Covalent linkage of the DNA repair template to the CRISPR/Cas9 complex enhances homology-directed repair

The CRISPR/Cas9 targeted nuclease technology allows the insertion of genetic modifications with single base-pair precision. The preference of mammalian cells to repair Cas9-induced DNA double-strand breaks via non-homologous end joining (NHEJ) rather than via homology-directed repair (HDR) however leads to relatively low rates of correctly edited loci. Here we demonstrate that covalently linking the DNA repair template to Cas9 increases the ratio of HDR over NHEJ up to 23-fold, and therefore provides advantages for clinical applications where high-fidelity repair is needed.


Main text
The CRISPR/Cas9 system is a versatile genome-editing tool that enables the introduction of site-specific genetic modifications 1 . In its most widespread variant a programmable chimeric short guide RNA (sgRNA) directs the Cas9 nuclease to the genomic region of interest, where it generates a site-specific DNA double-strand break (DSB) 2 . In mammalian cells, DSBs are either repaired by non-homologous end joining (NHEJ), or by homology-directed repair (HDR) pathways 3 . While NHEJ is an error-prone process that produces random insertions or deletions (indels) 4 , HDR repairs DSBs accurately from template DNA, and enables the introduction of modifications with single base precision 5 .
Therapeutic applications of CRISPR/Cas9 generally require the precise correction of pathogenic mutations. In mammalian cells, however, DSBs are predominantly repaired by NHEJ. As the thereby induced indels inhibit the CRISPR/Cas9 complex from retargeting the locus, NHEJ directly competes with HDR and reduces precise correction rates. In addition, if the targeted allele is a hypomorph with residual gene function, indels generated by NHEJ could further worsen the clinical phenotype of the disease. In recent years, several attempts have been made to enhance DSB repair by the HDR pathway. These include: i) synchronizing cells in the M phase of the cell cycle prior to CRISPR/Cas9 delivery 6 , ii) limiting Cas9 expression to the S/G2 phase of the cell cycle 7 , iii) chemically modulating the NHEJ and HDR pathways [8][9][10][11] , and iv) rationally designing DNA repair templates with optimal homology arm lengths 12 . In addition, it has been proposed that the availability of the DNA repair template might present a rate-limiting factor for HDR, and that bringing it in close spatial proximity to the DSBs could therefore enhance HDR editing rates 13,14 . Based on this hypothesis, we here generated and tested novel CRISPR/Cas variants, in which the DNA repair template is covalently conjugated to Cas9 via 'click chemistry' (Fig 1a).
To be able to easily test HDR based editing efficiencies of novel CRISPR/Cas9 variants in a high throughput manner, we first generated a fluorescent reporter system that allowed us to quantify HDR and NHEJ editing frequencies in mammalian cells by FACS (Fig.   1b). In brief, the reporter expresses a green fluorescent protein (GFP), which is preceded by an inactive version of a mutant red fluorescent protein (mutRFP). While precise correction of the mutation via HDR leads to re-activation of RFP activity, the generation of frame shifts via NHEJ leads to loss of GFP activity (Fig. 1b). To test the functionality of the reporter and to determine the optimal length of DNA repair templates, we transfected mammalian cells that stably express a single copy of the reporter with Cas9-sgRNA ribonucleoprotein (RNP) complexes and repair templates of different lengths (Fig. Sup. 1a). In line with previous studies 15 , we found that maximal HDR efficiencies of DSBs are reached with DNA oligonucleotides (oligos) of approximately 80 bases. Nevertheless, as we reasoned that if repair templates are brought in close proximity to DSBs also shorter homology arms could be sufficient, we continued our study with 65-nucleotide (65-mers) and 81-nucleotide (81-mers) DNA repair templates.
In order to link repair oligos to Cas9, we used the SNAP-tag technology, which allows covalent binding of O 6 -benzylguanine (BG)-labeled molecules to SNAP-tag fusion proteins 16 .
To generate O 6 -benzylguanine (BG)-linked DNA repair templates, we first synthesized amine-modified oligos, and coupled them to commercially available amine-reactive BG building blocks (Fig. 1d, Suppl. Fig. 1b). The BG-linked oligos were further separated from unlinked oligos by HPLC (Fig. 1e), and analyzed by mass spectrometry to confirm purity (Suppl. Fig. 1c). Next, we produced recombinant Cas9 proteins with SNAP-tag fused to the C-terminus (Fig. 1f,g). The fusion proteins were then complexed with the BG-coupled oligonucleotides, and covalent binding was confirmed by SDS-PAGE ( Fig. 1 h-k). The protein-oligo conjugate was finally mixed with in vitro transcribed sgRNAs targeting the mutRFP locus (Suppl. Fig. 1f,g), generating the Cas9 ribonucleoprotein-DNA (RNPD) complex.
To test if linking the repair template to Cas9 changes the ratio between NHEJ and HDR, we used our reporter system to compare S. pyogenes (Sp)Cas9 complexes with coupled repair oligos to SpCas9 complexes with uncoupled repair oligos (Fig. 2a). Notably, the correction efficiency (percentage of HDR in edited cells) with coupled complexes was significantly enhanced, reaching 22% with the 65-mers and 26% with the 81-mers (Fig. 2b, Suppl. Fig. 2a,b). In comparison to uncoupled complexes this represented a 12-and 4-fold increase, respectively (Fig. 2c).
Since it is conceivable that that the RNPD complex could dissociate from the target locus before repair of the DSB is initiated, we designed a two-component system in which the DSB is induced by the SpCas9 RNP complex, and the repair template is linked to a catalytically inactive Staphylococcus aureus (Sa)dCas9 that binds in close proximity to the targeted locus. As the inactive SadCas9 does not induce DSBs, its target sequence is not destroyed, thus avoiding dissociation of the repair template from the locus (Fig. 2d). To test whether this two-component system further enhances gene-repair efficiency, we cotransfected both complexes into the reporter cell line, and quantified HDR and NHEJ rates.
Notably, the correction efficiency increased to 30% with 65-mers, and to 33% with 81-mers ( Fig. 2e, Suppl. Fig. 2c,d), confirming our hypothesis that longer retention of the repair template at the DSB further enhances HDR rates.
In vivo, the delivery efficiency of RNPs and oligos is generally lower than in vitro.
Thus, if the repair template is not bound to Cas9, there is substantial probability that only one of the two components is delivered into the cell. In addition, at lower transfection efficiencies fewer repair templates are present in the nucleus and in close proximity to the targeted locus, potentially decreasing HDR rates. As we presumed that linking the repair oligo to Cas9 should largely overcome these issues, we investigated whether the repair efficiency with RNPD complexes is affected when transfected at 5-fold lower concentrations. Importantly, although under these conditions the correction efficiencies were generally lower with both bound-and unbound Cas9 complexes, the difference between the two systems was even more pronounced. Compared to the uncoupled RNP complex, the RNPD system yielded a 21-fold and a 4-fold increase in repair efficiency with 65-mer and 81-mer repair template oligos, respectively. Similarly, the two-component RNP-RNPD system led to a 23-fold increase with 65-mers and a 6-fold increase with 81-mers ( Fig. 2f,g, Suppl. Fig. 2e,f). Our results suggest that linking the repair template to the Cas9 complex leads to improved correction efficiency compared to the classical CRISPR/Cas system, and that this effect is   Mann-Whitney test was used for comparisons.

Methods
Please see Supplementary Tables 1-4 for a list of the DNA sequences used in this manuscript.
Plasmids: All plasmids used in this study are listed in Supplementary Table 4.
The green fluorescence signal of the SNAP-tag was detected with a UV transilluminator (Biorad). Subsequently, silver staining was completed using the Pierce™ Silver Stain Kit (Thermo Scientific) according to manufacturer instructions, and imaged with a UV transilluminator (Biorad).
Production of sgRNAs: sgRNAs were generated from DNA templates using the T7 RNA Polymerase (Roche) in vitro transcription (IVT) kit. In short, sgRNA specific primers that also contain the T7 sequence were annealed with a common reverse primer that contains the sequence of the sgRNA scaffold (final concentrations 10 µM). DNA was purified with the QIAquick purification (Qiagen) kit and eluted in DEPC-treated water. PCR products were run on agarose to estimate concentration and to confirm amplicon size. In vitro transcription was performed at 37°C overnight. For purification, DNase I was added to the sgRNAs and incubated for 15 minutes at 37°C, and subsequently ethanol precipitation was performed overnight at -20°C. The sgRNAs were then further purified using RNA Clean & Concentrators (Zymo Research). Before use, all sgRNAs were checked on denaturing 2% MOPS gels.
Complete sequences for all sgRNA protospacers and IVT primers can be found in Supplementary Table 1