Rapid Evolution of Manifold CRISPR Systems for Plant Genome Editing

Abstract

Advanced CRISPR-Cas9 based technologies first validated in mammalian cell systems are quickly being adapted for use in plants. These new technologies increase CRISPR-Cas9's utility and effectiveness by diversifying cellular capabilities through expression construct system evolution and enzyme orthogonality, as well as enhanced efficiency through delivery and expression mechanisms. Here, we review the current state of advanced CRISPR-Cas9 and Cpf1 capabilities in plants and cover the rapid evolution of these tools from first generation inducers of double strand breaks for basic genetic manipulations to second and third generation multiplexed systems with myriad functionalities, capabilities, and specialized applications. We offer perspective on how to utilize these tools for currently untested research endeavors and analyze strengths and weaknesses of novel CRISPR systems in plants. Advanced CRISPR functionalities and delivery options demonstrated in plants are primarily reviewed but new technologies just coming to the forefront of CRISPR development, or those on the horizon, are briefly discussed. Topics covered are focused on the expansion of expression and delivery capabilities for CRISPR-Cas9 components and broadening targeting range through orthogonal Cas9 and Cpf1 proteins.

Keywords: CRISPR; Cas9; Cpf1; RNA-guided endonucleases; plant genome editing; sequence specific nucleases.

Figure 1

Diversified plant CRISPR-Cas9 expression systems. (A) The most common expression system is the mixed dual promoter system (Pol II:Cas9/Pol III:gRNA) where Cas9 is expressed from RNA polymerase II (Pol II) based promoters and gRNAs are expressed from RNA polymerase III promoters (Pol III promoters—such as U6 and U3). Top vector shows the canonical arrayed gRNA cassette expression system which expresses each gRNA with its own Pol III promoter, gRNA spacer, scaffold, and terminator sequences. Cas9 is expressed from a Pol II promoter and typically has N and C terminal nuclear localization signals (NLS), a FLAG tag for immunodetection, and a 3′ transcriptional terminator sequence. Middle vector shows the polycistronic tRNA processing gRNA expression system which expresses Cas9 using Pol II and gRNAs from a polycistronic Pol III driven transcript. tRNA encoding sequences act to process out RNA sequences they flank using endogenous tRNA processing ribonucleases. Bottom vector is the polycistronic Csy4 processing gRNA expression system that has been tested to function in mammalian cells, but not yet in plants. The Csy4 system utilizes the CRISPR type III ribonuclease, Csy4, to cleave the (28 bp) sequence which cuts out RNA sequences flanked by these sequence elements. A poly A tail (pA tail) is used to stabilize the 3′ RNA transcript sequence after processing. (B) The Dual (Pol II) promoter system (Pol II:Cas9/Pol II:gRNA) uses RNA polymerase II promoters to drive both Cas9 and gRNA expression. Different Pol II promoters can be used simultaneously to express the separate Cas9 or gRNA transcripts providing great flexibility and capacity for constitutive or inducible RNA expression. Top vector shows the polycistronic ribozyme self-processing gRNA expression system which processes out gRNAs from Pol II primary transcripts using the hammerhead (HH) and (HDV) ribozyme cleavage sequence elements. Middle shows the polycistronic tRNA processing gRNA expression system from (A) but adapted for the dual promoter system by Pol II promoter controlled transcription. Bottom shows the Csy4 processing gRNA expression system as above but under the control of two separate Pol II promoters. Note that Csy4 ribonuclease must be expressed from the Cas9 transcript using the translational viral cleavage sequence (T2A) which allows for two functional polypeptides to be produced from a single transcript. (C) Shows the polycistronic ribozyme self-processing system, the tRNA processing and Csy4 processing gRNA expression systems under the the control of a single Pol II promoter. Note that the single promoter system is the most compact of all the systems. ^*Experimentally validated function in plants is denoted below vector.

Figure 2

Delivery of CRISPR reagents to plant cells and tissues. (A) Floral dip transformation of Arabidopsis with transgenic T-DNA carrying Agrobacteria. (B) Transient inoculation of plant leaf tissue or calli with Agrobacteria harboring Cas9 and gRNA T-DNA. (C) Viral vector delivery causes a transiently transformed plant (at left) to develop systemic infection upon viral capsid replication after initial transformation of vector DNA. (D) Transient particle bombardment of plant leaf tissue using a gene gun with Cas9 and gRNA or (E) gRNAs only to stable Cas9-expressing transgenic plants. (F) Ribonucleoprotein (RNP) complex delivery directly to protoplasts using PEG transformation or (G) RNA delivery directly to protoplasts (shown here) using PEG transformation or calli using “gene gun” as in (D).

Figure 3

Potentially beneficial features of Cpf1 vs. Cas9 for RGEN use in plants. (Top) Cpf1-gRNA enzyme complex cleaving target DNA. Yellow staggered line with stars indicates overhanging DNA cleavage at sites distal to PAM. (A) Cpf1 creates staggered overhanging DNA cleavage where Cas9 creates blunt end DSBs. (B) DNA cleavage is distal to the thiamine-rich PAM recognition sequences opening up the prospect for enhanced HDR frequencies as recognition sites may not be abolished after NHEJ induced mutations distal to PAM. (C) Cpf1 gRNA is roughly half the size of Cas9, making delivery more compact and potentially efficient, especially for viral delivery methods. (D) Overhanging sticky ends after cleavage create the possibility for NHEJ mediated insertion of transgenes with directionality.