The DOI matches its Crossref record; 73 of the 74 references it cites were found in a registry or an index; 29 of the 37 sources its claims cite were checked for support.
A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells
Open accessAbstract
Translation of the CRISPR–Cas9 system to human therapeutics holds high promise. However, specificity remains a concern especially when modifying stem cell populations. We show that existing rationally engineered Cas9 high-fidelity variants have reduced on-target activity when using the therapeutically relevant ribonucleoprotein (RNP) delivery method. Therefore, we devised an unbiased bacterial screen to isolate variants that retain activity in the RNP format. Introduction of a single point mutation, p.R691A, in Cas9 (high-fidelity (HiFi) Cas9) retained the high on-target activity of Cas9 while reducing off-target editing. HiFi Cas9 induces robust AAV6-mediated gene targeting at five therapeutically relevant loci (HBB, IL2RG, CCR5, HEXB, and TRAC) in human CD34+ hematopoietic stem and progenitor cells (HSPCs) as well as primary T cells. We also show that HiFi Cas9 mediates high-level correction of the sickle cell disease (SCD)-causing p.E6V mutation in HSPCs derived from patients with SCD. We anticipate that HiFi Cas9 will have wide utility for both basic science and therapeutic genome-editing applications. A bacterial screen yields a Cas9 variant that retains high on-target activity when delivered in the RNP format. As proof of principle, this Cas9 variant enables high-level correction of the sickle cell disease mutation in patient-derived HSPCs.
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Claims the paper makes
12 Own result · 6 Partly supported · 4 Supported · 1 No full text of the sources · 1 Own method · accuracy not yet measured
The cited sources support only part of this claim — read each source’s verdict below.
In the paper
“Repurposing bacterial “CRISPR” systems to facilitate RNA-guided cleavage of complementary DNA has revolutionized the field of gene editing in mammalian cells 1–4.”
§ A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human haematopoietic stem and progenitor cells > INTRODUCTION
What the sources say
- Partly supportedcites [1] Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012). sourceExperimental — accuracy not yet measured
The paper’s Abstract reports RNA-guided Cas9 cleavage of complementary DNA, and its Conclusions propose the system’s potential for genome editing. It does not report editing in mammalian cells or state that this application had revolutionized the field; the reported cleavage experiments used DNA substrates in vitro. (LLM, conf=0.88)
- Supportedcites [2] Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013). source
The Abstract reports engineering prokaryotic CRISPR/Cas systems so that RNA-guided Cas9 cleaves genomic targets in human and mouse cells. It describes this as a new class of genome-engineering tools with wide applicability in mammalian cells; “revolutionized” is the citing authors’ characterization of that significance. (LLM, conf=0.88)
- Not checked — no full text of the sourcecites [3] Doudna, J. A. & Charpentier, E. Genome editing. The new frontier of genome engineering with CRISPR–Cas9. Science 346, 1258096 (2014). source
- Supportedcites [4] Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPR–Cas9 for genome engineering. Cell 157, 1262–1278 (2014). source
The Introduction identifies Cas9 as an RNA-guided nuclease derived from a microbial CRISPR system. “CRISPR-Cas9: From Yogurt to Genome Editing” reports that bacterial CRISPR systems were engineered to direct DNA cleavage and genome editing in mammalian cells, followed by rapid adoption. The paper thus supports the citing sentence’s broad characterization, though it does not use the word “revolutionized.” (LLM, conf=0.91)
- Partly supported
Every source the paper cites for this claim was checked and supports it.
In the paper
“If a homologous donor DNA template is provided during the DSB repair, the homologous recombination (HR) repair pathway can generate more precise changes to the target DNA, including single nucleotide changes to insertion of large gene cassettes8,9.”
§ A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human haematopoietic stem and progenitor cells > INTRODUCTION
What the sources say
- Supportedcites [8] Porteus, M. H. Towards a new era in medicine: therapeutic genome editing. Genome Biol. 16, 286 (2015). source
The development of genome editing and the contemporary toolbox states that HR repair of a double-strand break using a provided donor can make precise changes ranging from a single base to a large gene cassette. Box 1 explains that the donor provides the homologous template used during repair. (LLM, conf=0.94)
- Supportedcites [9] Dever, D. P. & Porteus, M. H. The changing landscape of gene editing in hematopoietic stem cells: a step towards Cas9 clinical translation. Curr. Opin. Hematol. 24, 481–488 (2017). source
The section on genome-editing reagents states that HR repair of a double-strand break using an exogenous donor with homology arms can introduce specific DNA changes. It also says an rAAV6 donor can mediate precise nucleotide changes and insert gene cassettes up to 5.7 kb. No page number is provided in our copy. (LLM, conf=0.93)
- Supported
Every source the paper cites for this claim was checked and supports it.
In the paper
“The GUIDE-seq procedure10 can be used to empirically identify real off-target sites in cells.”
§ A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human haematopoietic stem and progenitor cells > RESULTS > Quantitative reduction in off-target editing using the HiFi mutant
What the sources say
- Supportedcites [10] Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR–Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015). source
The Discussion describes GUIDE-Seq as an experimental method that identifies bona fide off-target sites genome-wide. It also states that GUIDE-Seq is performed on living cells, matching the claim that it can empirically identify actual off-target sites in cells. (LLM, conf=0.94)
- Supported
The cited sources support only part of this claim — read each source’s verdict below.
In the paper
“RNP delivery results in an initially high level of the genome editing machinery followed by rapid decay, leading to a “fast on, fast off” strategy that results in highly efficient editing while minimizing OTEs13–19.”
§ A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human haematopoietic stem and progenitor cells > INTRODUCTION
What the sources say
- Partly supportedcites [13] Gundry, M. C. et al. Highly efficient genome editing of murine and human hematopoietic progenitor cells by CRISPR/Cas9. Cell Reports 17, 1453–1461 (2016). sourceExperimental — accuracy not yet measured
The Discussion (page unavailable) describes RNP electroporation as a transient “hit-and-run” approach that enables efficient editing and reduces concerns about off-target cleavage. The paper does not establish the claimed initially high level followed by rapid decay of editing machinery, or demonstrate that this time course causes fewer off-target effects. (LLM, conf=0.85)
- Supportedcites [14] Gundry, M. C. et al. Technical considerations for the use of CRISPR/Cas9 in hematology research. Exp. Hematol. 54, 4–11 (2017). source
The ‘Cas9 and sgRNA reagents and delivery’ section says RNP delivery produces editing rates exceeding 85% in hematopoietic cells and may minimize off-target effects by delivering only a brief pulse of Cas9. The ‘CRISPR/Cas9 for gene disruption’ summary likewise says the RNP’s short half-life minimizes off-target cleavage. No page numbers are provided in our copy. (LLM, conf=0.91)
- Partly supportedcites [15] Dever, D. P. et al. CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells. Nature 539, 384–389 (2016). sourceExperimental — accuracy not yet measured
The Abstract reports efficient HBB editing in human HSPCs using Cas9 RNPs, and the Extended Data report greater on-target versus off-target specificity for RNP delivery than for an all-RNA system. Neither passage establishes an initially high level of editing machinery followed by rapid decay, or shows that this timing causes the improved specificity. (LLM, conf=0.86)
- Partly supportedcites [16] DeWitt, M. A. et al. Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells. Sci. Transl. Med. 8, 360ra134 (2016). sourceExperimental — accuracy not yet measured
The Discussion reports efficient editing in human HSPCs with minimal genic off-target activity and notes that RNP delivery has been associated with reduced off-target effects. But our copy does not establish an initial high level followed by rapid decay, or show that such a pulse causes fewer off-target effects. The Results also report substantial activity at one intergenic off-target site, so the off-target finding needs that qualification. (LLM, conf=0.86)
- Supportedcites [17] Hultquist, J. F. et al. A Cas9 ribonucleoprotein platform for functional genetic studies of HIV–host interactions in primary human t cells. Cell Reports 17, 1438–1452 (2016). source
The Introduction (page unavailable) describes Cas9 RNP delivery as transient, says it enables high-efficiency editing, and identifies low rates of off-target editing as an advantage over traditional approaches. This supports the attributed brief-delivery, efficient-editing, low-off-target account. (LLM, conf=0.88)
- Partly supportedcites [18] Schumann, K. et al. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. Proc. Natl. Acad. Sci. USA 112, 10437–10442 (2015). sourceExperimental — accuracy not yet measured
The Discussion (page unavailable) describes fast editing, rapid Cas9 turnover and efficient editing in human T cells. The Results (page unavailable) found rare indels at two predicted off-target sites at rates comparable to controls. But the paper does not establish that RNP delivery minimizes off-target effects compared with other delivery methods; the Discussion says further testing is needed. (LLM, conf=0.86)
- Supportedcites [19] Kim, S., Kim, D., Cho, S. W., Kim, J. & Kim, J. S. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24, 1012–1019 (2014). source
The Results sections on kinetics and off-target effects report that RNPs begin editing almost immediately, Cas9 is barely detectable after 24 hours, and editing remains highly efficient while off-target mutations are reduced relative to plasmid delivery. This supports the citing sentence’s “fast on, fast off” paraphrase. (LLM, conf=0.94)
- Partly supported
Every source the paper cites for this claim was checked and supports it.
In the paper
“While cleavage at sites with perfect complementarity to the protospacer domain of the gRNA typically occurs with highest efficiency, cleavage at sites having one to several bases of mismatch with the gRNA can occur. These undesired cleavage events are called “off-target effects” (OTEs)10, and their existence should be minimized for therapeutic applications of genome editing technologies.”
§ A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human haematopoietic stem and progenitor cells > INTRODUCTION
What the sources say
- Supportedcites [10] Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR–Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015). source
The Abstract describes unintended cleavage at off-target sites with protospacer mismatches and says therapeutic use requires understanding off-target effects to minimize harmful outcomes. The Results section on off-target sequence characteristics likewise reports cleavage at sites with several guide–protospacer mismatches. The paper also identifies other kinds of off-target sites, so mismatched sites are an example rather than an exhaustive definition. (LLM, conf=0.91)
- Supported
The cited sources support only part of this claim — read each source’s verdict below.
In the paper
“AAV6 homologous donor DNA templates are an efficient way to deliver donor templates for sensitive primary immune cells, including HSPCs and T-Cells15,35–40.”
§ A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human haematopoietic stem and progenitor cells > RESULTS > The R691A HiFi Cas9 mutant delivered as RNP is highly active and specific in CD34+ HSPCs
What the sources say
- Partly supportedcites [15] Dever, D. P. et al. CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells. Nature 539, 384–389 (2016). sourceExperimental — accuracy not yet measured
The section “CRISPR/Cas9 targeted HBB gene editing in HSPCs” reports high HSPC transduction and stable GFP expression in an average of 29% of HSPCs after delivery of homologous ssAAV6 donors. The paper supports the HSPC part of the claim, but the passages found do not establish efficient donor delivery in T cells. (LLM, conf=0.88)
- Partly supportedcites [35] Wang, J. et al. Homology-driven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors. Nat. Biotechnol. 33, 1256–1263 (2015). sourceExperimental — accuracy not yet measured
The Results section ('HDR-mediated editing in HSPCs by ZFN mRNA and AAV6 donors'; page unavailable) identifies AAV6 as an effective vehicle for delivering homologous donor templates to CD34+ HSPCs. The paper also reports T cells arising after edited HSPCs were transplanted, but does not establish efficient donor-template delivery directly into T cells. That extension is not supported by this source. (LLM, conf=0.88)
- Partly supportedcites [36] Wang, J. et al. Highly efficient homology-driven genome editing in human T cells by combining zinc-finger nuclease mRNA and AAV6 donor delivery. Nucleic Acids Res. 44, e30 (2016). sourceExperimental — accuracy not yet measured
The Results (page unavailable) state that AAV6 vectors effectively delivered homologous donor DNA templates to CD4+ and CD8+ T cells. The paper does not establish the same result for HSPCs, so the claim’s inclusion of that cell population is unsupported by this source. (LLM, conf=0.88)
- Partly supportedcites [37] De Ravin, S. S. et al. Targeted gene addition in human CD34+ hematopoietic cells for correction of X-linked chronic granulomatous disease. Nat. Biotechnol. 34, 424–429 (2016). sourceExperimental — accuracy not yet measured
The Methods section (AAV6 Donor Construction and Virus Production; page unavailable) describes AAV donor constructs flanked by homology arms. The results report efficient targeted integration in primary human HSPCs using AAV6 donor delivery. Our copy does not report AAV6 donor delivery in T cells, so it supports the HSPC example but not the T-cell example. (LLM, conf=0.87)
- Supportedcites [38] Sather, B. D. et al. Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template. Sci. Transl. Med. 7, 307ra156 (2015). source
The Discussion (page unavailable) identifies AAV6 as the optimal serotype for primary T cells and CD34+ progenitors and says it was used successfully to deliver rAAV donors in these cells. It also discusses donors with homology arms and reports approximately 14% HDR in CD34+ cells, though this was less robust than in T cells. (LLM, conf=0.90)
- Partly supportedcites [39] Eyquem, J. et al. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature 543, 113–117 (2017). sourceExperimental — accuracy not yet measured
The Methods (AAV and Gene targeting; page not provided) describe an AAV6 donor with homologous TRAC sequences delivered to primary T cells. A results passage indexed under Abstract (page not provided) reports more than 40% CAR knock-in, supporting efficiency in T cells. The paper does not demonstrate this approach in HSPCs, so that extension is unsupported by this source. (LLM, conf=0.88)
- Not checked — no full text of the sourcecites [40] Schiroli, G. et al. Preclinical modeling highlights the therapeutic potential of hematopoietic stem cell gene editing for correction of SCID-X1. Sci. Transl. Med. 9, eaan0820 (2017). source
- Partly supported
The cited sources support only part of this claim — read each source’s verdict below.
In the paper
“Recent studies have shown that disease-causing genetic errors can be corrected using CRISPR/Cas9 in long-term engrafting HSPCs that are preserved in downstream effector lineages15,16,24,25.”
§ A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human haematopoietic stem and progenitor cells > INTRODUCTION
What the sources say
- Partly supportedcites [15] Dever, D. P. et al. CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells. Nature 539, 384–389 (2016). sourceExperimental — accuracy not yet measured
The “HBB gene correction in SCD HSPCs” section reports correction of the disease-causing E6V mutation in patient-derived HSPCs and expression of corrected β-globin in erythrocytes differentiated from them in vitro. The “HBB targeting in long-term repopulating HSCs” section reports edited cells in downstream lineages and secondary transplant recipients, but those experiments tracked HBB targeting with reporter donors, not correction of the disease-causing mutation. The paper therefore does not show that mutation-corrected cells themselves persisted through long-term engraftment into downstream lineages. (LLM, conf=0.90)
- Partly supportedcites [16] DeWitt, M. A. et al. Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells. Sci. Transl. Med. 8, 360ra134 (2016). sourceExperimental — accuracy not yet measured
The Results sections report CRISPR/Cas9 correction of the sickle mutation in patient HSPCs and, separately, durable editing after HSPC engraftment in mice. But the engraftment experiment edited wild-type HSPCs to carry the sickle mutation, rather than testing corrected patient HSPCs in vivo. The paper therefore does not show that correction of a disease-causing mutation persisted in downstream lineages after long-term engraftment. (LLM, conf=0.91)
- Not checked — no full text of the sourcecites [24] De Ravin, S. S. et al. CRISPR–Cas9 gene repair of hematopoietic stem cells from patients with X-linked chronic granulomatous disease. Sci. Transl. Med. 9, eaah3480 (2017). source
- Partly supportedcites [25] Traxler, E. A. et al. A genome-editing strategy to treat β-hemoglobinopathies that recapitulates a mutation associated with a benign genetic condition. Nat. Med. 22, 987–990 (2016). sourceExperimental — accuracy not yet measured
The main text (page unavailable) reports CRISPR/Cas9 editing of CD34+ HSPCs and increased fetal hemoglobin in their cultured erythroid progeny. It does not show correction of the disease-causing mutation or long-term engraftment; the Methods describe in-vitro differentiation rather than a transplantation study. (LLM, conf=0.88)
- Partly supported
The cited sources support only part of this claim — read each source’s verdict below.
In the paper
“Accordingly, several reports have shown efficient Glu6Val correction in SCD-HSPCs, although correction efficiency can sometimes be linked with off-target INDELs in the same edited population15,16,45,46.”
§ A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human haematopoietic stem and progenitor cells > RESULTS > The R691A HiFi Cas9 delivered as RNP mediates high-level Glu6Val correction while reducing off-target activity in sickle cell disease-CD34+ HSPCs
What the sources say
- Partly supportedcites [15] Dever, D. P. et al. CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells. Nature 539, 384–389 (2016). sourceExperimental — accuracy not yet measured
The ‘HBB gene correction in SCD HSPCs’ section reports correction of an average of 50% of E6V alleles in patient-derived HSPCs, so this paper supports the efficient-correction part as one of the cited reports. The Extended Data describe off-target INDEL measurements in CD34+ HSPCs, but do not link those INDELs to correction in the same SCD-HSPC population; the INDELs reported alongside SCD correction were measured at the on-target HBB locus. No page numbers are available in our copy. (LLM, conf=0.85)
- Partly supportedcites [16] DeWitt, M. A. et al. Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells. Sci. Transl. Med. 8, 360ra134 (2016). sourceExperimental — accuracy not yet measured
The Results section on correcting SCD HSPCs reports up to 25% of alleles corrected to wild type. A separate Results section on editing CD34+ HSPCs reports off-target indels, including 44% at an intergenic site, but does not establish that those indels occurred in the same SCD-HSPC population whose mutation was corrected. The paper therefore supports efficient correction and an off-target concern, but not that specific linkage. (LLM, conf=0.83)
- Partly supportedcites [45] Hoban, M. D. et al. Correction of the sickle cell disease mutation in human hematopoietic stem/progenitor cells. Blood 125, 2597–2604 (2015). sourceExperimental — accuracy not yet measured
The Gene correction in sickle BM section reports correction of the sickle mutation in patient-derived HSPCs. The Comprehensive ZFN specificity assessment reports off-target modification at the δ-globin gene in SCD bone-marrow CD34+ cells, but the passages do not establish that those off-target modifications were specifically INDELs; the indels shown alongside correction in Figure 5 are at the target β-globin cut site. (LLM, conf=0.83)
- Not checked — no full text of the sourcecites [46] Hoban, M. D. et al. CRISPR/Cas9-mediated correction of the sickle mutation in human CD34+ cells. Mol. Ther. 24, 1561–1569 (2016). source
- Partly supported
The cited sources support only part of this claim — read each source’s verdict below.
In the paper
“The lesion can be healed by the error-prone Non-Homologous End-Joining (NHEJ) pathway, which often results in creation of an insertion/deletion (INDEL) of one to several bases7.”
§ A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human haematopoietic stem and progenitor cells > INTRODUCTION
What the sources say
- Partly supportedcites [7] Kass, E. M. & Jasin, M. Collaboration and competition between DNA double-strand break repair pathways. FEBS Lett. 584, 3703–3708 (2010). sourceExperimental — accuracy not yet measured
Section 4, “Repair by non-homologous end joining” (page not provided), says that processing DNA ends during NHEJ can produce insertions and deletions, making it more error-prone than homologous recombination. It does not establish that INDELs occur often or that they are typically one to several bases long. (LLM, conf=0.87)
- Partly supported
Every source the paper cites for this claim was checked and supports it.
In the paper
“Cas9/gRNA is directed to search for NGG protospacer adjacent motifs (PAMs) in the genome and if sufficient complementarity exists between the guide and the target, conformational changes occur and the now fully active Cas9 cleaves the DNA, resulting in a targeted double-stranded break (DSB)5,6.”
§ A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human haematopoietic stem and progenitor cells > INTRODUCTION
What the sources say
- Supportedcites [5] Dagdas, Y. S. et al. A conformational checkpoint between DNA binding and cleavage by CRISPR–Cas9. Sci. Adv. 3, eaao0027 (2017). source
The Introduction states that S. pyogenes Cas9 target recognition requires an NGG PAM and guide-RNA base pairing, and that its nuclease domains cleave both DNA strands. The Results (Fig. 4G) describe a conformational checkpoint: sufficient RNA–DNA complementarity permits the HNH domain to enter its catalytically active conformation before cleavage. (LLM, conf=0.92)
- Supportedcites [6] Sternberg, S. H., LaFrance, B., Kaplan, M. & Doudna, J. A. Conformational control of DNA target cleavage by CRISPR–Cas9. Nature 527, 110–113 (2015). source
The paper’s discussion of its model says Cas9 identifies potential targets through PAM binding and guide–DNA complementarity, then undergoes an HNH conformational change that enables coordinated cleavage of both DNA strands. Extended Data Table 2 identifies the PAM as 5′-NGG-3′. (LLM, conf=0.92)
- Supported
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
The source backs part of the claim, not all of it — or backs it for a different population, condition or comparison.
In the citing paper
“It worth mentioning that in several previous reports, LZ3 Cas9, Sniper Cas9, and HiFi Cas9 had comparable editing activity to the SpCas9-WT in human cells or other animal embryos.”
From Machine learning-based prediction models to guide the selection of Cas9 variants for efficient gene editing (2024) · cites this paper as [17]
Experimental — accuracy not yet measuredThe source backs part of the claim, not all of it — or backs it for a different population, condition or comparison.
In the citing paper
“To further increase on-target efficiency and minimize off-target editing, S.p. HiFi Cas9 Nuclease was used77.”
From The APOE-R136S mutation protects against APOE4-driven tau pathology, neurodegeneration, and neuroinflammation (2023) · cites this paper as [77]
Experimental — accuracy not yet measuredThe cited source says what the claim attributes to it.
In the citing paper
“Compared to SpCas9, HiFi Cas9 shows similar editing efficiency but lower off-target effects in some genome sites.”
From Machine learning-based prediction models to guide the selection of Cas9 variants for efficient gene editing (2024) · cites this paper as [17]
In the citing paper
“The Cas9 nuclease is directed by a single-guide RNA (sgRNA) to modify a specific chromosomal DNA sequence by inducing a sequence-specific double-strand break (DSB) (3, 4).”
From CRISPR-Cas9 genome editing using targeted lipid nanoparticles for cancer therapy (2020) · cites this paper as [4]
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
Each contradiction between two passages listed here was confirmed by an independent check.
Rated minor: it would not touch a result — a value no result rests on, a p-value that stays on the same side of the significance level, or a conflict that is clerical, resolved elsewhere in the paper, or outside its own findings.
In the paper
§ A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human haematopoietic stem and progenitor cells > RESULTS > The R691A high fidelity (HiFi) Cas9 mutant demonstrates superior on-target editing with RNP delivery
“HypaCas9 (R692A/M694A/Q695A/H698A)28”
§ A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human haematopoietic stem and progenitor cells > DISCUSSION
“HypaCas9, which included 4 mutations (N692A/M694A/Q695A/H698A) in the REC3 domain.”
Rated minor: it would not touch a result — a value no result rests on, a p-value that stays on the same side of the significance level, or a conflict that is clerical, resolved elsewhere in the paper, or outside its own findings.
In the paper
§ A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human haematopoietic stem and progenitor cells > RESULTS > The R691A high fidelity (HiFi) Cas9 mutant demonstrates superior on-target editing with RNP delivery
“HypaCas9 (R692A/M694A/Q695A/H698A)28.”
§ A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human haematopoietic stem and progenitor cells > DISCUSSION
“HypaCas9, which included 4 mutations (N692A/M694A/Q695A/H698A) in the REC3 domain.”
Citations
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Liu, Q., Li, X., Xu, H., Luo, Y., Cheng, L., Liang, J., He, Y., Liu, H., Fang, J., & Huang, J. (2025) · Advanced Biotechnology · doi:10.1007/s44307-024-00053-5 · Cited by 6
“…the functional reparation frequency of homozygous HBB CD41-42 (-TCTT) in patient-derived HSPCs was 49.92% ± 4.31% in vitro without selection, surpassing the HDR efficiency reported in most existing studies (Azhagiri et al. 2021; Park et al. 2019; Pavel-Dinu et al. 2019; Vakulskas et al. 2018).”
InfluentialNoel, E. A., Sahu, S. U., Wyman, S. K., Krishnappa, N., Jeans, C., & Wilson, R. C. (2025) · The CRISPR Journal · doi:10.1089/crispr.2024.0080 · Cited by 6
Mapping the therapeutic landscape of CRISPR-Cas9 for combating age-related diseases
He, Q., Wang, Y., Tan, Z., Zhang, X., Yu, C., & Jiang, X. (2025) · Frontiers in Genome Editing · doi:10.3389/fgeed.2025.1558432 · Cited by 6
Weaponizing CRISPR/Cas9 for selective elimination of cells with an aberrant genome
Tavella, S., di Lillo, A., Conti, A., Iannelli, F., Mancheno-Ferris, A., Matti, V., Micco, R. D., & di Fagagna, F. D. (2025) · DNA repair · doi:10.1016/j.dnarep.2025.103840 · Cited by 6
Chemical Modifications in Nucleic Acid Therapeutics
Lennox, K. A., Young, R. C., & Behlke, M. A. (2025) · Methods in molecular biology · doi:10.1007/978-1-0716-4742-4_3 · Cited by 8
Self-citationCD44-targeted lipid nanoparticles for enhanced CRISPR/Cas9 delivery in cancer gene editing
Zeng, Y., Zhao, G., Wu, S., Bochuan, H., Forn‐Cuní, G., Knol, R. A., Ghalbzouri, A. E., Snaar-Jagalska, E. B., & Kros, A. (2026) · Journal of Controlled Release · doi:10.1016/j.jconrel.2025.114598 · Cited by 6
Structural basis of supercoiling-induced CRISPR–Cas9 off-target activity
Smith, Q. M., Whittle, S., Aramayo, R. J., Rollins, D. E., Jalal, A. S. B., Egharevba, D. I., Morris, K. L., Pyne, A. L. B., & Rueda, D. (2026) · Nature · doi:10.1038/s41586-026-10255-7 · Cited by 10
“Understanding the molecular basis of off-target activity has led to the development of various high-fidelity Cas9 nucleases that reduce off-target effects 7,27,48,49 .”
Singh, S., Pugliano, C. M., Honaker, Y., Laird, A., DeGottardi, M. Q., Lopez, E., Lachkar, S., Stoffers, C. M., Sommer, K., Khan, I., et al. (2023) · Molecular Therapy — Methods & Clinical Development · doi:10.1016/j.omtm.2023.101183 · Cited by 7
Bhavanee, K. N. D., Krishnamoorthi, A., Rathva, H. M., Mareguddikar, S. C., Singh, A., Singh, B. P., Nageshwar, & Chittibomma, K. (2024) · Journal of Advances in Biology & Biotechnology · doi:10.9734/jabb/2024/v27i2702 · Cited by 11
CRISPR/nCas9-Edited CD34+ Cells Rescue Mucopolysaccharidosis IVA Fibroblasts Phenotype
Herreño-Pachón, A. M., Leal, A. F., Khan, S. A., Alméciga-Díaz, C. J., & Tomatsu, S. (2025) · International Journal of Molecular Sciences · doi:10.3390/ijms26094334 · Cited by 6
Gnanapragasam, M. N., Planutis, A., Glassberg, J., & Bieker, J. J. (2023) · Scientific Reports · doi:10.1038/s41598-023-34805-5 · Cited by 6
Gene and epigenetic editing in the treatment of primary ciliopathies
Molinari, E., & Sayer, J. A. (2021) · Progress in molecular biology and translational science · doi:10.1016/bs.pmbts.2021.01.027 · Cited by 6
Genome editing for blood disorders: state of the art and recent advances
Romito, M., Rai, R., Thrasher, A. J., & Cavazza, A. (2019) · Emerging Topics in Life Sciences · doi:10.1042/etls20180147 · Cited by 7
Reduced off-target effect of NG-BE4max by using NG-HiFi system
Shan, H., Liu, Z., Jia, Y., Chen, S., Chen, M., Song, Y., Sui, T., Lai, L., & Li, Z. (2021) · Molecular Therapy — Nucleic Acids · doi:10.1016/j.omtn.2021.05.012 · Cited by 6
Omura, S. N., & Nureki, O. (2025) · Methods in enzymology on CD-ROM/Methods in enzymology · doi:10.1016/bs.mie.2025.01.052 · Cited by 5
Kinney, K. J., Jia, K., Zhang, H., Schmaljohn, E., Osborne, T., Thommandru, B., Murugan, K., Peña, A. S., West, S. C., Chen, S., et al. (2026) · Nature Communications · doi:10.1038/s41467-026-74623-7 · Cited by 6
Self-citationYang, G., Deng-gao, W., Zhao, X., Hao, D., Zhang, Z.-Q., & Liu, D. (2025) · Molecular Therapy — Methods & Clinical Development · doi:10.1016/j.omtm.2025.101542 · Cited by 6
Skov, T. W., Wolff, J. H., Haslund, D., Revenfeld, A. L., van de Venn, L., Dorset, S. R., Ravendran, S., Jørgensen, S. E., Thomsen, M. K., Holm, M., et al. (2025) · Molecular Therapy · doi:10.1016/j.ymthe.2025.07.038 · Cited by 6
Self-citationWolff, J. H., Skov, T. W., Haslund, D., Dorset, S. R., Revenfeld, A. L., Aussel, C., Jørgensen, S. E., Holm, M., Thomsen, M. K., Ammann, S., et al. (2025) · Nature Communications · doi:10.1038/s41467-025-62738-2 · Cited by 9
Self-citationGuarding the genome: strategies for off-target detection and minimization in CRISPR/Cas9 system
Lotfi, M., Yazdi, Z. F., Hashemi, M. R., Abbaszadegan, M. R., & Sharif, S. (2025) · Expert Review of Molecular Diagnostics · doi:10.1080/14737159.2025.2581677 · Cited by 6
“Additional SpCas9 variations include SpCas9-HFI [103], eSpCas9 [104], Hypa-Cas9 [109], Sniper-Cas9 [113], HiFi Cas9 [108], SpG and PAM-less SpRY [108] have been engineered to minimize genome-wide off-target effects with high precision.”
Ali, A. M., Özdemirli, M., Hamilton, M. P., & Jain, M. D. (2026) · Nature Reviews Clinical Oncology · doi:10.1038/s41571-026-01147-w · Cited by 7
“A related but distinct approach involves transient delivery of genome-editing machinery, such as Cas9 ribonucleoprotein complexes, which achieves permanent genome editing while limiting the duration of nuclease activity in the cell, thereby reducing off-target cleavage risk 147 .”
Peña-Gutiérrez, I., Olalla-Sastre, B., Rı́o, P., & Rodríguez-Madoz, J. R. (2024) · Cytotherapy · doi:10.1016/j.jcyt.2024.10.010 · Cited by 5
Microfluidic tool for rapid functional characterization of CRISPR complexes
Peleg-Chen, D., Shuvali, G., Brio, L., et al. (2022) · New Biotechnology · doi:10.1016/j.nbt.2022.01.003 · Cited by 5
Trakarnsanga, K., Thongsin, N., Metheetrairut, C., et al. (2022) · Scientific Reports · doi:10.1038/s41598-022-19934-7 · Cited by 5
Enabling Precision Medicine with CRISPR-Cas Genome Editing Technology: A Translational Perspective
Ilahibaks, N. F., Hulsbos, M. J., Lei, Z., Vader, P., & Sluijter, J. P. G. (2022) · Advances in experimental medicine and biology · doi:10.1007/978-981-19-5642-3_20 · Cited by 7
CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation
Ma, L., Jang, L., Chen, J., Song, J. W., Yang, D., Zhang, J., Chen, Y. E., & Xu, J. (2019) · Journal of Visualized Experiments · doi:10.3791/59512 · Cited by 5
Nguyen, D. N., Roth, T. L., Li, J., Chen, P. A., Mamedov, M. R., Vo, L. T., Tobin, V. R., Apathy, R., Goodman, D. B., Shifrut, E., et al. (2019) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/591719 · Cited by 5
CRISPR-enhanced human adipocyte browning as cell therapy for metabolic disease
Tsagkaraki, E., Nicoloro, S. M. C., DeSouza, T., Solivan-Rivera, J., Desai, A., Lifshitz, L. M., Shen, Y., Kelly, M., Guilherme, A., Henriques, F. D. S., et al. (2021) · RePEc: Research Papers in Economics · Cited by 5
PAM-Flexible Genome Editing with an Engineered Chimeric Cas9Preprint
Koseki, S., Hong, L., Yudistyra, V., et al. (2023) · Research Square · doi:10.21203/rs.3.rs-2625838/v1 · Cited by 5
“To generate SpRYc, the N-terminal ORF of Sc++ (Addgene Plasmid #155011), corresponding to residues (1-1119) was PCR amplified and assembled using Gibson Assembly into the pCMV-T7-SpRY-P2A-EGFP backbone (Addgene Plasmid #139989), preserving residues 1111-1368 of SpRY's ORF.”
Luna, S. E., Camarena, J., Hampton, J. P., Majeti, K. R., Charlesworth, C. T., Soupène, E., Selvaraj, S., Jia, K., Sheehan, V., Cromer, M. K., et al. (2023) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2023.08.04.552064 · Cited by 5
Self-citationPharmacological control of CAR T cells through CRISPR-driven rapamycin resistancePreprint
Levesque, S., Carleton, G. A., Duque, V., Goupil, C., Fiset, J.-P., Villeneuve, S., Normandeau, É., Morin, G., Dumont, N., Nelson, B. H., et al. (2023) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2023.09.14.557485 · Cited by 4
CRISPR technology for diagnosis and treatment of human brucellosis
Lau, C., Li, X., Liang, Q., Guo, R., Ren, X., Xu, Z., Zhu, Y., Lai, Y., Liu, G., Huang, Y., et al. (2025) · Journal of Microbiological Methods · doi:10.1016/j.mimet.2025.107339 · Cited by 4
Viruses Are Part of the Holobiont’s Fitness and Evolution
ROSENBERG, E. B. (2021) · The microbiomes of humans, animals, plants, and the environment · doi:10.1007/978-3-030-65317-0_7 · Cited by 5
Zang, S.-S., Zhang, R., Zhang, J.-R., Zhang, X., & Li, J. (2025) · Human Gene Therapy · doi:10.1089/hum.2024.262 · Cited by 6
Optimizing gene editing in pigs: The role of electroporation and lipofection
Piñeiro-Silva, C., & Gadea, J. (2025) · Animal Reproduction Science · doi:10.1016/j.anireprosci.2025.107874 · Cited by 4
Gene delivery to tackle diabetic nephropathy
Kumawat, A., Raval, N., Reddy, G., et al. (2020) · Elsevier eBooks · doi:10.1016/b978-0-12-814455-8.00015-3 · Cited by 21
Shor, O., Rabinowitz, R., Offen, D., & Benninger, F. (2022) · Computational and Structural Biotechnology Journal · doi:10.1016/j.csbj.2022.04.026 · Cited by 4
“1) [27], SpCas9-HF1 [28], HypaCas9 [29], evoCas9 [30], Sniper-Cas9 [31], Hifi-Cas9 [32] and LZ3 Cas9 [26].”
Long, J. D., Trope, E. C., Yang, J., Rector, K., & Kuo, C. Y. (2022) · Hematology/Oncology Clinics of North America · doi:10.1016/j.hoc.2022.03.011 · Cited by 4
A Practical Guide for CRISPR-Cas9-Induced Mutations in Axolotls
Sousounis, K., Courtemanche, K., & Whited, J. L. (2022) · Methods in molecular biology · doi:10.1007/978-1-0716-2659-7_22 · Cited by 4
Frankenstein Cas9: engineering improved gene editing systems
Vos, P. D., Filipovska, A., & Rackham, O. (2022) · Biochemical Society Transactions · doi:10.1042/bst20220873 · Cited by 3
Pooled screening with next-generation gene editing tools
Zhou, L., Yang, L., Feng, Y., & Chen, S. (2023) · Current Opinion in Biomedical Engineering · doi:10.1016/j.cobme.2023.100479 · Cited by 4
The Development of SpCas9 Variants with High Specificity and Efficiency Based on the HH Theory
Wang, G., Wang, C., Wu, X. J., Chu, T., Huang, D., & Li, J. (2023) · Molecular Biology · doi:10.1134/s0026893324010187 · Cited by 4
Uchigashima, M., & Mikuni, T. (2024) · Frontiers in Neural Circuits · doi:10.3389/fncir.2024.1523614 · Cited by 5
Blood cancer therapy with synthetic receptors and CRISPR technology
Zhang, H., Zhong, M., Zhang, J., & Chen, C. (2025) · Leukemia Research · doi:10.1016/j.leukres.2025.107646 · Cited by 4
Zhang, D., Parth, F., da Silva, L. M., Ha, T.-C., Schambach, A., & Boch, J. (2025) · Genome biology · doi:10.1186/s13059-025-03478-w · Cited by 6
“To address the Cas-dependent off-targets, high-fidelity Cas9 variants can be incorporated into the CBE architectures to replace the wild-type nickase Cas9 [53– 58].”
Modelling the Cancer Phenotype in the Era of CRISPR-Cas9 Gene Editing
Stewart, J., Banerjee, S. N., Pettitt, S. J., & Lord, C. J. (2019) · Clinical Oncology · doi:10.1016/j.clon.2019.09.057 · Cited by 4
High level of fetal-globin reactivation by designed transcriptional activator-like effector
Zhan, J., Irudayam, M. J., Nakamura, Y., Kurita, R., & Nienhuis, A. W. (2020) · Blood Advances · doi:10.1182/bloodadvances.2019000482 · Cited by 4
CRISPR-Cas Gene Perturbation and Editing in Human Induced Pluripotent Stem Cells
van Essen, M. J., Riepsaame, J., & Jacob, J. (2021) · The CRISPR Journal · doi:10.1089/crispr.2021.0063 · Cited by 4
Matsuzaki, S., Sakuma, T., & Yamamoto, T. (2024) · In Vitro Cellular & Developmental Biology - Animal · doi:10.1007/s11626-024-00850-1 · Cited by 4
“2016; Vakulskas et al .”
CRISPR-Cas Based Precision Breeding in Date Palm: Future Applications
Sattar, M. N., Iqbal, Z., & Al-Khayri, J. M. (2021) · Compendium of plant genomes · doi:10.1007/978-3-030-73750-4_9 · Cited by 5
An overview of OECD activities related to modern techniques of biotechnology and genome editing
Kearns, P. (2019) · Transgenic Research · doi:10.1007/s11248-019-00131-8 · Cited by 4
“To secure the safety of these applications especially in medical treatments, a number of studies are being carried out to develop genome editing tools with higher accuracy and efficacy (Gaudelli et al. 2017; Kleinstiver et al. 2016; Kimberland et al. 2018; Vakulskas et al. 2018).”
BackgroundThe CRISPR System and Cancer Immunotherapy Biomarkers
Balan, V., & Wang, J. (2019) · Methods in molecular biology · doi:10.1007/978-1-4939-9773-2_14 · Cited by 4
Inter-cellular mRNA Transfer Alters Human Pluripotent Stem Cell StatePreprint
Yoneyama, Y., Zhang, R., Kimura, M., Cai, Y., Adam, M., Parameswaran, S., Masaki, H., Mizuno, N., Bhadury, J., Maezawa, S., et al. (2024) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2024.06.27.600209 · Cited by 4
“…pEF-SpCas9HiFi-2APuro-U6-gRNA, which is modified from the original pCS2-U6-gRNA-FN-Cas9-N-P2A-Puro(56) (kindly provided by S. Takahashi, The University of Tokyo, Japan) where the CMV promoter was swapped with EF1 a and introduction of R691A mutation into SpCas9 for high-fidelity Cas9 protein(57).”
Maroofi, N., Maleki, M. S. M., Tahmasebi, M., Khorshid, H. R. K., Modaberi, Y., Najafipour, R., & Banan, M. (2024) · Molecular Biotechnology · doi:10.1007/s12033-024-01155-0 · Cited by 3
“It has been determined that use of the engineered high fidelity (HiFi) Cas9 in an RNP format can greatly reduce off-target activity [43].”
Advances and challenges of CRISPR/Cas gene editing for corneal diseases
Han, Y., Chen, R., & Shentu, X. (2025) · Advances in Ophthalmology Practice and Research · doi:10.1016/j.aopr.2025.11.002 · Cited by 4
“103,104 Compact CRISPR/Cas9d system monitors sgRNA-DNA heteroduplex complementarity, resulting in a lower mismatch tolerance compared to SpCas9.”
Gong, J. D., Samarkhazan, H. S., Siavashi, M., Servatian, N., & Pirsavabi, F. (2025) · Molecular Biology Reports · doi:10.1007/s11033-025-11235-2 · Cited by 5
Lee, E.-C., Kim, K., Kim, S., Kim, M., & Kim, H. (2025) · Nucleic Acids Research · doi:10.1093/nar/gkaf1404 · Cited by 3
“…9] ribonucleoprotein (RNP) complexes containing Alt-R CRISPR/Cas9 guide RNAs (Integrated DNA Technologies, Inc.; Supplementary Table S4) and TrueCut™ Cas9 Protein v2 (Invitrogen, A36496) were delivered into CD4 + T cells using the 4D nucleofector™ platform (Lonza), as described previously [36].”
In vitro and in vivo base editing of CCR5 in hematopoietic stem cells confers HIV-1 resistance
Anderson, A. K., Georgakopoulou, A., Kuhlmann, A.-S., Wang, H., Riker, A. O., Karuppusamy, K. V., Radtke, S., Bui, J. K., Kiem, H., Lieber, A., et al. (2026) · Molecular Therapy · doi:10.1016/j.ymthe.2026.03.018 · Cited by 3
Omega-based mini prime- and base-editing systems in Escherichia coli
Liu, L.-H., Zhang, Y., Bo, X. U., YunXia, L. I., Guo, Y., Lu, X., Zhang, Z., Lai, S., Ge, Q., Zhang, K., et al. (2026) · Trends in biotechnology · doi:10.1016/j.tibtech.2026.02.002 · Cited by 3
Chen, Y., Banie, L., Breyer, B. N., Tan, Y. L., Wang, Z., Zhou, F., Wang, G., Lin, G., Liu, J., Qi, L. S., et al. (2022) · The CRISPR Journal · doi:10.1089/crispr.2022.0009 · Cited by 3
Regulatory Role of DNA Methylation and Its Significance in Plants
Rehman, R. S., Ali, M., Zafar, S. A., Bashir, H., Pasha, A. N., Javed, A., Ashraf, N. A., & Shah, W. A. (2022) · Asian Journal of Biochemistry Genetics and Molecular Biology · doi:10.9734/ajbgmb/2022/v11i330267 · Cited by 3
“induced (PSI) genes, these genes are characterized by their proximity to stress responsive genes [102,109,115].”
BackgroundEisen, B., & Binah, O. (2023) · International Journal of Molecular Sciences · doi:10.3390/ijms24108657 · Cited by 4
Simmons, D. A., Selvaraj, S., Chen, T., Cao, G., Camelo, T. S., McHugh, T. L. M., Gonzalez, S., Martin, R. M., Simanauskaite, J., Uchida, N., et al. (2025) · Molecular Therapy — Methods & Clinical Development · doi:10.1016/j.omtm.2025.101415 · Cited by 3
Self-citationCD33 epitope editing unlocks UM171-expanded cord blood grafts for AML immunotherapy
Lehnertz, B., Langouët, M., Corneau, S., MacRae, T., Joe, M., Schmidt, E. N., Spinella, J.-F., Mayotte, N., Moore, S., Tellechea, M. F., et al. (2025) · Molecular Therapy · doi:10.1016/j.ymthe.2025.11.010 · Cited by 3
RNA-based CRISPR-Mediated Loss-of-Function Mutagenesis in Human Pluripotent Stem Cells
Leung, A. W., Broton, C., Bogacheva, M. S., Xiao, A., García‐Castro, M. I., & Lou, Y. (2020) · Journal of Molecular Biology · doi:10.1016/j.jmb.2020.04.017 · Cited by 3
Editing of Endogenous Genes in Cellular Immunotherapies
Roth, T. L. (2020) · Current Hematologic Malignancy Reports · doi:10.1007/s11899-020-00587-0 · Cited by 3
Mechanism of the CRISPR/Cas9 System Specificity in Genome Editing
Kulishova, L. M., Vokhtantsev, I. P., Kim, D. V., & Zharkov, D. O. (2023) · Molecular Biology · doi:10.31857/s0026898423020155 · Cited by 3
Innovations in Artificial Induction of Plant Genetic Diversity
Sharma, V., Kordrostami, M., Maan, S. S., Sarsu, F., & Penna, S. (2024) · Sustainable development and biodiversity · doi:10.1007/978-981-99-5245-8_8 · Cited by 5
Panda, G., & Ray, A. (2024) · International Journal of Biological Macromolecules · doi:10.1016/j.ijbiomac.2024.137835 · Cited by 3
Shams, F., Sharif, E., Abbasi‐Kenarsari, H., Hashemi, N., Hosseini, M. S., Heidari, N., Noori, E., Amini, A. H., Bazrgar, M., Rouhani, M., et al. (2025) · Current Gene Therapy · doi:10.2174/0115665232357078250331180413 · Cited by 6
Tapping into the Unsung Potential of CRISPR/CAS Technology in Agriculture
Rehman, R. S., Ali, M., Zafar, S. A., Ahmad, M., Pasha, A. N., Bashir, H., Rashid, F., & Hussain, M. (2022) · Asian Journal of Biochemistry Genetics and Molecular Biology · doi:10.9734/ajbgmb/2022/v10i430249 · Cited by 3
CRISPR technology for immuno-oncology applications
Wang, J., Balan, V., & Marincola, F. M. (2019) · Methods in enzymology on CD-ROM/Methods in enzymology · doi:10.1016/bs.mie.2019.05.018 · Cited by 3
Ex vivo gene therapy for lysosomal storage disorders: future perspectives
Poletto, É., Silva, A. O., Weinlich, R., Martin, P. K. M., Torres, D. C., Giugliani, R., & Baldo, G. (2023) · Expert Opinion on Biological Therapy · doi:10.1080/14712598.2023.2192348 · Cited by 3
“For example, HiFi Cas9 (R691A) has been shown to possess reduced off-target activity, while being capable of sustaining similar on-target levels as the wild-type SpCas9 [28].”
BackgroundInfluential[Development of CRISPR technology and its application in bone and cartilage tissue engineering].
Chen, G., Cheng, D., & Chen, B. (2019) · Europe PMC (PubMed Central) · doi:10.12122/j.issn.1673-4254.2019.12.19 · Cited by 3
“[67]Vakulskas CA, Dever DP, Rettig GR, et al.”
BackgroundA slow-exchange conformational switch regulates off-target cleavage by high-fidelity Cas9Preprint
Paula, V. S. D., Dubey, A., Arthanari, H., & Sgourakis, N. G. (2020) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2020.12.06.413757 · Cited by 2
Frame Editors for Precise, Template-Free FrameshiftingPreprint
Nakade, S., Nakamae, K., Tang, T., Yu, D., Sakuma, T., Yamamoto, T., & Lu, T. K. (2022) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2022.12.05.518807 · Cited by 2
Rabinowitz, R., Shor, O., Zerbib, J., Herman, S., Zelikson, N., Madiwale, S., Yom-Tov, N., Ben‐David, U., Benninger, F., & Offen, D. (2023) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2023.04.11.536265 · Cited by 3
Chu, S. N., Soupène, E., Wienert, B., Yin, H., Sharma, D., McCreary, T., Jia, K., Homma, S., Hampton, J. P., Gardner, J. M., et al. (2023) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2023.09.01.555926 · Cited by 3
Self-citationReeves, D. B., Rigau, D. N., Romero, A., et al. (2024) · medRxiv · doi:10.1101/2024.02.13.24302704 · Cited by 3
Catalytic-state structure of Candidatus Hydrogenedentes Cas12b revealed by cryo-EM studies
Li, Y., Li, J. J., Pei, X., Wei, J., Gan, J., & Lin, J. (2025) · Nucleic Acids Research · doi:10.1093/nar/gkaf519 · Cited by 2
“As a result, several SpCas9 variants have been identified, such as eSpCas9, HiFi Cas9, and SpCa9-HF1 [ 10 , 19 ].”
Knockout of chitin synthase gene confers resistance to Bt toxin Vip3Aa in Helicoverpa zea
Heu, C. C., Schutze, I. X., LeRoy, D. M., et al. (2025) · Pest Management Science · doi:10.1002/ps.70248 · Cited by 2
Guan, Y., Wen, R., Zhao, C., Zhang, L., Wu, F., Yang, F., & Chen, G. (2025) · Chemical Engineering Journal · doi:10.1016/j.cej.2025.171411 · Cited by 2
Screening strategy to identify Cas9 variants with higher HDR activity based on diphtheria toxin
Matsumoto, D., Kubota, K., Sato, Y., Kato-Inui, T., Nigorikawa, K., Miyaoka, Y., & Nomura, W. (2025) · Journal of Biomedical Science · doi:10.1186/s12929-025-01197-9 · Cited by 2
Abasic CRISPR RNAs inherently harness fidelity of SpCas9 for genome editing
Gu, D., Kim, G.-W. D., Park, M., Park, A. D., Lee, H.-S., Eom, S., Weon, H., Park, J., Lee, J., Ahn, S. H., et al. (2026) · Nature Chemical Biology · doi:10.1038/s41589-026-02139-8 · Cited by 2
Enhancing the specificity of gene editing outcomes by using Cas9 variants in porcine embryos
Kim, J., Yoon, J. D., Chen, J., Lee, J., Lee, H. J., Whitworth, K. M., Redel, B. K., Prather, R. S., & Lee, K. (2026) · Journal of Animal Science · doi:10.1093/jas/skag030 · Cited by 2
“…K1003A, and R1060A(HNH and RuvC-III domain of nuclease lobe), HiFi Cas9—R691A (REC3 Recognition domain), LZ3 Cas9 - N690C (REC3), T769I, G915M, and N980K (L1 and L2 roop and RuvC-III on nuclease lobe) (Slaymaker et al. 2016; Vakulskas et al. 2018a; Schmid-Burgk et al. 2020; Allemailem et al. 2023).”
du Rand, A., Buttle, B., & Sheppard, H. M. (2026) · Drugs · doi:10.1007/s40265-026-02285-2 · Cited by 2
“…the commercially available hfCas9 nuclease (available from Integrated DNA Technologies [IDT]) encompassing a single point mutation (p.R691A) was demonstrated to significantly reduce off-target editing to ~1% across multiple off-target loci, which showed up to 27% editing with wild-type Cas9 [91].”
Zarrinrad, G., Burkhardt, L.-M., Beltran-Mestres, C., Romero‐Suárez, S., Wagner, D. L., Schlickeiser, S., Stein, M., Wendering, D. J., de la Cebosa, I. J. C. D., Ehlen, L., et al. (2026) · Molecular Therapy Advances · doi:10.1016/j.omta.2026.201735 · Cited by 2
Self-citationCho, E., Lee, J., Kim, J., Choi, J., Kang, M., & Song, J. (2026) · Journal of Biological Engineering · doi:10.1186/s13036-026-00673-6 · Cited by 2
Omura, S. N., Nakagawa, R., Kajimoto, S., Okazaki, S., Ishiguro, S., Mori, H., Onishi, K., Kashiwakura, Y., Hiramoto, T., Horinaka, K., et al. (2026) · Nature Communications · doi:10.1038/s41467-026-71626-2 · Cited by 2
Siddika, A., Rousseau, J., Veillette, F., Bouchard, C., Lu, Y., & Tremblay, J. P. (2026) · Cells · doi:10.3390/cells15100852 · Cited by 2
“Catalytically modified forms of Cas9, including nickase (nCas9) and nuclease-dead Cas9 (dCas9), reduce or eliminate double-strand cleavage, thereby improving safety profiles [147,148].”
CRISPR-Cas orthologs and variants
Scollan, M. E. (2021) · Elsevier eBooks · doi:10.1016/b978-0-12-817876-8.00008-5 · Cited by 2
Reduced Expression of TMEM16A Impairs Nitric Oxide-Dependent Cl− Transport in Retinal Amacrine Cells
Rodriguez, T., Zhong, L., Simpson, H., & Gleason, E. L. (2022) · Frontiers in Cellular Neuroscience · doi:10.3389/fncel.2022.937060 · Cited by 2
Comprehensive UHPLC- and CE-Based Methods for Engineered Cas9 Characterization
Camperi, J., Console, G., Zheng, L. Y., Stephens, N. M., Montti, M., Roper, B., Zheng, M., Moshref, M., Dagdas, Y. S., Holder, P. G., et al. (2022) · Talanta · doi:10.1016/j.talanta.2022.123780 · Cited by 2
Lee, J., Jung, M., Jeong, E., et al. (2019) · Journal of Visualized Experiments · doi:10.3791/59202-v · Cited by 2
Yao, Y., Cao, J., Wang, W., Liu, B., Pei, X., Zhang, L., & Rao, S. (2022) · The CRISPR Journal · doi:10.1089/crispr.2022.0046 · Cited by 2
Genome-Editing Technologies in Crop Improvement
Das, R., Kumar, P., Agrawal, S., Singh, K., Singh, N., Singh, S., Singh, A. K., Rajput, V. D., Shukla, P. K., Minkina, T., et al. (2024) · Interdisciplinary biotechnological advances · doi:10.1007/978-981-99-9338-3_4 · Cited by 2
Metadata
- Authors
- Vakulskas, C. A., Dever, D. P., Rettig, G. R., Turk, R., Jacobi, A. M., Collingwood, M. A., Bode, N. M., McNeill, M. S., Yan, S., Camarena, J., Lee, C. M., Park, S. H., Wiebking, V., Bak, R. O., Gomez-Ospina, N., Pavel-Dinu, M., Sun, W., Bao, G., Porteus, M. H., & Behlke, M. A.
- Affiliations
- Integrated DNA Technologies (United States) (US)
- Stanford Medicine (US)
- Rice University (US)
- Aarhus University (DK)
- Corresponding author
- Porteus, Matthew H.
- Behlke, Mark A.
- ORCID
- Vakulskas, Christopher A.
- Dever, Daniel P.
- Rettig, Garrett R.
- Turk, Rolf
- Jacobi, Ashley M.
- Collingwood, Michael A.
- Bode, Nicole M.
- McNeill, Matthew S.
- Yan, Shuqi
- Camarena, Joab
- Lee, Ciaran M.
- Wiebking, Volker
- Bak, Rasmus O.
- Gomez-Ospina, Natalia
- Pavel-Dinu, Mara
- Sun, Wenchao
- Bao, Gang
- Porteus, Matthew H.
- Behlke, Mark A.
- Venue
- Nature Medicine
- Published
- 2018-08
- Volume
- 24
- Issue
- 8
- Pages
- 1216-1224
- ISSN
- 1078-8956 (ISSN-L)
- 1546-170X
- PMC
- PMC6107069
- OpenAlex
- W2886819502
- Cited by
- 782
- References
- 74
- Citations by year
- 2026: 66 · 2025: 92 · 2024: 91 · 2023: 86 · 2022: 116 · 2021: 113 · 2020: 116 · 2019: 92 · 2018: 9
- FWCI
- 29.32
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Nature Medicine (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- Amon G. Carter Foundation
- Lundbeckfonden (R238‐2016‐3349)
- National Institutes of Health (5r01ai120766-02, R01-AI120766, AI120766)
- National Institute of Allergy and Infectious Diseases (R01-AI120766, R01AI097320)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Health Sciences › Medicine › Oncology › CAR-T cell therapy research
- Keywords
- ribonucleoprotein delivery, CRISPR/Cas9 genome editing, hematopoietic stem and progenitor cells, sickle cell disease, off-target editing, HBB, IL2RG, CCR5, HEXB, TRAC, T cells
- MeSH terms
- Gene Editing, CRISPR-Associated Protein 9, Anemia, Sickle Cell, Base Sequence, Escherichia coli, Hematopoietic Stem Cells, Humans, Mutation, Ribonucleoproteins, Antigens, CD34, HEK293 Cells
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