Multiplex CRISPR Research Articles

Construct design for CRISPR/Cas-based genome editing in plants.

CRISPR construct design is a key step in the practice of genome editing, which includes identification of appropriate Cas proteins, design and selection of guide RNAs (gRNAs), and selection of regulatory elements to express gRNAs and Cas proteins. Here, we review the choices of CRISPR-based genome editors suited for different needs in plant genome editing applications. We consider the technical aspects of gRNA design and the associated computational tools. We also discuss strategies for the design of multiplex CRISPR constructs for high-throughput manipulation of complex biological processes or polygenic traits. We provide recommendations for different elements of CRISPR constructs and discuss the remaining challenges of CRISPR construct optimization in plant genome editing.

The yeast platform engineered for synthetic gRNA-landing pads enables multiple gene integrations by a single gRNA/Cas9 system

Saccharomyces cerevisiae is a versatile microbial platform to build synthetic metabolic pathways for production of diverse chemicals. To expedite the construction of complex metabolic pathways by multiplex CRISPR-Cas9 genome-edit, eight desirable intergenic loci, located adjacent to highly expressed genes selected from top 100 expressers, were identified and fully characterized for three criteria after integrating green fluorescent protein (GFP) gene - CRISPR-mediated GFP integration efficiency, expression competency assessed by levels of GFP fluorescence, and assessing growth rates of GFP integrated strains. Five best performing intergenic loci were selected to build a multiplex CRISPR platform, and a synthetic 23-bp DNA comprised of 20-bp synthetic DNA with a protospacer adjacent motif (PAM) was integrated into the five loci using CRISPR-Cas9 in a sequential manner. This process resulted in five different yeast strains harbouring 1–5 synthetic gRNA-binding sites in their genomes. Using these pre-engineered yeast strains, simultaneous integrations of 2-, 3-, 4-, or 5-genes to the targeted loci were demonstrated with efficiencies from 85% to 98% using beet pigment betalain (3-gene pathway), hygromycin and geneticin resistance markers. Integrations of the multiple, foreign genes in the targeted loci with 100% precision were validated by genotyping. Finally, we further developed the strain to have 6th synthetic gRNA-binding site, and the resulting yeast strain was used to generate a yeast strain producing a sesquiterpene lactone, kauniolide by simultaneous 6-gene integrations. This study demonstrates the effectiveness of a single gRNA-mediated CRISPR platform to build complex metabolic pathways in yeast.

Efficient simultaneous mutagenesis of multiple genes in specific plant tissues by multiplex CRISPR.

CRISPR technology is an established tool for the generation of knockout plants (Zhang et al., 2019), yet limitations remain. First, the manipulation of individual genes may fail to produce phenotypes for groups of genes with redundant or synergistic functions. While this has been partially addressed by multiplexing guide RNAs (gRNAs), there is concern that as the number of targets increases, the chances of obtaining higher-order knockouts diminish (Zhang et al., 2016). Second, knocking out fundamentally important genes can cause severe pleiotropic phenotypes or lethality. Tissue-specific knockout of genes in somatic tissues can overcome this limitation (Decaestecker et al., 2019 ; Wang et al., 2020 ; Liang et al., 2019). However, the efficiency of simultaneously targeting more than three genes in a tissue-specific context is unexplored. Here, by multiplexing gRNAs in Arabidopsis thaliana plants expressing Cas9 either ubiquitously (pPcUBI) or root cap-specifically (pSMB), we show that six genes can be simultaneously mutated with high efficiency, generating higher-order mutant phenotypes already in the first transgenic generation (T1). The mutation frequencies for all target genes were positively correlated and unaffected by the order of the gRNAs in the vector, showing that efficient higher-order mutagenesis in specific plant tissues can be readily achieved.

Novel Prokaryotic CRISPR-Cas12a-Based Tool for Programmable Transcriptional Activation and Repression.

Transcriptional perturbation using inactivated CRISPR-nucleases (dCas) is a common method in eukaryotic organisms. While rare examples of dCas9-based tools for prokaryotes have been described, multiplexing approaches are limited due to the used effector nuclease. For the first time, a dCas12a derived tool for the targeted activation and repression of genes was developed. Therefore, a previously described SoxS activator domain was linked to dCas12a to enable the programmable activation of gene expression. A proof of principle of transcriptional regulation was demonstrated on the basis of fluorescence reporter assays using the alternative host organism Paenibacillus polymyxa as well as Escherichia coli. Single target and multiplex CRISPR interference targeting the exopolysaccharide biosynthesis of P. polymyxa was shown to emulate polymer compositions of gene knockouts. The simultaneous expression of 11 gRNAs targeting multiple lactate dehydrogenases and a butanediol dehydrogenase resulted in decreased lactate formation, as well as an increased butanediol production in microaerobic fermentation processes. Even though Cas12a is more restricted in terms of its genomic target sequences compared to Cas9, its ability to efficiently process its own guide RNAs in vivo makes it a promising tool to orchestrate sophisticated genetic reprogramming of bacterial cells or to screen for engineering targets in the genome. The developed tool will accelerate metabolic engineering efforts in the alternative host organism P. polymyxa and might be also applied for other bacterial cell factories.

No Evidence that Wnt Ligands Are Required for Planar Cell Polarity in Drosophila

SUMMARYThe frizzled (fz) and dishevelled (dsh) genes are highly conserved members of both the planar cell polarity (PCP) pathway and the Wnt signaling pathway. Given these dual functions, several studies have examined whether Wnt ligands provide a tissue-scale orientation cue for PCP establishment during development, and these studies have reached differing conclusions. Here, we re-examine this issue in the Drosophila melanogaster wing and notum using split-Gal4 co-expression analysis, multiplex somatic CRISPR, and double RNAi experiments. Pairwise loss-of-function experiments targeting wg together with other Wnt genes, via somatic CRISPR or RNAi, do not produce PCP defects in the wing or notum. In addition, somatic CRISPR against evi (aka wntless), which is required for the secretion of Wnt ligands, did not produce detectable PCP phenotypes. Altogether, our results do not support the hypothesis that Wnt ligands contribute to PCP signaling in the Drosophila wing or notum.

One-Day Construction of Multiplex Arrays to Harness Natural CRISPR-Cas Systems.

CRISPR-Cas systems are prokaryotic immune systems that have proliferated widely not only in bacteria and archaea, but also much more recently, in human biological research and applications. Much work to date has utilized synthetic sgRNAs along with the CRISPR nuclease Cas9, but the discovery of array-processing nucleases now allows the use of more compact, natural CRISPR arrays in heterologous hosts, in addition to organisms with endogenous systems. Unfortunately, the construction of multiplex natural CRISPR arrays remains technically challenging, expensive, and/or time-consuming. This limitation hampers research involving natural CRISPR arrays in both native and heterologous hosts. To address this problem, we present a method to assemble CRISPR arrays that is simple, rapid, affordable, and highly scalable-we assembled 9-spacer arrays with 1 day's worth of work. We used this method to harness the endogenous CRISPR-Cas system of the highly competent bacterium Acinetobacter baylyi, showing that while single spacers are not always completely effective at blocking DNA acquisition through natural competence, multiplex natural CRISPR arrays enable both nearly complete DNA exclusion and genome editing, including with multiple targets for both. In addition to demonstrating a CRISPR array assembly method that will benefit a variety of applications, we also find a potential bet-hedging strategy for balancing CRISPR defense versus DNA acquisition in naturally competent A. baylyi.

A novel cloning strategy for one-step assembly of multiplex CRISPR vectors

One key advantage of the CRISPR/Cas9 system in comparison with other gene editing approaches lies in its potential for multiplexing. Here, we describe an elaborate procedure that allows the assembly of multiple gRNA expression cassettes into a vector of choice within a single step, termed ASAP(Adaptable System for Assembly of multiplexed Plasmids)-cloning. We demonstrate the utility of ASAP-cloning for multiple CRISPR-mediated applications, including efficient multiplex gene editing, robust transcription activation and convenient analysis of Cas9 activity in the presence of multiple gRNAs.

One step generation of customizable gRNA vectors for multiplex CRISPR approaches through string assembly gRNA cloning (STAgR).

Novel applications based on the bacterial CRISPR system make genetic, genomic, transcriptional and epigenomic engineering widely accessible for the first time. A significant advantage of CRISPR over previous methods is its tremendous adaptability due to its bipartite nature. Cas9 or its engineered variants define the molecular effect, while short gRNAs determine the targeting sites. A majority of CRISPR approaches depend on the simultaneous delivery of multiple gRNAs into single cells, either as an essential precondition, to increase responsive cell populations or to enhance phenotypic outcomes. Despite these requirements, methods allowing the efficient generation and delivery of multiple gRNA expression units into single cells are still sparse. Here we present STAgR (String assembly gRNA cloning), a single step gRNA multiplexing system, that obtains its advantages by employing the N20 targeting sequences as necessary homologies for Gibson assembly. We show that STAgR allows reliable and cost-effective generation of vectors with high numbers of gRNAs enabling multiplexed CRISPR approaches. Moreover, STAgR is easily customizable, as vector backbones as well as gRNA structures, numbers and promoters can be freely chosen and combined. Finally, we demonstrate STAgR’s widespread functionality, its efficiency in multi-targeting approaches, using it for both, genome and transcriptome editing, as well as applying it in vitro and in vivo.

Multiplexed CRISPR/Cas9- and TAR-Mediated Promoter Engineering of Natural Product Biosynthetic Gene Clusters in Yeast.

The use of DNA sequencing to guide the discovery of natural products has emerged as a new paradigm for revealing chemistries encoded in bacterial genomes. A major obstacle to implementing this approach to natural product discovery is the transcriptional silence of biosynthetic gene clusters under laboratory growth conditions. Here we describe an improved yeast-based promoter engineering platform (mCRISTAR) that combines CRISPR/Cas9 and TAR to enable single-marker multiplexed promoter engineering of large gene clusters. mCRISTAR highlights the first application of the CRISPR/Cas9 system to multiplexed promoter engineering of natural product biosynthetic gene clusters. In this method, CRISPR/Cas9 is used to induce DNA double-strand breaks in promoter regions of biosynthetic gene clusters, and the resulting operon fragments are reassembled by TAR using synthetic gene-cluster-specific promoter cassettes. mCRISTAR uses a CRISPR array to simplify the construction of a CRISPR plasmid for multiplex CRISPR and a single auxotrophic selection to improve the inefficiency of using a CRISPR array for multiplex gene cluster refactoring. mCRISTAR is a simple and generic method for multiplexed replacement of promoters in biosynthetic gene clusters that will facilitate the discovery of natural products from the rapidly growing collection of gene clusters found in microbial genome and metagenome sequencing projects.

Selection of chromosomal DNA libraries using a multiplex CRISPR system.

The directed evolution of biomolecules to improve or change their activity is central to many engineering and synthetic biology efforts. However, selecting improved variants from gene libraries in living cells requires plasmid expression systems that suffer from variable copy number effects, or the use of complex marker-dependent chromosomal integration strategies. We developed quantitative gene assembly and DNA library insertion into the Saccharomyces cerevisiae genome by optimizing an efficient single-step and marker-free genome editing system using CRISPR-Cas9. With this Multiplex CRISPR (CRISPRm) system, we selected an improved cellobiose utilization pathway in diploid yeast in a single round of mutagenesis and selection, which increased cellobiose fermentation rates by over 10-fold. Mutations recovered in the best cellodextrin transporters reveal synergy between substrate binding and transporter dynamics, and demonstrate the power of CRISPRm to accelerate selection experiments and discoveries of the molecular determinants that enhance biomolecule function.

Multiplex engineering of industrial yeast genomes using CRISPRm.

Global demand has driven the use of industrial strains of the yeast Saccharomyces cerevisiae for large-scale production of biofuels and renewable chemicals. However, the genetic basis of desired domestication traits is poorly understood because robust genetic tools do not exist for industrial hosts. We present an efficient, marker-free, high-throughput, and multiplexed genome editing platform for industrial strains of S. cerevisiae that uses plasmid-based expression of the CRISPR/Cas9 endonuclease and multiple ribozyme-protected single guide RNAs. With this multiplex CRISPR (CRISPRm) system, it is possible to integrate DNA libraries into the chromosome for evolution experiments, and to engineer multiple loci simultaneously. The CRISPRm tools should therefore find use in many higher-order synthetic biology applications to accelerate improvements in industrial microorganisms.

Running the Numbers

CRISPR-Cas systems are prokaryotic immune systems that have proliferated widely not only in bacteria and archaea, but also much more recently, in human biological research and applications. Much work to date has utilized synthetic sgRNAs along with the CRISPR nuclease Cas9, but the discovery of array-processing nucleases now allows the use of more compact, natural CRISPR arrays in heterologous hosts, in addition to organisms with endogenous systems. Unfortunately, the construction of multiplex natural CRISPR arrays remains technically challenging, expensive, and/or time-consuming. This limitation hampers research involving natural CRISPR arrays in both native and heterologous hosts. To address this problem, we present a method to assemble CRISPR arrays that is simple, rapid, affordable, and highly scalable -- we assembled 9-spacer arrays with one day's worth of work. We used this method to harness the endogenous CRISPR system of the highly competent bacterium *Acinetobacter baylyi*, showing that while single spacers are not always completely effective at blocking DNA acquisition through natural competence, multiplex natural CRISPR arrays enable both nearly complete DNA exclusion and genome editing, including with multiple targets for both. In addition to demonstrating a CRISPR array assembly method that will benefit a variety of applications, we also find a potential bet-hedging strategy for balancing CRISPR defense vs. DNA acquisition in naturally competent *A. baylyi*.