Delivering CRISPR: a review of the challenges and approaches

In the paper

“Gene therapy may greatly benefit from CRISPR/Cas9 technology.”

What the sources say

• Supported

  The Abstract identifies CRISPR-associated Cas9 as a technology with therapeutic genome-editing potential. The Therapeutic Genome Editing Strategies section explains that targeted gene insertion can serve a purpose similar to viral gene therapy while offering better control over transgene copy number and expression. This supports the broad, hedged claim; the indexed passages provide no page numbers. (LLM, conf=0.88)

  cites Cox et al., 2015 source

In the paper

“Using a seventh-generation evolved transfer RNA adenosine deaminase attached to dCas9, researchers demonstrated conversion of A*T base pairs to G*C base pairs in a targeted fashion at efficiencies of around 50% with few undesired mutations in human cells.”

What the sources say

• Partly supported

  The Results (Characterization of Late-Stage ABEs; page unavailable) report 54% on-target editing by ABE7.10 in human cells and relatively low off-target editing. But the Results (Evolution of an adenine deaminase that processes DNA; page unavailable) say the mammalian editor replaces dCas9 with a Cas9 nickase. The claim’s dCas9 description therefore does not accurately describe the editor used for the human-cell result. (LLM, conf=0.88)

  cites Gaudelli et al., 2017 sourceExperimental — accuracy not yet measured

In the paper

“Increasing access to the NLS by adding a 32 amino acid spacer between the NLS and Cas9 was shown to increase DNA cleavage activity (Shen et al., 2013).”

What the sources say

• Supported

  The Dear Editor section states that adding a 32-amino-acid linker between the NLS and Cas9 enabled nuclear localization and that the resulting NLS-flag-linker-Cas9 showed enhanced cleavage activity. The proposed explanation—that the NLS had been buried or shielded—is presented as a possibility, not an established mechanism. (LLM, conf=0.93)

  cites Shen et al., 2013 source

In the paper

“A powerful advantage of the CRISPR/Cas9 system is the ability to target any ∼23-bp sequence that contains a PAM motif on either strand of DNA.”

What the sources say

• Partly supported

  The Abstract (page unavailable) says that Streptococcus pyogenes Cas9 targets a sequence complementary to a 20-nt guide followed by a 3-nt NGG PAM, supporting the approximately 23-bp target description. Our copy does not state that targets can be selected on either DNA strand, and it specifies NGG rather than an unspecified PAM motif. (LLM, conf=0.82)

  cites Ramakrishna et al., 2014 sourceExperimental — accuracy not yet measured

In the paper

“Several Cas9 proteins from differing species have been identified and used for gene editing, including Staphylococcus aureus (SaCas9) (Ran et al., 2013), Neisseria meningitidis (NmCas9) (Hou et al., 2013), and S. thermophiles (St1Cas9) (Kleinstiver et al., 2015).”

What the sources say

• ?Could not verify

  The Introduction identifies the Cas9 nuclease studied as coming from Streptococcus pyogenes, not Staphylococcus aureus. A search for Staphylococcus aureus and SaCas9 found no passage in our copy addressing whether SaCas9 had been identified or used for gene editing; the other species in the citing sentence have separate citations. (LLM, conf=0.76)

  cites Ran et al., 2013 sourceExperimental — accuracy not yet measured
• Supported

  The paper identifies NmCas9 as the Cas9 protein from Neisseria meningitidis and reports its use for genome editing in human pluripotent stem cells (opening section, passage 7; page not provided). The Hou et al. citation is attached specifically to NmCas9, not to the other species in the sentence. (LLM, conf=0.95)

  cites Hou et al., 2013 source
• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Kleinstiver et al., 2015

In the paper

“Cas9 mutants have also been designed to reduce non-specific DNA contacts by weakening binding of the target DNA strand (Kleinstiver et al., 2016) or the non-target DNA strand (Slaymaker et al., 2016) to Cas9 while maintaining robust on-target cleavage.”

What the sources say

• Supported

  The Abstract describes designing SpCas9 substitutions to disrupt non-specific contacts with the target DNA strand while retaining robust on-target activity. In “Alteration of SpCas9 DNA contacts,” the authors report that the tested variants had on-target activity comparable to wild-type SpCas9 in an EGFP assay. The Kleinstiver citation is attached to the target-strand part of the sentence, not the non-target-strand part. (LLM, conf=0.91)

  cites Kleinstiver et al., 2016 source
• Supported

  The cited paper supports the non-target-strand part of the sentence; the target-strand part is attributed to a different citation. Its reported findings state that mutations weakened Cas9 interactions with the non-target DNA strand, reduced off-target cutting, and preserved on-target activity. (LLM, conf=0.94)

  cites Slaymaker et al., 2016 source

In the paper

“Even in the presence of donor template DNA, NHEJ is the more frequent repair mechanism observed from CRISPR/Cas9 editing (Maruyama et al., 2015).”

What the sources say

• Supported

  The passage on baseline editing in mouse zygotes (indexed as Abstract; no page given) reports that insertional mutagenesis was less frequent than deletional mutagenesis in the same experiment. At both reported loci, deletion frequencies exceeded insertion frequencies, supporting the claim that NHEJ was more frequent despite the use of donor templates. (LLM, conf=0.92)

  cites Maruyama et al., 2015 source

In the paper

“Increasing the relative concentration of sgRNA to Cas9 protein was also shown in increase on-target cutting activity, presumably by ensuring all Cas9 proteins formed the active ribonucleoprotein complex (Kim et al., 2014).”

What the sources say

• Supported

  The Results section (‘Targeted mutagenesis in human cells using RGEN RNPs’) reports that up to a sixfold molar excess of sgRNA over Cas9 was required to maximize mutation frequencies. This supports the claim that increasing sgRNA relative to Cas9 increased on-target editing activity. (LLM, conf=0.92)

  cites Kim et al., 2014 source

In the paper

“The use of this system in mammalian cells reduced off-target cutting by three orders of magnitude with little to no reduction in on-target cutting efficacy (Mali et al., 2013; Ran et al., 2013; Cho et al., 2014).”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Mali et al., 2013
• Partly supported

  The Abstract reports that paired nicking reduced off-target activity by 50–1,000-fold in cell lines without sacrificing on-target cleavage efficiency. The Results also report comparable on-target editing, but a three-orders-of-magnitude reduction is the upper end of the reported range, not the reduction observed throughout. (LLM, conf=0.86)

  cites Ran et al., 2013 sourceExperimental — accuracy not yet measured
• Partly supported

  The Results ('Paired Cas9 nickases,' Fig. 5A–B; page not provided) report on-target indel frequencies comparable to paired nucleases and no detectable off-target indels from paired nickases at six sites where Cas9 nucleases caused mutations. They do not establish a three-orders-of-magnitude reduction in off-target cutting: the reported 990-fold figure compares on-target with off-target activity for one nickase pair, not nickase with nuclease off-target activity. (LLM, conf=0.88)

  cites Cho et al., 2014 sourceExperimental — accuracy not yet measured

In the paper

“However, several groups have shown good success with the use of Cas9:sgRNA RNP complexes (Zuris et al., 2015; Wang et al., 2016).”

What the sources say

• Partly supported

  The Results section ('Anionic sgRNA is necessary and sufficient for Cas9 delivery'; page not available) reports successful cationic lipid-mediated delivery of Cas9:sgRNA complexes, with up to 80% targeted gene disruption. The cited paper supports the success of lipid-mediated RNP delivery, but the passages describe Lipofectamine and RNAiMAX rather than establishing the more specific claim of lipid nanoparticle delivery. (LLM, conf=0.85)

  cites Zuris et al., 2015 sourceExperimental — accuracy not yet measured
• Supported

  The Results and Discussion report that bioreducible lipid nanoparticles delivered Cas9:sgRNA complexes into HEK cells, with several lipids producing more than 50% loss of EGFP expression. The Conclusions likewise describe nanoparticle delivery of these complexes for genome editing. This supports the success attributed to Wang et al.; the separate Zuris citation is not assessed here. (LLM, conf=0.94)

  cites Wang et al., 2016 source

In the paper

“If delivering Cas9 mRNA and sgRNA, this method is functionally like microinjection in result (Yin et al., 2016).”

What the sources say

• Partly supported

  The paper reports that lipid nanoparticles delivered Cas9 mRNA and enabled genome editing, but it delivered sgRNA by AAV, not lipid nanoparticles (Abstract; page unavailable). Its reported comparison is with high-pressure injection of DNA, not microinjection; our copy does not establish that the outcomes are functionally alike. (LLM, conf=0.86)

  cites Yin et al., 2016 sourceExperimental — accuracy not yet measured

In the paper

“Examples of LV delivery of CRISPR/Cas9 include modification of up to five genes using a single LV to deliver plasmid DNA encoding Cas9, sgRNA and a fluorescent marker to develop a mouse model of acute myeloid leukemia (Heckl et al., 2014)”

What the sources say

• Partly supported

  The indexed copy’s Abstract reports lentiviral CRISPR-Cas9 editing of up to five genes and the generation of mouse AML models. It also describes a single lentiviral vector carrying Cas9, an sgRNA and a fluorescent marker. But for multiplex editing, it specifies a two-vector system and a pool of sgRNA vectors, not modification of five genes using a single lentiviral vector; it does not describe the lentivirus as delivering plasmid DNA. (LLM, conf=0.91)

  cites Heckl et al., 2014 sourceExperimental — accuracy not yet measured

In the paper

“In addition, there is evidence that injection of Cas9 mRNA and sgRNA into the zygote cytoplasm is the most efficient method for yielding normal embryos and full-term mouse pups harboring the desired modification (Horii et al., 2014).”

What the sources say

• Supported

  The Methods identify the injected RNAs as Cas9 mRNA and gRNA. The Results report that cytoplasmic RNA injection produced the highest blastocyst and full-term developmental rates among the three methods tested, and the Discussion reports the best overall knockout efficiency. Thus the claim is supported within the methods compared in this study. (LLM, conf=0.91)

  cites Horii et al., 2014 source

In the paper

“target of herpes simplex virus-1 genome regions essential to viral protein expression during early and late phases of viral infection/reinfection to suppress infection and prevent new infection (Roehm et al., 2016)”

What the sources say

• Supported

  The Results section reports that lentiviral delivery of guide RNAs targeting HSV-1 genes suppressed immediate-early, early, and late viral protein expression and reduced viral production in infected cells. It also reports that lentiviral delivery of Cas9 and guide RNAs protected cultured cells against subsequent HSV-1 infection. These are cell-culture findings, not demonstrated prevention of infection in people. (LLM, conf=0.90)

  cites Roehm et al., 2016 source

In the paper

“Further, two microinjections, even when separated by several hours, typically results in non-viable cells (Yang et al., 2013).”

What the sources say

• Partly supported

  The Results section describes two injections into the same zygote two hours apart. It reports a low rate of targeted blastocysts from that procedure, but does not say that the injections typically made the embryos non-viable; targeting efficiency is not a measure of viability. No page number is available in our copy. (LLM, conf=0.86)

  cites Yang et al., 2013 sourceExperimental — accuracy not yet measured

In the paper

“and the use of LV to mutate genes in mouse primary immune cells (Platt et al., 2014).”

What the sources say

• Supported

  The Discussion explicitly says the study mutated genes in primary immune cells using lentivirus. The Results section describes infecting mouse bone-marrow-derived dendritic cells with lentivirus carrying sgRNAs targeting MyD88 or A20 and detecting indels. (LLM, conf=0.95)

  cites Platt et al., 2014 source

In the paper

“Shalem et al. (2014) delivered 64,751 unique sgRNA sequences by LV to human cancer cells”

What the sources say

• Supported

  The Abstract states that the authors used lentiviral delivery of a GeCKO library containing 64,751 unique guide sequences. It also says they screened for cell viability in cancer cells and drug resistance in a melanoma model, supporting the stated cell type. (LLM, conf=0.93)

  cites Shalem et al., 2014 source

In the paper

“Four months post treatment, Pten gene-edited mice showed massive hepatomegaly and features of NASH.”

What the sources say

• Supported

  The Abstract states that mice receiving the Pten gene-editing adenovirus vector showed massive hepatomegaly and features of NASH four months after infusion. The Introduction likewise reports hepatomegaly and steatohepatitis at four months. (LLM, conf=0.95)

  cites Wang et al., 2015 source

In the paper

“typical AdVs and LVs elicit strong immune responses (Follenzi et al., 2007; Ahi et al., 2011).”

What the sources say

• ?Not checked — no full text of the sourcecites Follenzi et al., 2007 source
• Partly supported

  Section 2 (Adenoviral Immunity) says first-generation adenoviral vectors stimulate cellular and humoral immune responses, and Section 3 describes immediate innate immune activation after systemic administration. The cited paper supports the AdV portion, but the passages do not establish the claim about lentiviral vectors (LVs). (LLM, conf=0.86)

  cites Ahi et al., 2011 sourceExperimental — accuracy not yet measured

In the paper

“Zuckermann et al. (2015) reported the use of PEI to deliver Cas9- and sgRNA-encoding plasmid DNA into mouse brains to generate a malignant brain tumor model (Figure 7(B)).”

What the sources say

• Supported

  The Methods (Animal studies; page unavailable) describe injecting plasmid DNA mixed with in vivo-jetPEI into mouse brains. The Results (Postnatal somatic CRISPR/Cas-mediated deletion of Ptch1; page unavailable) report that PEI delivery of Cas9 and a Ptch1-targeting guide RNA induced medulloblastoma in Trp53-knockout mice. (LLM, conf=0.93)

  cites Zuckermann et al., 2015 source

In the paper

“Some work with CPPs and CRISPR/Cas9 was accomplished as early as 2014 by Ramakrishna et al. (2014), who showed separate delivery of CPP–Cas9 and CPP–sgRNA to multiple human cell lines.”

What the sources say

• Supported

  The Results section, “Application of Cas9-m9R and sgRNA:9R to several human cell types” (page not provided), reports treating multiple human cell types with the two CPP-associated components. The Methods section describes how Cas9-m9R and sgRNA:9R were prepared as distinct components; for the multiple-cell-type experiments, they were applied simultaneously rather than one after the other. (LLM, conf=0.91)

  cites Ramakrishna et al., 2014 source

In the paper

“Lipofectamine has been used to deliver Cas9- and sgRNA-encoding plasmid DNA to human pluripotent stem cells to generate a model for Immunodeficiency, Centromeric region instability, Facial anomalies syndrome (ICF) syndrome with 63% transfection efficiency (Horii et al., 2013)”

What the sources say

• Partly supported

  The Transfection of Cells section says Lipofectamine 2000 was used to deliver Cas9- and sgRNA-expression plasmids into human iPS cells, and the Conclusions describe an ICF syndrome model. But the Abstract identifies 63% as the proportion of transfected clones with mutations in both DNMT3B alleles, not the transfection efficiency. (LLM, conf=0.93)

  cites Horii et al., 2013 sourceExperimental — accuracy not yet measured

In the paper

“transfect human cells with an all-in-one expression cassette with up to seven sgRNAs and a Cas9 nuclease/nickase (Sakuma et al., 2014)”

What the sources say

• Supported

  The Methods (Cell culture and transfection) state that Lipofectamine LTX was used to transfect HEK293T human cells with CRISPR/Cas9 plasmids. The Results report an all-in-one vector with seven gRNAs and Cas9 nuclease transfected into those cells, and a separate vector with six gRNAs and Cas9 nickase. (LLM, conf=0.94)

  cites Sakuma et al., 2014 source

In the paper

“iTOP was used to delivery CRISPR/Cas9 RNP into primary human KBM7 and H1 cells, conferring diphtheria toxin resistance with ∼70% gene knockout efficiency.”

What the sources say

• Partly supported

  In Protein-Mediated Gene Editing, the paper describes iTOP delivery of Cas9 protein and guide RNA to KBM7 and H1 human embryonic stem cells. Its DPH7 knockout results report diphtheria-toxin resistance in around 70% of clones for four of six guide RNAs. The claim overgeneralizes by calling both cell types primary and by presenting the approximately 70% efficiency without its guide-RNA qualifier; the paper describes KBM7 as a near-diploid clone. (LLM, conf=0.88)

  cites D'Astolfo et al., 2015 sourceExperimental — accuracy not yet measured

In the paper

“Ebina et al. (2013) used calcium phosphate transfection to deliver plasmid DNA encoding Cas9 and sgRNA into latent HIV-1-infected human 293 T cells.”

What the sources say

• Partly supported

  The Methods (‘TF and flow cytometry’; page not provided) say that 293 T cells were transfected by the calcium phosphate method using hCas9 and gRNA expression vectors. The Results (‘LTR-specific editing’; page not provided) show those cells carried an HIV-1 provirus model, but the paper identifies its latency-model cells as Jurkat clones, not 293 T cells. Thus the delivery method and plasmids are supported, while the description of the 293 T cells as latently infected is not. (LLM, conf=0.91)

  cites Ebina et al., 2013 sourceExperimental — accuracy not yet measured

In the paper

“They showed greater efficiencies for electroporation transfection versus lipofectamine”

What the sources say

• Supported

  The Discussion (page unavailable) states that electroporation produced the highest target-specific indel levels under the conditions tested. The Methods (section 2.5, page unavailable) identify Lipofectamine as the lipid-based transfection reagent used for comparison. The finding concerns genome-editing efficiency in the tested cell lines, not transfection efficiency in every setting. (LLM, conf=0.91)

  cites Liang et al., 2015 source

In the paper

“introduce modular ‘AND’ gate circuits based on CRISPR/Cas9 that detects bladder cancer cells, inhibits bladder cancer cell growth, induces apoptosis, and decreases cell motility (Liu et al., 2014)”

What the sources say

• Supported

  The Cell transfection methods in Additional information state that the plasmid mixtures were introduced into cells using Lipofectamine 2000. The Abstract reports that the modular CRISPR-Cas9 AND gate circuits detected bladder cancer cells, inhibited their growth, induced apoptosis, and decreased cell motility in vitro. (LLM, conf=0.94)

  cites Liu et al., 2014 source

In the paper

“By coating the nanoclew with PEI to induce endosomal escape (Figure 8(A-b)), the group demonstrated roughly 36% efficiency in delivery of CRISPR/Cas9 RNP with the nanoclew (compared with 5% with bare Cas9:sgRNA and PEI).”

What the sources say

• Supported

  The paper describes coating the Cas9/sgRNA-loaded nanoclew with PEI to help induce endosomal escape. In its EGFP cell assay, the nanoclew/PEI particles reduced fluorescence in approximately 36% of cells, versus 5% for Cas9/sgRNA/PEI without the nanoclew. These percentages measure loss of EGFP fluorescence, rather than direct uptake of the RNP. (LLM, conf=0.90)

  cites Sun et al., 2015 source

In the paper

“and deliver Cas9:sgRNA RNP in vivo to modify the hair cells within mouse inner ear (Zuris et al., 2015).”

What the sources say

• Supported

  The Results section (‘In vivo delivery of Cre recombinase and Cas9:sgRNA’) reports that Cas9:sgRNA complexed with Lipofectamine 2000 was delivered to mouse cochlea in vivo, with loss of GFP expression in 20 ± 3% of outer hair cells near the injection site. The Online Methods (‘Microinjection of proteins to mouse inner ear’) also specifies mixing Cas9:sgRNA with Lipofectamine 2000 before injection. (LLM, conf=0.94)

  cites Zuris et al., 2015 source

In the paper

“and lower off-target effects using RNP over plasmid DNA or mRNA cargo.”

What the sources say

• Supported

  Section 3.2 reports that, at both detected off-target sites in HEK293FT cells, the off-target-to-on-target mutation ratio was lower with Cas9 RNP than with either plasmid DNA or mRNA. The finding is limited to the sites tested; it does not establish a universal reduction across all off-target sites. (LLM, conf=0.89)

  cites Liang et al., 2015 source

In the paper

“this biological delivery system is composed of a mesoporous silica nanoparticle core and a lipid membrane shell (Liu et al., 2009).”

What the sources say

• Supported

  The paper describes liposome fusion onto a mesoporous silica particle core to create the delivery construct. It also reports a confocal image showing a green silica core and a red lipid shell, directly supporting the stated composition (passages 1 and 3). (LLM, conf=0.94)

  cites Liu et al., 2009 source

In the paper

“In addition, pore size, pore chemistry, and overall particle size can be individually tailored, allowing for the loading of various types of cargo (Du et al., 2014; Durfee et al., 2016).”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Du et al., 2014
• Supported

  The passage labeled Abstract says mesoporous silica nanoparticles have controlled size and shape, while pore size and surface chemistry can be varied widely to accommodate disparate cargos. The Synthesis Criteria for Monosized Protocells section also describes particle size and pore size as modular features. (LLM, conf=0.92)

  cites Durfee et al., 2016 source

In the paper

“To date, lipid-coated mesoporous silica particles have been loaded with a variety of imaging agents, chemotherapeutics, and phototherapy agents for both in vitro and in vivo work (Mackowiak et al., 2013; Wang et al., 2013; Durfee et al., 2016; Su et al., 2017).”

What the sources say

• ?Not checked — no full text of the sourcecites Mackowiak et al., 2013 source
• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Wang et al., 2013
• Partly supported

  The Protocell Cargo Loading and Delivery to Targeted Cells section reports fluorescent cargo delivery in vitro and in a live chick-embryo model, and the Conclusions report delivery of the chemotherapeutic gemcitabine in vitro. The cited paper does not establish loading with phototherapy agents, and it distinguishes its ex ovo embryo work from future in vivo leukemia studies. (LLM, conf=0.88)

  cites Durfee et al., 2016 sourceExperimental — accuracy not yet measured

In the paper

“It is important to note that both tragedies stemmed from the therapeutic delivery method (LaFountaine et al., 2015).”

What the sources say

• ?Not checked — no full text of the sourcecites LaFountaine et al., 2015 source

In the paper

“The focus then turns to the most difficult barrier to potential in vivo use of CRISPR/Cas9, delivery.”

In the paper

“The use of site-specific nucleases and NHEJ or HDR generally results in one of four gene editing products. Shown in Figure 2, these include gene knockout, deletion, correction, or addition.”

In the paper

“Type II CRISPR systems require only one protein, Cas9, to scan, bind and cleave the target DNA sequence (Makarova et al., 2011).”

What the sources say

• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites Makarova et al., 2011 source

In the paper

“We introduce several factors that influence CRISPR/Cas9 efficacy which must be addressed before effective in vivo human gene therapy can be realized.”

In the paper

“Of the 20 patents participating in the trial, 17 were successfully and stably cured (Cavazzana et al., 2016).”

What the sources say

• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites Cavazzana et al., 2016 source

In the paper

“In general, the incidence of HDR for DSB repair is extremely low compared with NHEJ, at least in instances where both pathways are equally available to an organism.”

In the paper

“In another tragedy, an 18-year-old male suffering from a partial deficiency of ornithine transcarbamylase (OTC) died after developing a massive inflammatory response to the genetic cargo delivery vehicle, an adenovirus vector, four hours after receiving the treatment (Marshall, 1999; Authors, 2002).”

What the sources say

• ?Not checked — no full text of the sourcecites Marshall, 1999 source
• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Authors, 2002

In the paper

“However, excessive sgRNA was also shown to increase off-target effects (Fu et al., 2013).”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Fu et al., 2013

In the paper

“Regarding CRISPR/Cas9 cargoes, there are three approaches that are commonly reported: (1) DNA plasmid encoding both the Cas9 protein and the guide RNA, (2) mRNA for Cas9 translation alongside a separate guide RNA, and (3) Cas9 protein with guide RNA (ribonucleoprotein complex).”

In the paper

“Additionally, directed evolution and structure-guided rational design has allowed for engineering of Cas9 variants with altered PAM sequence specificity (Kleinstiver et al., 2015; Anders et al., 2016; Hirano et al., 2016). As examples, the VGR, EQR, and VRER variants of SpCas9 have PAM sequences of 5′-NGAN-3′, 5′-NGAG-3′, and 5′-NGCG-3′, respectively, further reducing limits on genome target sele”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Kleinstiver et al., 2015
• Not checked yetcites Anders et al., 2016 source
• ?Not checked — no full text of the sourcecites Hirano et al., 2016 source

In the paper

“As delivery remains the major obstacle for use of CRISPR for in vivo applications, efforts addressing other factors will be briefly summarized here.”

In the paper

“This simplicity makes the CRISPR/Cas9 system the most convenient, simple, and flexible tool for site-directed gene editing currently available.”

In the paper

“Reports from several groups, however, have shown that target site selection and sgRNA design are not as simple as perhaps originally assumed.”

In the paper

“While the use of Scr7 and other inhibitors have resulted in increased HDR-mediated gene editing efficiency, these inhibitors may have toxic effects on the host cells.”

In the paper

“Thus, Cas9 is less like a catalytic enzyme and more like a single-shot actuator.”

In the paper

“Additionally, considerations for how tightly controlled the overall concentration of Cas9 is must also be made; by introducing Cas9 DNA instead of protein, it becomes more difficult to ascertain precisely how many functional Cas9 units are present in the system at any given timepoint.”

In the paper

“one cannot guarantee that the viral payload goes to the same precise location every time.”

In the paper

“Because of this, it is rare to see particularly high efficacies when delivery CRISPR/Cas9 components via lipid nanoparticles.”

In the paper

“This method also allows for the controlled delivery of known quantities of the cargo, improving control over off-target effects.”

In the paper

“CRISPR/Cas9 seems to be particularly well-suited to this type of delivery because Cas9 and the sgRNA as a ribonucleoprotein complex are highly anionic.”

In the paper

“genomics screening using CRISPR/Cas9 and LV was used to identify the genes essential for West-Nile-virus-induced cell death (Figure 6(e)) (Ma et al., 2015)”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Ma et al., 2015

In the paper

“Naturally, microinjection is best suited for in vitro and ex vivo work only, as the use of a microscope to target individual cells (and precisely inject cargoes to specific locations within them) precludes the use of microinjection in a true in vivo setting.”

In the paper

“This indicates the importance of also studying the immunogenicity of specific Cas9s for in vivo delivery of CRISPR.”

In the paper

“While this is within the realm of technical possibility, the SpCas9 and sgRNA are roughly 4.2kB in size, and the overall size of AAV (∼20 nm) only allows for ∼4.5–5 kb of genomic material to be packaged within it (Wu et al., 2010).”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Wu et al., 2010

In the paper

“It should be noted, however, that shorter Cas9 variants identified to date have longer PAM sequences and thus greater limitations on sequences available for targeting.”

In the paper

“it is not currently possible to completely eliminate the chances of integration into the host.”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Chen and Goncalves, 2016

In the paper

“With some exceptions, microinjection of CRISPR/Cas9 RNA components into cells results in a finite duration of action of the system, owing to the natural decay of mRNA within eukaryotic cells (Ross, 1995).”

What the sources say

• ?Not checked — no full text of the sourcecites Ross, 1995 source

In the paper

“and define a signal peptide pathway required by flaviviruses (Zhang et al., 2016).”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Zhang et al., 2016

In the paper

“Owing to the oftentimes-large amounts of voltage needed to be applied across cell membranes, however, electroporation is typically not suitable for in vivo applications.”

In the paper

“Wang et al. (2014) used LV to deliver a pool of 73,000 sgRNAs to two human cells lines that had been previously engineered to express Cas9.”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Wang et al., 2014

In the paper

“Leveraging the integration ability of LV, this system has also been used to create gene libraries for studying mechanisms of disease.”

In the paper

“The process of hydrodynamic delivery can be quite traumatic, resulting in potential physiological complications, including cardiac dysfunction, elevated blood pressure, and liver expansion (Suda et al., 2007; Bonamassa et al., 2011).”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Suda et al., 2007
• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Bonamassa et al., 2011

In the paper

“While none of these have been demonstrated in the literature for CRISPR/Cas9 delivery, their properties make them naturally amenable for use as CRISPR/Cas delivery vehicles.”

In the paper

“As CPPs are roughly 40-fold more efficient than transfection from bare plasmids, CPPs are a serviceable method for delivery of CRISPR/Cas9 components to cells.”

In the paper

“As shown above, however, naked plasmids can achieve tangible long-term effects with an efficiency rate of just 0.4%.”

In the paper

“Lee et al. (2017) also reported use of AuNPs to deliver Cas9:sgRNA RNP to mice suffering from DMD.”

What the sources say

• Not checked yetcites Lee et al., 2017 source

In the paper

“Delivery of CRISPR/Cas9 gene editing components has been reported using other nanocomplexes that generally rely on electrostatic interactions.”

In the paper

“Finally, lipid nanoparticles are like virus particles in that the nature and size of the cargo, along with the target cell type, highly affect transfection efficiency and the types of lipids that are appropriate or useful in the system.”

In the paper

“Because of the level of detail required for this optimization, CPPs are not generally currently utilized to deliver components in vivo.”

In the paper

“As this is a relatively new delivery technology, it has currently only been utilized in an in vitro setting.”

In the paper

“correct the cystic fibrosis transmembrane conductor receptor locus in cultured intestinal stem cells of cystic fibrosis patients (Schwank et al., 2013)”

What the sources say

• Not checked yetcites Schwank et al., 2013 source

In the paper

“PEI was also used to deliver CRISPR plasmid DNA to inhibit HBV replication and gene expression in HBV-infected mice (Zhen et al., 2015).”

What the sources say

• ?Not checked — no full text of the sourcecites Zhen et al., 2015 source

In the paper

“Typically, CPPs show low efficiency of the desired targeted mutation in cells – usually around 10–20%.”

In the paper

“In 2015, the group repurposed the nanoclew for CRISPR/Cas9 delivery by designing the palindromic sequences to be partially complementary to the sgRNA within the Cas9:sgRNA ribonucleoprotein complex (Figure 8(A-a)) (Sun et al., 2015).”

What the sources say

• Not checked yetcites Sun et al., 2015 source

In the paper

“Instead, multiple methods are seen for delivering CRISPR to cells.”

In the paper

“Further, the best gene editing results with minimal off-target effects are generally obtained from delivery of the ribonucleoprotein, as opposed to plasmid DNA or mRNA.”

In the paper

“MENDs, developed by the Harashima group at Hokkaido University, are a non-viral gene editing and therapeutic delivery system that is composed of condensed plasmid DNA, a PLL core, and a lipid film shell (Kogure et al., 2004).”

What the sources say

• ?Not checked — no full text of the sourcecites Kogure et al., 2004 source

In the paper

“MEND has been used to successfully deliver cargoes of plasmid DNA, short interfering RNA (siRNA), and Bacillus Calmette-Guerin (BCG) cell wall therapeutic agents.”

In the paper

“Addition of the cell-penetrating peptide stearyl octaarginine to the lipid shell increased transfection rates by 1000-fold over bare DNA/PLL.”

In the paper

“Walev et al. (2001) developed a system in 2001 to allow for this toxin to act in a reversible manner.”

What the sources say

• ?Not checked — no full text of the sourcecites Walev et al., 2001 source

In the paper

“Regarding gene cargo delivery systems, this remains the greatest obstacle for CRISPR/Cas9 use, and an all-purpose delivery method has yet to emerge.”

In the paper

“however, our understanding of off-target effects remains poor.”

In the paper

“and an all-purpose delivery method has yet to emerge.”

In the paper

“The silica core has a large internal surface area, leading to high cargo loading capacities.”

In the paper

“CRISPR/Cas9 components, whether in an RNP complex, as mRNA, or as DNA plasmids, are larger than other components that have been reported to load within the particles.”

In the paper

“Within the system itself, off-target cutting remains a problem.”

In the paper

“Packaging the DNA/PLL core with lipids increased transfection rates by ten-fold over bare DNA/PLL.”

What the sources say

• ?Not checked — no full text of the sourcecites Kogure et al., 2004 source

In the paper

“This allows for the delivery of proteins of up to 100 kDa to the cytosol of both adherent and non-adherent cells in culture without compromising overall viability.”

What the sources say

• ?Not checked — no full text of the sourcecites Walev et al., 2001 source