Multiplexed CRISPR technologies for gene editing and transcriptional regulation

Abstract

Multiplexed CRISPR technologies, in which numerous gRNAs or Cas enzymes are expressed at once, have facilitated powerful biological engineering applications, vastly enhancing the scope and efficiencies of genetic editing and transcriptional regulation. In this review, we discuss multiplexed CRISPR technologies and describe methods for the assembly, expression and processing of synthetic guide RNA arrays in vivo. Applications that benefit from multiplexed CRISPR technologies, including cellular recorders, genetic circuits, biosensors, combinatorial genetic perturbations, large-scale genome engineering and the rewiring of metabolic pathways, are highlighted. We also offer a glimpse of emerging challenges and emphasize experimental considerations for future studies.

Fig. 1. Basic overview of multiplexed CRISPR–Cas technologies.

Multiplexed CRISPR–Cas can be implemented by simultaneously expressing multiple gRNAs at once. By adding orthologous Cas enzymes, gene editing, transcriptional activation (CRISPRa) and transcriptional repression (CRISPRi) can be performed in tandem at numerous locations in the genome. Cas enzymes are represented by the shaded green shape, while green and red cylinders attached to the Cas enzymes represent activation and repression domains, respectively. Editing requires that a Cas enzyme cleave dsDNA, which is repaired by error-prone, non-homologous end joining. Transcriptional regulation occurs by targeting nuclease-null Cas enzymes to specific regions up- or downstream of a transcription start site to either occlude RNA polymerase binding sites, or to recruit transcription factors (via fused effector domains) for activation or repression of the target gene.

Fig. 2. General strategies to express multiple gRNAs in vivo.

The expression and processing of multiplexed gRNAs typically occur via three distinct mechanisms; arrayed sgRNA expression constructs, in which each construct contains a promoter, sgRNA, and terminator (green header), CRISPR arrays, wherein each gRNA is processed via a native CRISPR processing mechanism (blue headers), or synthetic gRNA arrays, in which each gRNA is flanked by RNA cleavage sites (red headers). a A common method to express numerous gRNAs in vivo is to express each gRNA from an individual promoter. b A strategy to process crRNA arrays, based on Type II CRISPR systems, is to flank each crRNA with a direct repeat, a repetitive sequence required for processing of pre-crRNA. tracrRNA is expressed separately. RNase III is an endogenous enzyme that removes these direct repeats in a tracrRNA-dependent manner. c Cas12a and Cas13a can process crRNA arrays and remove direct repeats, even in the absence of tracrRNA. d An engineering approach to process arrays involves flanking each gRNA with self-cleaving sequences, such as Hammerhead or HDV ribozymes. Arrays of this form have been expressed from both Pol II and Pol III promoters. e Csy4 is an enzyme that recognizes a 28-nt stem–loop sequence in RNA and cuts after the 20th nucleotide. Exogenous co-expression of Csy4 can be used to process gRNA arrays, provided that each gRNA is flanked by its recognition sequence. f gRNA arrays flanked by tRNAs can be transcribed (either by Pol II or Pol III promoters) and processed by endogenous RNase P and RNase Z, which cut the 5′ and 3′ ends, respectively, of pre-tRNAs, to produce functional gRNAs. For each sgRNA, the portion in gray is derived from the tracrRNA, while the portion in blue indicates the 20-nt spacer sequence.

Fig. 3. Assembly methods for synthetic gRNA arrays.

a Oligo-based assembly methods are frequently used to assemble arrays. This approach works by annealing oligonucleotides that encode each gRNA unit, and then ligating these units into digested vectors which already contain predesigned Type IIs restriction sites and 4-nt overhangs. Each gRNA-containing entry vector is then cloned into a destination vector, which contains a promoter and terminator to express the assembled array. Conversely, oligos can be annealed that already contain complementary overhangs for direct ligation into a digested vector (shown on the right side). b A second strategy, referred to as PCRs and Golden Gate, utilizes a single vector with a repetitive “processing” sequence (either direct repeats, tRNA, ribozymes, or a Csy4 recognition site) as a template for PCR. Primer extensions add the desired spacer sequence and Type IIs restriction sites with specified, 4-nt overhangs. Digestion of each PCR amplicon, followed by Golden Gate assembly into a predigested destination vector, is used to assemble the final array. This strategy is both inexpensive and adaptable but requires multiple steps for cloning. c A more versatile, but expensive, strategy uses direct synthesis and ligation to build arrays. Briefly, multiple gRNA units are synthesized in tandem, and the ends of each synthesized piece of DNA contain overhangs for Gibson Assembly, specified Type IIs restriction sites for Golden Gate, or some other sequence to mediate DNA joining. In some cases, DNA synthesis can be directly used to directly build a full gRNA array.

Fig. 4. Applications of multiplexed CRISPR–Cas technologies.

a Multiple gRNAs can be expressed, together with dCas9, to build complex logic circuits, including wired NOR gates, in which upstream gRNAs regulate the expression of downstream sgRNAs. Logic circuits can be used to produce a simple output signal, like GFP, or can be interfaced into cellular pathways to control phenotypes or behaviors. b Cas13a orthologs can be used to detect multiple viral pathogens at once. The viruses are lysed, their genomes are amplified, and the amplified RNA is then used as the input for Cas13a-based biosensors. Upon recognition of an RNA target, Cas13a collaterally cleaves nearby transcripts, a characteristic that can be exploited to release orthogonal, fluorescent outputs from ssRNA reporters. c Multiplexed gRNAs enable combinatorial mapping of genotype to phenotype. Pairs of gRNAs, each with a unique barcode, are programmed to target different genes involved in a known pathway or cellular process. These gRNA:barcode pairs are transformed into Cas9-expressing cells, and the barcodes of each cell in a population are sequenced to determine which gRNA pair each cell received. By measuring the frequency of the barcodes over multiple conditions, combinations of genes that modulate a given phenotype can be inferred. d Multi-event recording enables multiple signals to be detected and recorded in the genome of living cells. One gRNA is used to “write” each detected signal. Event recorders commonly use base editors and gRNAs that target a pre-defined locus, and recordings can be read out by sequencing the targeted loci. e Multiplexed CRISPR–Cas enables specific genomic rearrangements or modifications, including indels (which are produced by error-prone, non-homologous end joining) and insertions (via homology-directed repair, where donor DNA contains homology arms to the double-strand break), for rapid strain engineering. f Multiplexed CRISPR–Cas technologies can be used to perturb numerous parts of a pathway simultaneously, thus redirecting flux and enhancing the production of a desired compound. CRISPRi, CRISPRa, and editing of DNA can be achieved simultaneously, simply by expressing orthogonal dCas:gRNA pairs (one for activation and another for repression), together with Cas12a or Cas9 for editing.