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
29 of 37 cited sources checked · 12 supported · 0 flagged · accuracy not yet measured
- Background
The paper notes that adapting bacterial CRISPR systems for RNA-guided cleavage of complementary DNA transformed mammalian gene editing.
“Repurposing bacterial “CRISPR” systems to facilitate RNA-guided cleavage of complementary DNA has revolutionized the field of gene editing in mammalian cells 1–4.”
Quote in the paperNot yet classified3 of 4 sources checked- cites [1] Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012). source
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)
Experimental — accuracy not yet measured - cites [2] Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013). source
- cites [3] Doudna, J. A. & Charpentier, E. Genome editing. The new frontier of genome engineering with CRISPR–Cas9. Science 346, 1258096 (2014). source
- cites [4] Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPR–Cas9 for genome engineering. Cell 157, 1262–1278 (2014). source
- Background
It explains that supplying a homologous donor during break repair allows homologous recombination to introduce precise edits, from single-base changes to large cassette insertions.
“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.”
Quote in the paperNot yet classified2 of 2 sources checked- cites [8] Porteus, M. H. Towards a new era in medicine: therapeutic genome editing. Genome Biol. 16, 286 (2015). source
- cites [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
- Background
It describes GUIDE-seq as a method for empirically finding actual off-target sites in cells.
“The GUIDE-seq procedure10 can be used to empirically identify real off-target sites in cells.”
Quote in the paperNot yet classified1 of 1 sources checked- cites [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
- Background
It reports that ribonucleoprotein delivery gives a brief pulse of editing machinery, producing efficient editing with fewer off-target effects.
“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.”
Quote in the paperNot yet classified7 of 7 sources checked- cites [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). source
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)
Experimental — accuracy not yet measured - cites [14] Gundry, M. C. et al. Technical considerations for the use of CRISPR/Cas9 in hematology research. Exp. Hematol. 54, 4–11 (2017). source
- cites [15] Dever, D. P. et al. CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells. Nature 539, 384–389 (2016). source
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)
Experimental — accuracy not yet measured - cites [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). source
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)
Experimental — accuracy not yet measured - cites [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
- cites [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). source
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)
Experimental — accuracy not yet measured - cites [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
- Background
It defines off-target effects as cleavage at guide-mismatched sites and says they should be minimized for therapeutic editing.
“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.”
Quote in the paperNot yet classified1 of 1 sources checked- cites [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
- Background
It states that AAV6 donor templates with homology arms efficiently deliver donor DNA into sensitive primary immune cells such as HSPCs and T cells.
“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.”
Quote in the paperNot yet classified6 of 7 sources checked- cites [15] Dever, D. P. et al. CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells. Nature 539, 384–389 (2016). source
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)
Experimental — accuracy not yet measured - cites [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). source
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)
Experimental — accuracy not yet measured - cites [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). source
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)
Experimental — accuracy not yet measured - cites [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). source
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)
Experimental — accuracy not yet measured - cites [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
- cites [39] Eyquem, J. et al. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature 543, 113–117 (2017). source
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)
Experimental — accuracy not yet measured - cites [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
- Background
It cites prior work showing that CRISPR/Cas9 can correct disease-causing mutations in long-term engrafting HSPCs that persist in downstream lineages.
“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.”
Quote in the paperNot yet classified3 of 4 sources checked- cites [15] Dever, D. P. et al. CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells. Nature 539, 384–389 (2016). source
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)
Experimental — accuracy not yet measured - cites [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). source
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)
Experimental — accuracy not yet measured - cites [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
- cites [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). source
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)
Experimental — accuracy not yet measured
- Background
It notes that multiple reports have achieved efficient Glu6Val correction in SCD HSPCs, though off-target insertions or deletions can accompany correction.
“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.”
Quote in the paperNot yet classified3 of 4 sources checked- cites [15] Dever, D. P. et al. CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells. Nature 539, 384–389 (2016). source
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)
Experimental — accuracy not yet measured - cites [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). source
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)
Experimental — accuracy not yet measured - cites [45] Hoban, M. D. et al. Correction of the sickle cell disease mutation in human hematopoietic stem/progenitor cells. Blood 125, 2597–2604 (2015). source
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)
Experimental — accuracy not yet measured - cites [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
- Background
It states that non-homologous end joining repairs the break in an error-prone manner and frequently leaves small insertions or deletions.
“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.”
Quote in the paperNot yet classified1 of 1 sources checked- cites [7] Kass, E. M. & Jasin, M. Collaboration and competition between DNA double-strand break repair pathways. FEBS Lett. 584, 3703–3708 (2010). source
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)
Experimental — accuracy not yet measured
- Background
It describes that Cas9 with guide RNA scans for NGG PAMs and, upon sufficient guide-target complementarity, changes conformation and cuts DNA to make a double-strand break.
“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.”
Quote in the paperNot yet classified2 of 2 sources checked- cites [5] Dagdas, Y. S. et al. A conformational checkpoint between DNA binding and cleavage by CRISPR–Cas9. Sci. Adv. 3, eaao0027 (2017). source
- cites [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
- Finding
After erythroid differentiation, more than half of hemoglobin tetramers in HiFi-targeted cells were adult hemoglobin.
“Hemoglobin tetramer analyses confirmed the high frequency of Glu6Val correction using HiFi Cas9 by showing that >50% of hemoglobin tetramers produced in targeted differentiated erythrocytes were adult hemoglobin (HbA) (Figs. 6d and 6e).”
Quote in the paperNot yet classified - Finding
It reports that HiFi Cas9 supports robust AAV6-mediated gene targeting at five therapeutic loci in human CD34+ HSPCs and primary T cells.
“HiFi Cas9 induces robust AAV6-mediated gene targeting at five therapeutically-relevant loci (HBB, IL2RG, CCR5, HEXB, TRAC) in human CD34+ hematopoietic stem and progenitor cells (HSPCs) as well as primary T-cells.”
Quote in the paperNot yet classified - Finding
It reports that HiFi Cas9 achieves high-level correction of the sickle cell disease Glu6Val mutation in patient-derived HSPCs.
“We also show that the HiFi Cas9 mediates high-level correction of the sickle cell disease (SCD)-causing Glu6Val mutation in SCD patient derived HSPCs.”
Quote in the paperNot yet classified - Finding
When delivered as RNP in HSPCs, eSpCas9(1.1) and SpCas9-HF1 showed much lower on-target editing than wild-type Cas9.
“The previously described improved-specificity Cas9 mutants delivered as RNP showed markedly reduced on-target editing efficiencies, with the eSpCas9(1.1) showing less than ~50% activity of WT in four out of five genes and the SpCas9-HF1 Cas9 showing less than ~25% of WT efficiency.”
Quote in the paperNot yet classified - Finding
HiFi Cas9 kept on-target INDEL activity nearly the same while lowering INDEL frequency at the main off-target site by 20-fold.
“While maintaining nearly identical on-target INDEL activity, the HiFi Cas9 reduced INDEL frequency at the dominant off-target site by 20 fold (Figs. 5d, 5e and Supplementary Figs. 4b and 6c).”
Quote in the paperNot yet classified - Finding
In a 12-site HPRT comparison, HiFi Cas9 had a normalized median on-target editing efficiency of 82%, versus 20% for eSpCas9(1.1), 2% for SpCas9-HF1, and 1.7% for HypaCas9.
“The normalized median on-target editing efficiencies for each mutant was: 20% for eSpCas9(1.1), 2% for SpCas9-HF1, 1.7% for HypaCas9, and 82% for the R691A HiFi Cas9 mutant (Figs. 3b and 3c).”
Quote in the paperNot yet classified - Finding
The paper reports that previously engineered high-fidelity Cas9 variants lose on-target activity when delivered as ribonucleoproteins.
“We show that existing rationally-engineered Cas9 high fidelity variants have reduced on-target activity using the therapeutically relevant ribonucleoprotein (RNP) delivery method.”
Quote in the paperNot yet classified - Finding
In SCD HSPCs, HiFi Cas9 corrected about 70% of Glu6Val alleles versus about 64% for wild-type Cas9 while cutting off-target sites 20-fold less.
“Compared to WT Cas9 editing, the HiFi Cas9 converted a nearly equivalent number of Glu6Val alleles (~70% vs ~64%), while reducing off-target cutting by 20 fold (Figs. 6a and 6b).”
Quote in the paperNot yet classified - Finding
It reports that the single R691A mutation, called HiFi Cas9, preserved strong on-target activity while lowering off-target editing.
“Introduction of a single point mutation, R691A (HiFi Cas9), retained high on-target activity while reducing off-target editing.”
Quote in the paperNot yet classified - Finding
Across 11 independent experiments, HiFi Cas9 gave a median 28% homologous recombination at HBB compared with 30% for wild-type Cas9.
“Across 11 independent experiments, the HiFi Cas9 mediated a median of 28% HR in comparison to 30% HR for the WT Cas9 (Fig. 5c and Supplementary Fig. 6e).”
Quote in the paperNot yet classified - Finding
Across 48 additional guide sites, HiFi Cas9 averaged 92% of wild-type Cas9 on-target efficiency.
“A more extended comparison of WT Cas9 vs HiFi Cas9 RNP on-target activity was performed with guide RNAs that target an additional 48 sites, and these results showed a similar trend where the HiFi Cas9 mutant on average functioned at 92% the efficiency of WT Cas9 (Supplementary Fig. 3e).”
Quote in the paperNot yet classified - Finding
In CD34+ HSPCs, HiFi Cas9 produced near-wild-type INDEL levels across all five tested genes, about 75% of wild-type activity.
“The R691A HiFi Cas9 mutant demonstrated near-WT levels of INDEL formation across all sites (~75% of WT Cas9 activity for all five genes) (Fig. 5a and Supplementary Fig. 6a).”
Quote in the paperNot yet classified - Method
It describes using an unbiased bacterial screen to find Cas9 variants that keep activity in the RNP format.
“Therefore, we devised an unbiased bacterial screen to isolate variants that retain activity in the RNP format.”
Quote in the paperNot yet classified - Background
It notes that allogeneic HSPC transplantation can cure serious genetic blood diseases if an immunologically matched donor is available.
“Allogeneic hematopoietic stem and progenitor cell (HSPC) transplantation (allo-HSCT) can provide a one-time and life-long cure for patients with serious genetic hematological diseases such as primary immunodeficiencies20 and hemoglobinopathies21,22, if a suitable immunologically matched donor can be found23.”
Quote in the paperNot yet classified0 of 4 sources checked- cites [20] Gatti, R. A., Meuwissen, H. J., Allen, H. D., Hong, R. & Good, R. A. Immunological reconstitution of sex-linked lymphopenic immunological deficiency. Lancet 2, 1366–1369 (1968). source
- cites [21] Johnson, F. L. et al. Bone-marrow transplantation in a patient with sickle-cell anemia. N. Engl. J. Med. 311, 780–783 (1984). source
- cites [22] Lucarelli, G. et al. Allogeneic marrow transplantation for thalassemia. Exp. Hematol. 12, 676–681 (1984).
- cites [23] Naldini, L. Gene therapy returns to centre stage. Nature 526, 351–360 (2015). source
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?
In several previous reports, LZ3 Cas9, Sniper Cas9, and HiFi Cas9 had comparable editing activity to SpCas9-WT in human cells or other animal embryos.
“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]
The Results section (‘The R691A high fidelity (HiFi) Cas9 mutant demonstrates superior on-target editing with RNP delivery’; page not provided) reports that HiFi Cas9 retained editing efficiency comparable to WT Cas9. Our copy does not establish the claimed comparable activity for LZ3 Cas9 or Sniper Cas9, or address editing in animal embryos. (LLM, conf=0.88)
Experimental — accuracy not yet measuredS.p. HiFi Cas9 Nuclease was used to increase on-target efficiency and minimize off-target editing.
“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]
The Introduction reports that HiFi Cas9 reduces off-target editing while maintaining high on-target activity. The Results section on RNP delivery reports on-target efficiency averaging 92% of wild-type Cas9, not an increase over wild type. It does report higher on-target efficiency than the other high-fidelity mutants tested, so the efficiency claim depends on the comparator. (LLM, conf=0.86)
Experimental — accuracy not yet measuredCompared to SpCas9, HiFi Cas9 shows similar editing efficiency but lower off-target effects in some genome sites.
“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]
The Cas9 nuclease is directed by a single-guide RNA to modify a specific chromosomal DNA sequence by inducing a sequence-specific double-strand break.
“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.
HypaCas9 mutation is identified as two different substitutions
MinorOne section lists the first HypaCas9 substitution as R692A, while the discussion lists it as N692A. The two descriptions cannot both identify the same four-mutation variant.
“HypaCas9 (R692A/M694A/Q695A/H698A)28”
“HypaCas9, which included 4 mutations (N692A/M694A/Q695A/H698A) in the REC3 domain.”
HypaCas9 mutation is identified inconsistently
MinorThe Results identify the substitution at position 692 as R692A, while the Discussion identifies it as N692A for the same HypaCas9 variant.
“HypaCas9 (R692A/M694A/Q695A/H698A)28.”
“HypaCas9, which included 4 mutations (N692A/M694A/Q695A/H698A) in the REC3 domain.”
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Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors
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CRISPR-engineered T cells in patients with refractory cancer
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CRISPR technology: A decade of genome editing is only the beginning
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“To reduce the offtarget effects of CRISPR-Cas nucleases resulting from unintended binding and cleavage, researchers have utilized a combination of rational design and selection to develop highfidelity Cas variants [such as SpCas9-HF1 (101), evoCas9 (102), HiFiCas9 (103)] and the Cas9_ R63A/Q768A variant (104) and guid…”
MethodCRISPR-Cas9 genome editing using targeted lipid nanoparticles for cancer therapy
Rosenblum, D., Gutkin, A., Kedmi, R., Ramishetti, S., Veiga, N., Jacobi, A. M., Schubert, M. S., Friedmann-Morvinski, D., Cohen, Z. R., Behlke, M. A., et al. (2020) · Science Advances · doi:10.1126/sciadv.abc9450 · Cited by 614 · see citing works
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CRISPR Gene Therapy: Applications, Limitations, and Implications for the Future
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“for the DNA:RNA heteroduplex to induce DSBs, thereby reducing OTEs while maintaining editing efficacy (38, 39).”
BackgroundTuning the Antigen Density Requirement for CAR T-cell Activity
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Self-citationHighly efficient therapeutic gene editing of human hematopoietic stem cells
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Machine learning in healthcare
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CRISPR-Based Therapeutic Genome Editing: Strategies and In Vivo Delivery by AAV Vectors
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Base editing: advances and therapeutic opportunities
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“Several studies have described Cas9 variants evolved to increase the targeting specificity [32,33].”
BackgroundUnbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq
Wienert, B., Wyman, S. K., Richardson, C. D., Yeh, C. D., Akcakaya, P., Porritt, M. J., Morlock, M., Vu, J. T., Kazane, K. R., Watry, H. L., et al. (2019) · Science · doi:10.1126/science.aav9023 · Cited by 434 · see citing works
“We formed these RNPs using WT Cas9 and the HiFi Cas9 260 R691A mutant (38) and compared the DISCOVER-Seq profiles of each experiment.”
MethodLatest Developed Strategies to Minimize the Off-Target Effects in CRISPR-Cas-Mediated Genome Editing
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DNA Repair Pathway Choices in CRISPR-Cas9-Mediated Genome Editing
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Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR/Cas9 genome editing
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CRISPR in cancer biology and therapy
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High prevalence of Streptococcus pyogenes Cas9-reactive T cells within the adult human population
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The emerging and uncultivated potential of CRISPR technology in plant science
Zhang, Y., Malzahn, A. A., Sretenovic, S., & Qi, Y. (2019) · Nature Plants · doi:10.1038/s41477-019-0461-5 · Cited by 404 · see citing works
Base editing of haematopoietic stem cells rescues sickle cell disease in mice
Newby, G. A., Yen, J. S., Woodard, K. J., Mayuranathan, T., Lazzarotto, C. R., Li, Y., Sheppard, H., Porter, S. N., Yao, Y., Mayberry, K. D., et al. (2021) · Nature · doi:10.1038/s41586-021-03609-w · Cited by 390 · see citing works
Increasing the specificity of CRISPR systems with engineered RNA secondary structures
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Optimized CRISPR guide RNA design for two high-fidelity Cas9 variants by deep learning
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“An initial 18 cycle PCR reaction using previously validated primers 14 (see Supplementary Table 1) to an approximately 150– 250 bp region centered on the predicted gRNA cut sites.”
Targeting a cytokine checkpoint enhances the fitness of armored cord blood CAR-NK cells
Daher, M., Başar, R., Gokdemir, E., Baran, N., Uprety, N., Cortes, A. K. N., Mendt, M. C., Kerbauy, L. N., Banerjee, P. P., Shanley, M., et al. (2020) · Blood · doi:10.1182/blood.2020007748 · Cited by 303 · see citing works
Self-citationRecent Advances in CRISPR/Cas9 Delivery Strategies
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CHANGE-seq reveals genetic and epigenetic effects on CRISPR–Cas9 genome-wide activity
Lazzarotto, C. R., Malinin, N. L., Li, Y., Zhang, R., Yang, Y., Lee, G., Cowley, E., He, Y., Lan, X., Jividen, K., et al. (2020) · Nature Biotechnology · doi:10.1038/s41587-020-0555-7 · Cited by 283 · see citing works
A New Class of Medicines through DNA Editing
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Self-citationAdvances in stem cell research and therapeutic development
Luca, M. D., Aiuti, A., Cossu, G., Parmar, M., Pellegrini, G., & Robey, P. G. (2019) · Nature Cell Biology · doi:10.1038/s41556-019-0344-z · Cited by 236 · see citing works
Quantitative evaluation of chromosomal rearrangements in gene-edited human stem cells by CAST-Seq
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Self-citationAssessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA editing
Tao, J., Bauer, D. E., & Chiarle, R. (2023) · Nature Communications · doi:10.1038/s41467-023-35886-6 · Cited by 215 · see citing works
Engineered materials for in vivo delivery of genome-editing machinery
Tong, S., Moyo, B., Lee, C. M., Leong, K. W., & Bao, G. (2019) · Nature Reviews Materials · doi:10.1038/s41578-019-0145-9 · Cited by 206 · see citing works
Self-citationHighly Parallel Profiling of Cas9 Variant Specificity
Schmid-Burgk, J. L., Gao, L., Li, D., Gardner, Z., Strecker, J., Lash, B., & Zhang, F. (2020) · Molecular Cell · doi:10.1016/j.molcel.2020.02.023 · Cited by 197 · see citing works
Improving Plant Genome Editing with High-Fidelity xCas9 and Non-canonical PAM-Targeting Cas9-NG
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Gene editing and CRISPR in the clinic: current and future perspectives
Hirakawa, M. P., Krishnakumar, R., Timlin, J. A., Carney, J. P., & Butler, K. S. (2020) · Bioscience Reports · doi:10.1042/bsr20200127 · Cited by 197 · see citing works
CRISPR-Cas Tools and Their Application in Genetic Engineering of Human Stem Cells and Organoids
Hendriks, D. F. G., Clevers, H. C., & Artegiani, B. (2020) · Cell stem cell · doi:10.1016/j.stem.2020.10.014 · Cited by 196 · see citing works
Gene correction for SCID-X1 in long-term hematopoietic stem cells
Pavel-Dinu, M., Wiebking, V., Dejene, B. T., Srifa, W., Mantri, S., Nicolas, C. E., Lee, C. M., Bao, G., Kildebeck, E., Punjya, N., et al. (2019) · Nature Communications · doi:10.1038/s41467-019-09614-y · Cited by 187 · see citing works
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“Therefore, we used RNP nucleofection and an AAV6 HDR donor for precise gene KI, which reportedly achieves high editing efficiency (24,27,28).”
MethodDevelopment of CRISPR-Cas systems for genome editing and beyond
Zhang, F. (2019) · Quarterly Reviews of Biophysics · doi:10.1017/s0033583519000052 · Cited by 185 · see citing works
“…of groups have subsequently used structural information or directed evolution to develop additional high-specificity variants of Cas9 (Kleinstiver et al., 2016a; Chen et al., 2017; Casini et al., 2018; Hu et al., 2018; Lee et al., 2018; Vakulskas et al., 2018) and Cas12a (Kleinstiver et al., 2019).”
BackgroundApplication of CRISPR/Cas9-Based Gene Editing in HIV-1/AIDS Therapy
Xiao, Q., Guo, D., & Chen, S. (2019) · Frontiers in Cellular and Infection Microbiology · doi:10.3389/fcimb.2019.00069 · Cited by 184 · see citing works
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Self-citationAsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines
Zhang, L., Zuris, J. A., Viswanathan, R., Edelstein, J., Turk, R., Thommandru, B., Rube, H. T., Glenn, S. E., Collingwood, M. A., Bode, N. M., et al. (2021) · Nature Communications · doi:10.1038/s41467-021-24017-8 · Cited by 161 · see citing works
Self-citationBalderston, S., Taulbee, J. J., Celaya, E., Fung, K., Jiao, A., Smith, K., Hajian, R., Gasiūnas, G., Kutanovas, S., Kim, D., et al. (2021) · Nature Biomedical Engineering · doi:10.1038/s41551-021-00706-z · Cited by 162 · see citing works
Mitigation of off-target toxicity in CRISPR-Cas9 screens for essential non-coding elements
Tycko, J., Wainberg, M., Marinov, G. K., Ursu, O., Hess, G. T., Ego, B. K., Aradhana, Li, A., Truong, A., Trevino, A. E., et al. (2019) · Nature Communications · doi:10.1038/s41467-019-11955-7 · Cited by 144 · see citing works
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“The use of blocked-cleavage primers requires that the primers correctly hybridize to the target sequence prior to amplification to reduce primer dimerization and increase specificity in multipexed PCR reactions (26).”
MethodResultInfluentialSelf-citationB cells engineered to express pathogen-specific antibodies protect against infection
Moffett, H. F., Harms, C. K., Fitzpatrick, K. S., Tooley, M. R., Boonyaratanakornkit, J. B., & Taylor, J. J. (2019) · Science Immunology · doi:10.1126/sciimmunol.aax0644 · Cited by 139 · see citing works
CRISPR-Cas-mediated transcriptional modulation: The therapeutic promises of CRISPRa and CRISPRi
Bendixen, L., Jensen, T. I., & Bak, R. O. (2023) · Molecular Therapy · doi:10.1016/j.ymthe.2023.03.024 · Cited by 144 · see citing works
Self-citationExploration of Plant-Microbe Interactions for Sustainable Agriculture in CRISPR Era
Shelake, R. M., Pramanik, D., & Kim, J. (2019) · Microorganisms · doi:10.3390/microorganisms7080269 · Cited by 141 · see citing works
“Some examples of the SpCas9 variants include: SpCas9(D1135E) [64], SpCas9(VQR) [86], SpCas9(EQR) [86], SpCas9(VRER) [86], SpCas9(QQR) [87], eSpCas9(1.0) [88], eSpCas9(1.1) [88], SpCas9-HF1 [89], HeFSpCas9 [90], HypaCas9 [91], evoCas9 [92] xCas9 [93], Cas9-NG [94], HiFiCas9 [95], Sniper Cas9 [96], iSpmacCas9 [95], eHF1…”
InfluentialNext-generation cell therapies: the emerging role of CAR-NK cells
Başar, R., Daher, M., & Rezvani, K. (2020) · Blood Advances · doi:10.1182/bloodadvances.2020002547 · Cited by 142 · see citing works
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An engineered ScCas9 with broad PAM range and high specificity and activity
Chatterjee, P., Jakimo, N., Lee, J., Amrani, N., Rodríguez, T., Koseki, S., Tysinger, E., Qing, R., Hao, S., Sontheimer, E. J., et al. (2020) · Nature Biotechnology · doi:10.1038/s41587-020-0517-0 · Cited by 128 · see citing works
Kararoudi, M. N., Nagai, Y., Elmas, E., Pereira, M. D. S. F., Ali, S. A., Imus, P. H., Wethington, D., Borrello, I., Lee, D. A., & Ghiaur, G. (2020) · Blood · doi:10.1182/blood.2020006200 · Cited by 131 · see citing works
Rivera, F. J. S., Diaz, B. J., Kastenhuber, E. R., Schmidt, H., Katti, A., Kennedy, M. C., Tem, V., Ho, Y.-J., Leibold, J., Paffenholz, S. V., et al. (2022) · Nature Biotechnology · doi:10.1038/s41587-021-01172-3 · Cited by 116 · see citing works
Romero, Z. G., Lomova, A., Said, S., Hoyt-Miggelbrink, A. M., Kuo, C. Y., Campo-Fernández, B., Hoban, M. D., Masiuk, K. E., Clark, D. N., Long, J. D., et al. (2019) · Molecular Therapy · doi:10.1016/j.ymthe.2019.05.014 · Cited by 123 · see citing works
Lei, T., Wang, Y., Zhang, Y., Yang, Y., Cao, J., Huang, J., Chen, J., Chen, H., Zhang, J., wang, L., et al. (2024) · Leukemia · doi:10.1038/s41375-024-02444-y · Cited by 141 · see citing works
Human genome-edited hematopoietic stem cells phenotypically correct Mucopolysaccharidosis type I
Gomez‐Ospina, N., Scharenberg, S. G., Mostrel, N., Bak, R. O., Mantri, S., Quadros, R. M., Gurumurthy, C. B., Lee, C. M., Bao, G., Suarez, C. J., et al. (2019) · Nature Communications · doi:10.1038/s41467-019-11962-8 · Cited by 119 · see citing works
Self-citationA Cas9 with PAM recognition for adenine dinucleotides
Chatterjee, P., Lee, J., Nip, L., Koseki, S., Tysinger, E., Sontheimer, E. J., Jacobson, J. M., & Jakimo, N. (2020) · Nature Communications · doi:10.1038/s41467-020-16117-8 · Cited by 121 · see citing works
Human genetic diversity alters off-target outcomes of therapeutic gene editing
Cancellieri, S., Zeng, J., Lin, L. Y., Tognon, M., Nguyen, M. A., Lin, J., Bombieri, N., Maitland, S. A., Ciuculescu, M.-F., Katta, V., et al. (2022) · Nature Genetics · doi:10.1038/s41588-022-01257-y · Cited by 122 · see citing works
Development of hRad51–Cas9 nickase fusions that mediate HDR without double-stranded breaks
Rees, H. A., Yeh, W.-H., & Liu, D. R. (2019) · Nature Communications · doi:10.1038/s41467-019-09983-4 · Cited by 122 · see citing works
Evaluation and Reduction of CRISPR Off-Target Cleavage Events
Vakulskas, C. A., & Behlke, M. A. (2019) · Nucleic Acid Therapeutics · doi:10.1089/nat.2019.0790 · Cited by 121 · see citing works
“For the present, HiFi Cas9 seems to be the primary choice if RNP methods are planned [47].”
BackgroundMethodInfluentialSelf-citationNelson, M. R., Liu, P., Agrawal, A., Yip, O., Blumenfeld, J., Traglia, M., Kim, M. J., Koutsodendris, N., Rao, A., Grone, B., et al. (2023) · Nature Neuroscience · doi:10.1038/s41593-023-01480-8 · Cited by 118 · see citing works
“To further increase on-target efficiency and minimize off-target editing, S.p. HiFi Cas9 Nuclease was used 77 .”
MethodJackow, J., Guo, Z., Hansen, C. S., Abaci, H. E., Doucet, Y., Shin, J. U., Hayashi, R., DeLorenzo, D., Kabata, Y., Shinkuma, S., et al. (2019) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1907081116 · Cited by 118 · see citing works
CRISPR-Cas9 Gene Editing in Lizards through Microinjection of Unfertilized Oocytes
Rasys, A. M., Park, S., Ball, R., Alcala, A. J., Lauderdale, J. D., & Menke, D. B. (2019) · Cell Reports · doi:10.1016/j.celrep.2019.07.089 · Cited by 116 · see citing works
Self-citationImproved genome editing by an engineered CRISPR-Cas12a
Ma, E., Chen, K., Shi, H., Stahl, E. C., Adler, B., Trinidad, M., Liu, J., Zhou, K., Ye, J., & Doudna, J. A. (2022) · Nucleic Acids Research · doi:10.1093/nar/gkac1192 · Cited by 110 · see citing works
Design of highly functional genome editors by modelling CRISPR–Cas sequences
Ruffolo, J. A., Nayfach, S., Gallagher, J. P., Bhatnagar, A., Beazer, J., Hussain, R., Russ, J., Yip, J., Hill, E., Pačesa, M., et al. (2025) · Nature · doi:10.1038/s41586-025-09298-z · Cited by 212 · see citing works
“In the future, it will be important to examine OpenCRISPR-1 activity across a range of experimental conditions, cell types and delivery methods to more thoroughly characterize robustness 11,37,47 .”
Wilkinson, A. C., Dever, D. P., Baik, R., Camarena, J., Hsu, I., Charlesworth, C. T., Morita, C., Nakauchi, H., & Porteus, M. H. (2021) · Nature Communications · doi:10.1038/s41467-021-20909-x · Cited by 111 · see citing works
Self-citationSelvaraj, S., Feist, W. N., Viel, S., Vaidyanathan, S., Dudek, A. M., Gastou, M., Rockwood, S. J., Ekman, F. K., Oseghale, A., Xu, L., et al. (2023) · Nature Biotechnology · doi:10.1038/s41587-023-01888-4 · Cited by 102 · see citing works
“69,70).”
Self-citationImproved gRNA secondary structures allow editing of target sites resistant to CRISPR-Cas9 cleavage
Riesenberg, S., Helmbrecht, N., Kanis, P., Maricic, T., & Pääbo, S. (2022) · Nature Communications · doi:10.1038/s41467-022-28137-7 · Cited by 109 · see citing works
Martin, R. M., Fowler, J. L., Cromer, M., Lesch, B., Ponce, E., Uchida, N., Nishimura, T., Porteus, M. H., & Loh, K. (2020) · Nature Communications · doi:10.1038/s41467-020-16455-7 · Cited by 100 · see citing works
Self-citationBanakar, R., Schubert, M. S., Collingwood, M. A., Vakulskas, C. A., Eggenberger, A. L., & Wang, K. (2020) · Rice · doi:10.1186/s12284-019-0365-z · Cited by 104 · see citing works
“There are mutant variants of Cas9 proteins from Streptococcus pyogenes available such as High Fidelity SpCas9 (HiFi Cas9) and Cas9 nickases (SpCas9 D10A and SpCas9 H840A) (Schiml et al. 2014; Shen et al. 2014; Vakulskas et al. 2018).”
BackgroundSelf-citationImproving homology-directed repair efficiency in human stem cells.
Skarnes, W., Pellegrino, E., & McDonough, J. (2019) · Methods · doi:10.1016/j.ymeth.2019.06.016 · Cited by 91 · see citing works
Schenke, D., & Cai, D. (2020) · iScience · doi:10.1016/j.isci.2020.101478 · Cited by 112 · see citing works
“, 2017), orHiFi Cas9 (Vakulskas et al., 2018), resulting in systems with almost no unintendedmutations.”
BackgroundCRISPR/Cas9 gene editing for curing sickle cell disease
Park, S. H., & Bao, G. (2021) · Transfusion and Apheresis Science · doi:10.1016/j.transci.2021.103060 · Cited by 110 · see citing works
“High-fidelity variants of SpyCas9 maintain on-target activities comparable to wild-type SpyCas9 with reduced offtarget activities in HSPCs [42].”
BackgroundSelf-citationCRISPR technologies for stem cell engineering and regenerative medicine
Hsu, M., Chang, Y., Truong, V. A., Lai, P., Nguyen, T. K. N., & Hu, Y. (2019) · Biotechnology Advances · doi:10.1016/j.biotechadv.2019.107447 · Cited by 105 · see citing works
TCR Redirected T Cells for Cancer Treatment: Achievements, Hurdles, and Goals
Manfredi, F., Cianciotti, B. C., Potenza, A., Tassi, E., Noviello, M., Biondi, A., Ciceri, F., Bonini, C., & Ruggiero, E. (2020) · Frontiers in Immunology · doi:10.3389/fimmu.2020.01689 · Cited by 105 · see citing works
“As for ZFNs (140–142) and TALENs (143, 144), a side effect of the CRISPR/Cas9 system is the risk of editing off-target genes.”
MethodInfluentialPrinciples of Genetic Engineering
Lanigan, T. M., Kopera, H. C., & Saunders, T. L. (2020) · Genes · doi:10.3390/genes11030291 · Cited by 104 · see citing works
CRISPR engineering in organoids for gene repair and disease modelling
Geurts, M. H., & Clevers, H. (2023) · Nature Reviews Bioengineering · doi:10.1038/s44222-022-00013-5 · Cited by 103 · see citing works
Lentiviral and genome-editing strategies for the treatment of β-hemoglobinopathies
Magrin, E., Miccio, A., & Cavazzana, M. (2019) · Blood · doi:10.1182/blood.2019000949 · Cited by 97 · see citing works
Pharmacological interventions enhance virus-free generation of TRAC-replaced CAR T cells
Kath, J., Du, W., Pruene, A., Braun, T., Thommandru, B., Turk, R., Sturgeon, M. L., Kurgan, G., Amini, L., Stein, M., et al. (2022) · Molecular Therapy — Methods & Clinical Development · doi:10.1016/j.omtm.2022.03.018 · Cited by 95 · see citing works
“GUIDE-seq GUIDE-Seq(68) for unbiased identification of CRISPR-Cas off targets was performed as previously described.(54,69) Briefly, SpCas9-expressing HEK293 cells were co-transfected with dsDNA oligonucleotides (GUIDE-seq tag; for sequence, see Tsai et al.”
MethodSelf-citationELAVL1 Primarily Couples mRNA Stability with the 3’UTRs of Interferon Stimulated Genes
Rothamel, K. L., Arcos, S., Kim, B., Reasoner, C., Lisy, S. M., Mukherjee, N., & Ascano, M. (2020) · Cell Reports · doi:10.1016/j.celrep.2021.109178 · Cited by 89 · see citing works
“The oligo duplex was then cooled at room temperature for 10 minutes prior to adding Cas 9 nuclease enzyme (IDT) to assemble RNPs (Vakulskas et al., 2018).”
CRISPR/Cas System and Factors Affecting Its Precision and Efficiency
Javaid, N., & Choi, S. (2021) · Frontiers in Cell and Developmental Biology · doi:10.3389/fcell.2021.761709 · Cited by 98 · see citing works
“Similarly, high-fidelity (HiFi) Cas9 variant was identified via unbiased bacterial screening approach in which a single point mutation (R691A) caused reduced off-targeting while maintaining higher on-target activity as a RNP complex (Vakulskas et al., 2018).”
BackgroundLiu, M., Gong, S., Yu, H., Jung, K., Johnson, K. A., & Taylor, D. W. (2020) · Nature Communications · doi:10.1038/s41467-020-17411-1 · Cited by 100 · see citing works
Park, S. H., Cao, M., Pan, Y., Davis, T., Saxena, L., Deshmukh, H., Fu, Y., Treangen, T. J., Sheehan, V., & Bao, G. (2022) · Science Advances · doi:10.1126/sciadv.abo7676 · Cited by 92 · see citing works
Self-citationAhmadi, S. E., Soleymani, M., Shahriyary, F., Amirzargar, M. R., Mahya, O., Fattahi, D., & Safa, M. (2023) · Cancer Gene Therapy · doi:10.1038/s41417-023-00597-z · Cited by 95 · see citing works
Tadić, V., Josipović, G., Zoldoš, V., & Vojta, A. (2019) · Methods · doi:10.1016/j.ymeth.2019.05.003 · Cited by 96 · see citing works
Epigenome engineering: new technologies for precision medicine
Sgro, A., & Blancafort, P. (2020) · Nucleic Acids Research · doi:10.1093/nar/gkaa1000 · Cited by 96 · see citing works
“The SniperSpCas9 (F539S, M763I, K890N) (224) and the high fidelity HiFi-SpCas9 (R691A) (222) variants have been delivered in a preassembled RNP format, the latter for the treatment of sickle cell disease.”
MethodRorα Enforces Stability of the T-Helper-17 Cell Effector Program
Hall, J. A., Pokrovskii, M. V., Kroehling, L., Kim, B., Kim, S. Y., Wu, L., Lee, J., & Littman, D. R. (2020) · Immunity · doi:10.1016/j.immuni.2022.09.013 · Cited by 99 · see citing works
“…Cas9:sgRNA) Alt-R (Integrated DNA Technologies, Inc) Cas9 RNP complex, and 20 μM Alt-R Cas9 Electroporation Enhancer (Integrated DNA Technologies, Inc) as described previously (Vakulskas et al., 2018). sgRNAs were designed using the Crispr guide design software (Integrated DNA Technologies, Inc).”
Discovery of Diverse CRISPR-Cas Systems and Expansion of the Genome Engineering Toolbox
Koonin, E. V., Gootenberg, J. S., & Abudayyeh, O. O. (2023) · Biochemistry · doi:10.1021/acs.biochem.3c00159 · Cited by 92 · see citing works
High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA
Oh, S. A., Senger, K., Madireddi, S., Akhmetzyanova, I., Ishizuka, I. E., Tarighat, S., Lo, J. H., Shaw, D., Haley, B., & Rutz, S. (2022) · Journal of Experimental Medicine · doi:10.1084/jem.20211530 · Cited by 88 · see citing works
“Several studies have subsequently reported high editing efficiencies using AAV-based repair templates (Choi et al., 2019; Vakulskas et al., 2018; Dai et al., 2019).”
MethodCRISPR-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) · Nature Communications · doi:10.1038/s41467-021-27190-y · Cited by 90 · see citing works
CRISPR off-target detection with DISCOVER-seq
Wienert, B., Wyman, S. K., Yeh, C. D., Conklin, B. R., & Corn, J. E. (2020) · Nature Protocols · doi:10.1038/s41596-020-0309-5 · Cited by 91 · see citing works
CRISPR/Cas9 Landscape: Current State and Future Perspectives
Tyumentsev, A. I., Tyumentsev, A. I., & Акимкин, В. Г. (2023) · International Journal of Molecular Sciences · doi:10.3390/ijms242216077 · Cited by 95 · see citing works
“Also, CRISPR/Cas9 was used to produce CAR-T cells in which a CAR or TCR cassette was introduced into the endogenous TCR gene locus to mitigate graft-versus-host disease, preventing random integration of the cassettes and ensuring uniform CAR (chimeric antigen receptor) expression [299–302].”
Design of highly functional genome editors by modeling the universe of CRISPR-Cas sequences
Ruffolo, J. A., Nayfach, S., Gallagher, J., Bhatnagar, A., Beazer, J., Hussain, R., Russ, J., Yip, J., Hill, E., Pacesa, M., et al. (2024) · doi:10.1101/2024.04.22.590591 · Cited by 90 · see citing works
Gene Therapy for Cystic Fibrosis: Progress and Challenges of Genome Editing
Maule, G., Arosio, D., & Cereseto, A. (2020) · International Journal of Molecular Sciences · doi:10.3390/ijms21113903 · Cited by 98 · see citing works
“Electroporation is the preferred method for CRISPR-Cas9 delivery ex-vivo which allows transient expression of the nuclease and limited off-target activity [58].”
MethodEfficient high-precision homology-directed repair-dependent genome editing by HDRobust
Riesenberg, S., Kanis, P., Macak, D., Wollny, D., Düsterhöft, D., Kowalewski, J., Helmbrecht, N., Maričić, T., & Pääbo, S. (2023) · Nature Methods · doi:10.1038/s41592-023-01949-1 · Cited by 87 · see citing works
Off-target effects in CRISPR-Cas genome editing for human therapeutics: Progress and challenges
Kalter, N., Fuster‐García, C., Silva, A., Ronco-Díaz, V., Roncelli, S., Turchiano, G., Gorodkin, J., Cathomen, T., Benabdellah, K., Lee, C. M., et al. (2025) · Molecular Therapy — Nucleic Acids · doi:10.1016/j.omtn.2025.102636 · Cited by 107 · see citing works
“54,55 Recently, large language models have entered protein engineering, including CRISPR proteins.”
Self-citationLopes, R., & Prasad, M. K. (2024) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2023.1339189 · Cited by 82 · see citing works
- 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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