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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Self-citationCRISPR/Cas-Based Modifications for Therapeutic Applications: A Review
Bharathkumar, N., Abraham, S., Meera, P., Aksah, S., Muthu, K., Saravanan, K. M., & Anand, T. (2021) · Molecular Biotechnology · doi:10.1007/s12033-021-00422-8 · Cited by 80
“Nmecas9, a CRISPR–Cas9 system based on Neisseria meningitidis, detects a sequence of 8-nt PAM (5-NNNNGATT) that can reduce off-target cleavage activity [27].”
BackgroundCRISPR Therapeutics for Duchenne Muscular Dystrophy
Erkut, E. J. N., & Yokota, T. (2022) · International Journal of Molecular Sciences · doi:10.3390/ijms23031832 · Cited by 82
“These modified Cas9 enzymes include eSpCas9(1.1) [39], SpCas9-HF1 [40], HypaCas9 [41], HeFSpCas9s [42], evoCas9 [43], xCas9 [44], HiFi Cas9 [45], Sniper-Cas9 [46], and SpCas9-NG [47].”
InfluentialAhmed, M. M., Hassan, H. K., Okesanya, O. J., Ukoaka, B. M., Eshun, G., Mourid, M. R., Adigun, O. A., Ogaya, J. B., Mohamed, Z., & Lucero‐Prisno, D. E. (2024) · Infection and Drug Resistance · doi:10.2147/idr.s494327 · Cited by 93
“114”
Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9Preprint
Agudelo, D., Carter, S., Velimirovic, M., Duringer, A., Rivest, J.-F., Levesque, S., Loehr, J., Mouchiroud, M., Cyr, D., Waters, P. J., et al. (2020) · Genome Research · doi:10.1101/gr.255414.119 · Cited by 75
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Expanding the genome-targeting scope and the site selectivity of high-precision base editors
Tan, J., Zhang, F., Karcher, D., & Bock, R. (2020) · Nature Communications · doi:10.1038/s41467-020-14465-z · Cited by 79
Atkins, A., Chung, C.-H., Allen, A. G., Dampier, W., Gurrola, T. E., Sariyer, I. K., Nonnemacher, M. R., & Wigdahl, B. L. (2021) · Frontiers in Genome Editing · doi:10.3389/fgeed.2021.673022 · Cited by 78
Gene Editing by Extracellular Vesicles
Kostyushev, D. S., Kostyusheva, A., Brezgin, S., Vv, S., Volchkova, E. V., Lukashev, A. N., & Chulanov, V. (2020) · International Journal of Molecular Sciences · doi:10.3390/ijms21197362 · Cited by 75
Vaidyanathan, S., Baik, R., Chen, L., Bravo, D. T., Suarez, C. J., Abazari, S. M., Salahudeen, A. A., Dudek, A. M., Teran, C. A., Davis, T., et al. (2021) · Molecular Therapy · doi:10.1016/j.ymthe.2021.03.023 · Cited by 70
Self-citationDonohoue, P. D., Pacesa, M., Lau, E., Vidal, B., Irby, M. J., Nyer, D. B., Rotstein, T., Banh, L., Toh, M. S., Gibson, J., et al. (2021) · Molecular Cell · doi:10.1016/j.molcel.2021.07.035 · Cited by 69
Bridge helix arginines play a critical role in Cas9 sensitivity to mismatches
Bratovič, M., Fonfara, I., Chylinski, K., Gálvez, E. J. C., Sullivan, T. J., Boerno, S. T., Timmermann, B., Boettcher, M., & Charpentier, E. (2020) · Nature Chemical Biology · doi:10.1038/s41589-020-0490-4 · Cited by 70
Shapiro, J., Iancu, O., Jacobi, A. M., McNeill, M., Turk, R., Rettig, G. R., Amit, I., Tovin, A., Yakhini, Z., Behlke, M. A., et al. (2020) · Molecular Therapy — Methods & Clinical Development · doi:10.1016/j.omtm.2020.04.027 · Cited by 72
“Data were analyzed using a custom-built pipeline (IDT, Coralville, IA, USA), as described previously.(34) The sequencing data were deposited to the SRA, under accession number: PRJNA628100.”
MethodInfluentialSelf-citationPrime Editing: Genome Editing for Rare Genetic Diseases Without Double-Strand Breaks or Donor DNA
Matsoukas, I. G. (2020) · Frontiers in Genetics · doi:10.3389/fgene.2020.00528 · Cited by 65
A highly efficient transgene knock-in technology in clinically relevant cell types
Allen, A. G., Khan, S., Margulies, C. M., Viswanathan, R., Lele, S. S., Blaha, L. C., Scott, S. N., Izzo, K. M., Gerew, A., Pattali, R. K., et al. (2023) · Nature Biotechnology · doi:10.1038/s41587-023-01779-8 · Cited by 71
“In brief, DNA sequences encoding wild-type or mutant AsCas12a 19 were cloned into pET28a vector by Gibson assembly 18 .”
Wiebking, V., Lee, C. M., Mostrel, N., Lahiri, P., Bak, R. O., Bao, G., Roncarolo, M. G., Bertaina, A., & Porteus, M. H. (2020) · Haematologica · doi:10.3324/haematol.2019.233882 · Cited by 73
Self-citationGenetic engineering meets hematopoietic stem cell biology for next-generation gene therapy
Ferrari, S., Valeri, E., Conti, A., Scala, S., Aprile, A., Micco, R. D., Kajaste‐Rudnitski, A., Montini, E., Ferrari, G., Aiuti, A., et al. (2023) · Cell stem cell · doi:10.1016/j.stem.2023.04.014 · Cited by 75
Cancellieri, S., Canver, M. C., Bombieri, N., Giugno, R., & Pinello, L. (2019) · Bioinformatics · doi:10.1093/bioinformatics/btz867 · Cited by 75
Strategies for genetic manipulation of adult stem cell-derived organoids
Menche, C., & Farin, H. F. (2021) · Experimental & Molecular Medicine · doi:10.1038/s12276-021-00609-8 · Cited by 72
Gene Editing of HIV-1 Co-receptors to Prevent and/or Cure Virus Infection
Allen, A. G., Chung, C.-H., Atkins, A., Dampier, W., Khalili, K., Nonnemacher, M. R., & Wigdahl, B. L. (2018) · Frontiers in Microbiology · doi:10.3389/fmicb.2018.02940 · Cited by 70
Chen, H., Victor, A. K., Klein, J. D., Tacer, K. F., Tai, D. J. C., de Esch, C. E. F., Nuttle, A., Temirov, J. P., Burnett, L. C., ROSENBAUM, M. S., et al. (2020) · JCI Insight · doi:10.1172/jci.insight.138576 · Cited by 66
“To minimize off-target editing and maximize efficiency, we incorporated high-fidelity Cas9 and ribonucleoprotein-based delivery as previously described (75).”
Gao, Q., Ouyang, W., Kang, B., Han, X., Xiong, Y., Ding, R., Li, Y., Wang, F., Huang, L., Chen, L., et al. (2020) · Theranostics · doi:10.7150/thno.42325 · Cited by 72
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Recent Advances in the Application of CRISPR/Cas9 Gene Editing System in Poultry Species
Khwatenge, C. N., & Nahashon, S. N. (2021) · Frontiers in Genetics · doi:10.3389/fgene.2021.627714 · Cited by 76
Kelley, C. P., Haerle, M. C., & Wang, E. T. (2022) · Cell Reports · doi:10.1016/j.celrep.2022.111226 · Cited by 61
CRISPR/Cas9 ribonucleoprotein-mediated genome and epigenome editing in mammalian cells
Bloomer, H., Khirallah, J., Li, Y., & Xu, Q. (2021) · Advanced Drug Delivery Reviews · doi:10.1016/j.addr.2021.114087 · Cited by 75
Recent Advances in Improving Gene-Editing Specificity through CRISPR–Cas9 Nuclease Engineering
Huang, X., Yang, D., Zhang, J., Xu, J., & Chen, Y. E. (2022) · Cells · doi:10.3390/cells11142186 · Cited by 68
Li, Z., Wang, J., Xu, J.-P., Yang, X. F., & Yang, X. F. (2023) · Military Medical Research · doi:10.1186/s40779-023-00447-x · Cited by 72
CRISPR/Cas9 for Sickle Cell Disease: Applications, Future Possibilities, and Challenges
Demirci, S., Leonard, A., Haro-Mora, J. J., Uchida, N., & Tisdale, J. F. (2019) · Advances in Experimental Medicine and Biology · doi:10.1007/5584_2018_331 · Cited by 50
Benchmarking of SpCas9 variants enables deeper base editor screens of BRCA1 and BCL2
Sangree, A. K., Griffith, A. L., Szegletes, Z. M., Roy, P., DeWeirdt, P. C., Hegde, M., McGee, A. V., Hanna, R. E., & Doench, J. G. (2022) · Nature Communications · doi:10.1038/s41467-022-28884-7 · Cited by 64
In-depth assessment of the PAM compatibility and editing activities of Cas9 variants
Zhang, W., Yin, J., Zhangding, Z., Xin, C., Liu, M., Wang, Y., Chen, A., & Hu, J. (2021) · Nucleic Acids Research · doi:10.1093/nar/gkab507 · Cited by 66
Mueller, K. P., Piscopo, N. J., Forsberg, M. H., Saraspe, L. A., Das, A., Russell, B., Smerchansky, M. E., Cappabianca, D., Shi, L., Shankar, K., et al. (2022) · Journal for ImmunoTherapy of Cancer · doi:10.1136/jitc-2021-004446 · Cited by 62
Cas9-directed immune tolerance in humans—a model to evaluate regulatory T cells in gene therapy?
Wagner, D. L., Peter, L., & Schmueck-Henneresse, M. (2021) · Gene Therapy · doi:10.1038/s41434-021-00232-2 · Cited by 63
Gene Editing and Genotoxicity: Targeting the Off-Targets
Blattner, G., Cavazza, A., Thrasher, A. J., & Turchiano, G. (2020) · Frontiers in Genome Editing · doi:10.3389/fgeed.2020.613252 · Cited by 64
“In the same way, the different gene editing strategies are shaping novel reagents, techniques and strategies to improve their safety and efficiency for clinical application (Miller et al., 2007; Kleinstiver et al., 2016; Casini et al., 2018; Gao et al., 2018; Vakulskas et al., 2018; Rai et al., 2020).”
BackgroundEvolution of CRISPR/Cas Systems for Precise Genome Editing
Hryhorowicz, M., Lipiński, D., & Zeyland, J. (2023) · International Journal of Molecular Sciences · doi:10.3390/ijms241814233 · Cited by 69
“Currently, numerous high-fidelity Cas9 variants have been engineered or developed, such as SpCas9-HF1 [16], HiFiCas9 [17], evoCas9 [18], eSpCas9 [19], HypaCas9 [20], Sniper-Cas9 [21], and xCas9 3.7 [22].”
InfluentialLi, J., Yu, Q., Liu, C., Zhang, N., & Xu, W. (2024) · Horticulture Research · doi:10.1093/hr/uhae366 · Cited by 67
Combinatorial mutagenesis en masse optimizes the genome editing activities of SpCas9
Choi, G. C. G., Zhou, P., Yuen, C. T. L., Chan, B. K. C., Xu, F., Bao, S., Chu, H. Y., Thean, D., Tan, K., Wong, K. H., et al. (2019) · Nature Methods · doi:10.1038/s41592-019-0473-0 · Cited by 63
Enhancing gene editing specificity by attenuating DNA cleavage kinetics
Miller, J. C., Patil, D. P., Xia, D. F., Paine, C. B., Fauser, F., Richards, H. W., Shivak, D. A., Bendaña, Y. R., Hinkley, S. J., Scarlott, N. A., et al. (2019) · Nature Biotechnology · doi:10.1038/s41587-019-0186-z · Cited by 63
GO: a functional reporter system to identify and enrich base editing activity
Katti, A., Foronda, M., Zimmerman, J., Diaz, B. J., Zafra, M. P., Goswami, S., & Dow, L. E. (2020) · Nucleic Acids Research · doi:10.1093/nar/gkaa124 · Cited by 60
“FNLS-HiFi was generated using InFusion assembly of 5′ and 3′ FNLS PCR amplicons, incorporating the R691A mutation (14) in the junction of these fragments.”
MethodSniper2L is a high-fidelity Cas9 variant with high activity
Kim, Y.-H., Kim, N., Okafor, I. C., Choi, S.-C., Min, S., Lee, J., Bae, S.-M., Choi, K., Choi, J., Harihar, V., et al. (2023) · Nature Chemical Biology · doi:10.1038/s41589-023-01279-5 · Cited by 70
The hidden risks of CRISPR/Cas: structural variations and genome integrity
Aussel, C., Cathomen, T., & Fuster‐García, C. (2025) · Nature Communications · doi:10.1038/s41467-025-62606-z · Cited by 72
Genome-wide detection of CRISPR editing in vivo using GUIDE-tag
Liang, S.-Q., Liu, P., Smith, J. L., Mintzer, E., Maitland, S., Dong, X., Yang, Q., Lee, J., Haynes, C. M., Zhu, L. J., et al. (2022) · Nature Communications · doi:10.1038/s41467-022-28135-9 · Cited by 54
PAM-flexible genome editing with an engineered chimeric Cas9
Zhao, L., Koseki, S. R. T., Silverstein, R. A., Amrani, N., Peng, C., Kramme, C., Savic, N., Pacesa, M., Rodríguez, T. C., Stan, T., et al. (2023) · Nature Communications · doi:10.1038/s41467-023-41829-y · Cited by 59
CRISPR-Cas gene editing technology and its application prospect in medicinal plants
Guo, M., Chen, H., Dong, S., Zhang, Z., & Luo, H. (2022) · Chinese Medicine · doi:10.1186/s13020-022-00584-w · Cited by 66
“HiFi Cas9 single point mutation R691A of SpCas9 NGG Enhanced specificity for ribonucleoprotein delivery [68]”
BackgroundCRISPR nuclease off-target activity and mitigation strategies
Wienert, B., & Cromer, M. K. (2022) · Frontiers in Genome Editing · doi:10.3389/fgeed.2022.1050507 · Cited by 66
“To address this, more specific versions of Cas9 have been engineered which reduce off-target activity by > 20-fold (Chen et al., 2017; Vakulskas et al., 2018; Bravo et al., 2022; Kulcsar et al., 2022).”
BackgroundThe CRISPR/Cas9 System Delivered by Extracellular Vesicles
Zhu, X., Gao, M., Yang, Y., Li, W., Bao, J., & Li, Y. (2023) · Pharmaceutics · doi:10.3390/pharmaceutics15030984 · Cited by 61
“Gene editing immediately begins to work through the delivery of the RNP form of the CRISPR/Cas9 system in the nucleus, which leads to a higher efficiency of gene editing and a lower rate of off-target effects than DNA and RNA forms of the CRISPR/Cas9 system [31].”
BackgroundInfluentialHigh-fidelity SaCas9 identified by directional screening in human cells
Xie, H., Ge, X., Yang, F., Wang, B., Li, S., Duan, J., Lv, X., Cheng, C., Song, Z., Liu, C., et al. (2020) · PLoS Biology · doi:10.1371/journal.pbio.3000747 · Cited by 59
“These include using Cas9 nickase mutants to create a pair of juxtaposed single-stranded DNA nicks [16], using a pair of catalytically inactive Cas9 nucleases, each fused to a FokI nuclease domain [17], delivering Cas9 as ribonucleoprotein (RNP) complexes [18], and truncating the guide sequence at the 5 end [19].”
Delivery Approaches for Therapeutic Genome Editing and Challenges
Ates, I., Rathbone, T., Stuart, C., Bridges, P. H., & Cottle, R. N. (2020) · Genes · doi:10.3390/genes11101113 · Cited by 63
“This gene-editing strategy involves isolating CD34+ HSPCs from patients, gene modifying the cells ex vivo, and infusing them back into the patient for the treatment of HIV, sickle cell anemia, and β-thalassemia.”
BackgroundInfluentialCas9 deactivation with photocleavable guide RNAs
Zou, R. S., Liu, Y., Wu, B., & Ha, T. (2021) · Molecular Cell · doi:10.1016/j.molcel.2021.02.007 · Cited by 61
Progress and challenges towards CRISPR/Cas clinical translation
Rosenblum, D., Gutkin, A., Dammes, N., & Peer, D. (2020) · Advanced Drug Delivery Reviews · doi:10.1016/j.addr.2020.07.004 · Cited by 61
Next-generation cell therapies: the emerging role of CAR-NK cells
Başar, R., Daher, M., & Rezvani, K. (2020) · Hematology · doi:10.1182/hematology.2020002547 · Cited by 57
“The use of ribonucleoprotein complexes and high-fidelity Cas9 was recently shown to significantly reduce the occurrence of such unwanted DNA changes.(56) Given the versatility of this gene editing technology, one can imagine its potential applications in cell therapy.”
MethodMa, L., Ruan, J., Song, J. W., Wen, L., Yang, D., Zhao, J., Xia, X., Chen, Y. E., Zhang, J., & Xu, J. (2020) · Nature Communications · doi:10.1038/s41467-020-19842-2 · Cited by 60
Rose, J. C., Popp, N. A., Richardson, C. D., Stephany, J. J., Mathieu, J., Wei, C. T., Corn, J. E., Maly, D. J., & Fowler, D. M. (2020) · Nature Communications · doi:10.1038/s41467-020-16542-9 · Cited by 61
Single-Base Resolution: Increasing the Specificity of the CRISPR-Cas System in Gene Editing
Rabinowitz, R., & Offen, D. (2020) · Molecular Therapy · doi:10.1016/j.ymthe.2020.11.009 · Cited by 58
Recent advances in therapeutic gene-editing technologies
Liu, D., Cao, D., & Han, R. (2025) · Molecular Therapy · doi:10.1016/j.ymthe.2025.03.026 · Cited by 70
Directed Evolution of CRISPR/Cas Systems for Precise Gene Editing
Liu, R., Liang, L., Freed, E. F., & Gill, R. T. (2020) · Trends in biotechnology · doi:10.1016/j.tibtech.2020.07.005 · Cited by 58
Dissecting ELANE neutropenia pathogenicity by human HSC gene editing
Rao, S., Yao, Y., de Brito, J. S., Yao, Q., Shen, A. H., Watkinson, R. E., Kennedy, A. L., Coyne, S. M., Ren, C., Zeng, J., et al. (2021) · Cell stem cell · doi:10.1016/j.stem.2020.12.015 · Cited by 57
Current Strategies for Increasing Knock-In Efficiency in CRISPR/Cas9-Based Approaches
Leal, A., Herreno-Pachón, A., Benincore-Flórez, E., Karunathilaka, A., & Tomatsu, S. (2024) · International Journal of Molecular Sciences · doi:10.3390/ijms25052456 · Cited by 58
“Even though some efforts have been made to increase the Cas9 specificity without size changes resulting in several Cas9 variants (see Table 1), they have a marginal effect on the knock-in efficiency increase [26,27,33].”
BackgroundBoosting genome editing efficiency in human cells and plants with novel LbCas12a variants
Zhang, L., Li, G., Zhang, Y., Cheng, Y., Roberts, N., Glenn, S. E., DeZwaan-McCabe, D., Rube, H. T., Manthey, J., Coleman, G. D., et al. (2023) · Genome biology · doi:10.1186/s13059-023-02929-6 · Cited by 58
Self-citationComputational Tools and Resources Supporting CRISPR-Cas Experiments
Śledziński, P., Nowaczyk, M., & Olejniczak, M. (2020) · Cells · doi:10.3390/cells9051288 · Cited by 57
High-level correction of the sickle mutation is amplified in vivo during erythroid differentiation
Magis, W., DeWitt, M. A., Wyman, S. K., Vu, J. T., Heo, S., Shao, S. J., Hennig, F., Romero, Z. G., Campo-Fernández, B., Said, S., et al. (2022) · iScience · doi:10.1016/j.isci.2022.104374 · Cited by 53
Self-citationMicronucleus is not a potent inducer of the cGAS/STING pathway
Sato, Y., & Hayashi, M. (2024) · Life Science Alliance · doi:10.26508/lsa.202302424 · Cited by 58
“For LentiCRISPR(HiFi) (Vakulskas et al, 2018), Lenti-enAsCas12a-HF1-2C (Kleinstiver et al, 2019), LentiGuide-sgRNA, and pLKO.1-shRNA, transduced cells were selected by 1 μ g/ml puromycin for 2 d after day 2 of transduction.”
MethodIkeda, A., Fujii, W., Sugiura, K., & Naito, K. (2019) · Communications Biology · doi:10.1038/s42003-019-0627-8 · Cited by 57
Hematopoietic Stem Cell Gene-Addition/Editing Therapy in Sickle Cell Disease
Germino-Watnick, P., Hinds, M., Le, A., Chu, R., Liu, X., & Uchida, N. (2022) · Cells · doi:10.3390/cells11111843 · Cited by 63
“Furthermore, HiFi Cas9 with a single point mutation (R691A) delivered via an AAV6 vector allowed for a 20-fold reduction in off-target effects as compared to a wild-type Cas9 [88].”
BackgroundMethodMetabolic engineering generates a transgene-free safety switch for cell therapy
Wiebking, V., Patterson, J. O., Martin, R. M., Chanda, M. K., Lee, C. M., Srifa, W., Bao, G., & Porteus, M. H. (2020) · Nature Biotechnology · doi:10.1038/s41587-020-0580-6 · Cited by 53
Self-citationTran, N. T., Danner, E., Li, X., Graf, R., Lebedin, M., de la Rosa, K., Kühn, R., Rajewsky, K., & Chu, V. T. (2022) · Science Advances · doi:10.1126/sciadv.abm9106 · Cited by 49
“In addition, off-target effects of Cas9 nuclease were shown to be lower when high-fidelity SpCas9 mutants were used ( 19 – 21 ).”
Allemailem, K. S., Almatroodi, S. A., Almatroudi, A., Alrumaihi, F., Abdulmonem, W. A., Al‐Megrin, W. A. I., Aljamaan, A. N., Rahmani, A. H., & Khan, A. A. (2023) · International Journal of Molecular Sciences · doi:10.3390/ijms24087052 · Cited by 53
“HiFiCas9 R691A 2018 human hematopoietic stem and progenitor cells [64], primary T cells [64]”
BackgroundAlves, C. R. R., Ha, L. L., Yaworski, R., Sutton, E. R., Lazzarotto, C. R., Christie, K. A., Reilly, A., Beauvais, A., Doll, R. M., de la Cruz, D., et al. (2023) · Nature Biomedical Engineering · doi:10.1038/s41551-023-01132-z · Cited by 72
“We generated ABE8e fusions to SpCas9 and SpRY in the presence of HF1 substitutions that were previously shown to eliminate nearly all off-target edits 47 , and the HiFi mutation that was previously shown to reduce levels of off-target editing 46 .”
Gutiérrez-Guerrero, A., Abrey-Recalde, M. J., Mangeot, P. E., Costa, C., Bernadin, O., Perian, S., Fusil, F., Froment, G., Martinez-Turtos, A., Krug, A., et al. (2021) · Frontiers in Genome Editing · doi:10.3389/fgeed.2021.604371 · Cited by 59
Clinical applications of CRISPR‐based genome editing and diagnostics
Foss, D. V., Hochstrasser, M. L., & Wilson, R. C. (2019) · Transfusion · doi:10.1111/trf.15126 · Cited by 51
“Furthermore, variants of Cas enzymes are being engineered with higher fidelity, which could limit OTEs to levels that are not of clinical concern.(106,107) The recent observation that Cas9 is capable of infrequently inducing larger genomic rearrangements stimulated significant apprehension.”
BackgroundModeling Psychiatric Disorder Biology with Stem Cells
Das, D., Feuer, K. L., Wahbeh, M. H., & Avramopoulos, D. (2020) · Current Psychiatry Reports · doi:10.1007/s11920-020-01148-1 · Cited by 49
Maldini, C. R., Gayout, K., Leibman, R. S., Dopkin, D. L., Mills, J. P., Shan, X., Glover, J. A., & Riley, J. L. (2020) · Molecular Therapy · doi:10.1016/j.ymthe.2020.05.012 · Cited by 50
Ex vivo editing of human hematopoietic stem cells for erythroid expression of therapeutic proteins
Pavani, G., Laurent, M., Fabiano, A., Cantelli, E., Sakkal, A., Corre, G., Lenting, P. J., Concordet, J., Toueille, M., Miccio, A., et al. (2020) · Nature Communications · doi:10.1038/s41467-020-17552-3 · Cited by 54
Kondo, K., & Taguchi, C. (2022) · Food Safety · doi:10.14252/foodsafetyfscj.d-21-00016 · Cited by 49
Kulcsár, P. I., Tálas, A., Tóth, E., Nyeste, A., Ligeti, Z., Welker, Z., & Welker, E. (2020) · Nature Communications · doi:10.1038/s41467-020-15021-5 · Cited by 45
Applications of Genome Editing Technology in Animal Disease Modeling and Gene Therapy
Li, Q., Qin, Z., Wang, Q., Xu, T., Yang, Y., & He, Z. (2019) · Computational and Structural Biotechnology Journal · doi:10.1016/j.csbj.2019.05.006 · Cited by 52
Liu, T., Lam, V., Thieme, E., Sun, D., Wang, X., Xu, F., Wang, L., Danilova, O. V., Xia, Z., Tyner, J. W., et al. (2021) · Clinical Cancer Research · doi:10.1158/1078-0432.ccr-21-0464 · Cited by 48
“Gene editing and overexpression Ribonucleoprotein (RNP) electroporation genome editing experiments in DLBCL cells (OCI-LY3 and VAL) were performed using the Lonza Amaxa Nucleofector II System (Lonza), 4 mmol/L (1:3, Cas9: sgRNA) Alt-R (Integrated DNA Technologies, Inc) Cas9 RNP complex, and 4 mmol/L Alt-R Cas9 Electro…”
MethodProgress and Perspective of CRISPR‐Cas9 Technology in Translational Medicine
Zheng, R., Zhang, L., Parvin, R., Su, L., Chi, J., Shi, K., Ye, F., & Huang, X. (2023) · Advanced Science · doi:10.1002/advs.202300195 · Cited by 56
Improved Double-Nicking Strategies for COL7A1-Editing by Homologous Recombination
Köcher, T., Wagner, R. N., Klausegger, A., Guttmann‐Gruber, C., Hainzl, S., Bauer, J. W., Reichelt, J., & Koller, U. (2019) · Molecular Therapy — Nucleic Acids · doi:10.1016/j.omtn.2019.09.011 · Cited by 48
“Since its inception, the CRISPR gene-editing toolbox has gone through a dramatic expansion, including the discovery of other targeting nucleases, most prominently Cas12a,(10) the repurposing of Streptococcus pyogenes Cas9 (from here on Cas9) for transcriptional modulation,(11) or the development of high-fidelity versi…”
BackgroundCurrent trends in gene recovery mediated by the CRISPR-Cas system
Jang, H., Song, B., Hwang, G., & Bae, S. (2020) · Experimental & Molecular Medicine · doi:10.1038/s12276-020-0466-1 · Cited by 49
Xu, H., Wu, X., Liu, Q., Yang, C., Shen, M., Wang, Y., Liu, S., Zhao, S., Xiao, T., Sun, M., et al. (2023) · ACS Nano · doi:10.1021/acsnano.3c07721 · Cited by 47
High-fidelity, hyper-accurate, and evolved mutants rewire atomic-level communication in CRISPR-Cas9
Skeens, E., Sinha, S., Ahsan, M., D’Ordine, A. M., Jogl, G., Palermo, G., & Lisi, G. P. (2024) · Science Advances · doi:10.1126/sciadv.adl1045 · Cited by 53
Functional profiling of single CRISPR/Cas9-edited human long-term hematopoietic stem cells
Wagenblast, E., Azkanaz, M., Smith, S. A., Shakib, L., McLeod, J. L., Krivdova, G., Araújo, J., Shultz, L. D., Gan, O. I., Dick, J. E., et al. (2019) · Nature Communications · doi:10.1038/s41467-019-12726-0 · Cited by 49
Okafor, I. C., Singh, D., Wang, Y., Jung, M., Wang, H., Mallon, J. P., Bailey, S., Lee, J. K., & Ha, T. (2019) · Nucleic Acids Research · doi:10.1093/nar/gkz1058 · Cited by 45
CRISPR/Cas9-Based Lateral Flow and Fluorescence Diagnostics
Osborn, M. J., Bhardwaj, A., Bingea, S. P., Knipping, F., Feser, C. J., Lees, C. J., Collins, D. P., Steer, C. J., Blazar, B. R., & Tolar, J. (2021) · Bioengineering · doi:10.3390/bioengineering8020023 · Cited by 47
“Either wild-type or a high-fidelity version of Cas9 (SpyFiTM) [37] were employed, and each showed differential fluorescence signal between matched and mismatched; however, it was not statistically significant (Figure S6A,B, in the Supplementary materials).”
BackgroundGene Editing of Hematopoietic Stem Cells: Hopes and Hurdles Toward Clinical Translation
Ferrari, S., Vavassori, V., Canarutto, D., Jacob, A., Castiello, M. C., Omer, A., & Genovese, P. (2021) · Frontiers in Genome Editing · doi:10.3389/fgeed.2021.618378 · Cited by 52
“Recently, other highly specific SpCas9 variants, such as EvoCas9 (Casini et al., 2018), SniperCas9 (Lee et al., 2018) and HiFi-Cas9 (Vakulskas et al., 2018) were discovered by directed evolution approaches.”
BackgroundInfluentialEngineered Cas12a-Plus nuclease enables gene editing with enhanced activity and specificity
Huang, H., Huang, G., Tan, Z., Hu, Y., Shan, L., Zhou, J., Zhang, X., Ma, S., Lv, W., Huang, T., et al. (2022) · BMC Biology · doi:10.1186/s12915-022-01296-1 · Cited by 46
“In particular, protein engineering is an efficient and widely used approach for the development of high-fidelity Cas-nuclease variants, which has been well-proved in SpCas9, such as the unbiased engineered variants HiFi-Cas9 [17], evoCas9 [18], xCas9 [19], Sniper-Cas9 [20], LZ3-Cas9 [21], etc., and the structure-guide…”
MethodOcular delivery of CRISPR/Cas genome editing components for treatment of eye diseases
Yu, W., & Wu, Z. (2020) · Advanced Drug Delivery Reviews · doi:10.1016/j.addr.2020.06.011 · Cited by 48
Fix, S. M., Forget, M., Sakellariou-Thompson, D., et al. (2022) · Journal for ImmunoTherapy of Cancer · doi:10.1136/jitc-2021-003750 · Cited by 45
Variability in Genome Editing Outcomes: Challenges for Research Reproducibility and Clinical Safety
Teboul, L., Hérault, Y., Wells, S. E., Qasim, W., & Pavlovic, G. (2020) · Molecular Therapy · doi:10.1016/j.ymthe.2020.03.015 · Cited by 47
“A wide variety of strategies have been tried, including the engineering of nucleases with higher specificity 111,112 , the coupling of Cas9 fusions with specific protein domains to enhance the frequency of DSB repair by homology directed repair (see 113 for review), and the abolition of all…”
Amit, I., Iancu, O., Levy-Jurgenson, A., Kurgan, G., McNeill, M., Rettig, G. R., Allen, D. G., Breier, D., Haim, N. B., Wang, Y., et al. (2021) · Nature Communications · doi:10.1038/s41467-021-22417-4 · Cited by 43
“The HPRT and AR sequencing data was obtained from Vakulskas et al., 2018, SRA accession: SRP150376.”
MethodSelf-citationTherapy Development by Genome Editing of Hematopoietic Stem Cells
Koniali, L., Lederer, C. W., & Kleanthous, M. (2021) · Cells · doi:10.3390/cells10061492 · Cited by 50
“20 A>T) CRISPR/Cas9 RNP AAV6 70% HDR [142]”
Genome Editing for Mucopolysaccharidoses
Poletto, É., Baldo, G., & Gomez‐Ospina, N. (2020) · International Journal of Molecular Sciences · doi:10.3390/ijms21020500 · Cited by 45
Self-citationSystematic in vitro profiling of off-target affinity, cleavage and efficiency for CRISPR enzymes
Zhang, L., Rube, H. T., Vakulskas, C. A., Behlke, M. A., Bussemaker, H. J., & Pufall, M. A. (2020) · Nucleic Acids Research · doi:10.1093/nar/gkaa231 · Cited by 45
“canis Cas9 (ScCas9), has been reported to have a singlebase specificity within the PAM (5′-NNG-3′), but otherwise similar activity to SpCas9 (19).”
BackgroundSelf-citationKulcsár, P. I., Tálas, A., Ligeti, Z., Krausz, S. L., & Welker, E. (2022) · Nature Communications · doi:10.1038/s41467-022-34527-8 · Cited by 46
Cromer, M. K., Majeti, K. R., Rettig, G. R., Murugan, K., Kurgan, G. L., Bode, N. M., Hampton, J. P., Vakulskas, C. A., Behlke, M. A., & Porteus, M. H. (2023) · Molecular Therapy · doi:10.1016/j.ymthe.2023.02.011 · Cited by 42
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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