CRISPR-Mediated Genome Editing Using Golden Gate and Gateway Cloning

CRISPR-Cas-based genome editing technologies have changed every facet of basic and translational biological research as we know it. Though multiple Cas systems have been described, the CRISPR-Cas9 effector nuclease from a class 2 bacterial CRISPR system has been the most extensively utilized in eukaryotes [1]. This Cas9 system provides specific and precise genome editing through the initial binding of 20-nucleotide sequence guide RNAs (gRNAs). The Cas9 enzyme will subsequently cleave the target DNA at 3 base pairs upstream from the Protospacer Adjacent Motif (PAM). This Cas-enzyme mediated cut results in a double strand break of the target DNA template [2]. This break can subsequently be repaired by either by error-prone DNA repair pathways such as classical non-homologous end joining (NHEJ) or through a template DNA-dependent pathway (homology directed repair, or HDR) to provide precise sequence editing [2]. The versatility of CRISPR-Cas9 to manipulate any target within the genome is realized through manipulating the nucleotide sequence of the exogenous guide RNA to direct the exogenous Cas9 system to the desired sequence target. Through these mechanisms, distinct insertions, deletions, or point mutations can be introduced through DNA repair mechanisms [2]. Given the utility and potential broad application of this methodology, CRISPR-Cas9 has been utilized extensively from basic research to generation of novel therapeutics.

Plant genome engineering using CRISPR-Cas systems has been successfully reported in plants for a multitude of purposes including agriculture, bioenergy, bioproduct development, as well as fundamental academic research [3]. However, technical challenges exist that can limit this technology from reaching its full potential in plant genome editing. For example, significant expertise in plant molecular biology is required to engineer functional expression constructs that allow simultaneous editing and transcriptional regulation. Although the CRISPR/Cas9 system offers significant advantages relative to other genome-editing methodologies, this approach becomes exceedingly more difficult in the context of multiple genomic loci, or in cases where multiplexing is required. Thus, the design of the CRISPR system elements, especially the generation of the gRNAs, need to be carefully considered to avoid potential complications or downstream issues. These considerations need to encompass the cloning strategy and methods utilized to generate the final vectors that deliver the CRISPR elements. To this end, both Gateway cloning and Golden Gate cloning methods allows for quick and efficient assembly of functional CRISPR/Cas9 transfer DNA (T-DNA) constructs for both monocot and dicot plants.

Gateway cloning methodology has been widely adopted by researchers as it allows for relatively simple simultaneous engineering of multiple complex expression vectors for delivery into the desired plant species [3]. As a result, an extensive library of Gateway destination vectors for multiple plants have been developed and validated with necessary plant-specific elements that can be utilized by other technologies, including CRISPR-based genome editing [4]. Gateway cloning technology is based on integration and excision recombination and is well suited for this as well as other cloning applications. This method offers high cloning efficiencies, without the use of restriction enzymes (RE). Gateway cloning starts by adding flanking attB sequences to gene of interest or DNA fragment. This fragment is subsequently cloned into an entry vector that contains the lethal ccdB gene sequence, flanked by 2 attP sequences through the BP Reaction. This reaction is catalyzed by the BP Clonase enzyme mix and produces the gene of interest flanked by attL sites in the entry clone and a free DNA fragment that contains the ccdB sequence. This vector is subsequently used to create the expression clone when the LR Reaction is started. This reaction involves the attL sites in the entry clone and the attR sites of a destination vector, which is catalyzed by the LR Clonase mix.

In the case of multigene assembly/modification, molecular tool kits have been described to facilitate the use of CRISPR in plants. Golden Gate cloning technology has the potential to circumvent some of the hurdles encountered with conventional restriction enzyme cloning in that multiple hands-on steps, such as the need for agarose gel purification, can be eliminated. Extremely useful in cases where large numbers of DNA parts need to be combined in a single pot assembly reaction, the advantages of Golden Gate cloning are realized via Type IIS restriction enzymes. These are a special class of RE that cut DNA at a defined distance downstream of the recognition sequence. Special sequence requirements downstream of the recognition sequence do not exist, so the sequences beyond the recognition site can be any nucleotide combination. With up to 256 potential overhang sequences possible, the digested DNA fragments and the final assembly no longer contain the Type IIS RE site. Thus, cutting and ligation can be performed simultaneously, and the assembled product accumulates over time.

Real world examples have demonstrated the utility and efficiency of both cloning methodologies in CRISPR/Cas9 cassette generation for genome editing in plants. Luo et al. demonstrated a combined system, known as pGate vectors (see Figure 1), for rapid evaluation of CRISPR/Cas9-mediated genome editing in plants [5]. By combining both Golden Gate cloning and Gateway cloning strategies, pGate vectors can serve as recipient vectors to engineer various DNA fragments but also work as entry vectors to generate the final destination vectors [5]. Additionally, a rapid and efficient use of CRISPR/Cas9-based toolbox was described for the engineering of multiple gene loci mutations that was dependent on the combining Golden Gate cloning and Gateway cloning strategies [5].

Independently, both Golden Gate and Gateway cloning methods have proven to be useful in making cloning approachable and scalable across diverse applications. Collectively, Golden Gate and Gateway cloning methodologies offer advantageous strategies for the generation of CRISPR cassettes for plant genome engineering. As shown in relatively recent publications, these cloning methods can serve across the CRISPR workflow to design and deliver multiple expression vectors for multiple gene loci editing. Making genome editing in plants more approachable and scalable will help support scientists to quickly evaluate the effects of adding, deleting, or altering specific genes in crop plants, when developing crops that address the needs of climate change and a growing world population.

1. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012). A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. science, 337(6096), 816–821. DOI: 10.1126/science.122582
2. Weiss T, Crisp PA, Rai KM, Song M, Springer NM, Zhang F. Epigenetic features drastically impact CRISPR-Cas9 efficacy in plants. Plant Physiol. 2022 Sep 28;190(2):1153–1164. DOI: 10.1093/plphys/kiac285
3. Pan C, Wu X, Markel K, Malzahn AA, Kundagrami N, Sretenovic S, Zhang Y, Cheng Y, Shih PM, Qi Y. CRISPR-Act3.0 for highly efficient multiplexed gene activation in plants. Nat Plants. 2021 Jul;7(7):942–953. DOI: 10.1038/s41477-021-00953-7
4. Alok A, Chauhan H, Upadhyay SK, Pandey A, Kumar J, Singh K. Compendium of Plant-Specific CRISPR Vectors and Their Technical Advantages. Life (Basel). 2021 Sep 28;11(10):1021. DOI: 10.3390/life11101021
5. Luo Y, Qiu Y, Na R, Meerja F, Lu QS, Yang C, Tian L. A Golden Gate and Gateway double-compatible vector system for high throughput functional analysis of genes. Plant Sci. 2018 Jun;271:117–126. DOI: 10.1016/j.plantsci.2018.03.023