CRISPR‐LbCas12a‐mediated modification of citrus

Recently, CRISPR‐Cas12a (Cpf1) from Prevotella and Francisella was engineered to modify plant genomes. In this report, we employed CRISPR‐LbCas12a (LbCpf1), which is derived from Lachnospiraceae bacterium ND2006, to edit a citrus genome for the first time. First, LbCas12a was used to modify the CsPDS gene successfully in Duncan grapefruit via Xcc‐facilitated agroinfiltration. Next, LbCas12a driven by either the 35S or Yao promoter was used to edit the PthA4 effector binding elements in the promoter (EBE_PthA4‐CsLOBP) of CsLOB1. A single crRNA was selected to target a conserved region of both Type I and Type II CsLOBPs, since the protospacer adjacent motif of LbCas12a (TTTV) allows crRNA to act on the conserved region of these two types of CsLOBP. CsLOB1 is the canker susceptibility gene, and it is induced by the corresponding pathogenicity factor PthA4 in Xanthomonas citri by binding to EBE_PthA4‐CsLOBP. A total of seven 35S‐LbCas12a‐transformed Duncan plants were generated, and they were designated as #D₃₅s1 to #D₃₅s7, and ten Yao‐LbCas12a‐transformed Duncan plants were created and designated as #D_yao1 to #D_yao10. LbCas12a‐directed EBE_PthA4‐CsLOBP modifications were observed in three 35S‐LbCas12a‐transformed Duncan plants (#D₃₅s1, #D₃₅s4 and #D₃₅s7). However, no LbCas12a‐mediated indels were observed in the Yao‐LbCas12a‐transformed plants. Notably, transgenic line #D₃₅s4, which contains the highest mutation rate, alleviates XccΔpthA4:dCsLOB1.4 infection. Finally, no potential off‐targets were observed. Therefore, CRISPR‐LbCas12a can readily be used as a powerful tool for citrus genome editing.     

CRISPR‐Cas9‐mediated efficient directed mutagenesis and RAD51‐dependent and RAD51‐independent gene targeting in the moss Physcomitrella patens

The ability to address the CRISPR‐Cas9 nuclease complex to any target DNA using customizable single‐guide RNAs has now permitted genome engineering in many species. Here, we report its first successful use in a nonvascular plant, the moss Physcomitrella patens. Single‐guide RNAs (sgRNAs) were designed to target an endogenous reporter gene, PpAPT, whose inactivation confers resistance to 2‐fluoroadenine. Transformation of moss protoplasts with these sgRNAs and the Cas9 coding sequence from Streptococcus pyogenes triggered mutagenesis at the PpAPT target in about 2% of the regenerated plants. Mainly, deletions were observed, most of them resulting from alternative end‐joining (alt‐EJ)‐driven repair. We further demonstrate that, in the presence of a donor DNA sharing sequence homology with the PpAPT gene, most transgene integration events occur by homology‐driven repair (HDR) at the target locus but also that Cas9‐induced double‐strand breaks are repaired with almost equal frequencies by mutagenic illegitimate recombination. Finally, we establish that a significant fraction of HDR‐mediated gene targeting events (30%) is still possible in the absence of PpRAD51 protein, indicating that CRISPR‐induced HDR is only partially mediated by the classical homologous recombination pathway.     

DnaQ exonuclease‐like domain of Cas2 promotes spacer integration in a type I‐E CRISPR‐Cas system

CRISPR‐Cas systems constitute an adaptive immune system that provides acquired resistance against phages and plasmids in prokaryotes. Upon invasion of foreign nucleic acids, some cells integrate short fragments of foreign DNA as spacers into the CRISPR locus to memorize the invaders and acquire resistance in the subsequent round of infection. This immunization step called adaptation is the least understood part of the CRISPR‐Cas immunity. We have focused here on the adaptation stage of Streptococcus thermophilus DGCC7710 type I‐E CRISPR4‐Cas (St4) system. Cas1 and Cas2 proteins conserved in nearly all CRISPR‐Cas systems are required for spacer acquisition. The St4 CRISPR‐Cas system is unique because the Cas2 protein is fused to an additional DnaQ exonuclease domain. Here, we demonstrate that St4 Cas1 and Cas2‐DnaQ form a multimeric complex, which is capable of integrating DNA duplexes with 3′‐overhangs (protospacers) in vitro. We further show that the DnaQ domain of Cas2 functions as a 3′–5′‐exonuclease that processes 3′‐overhangs of the protospacer to promote integration.     

One‐step CRISPR/Cas9 method for the rapid generation of human antibody heavy chain knock‐in mice

Here, we describe a one‐step, in vivo CRISPR/Cas9 nuclease‐mediated strategy to generate knock‐in mice. We produced knock‐in (KI) mice wherein a 1.9‐kb DNA fragment bearing a pre‐arranged human B‐cell receptor heavy chain was recombined into the native murine immunoglobulin locus. Our methodology relies on Cas9 nuclease‐induced double‐stranded breaks directed by two sgRNAs to occur within the specific target locus of fertilized oocytes. These double‐stranded breaks are subsequently repaired via homology‐directed repair by a plasmid‐borne template containing the pre‐arranged human immunoglobulin heavy chain. To validate our knock‐in mouse model, we examined the expression of the KI immunoglobulin heavy chains by following B‐cell development and performing single B‐cell receptor sequencing. We optimized this strategy to generate immunoglobulin KI mice in a short amount of time with a high frequency of homologous recombination (30–50%). In the future, we envision that such knock‐in mice will provide much needed vaccination models to evaluate immunoresponses against immunogens specific for various infectious diseases.     

Structural features of Cas2 from Thermococcus onnurineus in CRISPR‐cas system type IV

CRISPR‐Cas is RNA‐based prokaryotic immune systems that defend against exogenous genetic elements such as plasmids and viruses. Cas1 and Cas2 are highly conserved components that play an essential part in the adaptation stage of all CRISPR‐Cas systems. Characterization of CRISPR‐Cas genes in Thermococcus onnurineus reveals the association of the Cas2 gene with the putative type IV system that lacks Cas1 or its homologous genes. Here, we present a crystal structure of T. onnurineus Cas2 (Ton_Cas2) that exhibits a deep and wide cleft at an interface lined with positive residues (Arg16, Lys18, Lys19, Arg22, and Arg23). The obvious DNA recognizing loops in Cas2 from E. coli (Eco_Cas2) are absent in Ton_Cas2 and have significantly different shapes and electrostatic potential distributions around the putative nucleotide binding region. Furthermore, Ton_Cas2 lacks the hairpin motif at the C‐terminus that is responsible for Cas1 binding in Eco_Cas2. These structural features could be a unique signature and indicate an altered functional mechanism in the adaptation stage of Cas2 in type IV CRISPR‐Cas systems.     

Depolarization‐induced suppression of a glycinergic synapse in the superior olivary complex by endocannabinoids

The neuronal endocannabinoid system is known to depress synaptic inputs retrogradely in an activity‐dependent manner. This mechanism has been generally described for excitatory glutamatergic and inhibitory GABAergic synapses. Here, we report that neurones in the auditory brainstem of the Mongolian gerbil (Meriones unguiculatus) retrogradely regulate the strength of their inputs via the endocannabinoid system. By means of whole‐cell patch‐clamp recordings, we found that retrograde endocannabinoid signalling attenuates both glycinergic and glutamatergic post‐synaptic currents in the same types of neurones. Accordingly, we detected the cannabinoid receptor 1 in excitatory and inhibitory pre‐synapses as well as the endocannabinoid‐synthesising enzymes (diacylglycerol lipase α/β, DAGLα/β) post‐synaptically through immunohistochemical stainings. Our study was performed with animals aged 10–15 days, that is, in the time window around the onset of hearing. Therefore, we suggest that retrograde endocannabinoid signalling has a role in adapting inputs during the functionally important switch from spontaneously generated to sound‐related signals.

     

Engineering large viral DNA genomes using the CRISPR‐Cas9 system

Manipulation of viral genomes is essential for studying viral gene function and utilizing viruses for therapy. Several techniques for viral genome engineering have been developed. Homologous recombination in virus‐infected cells has traditionally been used to edit viral genomes; however, the frequency of the expected recombination is quite low. Alternatively, large viral genomes have been edited using a bacterial artificial chromosome (BAC) plasmid system. However, cloning of large viral genomes into BAC plasmids is both laborious and time‐consuming. In addition, because it is possible for insertion into the viral genome of drug selection markers or parts of BAC plasmids to affect viral function, artificial genes sometimes need to be removed from edited viruses. Herpes simplex virus (HSV), a common DNA virus with a genome length of 152 kbp, causes labialis, genital herpes and encephalitis. Mutant HSV is a candidate for oncotherapy, in which HSV is used to kill tumor cells. In this study, the clustered regularly interspaced short palindromic repeat‐Cas9 system was used to very efficiently engineer HSV without inserting artificial genes into viral genomes. Not only gene‐ablated HSV but also gene knock‐in HSV were generated using this method. Furthermore, selection with phenotypes of edited genes promotes the isolation efficiencies of expectedly mutated viral clones. Because our method can be applied to other DNA viruses such as Epstein–Barr virus, cytomegaloviruses, vaccinia virus and baculovirus, our system will be useful for studying various types of viruses, including clinical isolates.     

GTSF‐1 is required for formation of a functional RNA‐dependent RNA Polymerase complex in Caenorhabditis elegans

Argonaute proteins and their associated small RNAs (sRNAs) are evolutionarily conserved regulators of gene expression. Gametocyte‐specific factor 1 (Gtsf1) proteins, characterized by two tandem CHHC zinc fingers and an unstructured C‐terminal tail, are conserved in animals and have been shown to interact with Piwi clade Argonautes, thereby assisting their activity. We identified the Caenorhabditis elegans Gtsf1 homolog, named it gtsf‐1 and characterized it in the context of the sRNA pathways of C. elegans. We report that GTSF‐1 is not required for Piwi‐mediated gene silencing. Instead, gtsf‐1 mutants show a striking depletion of 26G‐RNAs, a class of endogenous sRNAs, fully phenocopying rrf‐3 mutants. We show, both in vivo and in vitro, that GTSF‐1 interacts with RRF‐3 via its CHHC zinc fingers. Furthermore, we demonstrate that GTSF‐1 is required for the assembly of a larger RRF‐3 and DCR‐1‐containing complex (ERIC), thereby allowing for 26G‐RNA generation. We propose that GTSF‐1 homologs may act to drive the assembly of larger complexes that act in sRNA production and/or in imposing sRNA‐mediated silencing activities.     

An Agrobacterium‐delivered CRISPR/Cas9 system for high‐frequency targeted mutagenesis in maize

CRISPR/Cas9 is a powerful genome editing tool in many organisms, including a number of monocots and dicots. Although the design and application of CRISPR/Cas9 is simpler compared to other nuclease‐based genome editing tools, optimization requires the consideration of the DNA delivery and tissue regeneration methods for a particular species to achieve accuracy and efficiency. Here, we describe a public sector system, ISU Maize CRISPR, utilizing Agrobacterium‐delivered CRISPR/Cas9 for high‐frequency targeted mutagenesis in maize. This system consists of an Escherichia coli cloning vector and an Agrobacterium binary vector. It can be used to clone up to four guide RNAs for single or multiplex gene targeting. We evaluated this system for its mutagenesis frequency and heritability using four maize genes in two duplicated pairs: Argonaute 18 (ZmAgo18a and ZmAgo18b) and dihydroflavonol 4‐reductase or anthocyaninless genes (a1 and a4). T₀ transgenic events carrying mono‐ or diallelic mutations of one locus and various combinations of allelic mutations of two loci occurred at rates over 70% mutants per transgenic events in both Hi‐II and B104 genotypes. Through genetic segregation, null segregants carrying only the desired mutant alleles without the CRISPR transgene could be generated in T₁ progeny. Inheritance of an active CRISPR/Cas9 transgene leads to additional target‐specific mutations in subsequent generations. Duplex infection of immature embryos by mixing two individual Agrobacterium strains harbouring different Cas9/gRNA modules can be performed for improved cost efficiency. Together, the findings demonstrate that the ISU Maize CRISPR platform is an effective and robust tool to targeted mutagenesis in maize.     

Lignin characterization of rice CONIFERALDEHYDE 5‐HYDROXYLASE loss‐of‐function mutants generated with the CRISPR/Cas9 system

The aromatic composition of lignin is an important trait that greatly affects the usability of lignocellulosic biomass. We previously identified a rice (Oryza sativa) gene encoding coniferaldehyde 5‐hydroxylase (OsCAld5H1), which was effective in modulating syringyl (S)/guaiacyl (G) lignin composition ratio in rice, a model grass species. Previously characterized OsCAld5H1‐knockdown rice lines, which were produced via an RNA‐interference approach, showed augmented G lignin units yet contained considerable amounts of residual S lignin units. In this study, to further investigate the effect of suppression of OsCAld5H1 on rice lignin structure, we generated loss‐of‐function mutants of OsCAld5H1 using the CRISPR/Cas9‐mediated genome editing system. Homozygous OsCAld5H1‐knockout lines harboring anticipated frame‐shift mutations in OsCAld5H1 were successfully obtained. A series of wet‐chemical and two‐dimensional NMR analyses on cell walls demonstrated that although lignins in the mutant were predictably enriched in G units all the tested mutant lines produced considerable numbers of S units. Intriguingly, lignin γ‐p‐coumaroylation analysis by the derivatization followed by reductive cleavage method revealed that enrichment of G units in lignins of the mutants was limited to the non‐γ‐p‐coumaroylated units, whereas grass‐specific γ‐p‐coumaroylated lignin units were almost unaffected. Gene expression analysis indicated that no homologous genes of OsCAld5H1 were overexpressed in the mutants. These data suggested that CAld5H is mainly involved in the production of non‐γ‐p‐coumaroylated S lignin units, common in both eudicots and grasses, but not in the production of grass‐specific γ‐p‐coumaroylated S units in rice.     

Generation of virus‐resistant potato plants by RNA genome targeting

CRISPR/Cas systems provide bacteria and archaea with molecular immunity against invading phages and foreign plasmids. The class 2 type VI CRISPR/Cas effector Cas13a is an RNA‐targeting CRISPR effector that provides protection against RNA phages. Here we report the repurposing of CRISPR/Cas13a to protect potato plants from a eukaryotic virus, Potato virus Y (PVY). Transgenic potato lines expressing Cas13a/sgRNA (small guide RNA) constructs showed suppressed PVY accumulation and disease symptoms. The levels of viral resistance correlated with the expression levels of the Cas13a/sgRNA construct in the plants. Our data further demonstrate that appropriately designed sgRNAs can specifically interfere with multiple PVY strains, while having no effect on unrelated viruses such as PVA or Potato virus S. Our findings provide a novel and highly efficient strategy for engineering crops with resistances to viral diseases.     

Development of germ‐line‐specific CRISPR‐Cas9 systems to improve the production of heritable gene modifications in Arabidopsis

The Streptococcus‐derived CRISPR/Cas9 system is being widely used to perform targeted gene modifications in plants. This customized endonuclease system has two components, the single‐guide RNA (sgRNA) for target DNA recognition and the CRISPR‐associated protein 9 (Cas9) for DNA cleavage. Ubiquitously expressed CRISPR/Cas9 systems (UC) generate targeted gene modifications with high efficiency but only those produced in reproductive cells are transmitted to the next generation. We report the design and characterization of a germ‐line‐specific Cas9 system (GSC) for Arabidopsis gene modification in male gametocytes, constructed using a SPOROCYTELESS (SPL) genomic expression cassette. Four loci in two endogenous genes were targeted by both systems for comparative analysis. Mutations generated by the GSC system were rare in T1 plants but were abundant (30%) in the T2 generation. The vast majority (70%) of the T2 mutant population generated using the UC system were chimeras while the newly developed GSC system produced only 29% chimeras, with 70% of the T2 mutants being heterozygous. Analysis of two loci in the T2 population showed that the abundance of heritable gene mutations was 37% higher in the GSC system compared to the UC system and the level of polymorphism of the mutations was also dramatically increased with the GSC system. Two additional systems based on germ‐line‐specific promoters (pDD45‐GT and pLAT52‐GT) were also tested, and one of them was capable of generating heritable homozygous T1 mutant plants. Our results suggest that future application of the described GSC system will facilitate the screening for targeted gene modifications, especially lethal mutations in the T2 population.     

Diversity in CRISPR‐based immunity protects susceptible genotypes by restricting phage spread and evolution

Diversity in host resistance often associates with reduced pathogen spread. This may result from ecological and evolutionary processes, likely with feedback between them. Theory and experiments on bacteria–phage interactions have shown that genetic diversity of the bacterial adaptive immune system can limit phage evolution to overcome resistance. Using the CRISPR–Cas bacterial immune system and lytic phage, we engineered a host–pathogen system where each bacterial host genotype could be infected by only one phage genotype. With this model system, we explored how CRISPR diversity impacts the spread of phage when they can overcome a resistance allele, how immune diversity affects the evolution of the phage to increase its host range and if there was feedback between these processes. We show that increasing CRISPR diversity benefits susceptible bacteria via a dilution effect, which limits the spread of the phage. We suggest that this ecological effect impacts the evolution of novel phage genotypes, which then feeds back into phage population dynamics.