The powerful CRISPR technologies have been widely applied in various biological systems and are greatly changing the field of genome engineering (Cong et al., 2013; Jinek et al., 2013; Hsu et al., 2014; Komor et al., 2017; Knott and Doudna, 2018). Cas12a(Cpf1), a member of the Class 2 Type V CRISPR-associated endonuclease family, differs from Cas9 in several ways (Zetsche et al., 2015): it has a T-rich PAM recognition site (TTTV) while Cas9 PAM site is G-rich (NGG) (Zetsche et al., 2015; Yamano et al., 2016); its guide RNA is shorter in sequence than Cas9 (Zetsche et al., 2015); Cas12a cuts DNA distal and downstream relative to the PAM sequence and results in staggered ends while Cas9 cuts DNA proximal and upstream and results in blunt ends; and Cas12a is reported to be more precise and induces less off-target activities than Cas9 (Zetsche et al., 2015; Kim et al., 2016; Kleinstiver et al., 2016; Yamano et al., 2016; Swarts et al., 2017). Moreover, due to its intrinsic ribonuclease activity that processes its own crRNA, Cas12a is capable of multiplex editing from a single RNA transcript (Zetsche et al., 2015; Zetsche et al., 2017).

Multiplex engineering can be useful in understanding gene-gene interactions, combinatorial effects of different genetic mutations, and biological pathways with redundancy between individual genes (Tak et al., 2017; Li et al., 2019; McCarty et al., 2020). Along with its other beneficial properties, Cas12a holds great potential in serving as a tool to solve genome engineering problems. However, similar to Cas9, Cas12a relies on endogenous homology-directed repair (HDR) mechanisms to achieve precise gene modifications, but the HDR efficiency is often very limited due to the competing error-prone repair pathways which happen much more frequently (Pickar-Oliver and Gersbach, 2019; Anzalone et al., 2020). While many previous studies have been done to improve the efficiency of base editing and short genomic alterations, large-scale genome editing using Cas12a still remains a challenge (Li et al., 2018; Kleinstiver et al., 2019; Wang et al., 2020).

Previously we have identified that phage single-stranded annealing proteins (SSAPs) could enhance Cas9-based gene editing efficiencies in mammalian cells, and among all SSAPs screened, E. coli RecT exhibited the largest increase (Wang et al., 2021). We termed this high-efficiency Cas9 editing method as RecT Editor via Designer-Cas9-Initiated Targeting (REDIT). Here, combining the efficiency of REDIT and the multi-target editing ability of Cas12a, we developed a new Cas12a-REDIT method.

We fused RecT to Cas12a via the previously reported SunTag-based recruitment system (Tanenbaum et al., 2014; Huang et al., 2017). In our design, Cas12a linking a tandem repeat of 10 copies of GCN4 peptides were cloned into a mammalian expression vector. The RecT protein (GFP protein in control) linking a single chain variable fragment (ScFv) of the anti-GCN4 antibody, was cloned into another vector. Upon the co-expression of two plasmids, ScFv antibodies would bind to their cognate peptide pair GCN4, recruiting RecT to the Cas12a protein (Figure 1A).

We tested our system via a genomic knock-in assay where a repair template of a T2A-mKate cassette (∼1 kb in length) was provided. The template bears homology arms (HA) on both sides that match the last exon (in-frame before the stop codon) of the target genes (Figure 1B). HDR efficiency was then quantified by fluorescence-activated cell sorting (FACS) where the percentage of mKate-positive cells represents successful knock-in.

Many types of Cas12a orthologs have been found across different bacteria; among those, Acidaminococcus sp. Cas12a (AsCas12a) and L. bacterium Cas12a (LbCas12a) are the two that are most efficient and commonly used (Zetsche et al., 2015). Therefore, we tested our design using these two orthologs in the HEK293T cell line. As expected, across two different gene targets (DYNLT1 and AASV1), there were significant increases of HDR efficiency when RecT was recruited compared to the control in both of the Cas12a variants. Moreover, AsCas12a performed slightly better compared to LbCas12a. Hence, we further optimized our design using AsCas12a in the rest of study (Figure 1C).

As we have previously shown that the template HA length is a deciding factor of the HDR efficiency (Wang et al., 2021), we investigated such influence in Cas12a-REDIT by testing different HA designs. We found that symmetric HA performed better than asymmetric HA in general, and while our design could work with as short as ∼100 bp template HA on each side, the efficiency increased as the HA length increased. A template that had ∼400 bp HA on both sides performed the best among all six designs tested (Figure 1D).

Next, we examined our designs in two more different human cell lines, Hela (cervix-derived) and HepG2 (liver-derived), in order to evaluate the generalizability of our system. As expected, there were notable improvements of nearly 2-fold in the HDR efficiency (Figure 1E).

To investigate whether the improvement persisted in the case of multi-target editing, two genes DYNLT1 and EMX1 were targeted at the same time where mKate was inserted into DYNLT1 and a 16bp insertion was made into EMX1. For the latter, next-generation sequencing was performed to assay the editing efficiency. There was a nearly four-fold increase of editing efficiency for both long and short knock-in editing when using the Cas12a REDIT method (Figure 2A). This enhancement of editing efficiencies in multiple-target settings were further validated by changing DYNLT1 locus to AAVS1 (Figure 2B).

Multiple genes can be edited by expressing a single CRISPR-Cas12a crRNA array together with Cas12a protein, taking advantage of its dual DNase and RNase activity (Zetsche et al., 2017). To further understand the potential of multiple-target editing using this system, we then targeted four genes (AAVS1, DYNLT1, EMX1, DNMT1) using a crRNA array and inserted mKate into DYNLT1 locus and GFP into AAVS1 locus at the same time. Focusing on fluorescent tag knock-in editing, we tested four different array designs with four guides in distinct orders, and achieved successful simultaneous HDR editing of both mKate and GFP with an approximate 2–4.5-fold increase in efficiency using Cas12a-REDIT (Figure 2C). While we maintained the guide sequence across the designs, we observed different editing efficiencies (Figure 2C). This is likely due to the varying processing efficiencies of crRNA array by Cas12a protein. This processing could be influenced by multiple factors. On one hand, the positioning of the crRNA within an array could influence the processing efficiencies, thus leading to variable expression levels of individual guide depending on the position, explaining the results we observed (Campa et al., 2019). On the other hand, recent work (Magnusson et al., 2021) also reported that the guide sequence could also influence the processing efficiencies of Cas12a crRNA arrays, which could also contribute to the differences in editing outcomes when we change the position of crRNAs within an array.

After validating the multi-target editing of the Cas12a-REDIT method, we additionally assayed the off-target effect of our design by profiling the editing events on potential off-target sites (OTS). We examined two genes, EMX1 and VEGFA, and OTS were predicted using http://crispor.org. Based on the score ranking, five EMX1 OTS and six VEGFA OTS were selected (sequences listed in Supplementary Table), and editing efficiencies on these OTS were determined by next-generation sequencing. For both EMX1 and VEGFA, our system exhibited no markable increase in the off-target editing (Figures 2D,E).

In summary, by employing the SSAP activity of phage RecT, we engineered a Cas12a-based system that can increase the efficiency of kilo-base scale HDR editing without compromising its specificity, and such improvements could also be achieved in multiplex genome editing.

In the longer term, additional method development could enhance the utility of this multiplex genome knock-in system. Specifically, many emerging areas of gene-editing research will have significant synergy with our work: protein engineering to define minimal-functional Cas12a-RecT editor including to optimize its efficiency; more powerful delivery systems including for the repair templates, which is very challenging for in vivo application of this multiplex editor. These additional advancements will further enhance our approach and help to achieve high-efficiency, multi-target long-sequence editing in human cells.