Typical single guide RNA (sgRNA) cloning into Cas9 vectors entails a lengthy process that requires specialized reagents (e.g., specific type IIS restriction enzymes) and multiple cloning steps such as incorporation of new restriction sites, restriction digests, and ligations. The traditional approach is further confounded by low cloning efficiency, resulting in additional time-consuming screens to identify a colony containing your sgRNA of interest. These bottlenecks also render it difficult to move sgRNAs from one vector to another and make it nearly impossible to multiplex several sgRNAs in one plasmid.
However, in a recent publication, Khan et al. were able to improve and streamline their Cas9/sgRNA plasmid construction using In-Fusion Cloning. The authors were able to produce ready-to-transfect single or multiplexed sgRNA constructs in less than 3 days.
Results
Cloning of a single guide RNA
To facilitate streamlined and rapid cloning of sgRNAs into a plant-specific expression vector (pDE-Cas9), the authors devised a cloning strategy that employed one set of target-specific primers and a second set of universal primers (Figure 1). Each primer set generated two fragments: Fragment A, consisting of a U6-26(P) promoter and a 20-nt protospacer sequence at the 3' end; and Fragment B, consisting of an sgRNA and a 15-bp overlap of the 3' end of the protospacer (required for In-Fusion-directed homologous recombination) that extended to the 5' end of the fragment. Fragments A and B were then assembled and placed into the linearized pDE-Cas9 vector in a single-step, single-tube In-Fusion cloning reaction.
Figure 1. Schematic for cloning a single sgRNA into a linearized vector using In-Fusion Cloning HD Plus. Fragments A and B were generated by PCR, then fused and directionally cloned into a linearized pDE-Cas9 vector in a one-step In-Fusion Cloning reaction.
Following cloning, plasmids were isolated from four colonies for restriction digest screens. While ligase-based methods can require screening large numbers of colonies to find a positive clone, all four In-Fusion clones gave the expected digest patterns. Subsequent sequencing of one vector (pDE-Cas9-gYFP1) demonstrated successful integration of the fragments in the correct orientation. These results indicate the successful generation of multiple, ready-to-transfect vectors in less than three days' time.
Cloning of multiple guide RNAs
The authors then modified this method to allow them to clone two sgRNAs into the pDE-Cas9 vector by using two sets of target-specific primers and two sets of universal primers (Figure 2). A first round of PCR generated four fragments—A, B, C, and D. Fragments A and C each consisted of a U6-26(P) promoter and a 20-nt protospacer, while fragments B and D each represented a single sgRNA sequence. In a second round of PCR, Fragments A and B were fused into fragment AB, while Fragments C and D were fused into fragment CD. Following second-round PCR, fragments AB and CD were gel purified, then assembled and inserted into the linearized pDE-Cas9 vector in a single-step, single-tube In-Fusion Cloning reaction.
Figure 2. Schematic for cloning multiple sgRNAs into a linearized vector with In-Fusion Cloning HD Plus. Fragments A, B, C, and D were amplified (Panel A) and fused into fragments AB and CD (Panel B) by PCR. Fragments AB and CD were then fused and directionally cloned into a linearized pDE-Cas9 vector in a one-step In-Fusion Cloning reaction.
Following cloning, five colonies were analyzed by restriction digestion and all five clones gave the expected digestion pattern. Subsequent sequencing of each clone revealed the successful integration of fragments in the correct orientation 100% of the time. These findings demonstrate the utility of this protocol in efficiently generating ready-to-transfect constructs with both single and multiple sgRNAs in less than three days' time.
Conclusions
Traditional sgRNA cloning methods are inefficient, time- and labor-intensive, require screening of large numbers of colonies to identify successful inserts, and cannot be easily shuttled between expression vectors. The In-Fusion Cloning methods described here eliminate these difficulties and allow the rapid, highly efficient generation of vectors containing single or multiple sgRNAs in a single-step, single-tube cloning reaction. We also note that the authors' methods may be adapted to your vector(s) of choice by simply modifying the universal primer sequences, providing a way to quickly and efficiently make constructs for your CRISPR/Cas-9 experiments.
To view more resources on sgRNA cloning with In-Fusion technology, visit our overview page.
Methods
In-Fusion seamless cloning reactions were performed using a wide variety of insert to vector ratios, all of which were successful (though we recommend a 2:1 ratio of insert:vector). Cloning reaction mixes were assembled using fragment AB or fragments AB and CD (15–180 fmoles and 0.5 µl each fragment), 0.5 µl linearized pDE-Cas9 vector (15–90 fmoles), and 0.3 µl 5X In-Fusion HD Cloning Plus mix. These reactions were incubated for 15 minutes at 50°C, inactivated for 10 minutes at 80°C, then 1 µl of the reaction mix was transformed into 20 µl of Stellar Competent Cells. The authors noted that using gel-purified fragments resulted in a 44-fold increase of colonies compared to unpurified fragments: ~200 per reaction gel-purified, compared to ~4.5 per reaction for unpurified fragments. The authors selected four single sgRNA or five multiple sgRNA clones, all of which contained the expected fragment sizes restriction fragment length polymorphism analyses. Sequencing analyses of these constructs revealed 100% of the clones contained the correct sequence and orientation of the targeted inserts.
References
Khan, A. A. et al. A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants. Plant Methods13, 86 (2017).
