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HomeBiotechnologyHideRNAs defend in opposition to CRISPR-Cas9 re-cutting after profitable single base-pair gene...

HideRNAs defend in opposition to CRISPR-Cas9 re-cutting after profitable single base-pair gene enhancing


Re-cutting after profitable base-pair substitution

As a reporter for single base-pair substitution, we used mouse Embryonic Stem Cells (mESCs) carrying a single-copy sequence that encodes inexperienced fluorescent protein (GFP) however lacks a begin codon. Nevertheless, an in body AAG triplet is current, which permits for GFP expression upon conversion into ATG (Fig. 1A)26.

Determine 1
figure 1

Re-cutting after CRISPR-Cas9-mediated single-nucleotide substitution. (A) Within the Rosa26 locus of mouse Embryonic Stem Cells, the improved GFP gene (gray arrow) is built-in, whose exercise relies on conversion of an in-frame upstream AAG triplet (inexperienced rectangle) into ATG. A part of the reporter sequence is proven, with the AAG triplet marked in inexperienced. The underside sequence exhibits the PAM (underlined); the higher stand is hybridized to the gRNA spacer sequence (gentle grey). The 5′ G (marked in purple) of the RNA spacer doesn’t hybridize to the goal sequence. Grey triangles point out the place of the double strand break. (B) Cas9 nuclease supply system and ssODN restore templates. A puromycin acetyltransferase gene (encoding resistance to puromycin) was inserted into the Cas9/gRNA expression vector from Cong et al.1. Two 120 nt ssODN HDR templates have been used: ‘ATG’ instructs conversion of the AAG into ATG; ‘ATG + PAM’ is equivalent to ‘ATG’ but in addition instructs disruption of the PAM. (C) Proportion of GFP optimistic cells decided by Movement cytometry as a operate of the puromycin focus used to pick for transfected cells. Blue diamonds signify GFP percentages obtained with the ATG ssODN restore template; orange squares signify GFP percentages obtained with the ATG + PAM ssODN restore template. Error bars denote normal error of imply (n = 4). (D) Reconstruction of the goal sequence after HDR-templated base-pair substitution. The highest half exhibits that restore with ATG ssODN template nonetheless permits gRNA hybridization regardless of the one T-U mismatch. Restore with the ATG + PAM ssODN template induced PAM disruption, precluding Cas9-mediated re-cutting.

To check single base-pair substitution by single-stranded oligodeoxyribonucleotide (ssODN)-templated homology-directed restore (HDR) of a DSB, we designed a single information RNA (i.e., a fusion between crRNA and tracrRNA, right here abbreviated as gRNA) that directs SpCas9 cleavage near the AAG triplet (Fig. 1A). This gRNA was expressed from a vector that additionally encoded SpCas9 and puromycin acetyltransferase (the latter permitting enrichment of vector-transfected cells by puromycin choice) (Fig. 1B). We designed a 120 nt ssODN to template restore and direct the conversion of AAG to ATG. This conversion alters the protospacer by 1 bp (Fig. 1B). We transfected the vector and the ssODN restore template into the mESC reporter line, making use of totally different concentrations of puromycin (Fig. 1B). Quantification of the fraction of GFP-positive cells by circulation cytometry confirmed a rise in HDR effectivity with elevated puromycin concentrations as much as 3.6 μg/ml (Fig. 1C). Nevertheless, HDR effectivity decreased when additional rising puromycin focus.

Cas9 can induce DSBs at goal sequences that differ barely from the protospacer sequence, and that is stimulated by elevated Cas9 concentrations. In our system, excessive concentrations of puromycin might choose for prime Cas9 ranges, which can promote re-cutting of the ATG containing sequence regardless of the imperfect match with the guideRNA (Fig. 1D). Whereas profitable conversion of AAG into ATG by HDR will activate GFP, subsequent re-cutting and NHEJ-mediated restore might disrupt this sequence and shift the ATG out of body or take away it fully, leading to lack of GFP-expressing cells. To forestall this, the experiment was repeated with an ssODN template instructing an extra, PAM disrupting mutation (Fig. 1B). We now noticed elevated nucleotide substitution ranges that didn’t decline at elevated puromycin concentrations (Fig. 1C). This experiment demonstrates {that a} Cas9 goal web site of which the protospacer had been modified by HDR might nonetheless be cleaved by Cas9, and repaired in an error susceptible trend. Nevertheless, this may be prevented by the introduction of an extra Cas9 goal web site disrupting (TSD) mutation.


The introduction of a PAM mutation is very efficient in stopping re-cutting, however is usually undesirable, e.g., when it causes a non-synonymous codon substitution. We due to this fact sought alternative routes to guard the efficiently modified goal web site from promiscuous Cas9 exercise. We hypothesized {that a} guideRNA with a truncated spacer sequence that completely matches the modified goal sequence might promote tight binding of inactive Cas9 to the mutated sequence, thus conferring efficient safety in opposition to re-cutting (Fig. 2A). To research if this technique certainly prevents re-cutting and disruption of the repaired GFP reporter, we designed 4 truncated gRNAs that we name “hideRNAs” (hRNAs). The hRNAs consisted of truncated spacer sequences that have been on the identical strand as the unique full-length gRNA, however included the AAG > ATG mutation (Fig. 2B). The protospacers of hRNAs 1 and a pair of (size 12 and 16 nt), have been related to the identical PAM because the gRNA (PAM1), whereas hRNAs 3 (10 nt) and 4 (15 nt) have been related to the adjoining PAM2 (Fig. 2B).

Determine 2
figure 2

Safety in opposition to re-cutting by hRNAs. (A) Higher half: Cas9 in advanced with a full-length gRNA (represented as purple, curving line) can nonetheless induce a break regardless of the presence of a spacer-protospacer mismatch (blue rectangle). Decrease half: a gRNA with truncated protospacer that has full homology to the goal sequence from the highest half stably binds this sequence, however doesn’t induce a break. It hides the goal web site from Cas9 certain to a full-length gRNA. (B) hRNAs with matching, truncated protospacer sequence defend the modified reporter sequence from re-cutting. hRNAs 1 and a pair of use the unique PAM (higher half); hRNAs 3 and 4 use an adjoining PAM. The beginning codon is marked inexperienced/purple. Notice that the sequence of the hRNA protospacers permits hybridization to the T of the ATG, (marked in inexperienced), whereas a T-U mismatch is shaped with the protospacer of the gRNA (indicated in gentle gray). (C) Experimental setup for evaluating the re-cutting prevention functionality of hRNAs. A vector expressing a hRNA and a gRNA is mixed with an ATG-inducing ssODN. (D) GFP percentages obtained with 6 totally different circumstances: gRNA + re-cutting-sensitive (ATG) or -protected (ATG + PAM) template; gRNA + hRNA 1–4 plus re-cutting delicate (ATG) template. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Welch one-sided take a look at comparability with ‘ATG ssODN’ situation, shade signifies for which situation this is applicable). Error bars denote normal error of imply (n = 3). (E) The safety rating was calculated as follows: (Gi − Gsens)/(GPAM − Gsens), with Gi the proportion of GFP optimistic cells for a pattern, Gsens the proportion of GFP optimistic cells obtained with the gRNA and the re-cutting delicate template, and GPAM the proportion of GFP optimistic cells obtained with the gRNA and the re-cutting-protected template. Colours of every bar as in (D).

The 4 hRNAs have been cloned individually into the vector from Fig. 1B adjoining to the gRNA expression cassette (Fig. 2C). Along with an HDR template instructing the ATG-creating nucleotide change however not the PAM-disrupting nucleotide change, we launched these vectors into mESCs and diverse Cas9/gRNA/hRNA ranges by choosing with 3 totally different puromycin concentrations (Fig. 2C). We then in contrast the variety of GFP-positive cells to that obtained with the experimental circumstances from Fig. 1B (solely gRNA + ATG or ATG + PAM ssODN). Strikingly, we noticed that the presence of hRNAs strongly improved the restoration of GFP-positive cells with the ATG ssODN, reaching ranges as excessive as obtained with the ATG + PAM ssODN (Fig. 2D). This impact was not brought on by the utilization of plasmid-borne hideRNA encoding sequences as a template for HDR as omission of the ssODN didn’t yield GFP-positive cells above background (Supplementary Fig. 1). We calculated a “safety rating” for every hRNA, 0 being no safety and 1 being as a lot safety as a PAM disrupting mutation (Fig. 2E). Whereas we discovered safety scores round 0.5 for the totally different hRNAs at 1.8 μg/ml puromycin, safety was maximal (round 1) for the best puromycin focus examined. This demonstrates that the launched ATG mutation might successfully be protected in opposition to re-cutting by any of the 4 hRNAs, obviating the requirement for an extra PAM-disrupting mutation.

HideRNA parameters

The 4 hRNAs examined appeared to carry out equally effectively, regardless of being related to two totally different PAMs and having totally different protospacer lengths. To determine parameters for finest hRNA safety, we designed extra hRNAs for the PAM utilized by the unique gRNA sequence, and two different PAMs on the identical strand (Fig. 3A) and diverse the size of the spacer. The safety scores (Fig. 3B) of those hRNAs have been decided in the identical means as in Fig. 2E. At intermediate puromycin focus (3.6 μg/ml), most hRNAs had safety scores round 0.6. On the highest puromycin focus (10.8 μg/ml), we noticed safety scores approaching 1 for hRNAs related to the 2 PAMs closest to the mutation. Spacer size appeared much less crucial (8–15 nucleotides have been equally efficient). The safety rating of hRNAs related to the PAM most distal from the mutation was not elevated at this puromycin focus. Curiously, a pointy cut-off was seen for hRNAs with spacer lengths > 15 nt, exhibiting no safety (16 nt) and even destructive safety scores (17 nt). Presumably this displays a DSB inducing functionality of those hRNAs or elevated binding to the non-modified sequence inflicting competitors with the gRNA.

Determine 3
figure 3

Parameters affecting hRNA efficiency. (A) Three hRNAs utilizing totally different PAMs (1, 2 and three, marked blue, purple and inexperienced, respectively) and ranging protospacer lengths have been designed to guard the activated GFP reporter in opposition to re-cutting. A part of the GFP reporter sequence after restore with the ATG solely template is proven (T of ATG marked inexperienced) and the PAMs. A part of the spacer sequence of the hRNAs that concentrate on PAMs 1–3 is proven, with the A that marks their specificity marked in inexperienced. (B) Safety scores of hRNAs 1–3 with totally different spacer lengths decided at three totally different puromycin concentrations. If the primary nucleotide of the protospacer was not a G, a G was added to the sequence. The hRNA protospacer size doesn’t take a mismatching 5′ G into consideration. Error bars denote normal error of imply (n = 3).

Apart from hiding the modified goal sequence, two non-specific mechanisms might contribute to lowered re-cutting. First, hideRNA-bound Cas9 is unavailable for binding gRNA. Thus, competitors between hRNA and gRNA binding to Cas9 might cut back Cas9:gRNA re-cutting exercise, regardless of the protospacer sequence of the hRNA. To find out if this “sponge impact” existed, we examined the safety effectivity of 6 hRNAs with protospacer sequences unrelated to the unique goal web site. Two hRNAs confirmed roughly 20% of the complete safety obtained with a PAM-disrupting mutation, albeit at solely one of many puromycin concentrations used (Supplementary Fig. 2). This indicated that often competitors for Cas9 binding between hRNA and gRNA might decrease re-cutting incidence, though the contribution of this mechanism is modest. The second cause for decreased re-cutting exercise could also be associated to cytotoxic or growth-retarding results incited by the binding of enormous numbers of inactive Cas9 complexes to the DNA. Upon expression of some, however not all hRNAs, we observed decreased numbers of dwelling cells (Supplementary Fig. 3). If progressively greater mobile hRNA expression results in reducing cell viability, the surviving cell inhabitants might harbor a decrease mobile focus of catalytically energetic RNP with an in accordance discount in re-cutting propensity. Nevertheless, we didn’t observe a correlation between safety effectivity and cell numbers, indicating this impact didn’t considerably contribute to hRNA safety.

These outcomes point out that other than sequence specificity, additional necessities for efficient hRNAs are relaxed: totally different spacer lengths and totally different PAMs carry out equally effectively in opposition to re-cutting. In lots of circumstances, solely the PAM that’s a part of the unique goal web site will probably be out there and this presents good safety. Safety appears most effective at excessive nuclease concentrations.

Safety of a mutation exterior the unique goal web site

We subsequent investigated whether or not hRNAs might additionally defend mutations that don’t modify the unique Cas9 goal web site. We designed an ssODN restore template to insert a TGG triplet that creates an in-frame ATG upstream of the gRNA goal web site thus leaving the PAM and protospacer sequence intact (Fig. 4A). We designed an analogous oligonucleotide template with extra PAM disrupting mutations (ATG + PAM). We hypothesized that HDR templated by the ‘ATG’ solely ssODN would end in GFP expression, however subsequent re-cutting within the downstream unaltered Cas9 goal web site might end in end-joining-mediated disruption of GFP expression. Certainly, in comparison with the unprotected ATG template, the ‘ATG + PAM’ template elevated the restoration of GFP-positive cells roughly twofold (Fig. 4B). We designed two hRNAs (A and B) utilizing totally different PAMs (Fig. 4A) and examined if these have been in a position to forestall re-cutting. Strikingly, we noticed safety by hRNA A was as environment friendly as that obtained with the ATG + PAM template (Fig. 4B). hRNA B, which utilized a PAM that was 3 bp additional away didn’t defend the modified allele from re-cutting. These outcomes show that hRNAs will be utilized to guard mutations which can be created exterior of the Cas9 goal web site.

Determine 4
figure 4

hRNAs defending a mutation exterior the goal web site. (A) High: sequences of the reporter earlier than (WT) and after ssODN-directed insertion of TGG (ATG) or TGG insertion plus PAM disruption (ATG + PAM). The ‘ATG’ template leaves the whole Cas9 goal web site intact and thus delicate to re-cutting. Backside: gRNA and hRNA A and B spacers are proven, interacting with the goal sequence after TGG insertion. (B) HideRNAs can defend a mutation that’s exterior of the Cas9 goal web site. The chances of GFP-expressing cells are scored after puromycin choice at 3 totally different concentrations for combos of Cas9 and the gRNA, the ATG, ATG + PAM ssODN template and hRNA (A) or (B). Error bars denote normal error of imply (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Welch one-sided take a look at, comparability with the ATG ssODN template).

HideRNA effectivity at endogenous genes

We subsequent assessed the generalizability of hRNAs for the safety of small mutations launched with CRISPR-Cas9. We chosen 15 naturally occurring variants within the human DNA MMR genes Mut S homolog 2 and 6 (MSH2, MSH6) and Mut L homolog 1 (MLH1) that may be generated by 1 nt adjustments that additionally create Sort II restriction enzyme websites. For every mutation, information RNAs have been designed to introduce a DSB near the meant mutation. We designed ‘unprotected templates’: 90 nucleotide lengthy ssODN templates equivalent to the goal sequence apart from the specified single nucleotide substitution (SNS). We additionally designed templates with a goal web site disrupting mutation (TSD), which have been equivalent to the SNS templates however included extra PAM or protospacer disrupting mutations (exemplified in Fig. 5A). These extra mutations didn’t disrupt the restriction enzyme web site, and the place inside 5 bp of the DSB, as we beforehand discovered that longer distance might negatively have an effect on the effectivity with which these extra mutations are launched into the genome. TSDs have been synonymous in 8 circumstances, non-synonymous in 5 circumstances and affecting an intronic sequence in 2 circumstances. As well as, we designed hRNAs for cover of every SNS. These hRNAs had the identical PAM sequence because the gRNA and their protospacer was shortened to a ten–15 nucleotide sequence previous the PAM and preferentially beginning with a guanosine. If no pure guanosine was current, a 12 or 14 nucleotide sequence was chosen and supplied with a guanosine on the 5’ facet. Cassettes expressing these hRNAs have been cloned into their respective gRNA vectors to yield 14 vectors that expressed a gRNA and a hRNA. We then transfected 293FT cells with gRNA expressing vectors and ssODN templates with the meant SNS solely or with an extra TSD. As well as, we transfected the 293FT cells with the gRNA- and hRNA-encoding vector and the SNS-only template (Fig. 5B). After transfection, we chosen for uptake of the vector with 7.2 μl/ml puromycin. PCR fragments containing the goal locus have been incubated with the suitable restriction enzyme, and the fraction of DNA that contained the modification was quantified (Fig. 5C). Digestion of the goal locus, indicating HDR occasions had taken place ranged from 1 to 12% (the proportion of digested DNA of the overall DNA). For every locus, we calculated a max-normalized HDR effectivity for the three totally different circumstances, which is proven in Fig. 5D.

Determine 5
figure 5

hRNA safety at endogenous loci. (A) Introduction of 1 nt substitution within the MSH6 DNA MMR gene that ends in a motif acknowledged by the Sort II restriction endonuclease NaeI. High: the wild-type sequence with single-letter amino acid code. The PAM is indicated with an orange line. Center: one nucleotide change (in blue) altering the encoded amino acid from proline to alanine. As well as, an NaeI web site is created (indicated with a black line). Backside: extra, silent mutations have been instructed to disrupt the protospacer. Notice that PAM disruption on this case would have disrupted the NaeI web site. (B) Three totally different transfections have been carried out for every mutation: gRNA + template instructing a single nucleotide change (SNS), gRNA + template instructing SNS and extra Cas9 goal web site disrupting (TSD) mutations, or gRNA + hRNA + template instructing SNS. (C) Experimental workflow. Quantification of the response merchandise was carried out utilizing the Caliper GX. (D) HideRNAs can be utilized to guard mutations generated at endogenous loci. On the left, the gene identify and meant codon substitution is proven. For every mutation, 4 sequences are proven: the wildtype genomic CRISPR-Cas9 goal sequence (WT), the sequence the place the SNS has been launched (marked with a blue ‘M’), the sequence with the SNS plus extra goal web site disruption (marked with purple ‘TSD’), the hRNA spacer sequence. Dots denote sequence identification; dashes point out that this sequence isn’t a part of the hRNA protospacer. The scores are calculated based mostly on the chances of digested DNA. Quantities of minimize and uncut DNA have been quantified on the labchip caliper GX, and the fraction of DNA that contained a Sort II restriction web site was calculated as: (minimize DNA/all DNA)*100%. The experiment was carried out thrice. Inside every replicate, a ‘max-normalization’ was carried out—fraction of DNA was divided by the worth of the pattern with the biggest quantity of digested DNA. Max normalizations for all replicates have been averaged and proven with SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Welch one-sided take a look at comparability with ‘M’ (no safety) situation, except in any other case indicated).

In solely two circumstances (MSH6 V878A and MSH2 H639Q), the restoration of HDR occasions didn’t profit from TSD or hRNA safety. The remaining circumstances benefited from TSD (6/14 circumstances) and/or from hRNA safety (8/14 circumstances). Considerably remarkably, solely 2 circumstances overlapped, i.e., benefited from each, TSD and hRNA safety (MLH1 K52E and G67V). Thus, in 4 circumstances (MSH2 E188Q, MLH1 Q346R, MSH2 T33P and MSH6 G39E) hRNAs didn’t attain the advance achieved by TSD. Conversely, in 7 circumstances (MLH1 K52E, N338S, F99I, G67V and L555P, and MSH6 P623A, and MSH2 D38A) hRNAs provided the perfect safety, yielding 2–10fold enchancment over the unprotected state of affairs. Importantly, whereas in two circumstances goal web site disruption appeared to scale back effectivity with respect to the unprotected state of affairs (MSH6 V878A: p = 0.07; MLH1 F99I; p = 0.11), the addition of a hRNA both gave the identical effectivity (6/14 circumstances) or improved the effectivity 2–10fold (8/14 circumstances).

HideRNAs enhance the fraction of HDR in mouse zygotes

Delicate gene modification in mice can these days effectively be achieved by injecting zygotes with Cas9 ribonucleoprotein complexes concentrating on the genomic DNA together with oligonucleotides templating the site-specific modification. Additionally right here, TSDs are sometimes added to enhance the restoration of gene-edited animals, however this may occasionally have undesired results. We due to this fact investigated whether or not hRNAs might be used to extend the frequency of ssODN-templated single-nucleotide substitution in murine zygotes. We designed guideRNAs, oligonucleotides and hRNAs to generate three totally different level mutations within the murine Mlh1 gene, representing variants of unsure significance present in suspected Lynch Syndrome sufferers (Fig. 6A). GuideRNA and hRNA Cas9 ribonucleoprotein (RNP) complexes have been assembled in separate reactions. For each level mutation, we carried out two rounds of mouse zygote injections: guideRNA RNPs with oligonucleotide and guideRNA plus hRNA RNPs with oligonucleotide (Fig. 6B). Injected zygotes have been cultured till blastocyst stage and the fraction of HDR modification among the many recovered alleles was decided by Sanger sequencing. It was obvious that almost all of chromatograms consisted of blended sequences. To quantify the contribution of the sequence ensuing from HDR, we algorithmically deconvoluted chromatograms with TIDER (Fig. 6C)27 and ICE (Supplementary Fig. 4A) (Hsiau et al., bioRxiv 251082, accessed 20190609). Each algorithms decide fractions of wildtype, HDR-derived and indel-containing sequences. There was appreciable overlap between the group of blastocysts that might be efficiently deconvoluted by both algorithm (Supplementary Fig. 4B), however the variety of deconvolutions with an R2 exceeding 0.8 was greater for the TIDER algorithm. The proportion of HDR alleles might be decided for 109 blastocysts out of 239 by each algorithms with an R2 of at the least 0.8 for every particular person chromatogram (Supplementary Fig. 4B), whereas the settlement between the 2 algorithms had an R2 of 0.91 (Supplementary Fig. 4C). Nevertheless, in some circumstances, TIDER detected HDR alleles as much as 20% that weren’t seen with ICE (Supplementary Fig. 4C).

Determine 6
figure 6

hideRNA safety in zygotes. (A) Sequences of wildtype (WT), oligonucleotide template (ssODN) and information and conceal RNAs for 3 totally different Mlh1 mutations. (B) For every mutation two rounds of zygote injections have been carried out: ssODN plus RNP with guideRNA or ssODN plus RNP with guideRNA and RNP with hRNA. Injected zygotes have been cultured to blastocyst stage and analyzed for the presence of the mutant allele by Sanger sequencing. (C) The proportion of HDR-deriving alleles was calculated per embryo by the TIDER algorithm. Horizontal bars point out the typical share of mutant allele. Variety of blastocysts analyzed: R691W information, n = 24; R691W information + conceal, n = 29; A21V information, n = 12; A21V information + conceal, n = 21; D304V information, n = 7; D304V information + conceal, n = 31. ****p < 0.00001 (Welch one-sided take a look at). (D) Examples of the chromatograms produced by Sanger sequencing blastocyst gDNA from guide-only and guideRNA + hRNA injections to make the D304V mutation. The arrow signifies the place of the base-change achieved upon HDR with the oligonucleotide template and the black line signifies the PAM of the information and hRNA (the orientation of the chromatogram sequence is complementary to the one depicted in A). With information solely, only one blastocyst confirmed HDR.

The proportion of HDR alleles per blastocyst discovered by TIDER is proven in Fig. 6C, whereas an analogous graph with ICE information, making use of to fewer blastocysts, is proven in Supplementary Fig. 4A). Usually the proportion of HDR alleles was decrease than 50%, indicating that HDR occasions or re-cutting occasions disrupting HDR alleles occurred at or past the 2-cell stage. Each, TIDER and ICE have a detection restrict round 5%. Once we solely take into account HDR percentages above 5%27 as reliably indicating profitable base substitution, in response to each, TIDER and ICE, the D304V substitution strongly benefited from the co-injection of a hRNA, with the presence of HDR alleles above 5% rising from 0 (0 out of seven) to ± 45% of the blastocysts (Fig. 6C and Supplementary Fig. 4A). The chromatograms in Fig. 6D spotlight the hideRNA impact on the extent of particular person blastocysts. The HDR mutation is simply seen in one of many chromatograms of the three best-scoring blastocysts from the hideRNA-free situation (Information solely). Conversely, within the chromatograms of the three best-scoring blastocysts situation with hideRNA supplementation, the wildtype nucleotide is nearly absent (Information plus conceal). The profit for R691W and A21V was modest, if current, with TIDER suggesting a slight profit for A21V (2/22 to 4/21) (Fig. 6C) and ICE suggesting a slight profit for R691W (1/19 to five/29) (Supplementary Fig. 4A).

These outcomes show that hRNAs not solely can work with plasmid-based supply of CRISPR-Cas9 elements, but in addition with RNP complexes injected into zygotes, albeit not with 100% effectivity. The addition of a hRNA might thus be helpful for the technology of transgenic mice carrying solely a single base-pair substitution.




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