Two strategies to generate cellular chain reactions using nCas9-CDA
We designed two systems to generate chain reactions using nCas9-CDA. In the first system, the T > C mutations are introduced in the scaffold region of sgRNA to disrupt its structure. First sgRNA without mutation (sgRNA-1) and nCas9-CDA correct the T > C mutations in the second sgRNA (sgRNA-2). The corrected sgRNA-2 in turn corrects the T > C mutations in the third sgRNA (sgRNA-3) in cooperation with nCas9-CDA (Fig. 1A). Because the sequence and the length of the stem-loop region in the sgRNA scaffold are not restrictive4,5, we can design multiple sgRNAs by introducing various sequences in the stem-loop region (XXXXX in sgRNA-2-MT, YYYYY in sgRNA-3-MT) (Fig. 1B).
Screening of inactive sgRNAs with scaffold mutations in yeast. (A) Schematic representation of the chain reaction with nCas9-CDA and sgRNAs with scaffold mutations. (B) Schematic representation of the sgRNA structure with the T > C mutation and specific sequences in the stem-loop region. (C) Schematic representation of TATAloxP structure and sequences. (D) Schematic representation of chain reaction with nCas9-CDA and TATAloxP with T > C mutations.
In the second system, the T > C mutations are introduced in the TATAloxP sequence (TATAloxP-MT). TATAloxP forms a 13-8-13 structure, which is composed of an 8-bp spacer sequence containing TATA box flanked on either side by two 13-bp inverted repeats (Fig. 1C). Cre recombinase binds to the 13 bp repeats of the TATAloxP, and importantly, this TATA box containing sequence can be incorporated into the U6 promoter to induce sgRNA expression6. The T > C mutations in the TATAloxP are corrected by the nCas9-CDA and the sgRNA (sgRNA-1) targeting the mutations and adjacent sequences (XXXXX in Fig. 1D). After the correction, a loxP flanked stop cassette is excised by Cre recombinase, which drives expression of the second sgRNA (sgRNA-2). The sgRNA-2 targets the next mutation and adjacent sequences (YYYYY in Fig. 1D), resulting in expression of the third sgRNA (sgRNA-3) (Fig. 1D). Because there is no restriction for the adjacent sequences, we can design multiple sgRNAs with various adjacent sequences that can be activated during the chain reaction.
Generation of inactive sgRNAs with T > C mutations in the scaffold region
To establish the first system, we introduced T > C mutations into the template sequence of the sgRNA scaffold region to inactivate it. Because nCas9-CDA was shown to efficiently induce C > T substitution in DNA when the cytosine was located 18 bp upstream of PAM sequence3, we also introduced PAM sequence (NGG) 18 bp downstream of the T > C mutation in each sgRNA template (Fig. 2A,B). We then evaluated the activity of each sgRNA with the T > C mutations and PAM insertion using the canavanine assay. Canavanine is a toxic analog of arginine and is imported into yeast cells via a transporter Can1. Therefore, expression of Cas9 and Can1-targeting sgRNA results in depletion of Can1 and decreases the sensitivity of yeast to canavanine (Fig. 2C)7. We used this assay to evaluate the effects of each T > C mutation on sgRNA inactivation and found that the T > C mutation at the fourth base resulted in sgRNA inactivation in yeast. Insertion of the PAM sequence did not inhibit sgRNA function in yeast (Fig. 2D,E).
Screening of inactive sgRNAs with scaffold mutations in yeast. (A) Schematic representation of the scaffold region of sgRNAs. (B) Schematic representation of part of the scaffold region of sgRNAs with T > C mutations and PAM insertion. (C–E) Schematic representation (C) and results of the canavanine assay with nCas9-CDA (D) or Cas9 (E). Yeasts transduced with the indicated sgRNA were grown on plates with (right column) or without (left column) canavanine.
We then examined the effects of T > C mutations on sgRNA function in mammalian cells. An EGFP mutant was used in which the start codon was mutated from ATG to GTG8. In this EGFP mutant, the template strand has a T > C mutation at the start codon and a PAM sequence at its 18 bp downstream. Therefore, nCas9-CDA and active sgRNA can correct the T > C mutation in the EGFP mutant and induce EGFP expression (Fig. 3A,B). However, the sgRNA did not work when a PAM sequence was inserted into the scaffold region of sgRNA, in contrast to the results of the canavanine assay (Fig. 3C). Because the weak interaction between G and U is known to be important for the maintenance of RNA structure9, we suspected that the loss of G-U interaction at the seventh base caused by PAM insertion might affect sgRNA function (Fig. 3D). Therefore, we introduced additional sequences near the PAM sequence to maintain the G-U interaction at the 7th base, because extending the stem-loop sequences in the sgRNA scaffold does not affect sgRNA function4,5. As expected, this optimized PAM-inserted sgRNA maintained the G-U interaction at the 7th base (Fig. 3D) and efficiently induced EGFP expression when expressed with nCas9-CDA and mutant EGFP in 293 T cells (Fig. 3E). We next examined whether the T > C mutation at the 4th base, which decreased sgRNA activity in yeast, also inactivated sgRNA in mammalian cells. Again, we obtained different results in mammalian cells from those in the canavanine assay. The sgRNA with a single T > C mutation at the fourth base did not inactivate GFP expression, indicating that it retained its normal function. Therefore, in addition to the 4th base, we introduced T > C mutations at 2, 3, or 5 bases into the sgRNA template. These sgRNAs with double T > C mutations at 2/4th, 3/4th, 4/5th bases lost the function to restore GFP expression in 293 T cells (Fig. 3F). We then performed similar experiments with the PAM-inserted sgRNAs and confirmed that the double T > C mutations at 3/4 bases, but not the single T > C mutation at the 4th base, resulted in loss of sgRNA function (Fig. 4A). Thus, we generated an inactive sgRNA with T > C mutations and PAM sequence in the scaffold region whose function can theoretically be restored by nCas9-CDA-induced base editing in mammalian cells.
Screening of inactive sgRNAs with scaffold mutations in mammalian cells. (A) Schematic representation of wild-type and mutant (ATG to GTG) EGFP with PAM sequence insertion. (B) Experimental scheme used in (C,E–F) and Fig. 4A. 293 T cells were transduced with the EGFP mutant. The mutant EGFP-expressing 293 T cells were then transfected with nCas9-CDA and sgRNAs targeting the T > C mutation. (C) Fluorescence images (left) and FACS plots (right) of 293 T cells expressing mutant EGFP and nCas9-CDA together with wild-type (top), PAM-inserted (middle), or no (bottom) sgRNA. (D) Schematic representation of wild-type (left) and PAM-inserted sgRNAs (original version: middle, optimized version: right). Note that the G-U interaction at the 7th base is lost in the original PAM-inserted sgRNA. The optimized version of the PAM-inserted sgRNA contains additional sequences in the stem-loop region to preserve the G-U interaction at the seventh base. (E) Fluorescence images (left) and FACS plots (right) of 293 T cells expressing mutant EGFP and nCas9-CDA together with wild-type (top), PAM-inserted (middle), or no (bottom) sgRNA. (F) Fluorescence images (left) and FACS plots (right) of 293 T cells expressing mutant EGFP and nCas9-CDA together with different sgRNAs. The indicated T > C mutations were introduced into the control sgRNA. mut; mutation.
Cellular chain reaction systems through sequential repair of sgRNA scaffold mutations. (A) Fluorescence images (left) and FACS plots (right) of 293 T cells expressing mutant EGFP and nCas9-CDA together with different sgRNAs. The indicated T > C mutations were introduced into the optimized PAM-inserted sgRNA. mut; mutation. (B) Schematic representation of the chain reaction by repair of sgRNA scaffold mutations to express EGFP in 293 T cells. (C) Experimental scheme as in (D). 293 T cells were transduced with mutant EGFP, nCas9-CDA and an EGFP-targeting sgRNA, then transfected with another sgRNA targeting the T > C mutation. (D) Fluorescence images (left) and FACS plots (right) of 293 T cells expressing mutant EGFP and nCas9-CDA together with the indicated sgRNAs. The sgRNA-1 converted the inactive sgRNA-2 to an active form, and the sgRNA-2 corrected the EGFP mutation to induce EGFP expression.
Establishment of a chain reaction by repair of sgRNA scaffold mutations
Next, we investigated whether the inactive sgRNA with T > C mutations can be converted to the active form by nCas9-CDA-induced base editing in mammalian cells. First, we transduced nCas9-CDA, the EGFP mutant, a PAM-inserted control or inactive sgRNA targeting the EGFP mutation, with or without the second sgRNA in 293 T cells. The second sgRNA was designed to correct the T > C mutations in the EGFP-targeting sgRNA and restore it to an active form (Fig. 4B,C). As expected, the optimized version of the PAM-inserted sgRNA efficiently corrected the EGFP mutation and induced GFP expression, whereas the version with T > C mutation did not. Importantly, coexpression of the second sgRNA restored the function of the inactive sgRNA and resulted in robust GFP expression in 293 T cells (Fig. 4D). Thus, the chain reaction was successfully established in mammalian cells with nCas9-CDA and two sgRNAs targeting the T > C mutation or EGFP mutation.
Generation of non-responsive TATAloxP sequences with T > C mutations
To establish the second system, we introduced T > C mutations into the TATAloxP sequences to make them insensitive to Cre-induced recombination. We generated several TATAloxP mutants in which a T base in the 13-bp repeat sequence (Fig. 1C) was replaced by C. These DNAs with different TATAloxP sequences at the 5′ or 3′ side (approximately 3500 bp-vector) were linearized and incubated with Cre recombinase. In this in vitro assay, Cre-induced recombination results in linear DNA of approximately 7000 bp when Cre recombinase can recognize the TATAloxP sites (Fig. 5A). As shown in Fig. 5B,C, the T > C mutation at the 13th base resulted in reduced recombination in both mutant/mutant and mutant/wild-type incubations.
In vitro Screening of non-responsive TATAloxP with T > C mutations. (A) Schematic representation of the in vitro recombination assay with TATAloxP sequences. (B,C) Linear DNAs with different TATAloxP sequences containing the indicated mutations were incubated with Cre recombinase. Note that TATAloxP with the T > C mutation at the first base probably did not efficiently produce recombined DNA in the first experiment because of technical errors (B), but did so very well in the second experiment (C). (NC; No loxP, PC; positive control DNA in Cre recombinase (NEB Catlog# M0298S)).
We next investigated whether the TATAloxP sequences containing the T > C mutations are resistant to Cre-mediated recombination in mammalian cells. We generated an expression vector containing a polyA signal flanked by two TATAloxP sites and a downstream EGFP cassette under the EF1α promoter in which one of the TATAloxP sites has the T > C mutations. If the mutated TATAloxP site is resistant to Cre-mediated recombination, EGFP is not expressed even in the presence of Cre recombinase (Fig. 6A). We expressed the TATAloxP (wild-type or mutant)-EGFP constructs in 293 T cells together with Cre-R32V, a mutant Cre recombinase with improved fidelity10 (Fig. 6B). However, in contrast to the results of the in vitro experiments, the T > C mutation at 13 base did not prevent Cre-R32V-induced EGFP expression in either or both 13-bp arms (Fig. 6C,D). Thus, the T > C mutation at the 13th base was not sufficient to render the TATAloxP sequence insensitive to Cre recombinase in mammalian cells. Because a previous report showed that double mutations in both 13-bp arms, particularly at the 7th, 8th, 11th, 12th, and 13th bases efficiently disrupted the loxP structure11, we subsequently examined the effect of double T > C mutations at the 11th and/or 13th bases on their responsiveness to Cre recombinase. Among the different combinations, we found that double T > C mutations at the 11th and 13th bases became resistant to Cre-induced EGFP expression in both arms (Fig. 6E,F,G). In this way, we generated a TATAloxP mutant which does not respond to Cre but will resume the responsiveness to Create by nCAS9-CDA-induced base editing in mammalian cells.
Screening of non-responsive TATAloxP with T > C mutations in mammalian cells. (A) Schematic representation of the EGFP cassette with mutated TATAloxP and polyA sequences under the EF1α promoter. (B) Experimental scheme used in (C,D). 293 T cells were transduced with the EGFP/TATAloxP cassette, and the mutant EGFP-expressing 293 T cells were subsequently transfected with Cre-R32V. (C,D) Fluorescence images of 293 T cells expressing EGFP/ various TATAloxP with the indicated T > C mutations and Cre-R32V. (E) Experimental scheme as in (F,G). 293 T cells were transfected with EGFP/TATAloxP cassette and Cre-R32V. (F,G) Fluorescence images (F) and FACS plots (G) of 293 T cells expressing EGFP/various TATAloxP with the indicated T > C mutations and Cre-R32V. WT; wild type.
Inefficient repair of TATAloxP mutations in the chain reaction system
Finally, we examined whether we could establish the cellular chain reaction system using the TATAloxp mutant. We transduced wild-type and nonresponsive TATAloxP-EGFP, Cre-R32V, and nCas9-CDA into 293 T cells with or without the second sgRNA. The second sgRNA was designed to correct the T > C mutations in the nonresponsive TATAloxP and convert it to a responsive form (Fig. 7A,B). Consistent with previous results, Cre-R32V induced recombination of only the wild-type TATAloxP but not the mutant TATAloxP to induce EGFP expression in 293 T cells (Fig. 7C). Unfortunately, coexpression of the second sgRNA failed to restore EGFP expression, indicating that the TATAloxP mutant with double T > C mutations at bases 11 and 13 is resistant to nCas9 DNA-mediated base editing. Thus, this TATAloxP system in its current form is not suitable for cellular chain reactions.
Cellular chain reaction systems through sequential repair of loxP mutations. (A) Schematic representation of the chain reaction by repair of TATAloxP mutations to express EGFP in 293 T cells. (B) Experimental scheme used in (C). 293 T cells were transduced with the EGFP/different TATAloxP cassette with the indicated T > C mutations, then infected with Cre-R32V, and then transfected with nCas9-CDA and sgRNA targeting the T > C mutation. (C) Fluorescence images of 293 T cells expressing EGFP/TATAloxP cassette, Cre-R32V, and nCas9-CDA along with the indicated sgRNAs. When the EGFP/TATAloxP cassette was used with T > C mutations at the 11th and 13th bases in both arms, no EGFP expression was detected, even in the presence of the TATAloxP-targeting sgRNA (bottom right). (D) Schematic representation of barcoding method.
