of CRISPR–Cas systems beyond adaptive immunity and discuss the implications for the development of CRISPR-based applications