Delivering CRISPR: a review of the challenges and approaches

May combine two claims.

In the SCID X-1 trial, five children subsequently developed T-cell leukemia, with one child dying from chemotherapy-refractory leukemia.

“However, five children subsequently developed T-cell leukemia, with one child dying from chemotherapy-refractory leukemia.”

May combine two claims.

NHEJ repairs double-stranded breaks without a homologous DNA template, regularly leading to mutations and deletions, and is error-prone.

“In NHEJ, protein factors re-ligate the broken DNA strand either directly or by including nucleotide insertions or deletions (indels) (Hefferin & Tomkinson, 2005). This process occurs without a homologous DNA template, regularly leading to mutations and deletions in the repaired strand (Bibikova et al., 2002), as shown in Figure 1(A). NHEJ is, therefore, characterized as error-prone.”

• cites Hefferin & Tomkinson, 2005Not checked yet

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• cites Bibikova et al., 2002 sourceNot checked yet

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May combine two claims.

Discovery of CRISPR, its function as an adaptive prokaryotic immune system, and its development into a genomic editing tool has revolutionized molecular biology.

“Discovery of the Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR) (Ishino et al., 1987; Mojica et al., 1993; van Soolingen et al., 1993), their function as part of an adaptive prokaryotic immune system (CRISPR-associated system, Cas) (Bolotin et al., 2005; Mojica et al., 2005; Pourcel et al., 2005; van der Oost et al., 2009), and subsequent development into a genomic editing tool”

• cites Ishino et al., 1987Not checked yet

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• cites Jinek et al., 2012Not checked yet

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• cites Mali et al., 2013Not checked yet

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May combine two claims.

TALENs are substantially larger than ZFNs, requiring 3 kb of cDNA per TALEN versus 1 kb per ZFN, making delivery of a pair of TALENs more challenging due to cargo size limitations.

“TALENs, however, are substantially larger than ZFNs, requiring 3 kb of cDNA encoding for one TALEN versus just 1 kb for a single ZFN. This makes delivery of a pair of TALENs more challenging than a pair of ZFNs due to delivery vehicle cargo size limitations.”

May combine two claims.

CRISPR/Cas9 avoids the need for protein engineering to target a specific DNA sequence, requiring only synthesis of new RNA, which simplifies and reduces the time needed for gene editing design and implementation.

“The use of CRISPR/Cas9 avoids the need for protein engineering to develop a site-specific nuclease against a specific DNA target sequence, requiring only the synthesis of a new piece of RNA. This dramatically simplifies and greatly reduces the time needed for gene editing design and implementation.”

May combine two claims.

Single mismatches, and sometimes multiple mismatches, can be tolerated by the Cas9:RNA complex, with mismatches more tolerated downstream of the seed region.

“Single mismatches, and sometimes multiple mismatches, can be tolerated by the Cas9:RNA complex, with mismatches being more tolerated in regions downstream of the seed region (Cong et al., 2013; Sternberg et al., 2014).”

• cites Cong et al., 2013Not checked yet

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• cites Sternberg et al., 2014Not checked yet

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May combine two claims.

TALEN design and engineering is simpler and faster than ZFNs, and TALENs are less limited in target site selection.

“Unlike ZFNs, design and engineering of TALENs is much simpler and can be done in a shorter time (Cermak et al., 2011; Reyon et al., 2012). TALENs are also not as limited as ZFNs in target site selection due to the 1:1 TALE–DNA binding (Zhu et al., 2013).”

• cites Cermak et al., 2011Not checked yet

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• cites Reyon et al., 2012Not checked yet

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• cites Zhu et al., 2013Not checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

Over 3000 genes have been associated with disease-causing mutations.

“To date, over 3000 genes have been associated with disease-causing mutations (Cox et al., 2015).”

• cites Cox et al., 2015Not checked yet

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May combine two claims.

In all cases of leukemia in the SCID X-1 trial, the correcting gene inserted within or near tumor-promoting genes and caused transcriptional activation.

“In all cases of leukemia, the SCID X-1 correcting gene had inserted into the patient genome within or near tumor-promoting genes and caused transcriptional activation (Check, 2002; Kaiser, 2003; Thomas et al., 2003).”

• cites Check, 2002Not checked yet

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• cites Kaiser, 2003Not checked yet

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• cites Thomas et al., 2003Not checked yet

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May combine two claims.

HDR uses a homologous repair template to precisely repair double-stranded breaks and typically occurs in late S- or G2-phase.

“In contrast, HDR uses a homologous repair template to precisely repair the DSB (Capecchi, 1989; Takata et al., 1998) (see Figure 1(B)). HDR typically occurs in late S- or G2-phase, when a sister chromatid can serve as the repair template.”

• cites Capecchi, 1989Not checked yet

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• cites Takata et al., 1998Not checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

The simplicity and flexibility of the CRISPR/Cas9 site-specific nuclease system has led to its widespread use in many biological research areas, including development of model cell lines, discovering mechanisms of disease, identifying disease targets, development of transgene animals and plants, and transcriptional modulation.

“Additionally, the simplicity and flexibility of the CRISPR/Cas9 site-specific nuclease system has led to its widespread use in many biological research areas including development of model cell lines, discovering mechanisms of disease, identifying disease targets, development of transgene animals and plants, and transcriptional modulation.”

May combine two claims.

The Cas9:RNA complex randomly interrogates DNA, searches for a PAM, unwinds the seed region, and cleaves the target and non-target DNA strands via its HNH and RuvC domains.

“The Cas9:RNA complex randomly interrogates DNA in the cell, searching first for the appropriate protospacer adjacent motif (PAM), a short motif (5′-NGG-3′ for Cas9 from Streptococcus pyogenes) adjacent to the target sequence (Chylinski et al., 2013).”

• Also stated in: § Biological mechanism of CRISPR · plain

  “If the interrogated DNA sequence matches the crRNA target sequence, the HNH nuclease domain of the Cas9 nuclease lobe cleaves the target strand while the RuvC-like nuclease domain of the Cas9 α-helical lobe cleaves the non-target strand (Anders et al., 2014; Jinek et al., 2014; Nishimasu et al., 2015).”

• cites Chylinski et al., 2013Not checked yet

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• cites Anders et al., 2014Not checked yet

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• cites Jinek et al., 2014Not checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

ZFNs are a fusion protein of Cys2-His2 zinc finger proteins and a non-specific FokI endonuclease.

“ZFNs are a fusion protein of Cys2-His2 zinc finger proteins (ZFPs) and a non-specific DNA restriction enzyme derived from FokI endonucleases, as shown in Figure 3(A).”

• cites Bibikova et al., 2002, 2003Not matched

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

May combine two claims.

In one side-by-side comparison study, TALENs were more specific and less cytotoxic than ZFNs.

“While off-target cutting remains a concern, TALENs have been shown in one side-by-side comparison study to be more specific and less cytotoxic than ZFNs (Mussolino et al., 2014).”

• cites Mussolino et al., 2014Not checked yet

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May combine two claims.

Challenges with ZFNs include design and engineering of the zinc finger protein for high-affinity binding, limited target site selection, and off-target cutting.

“Challenges with ZFNs include design and engineering of the ZFP for high-affinity binding of the desired sequence, which can prove non-trivial (Ramirez et al., 2008). Also, not all sequences are available for ZFP binding, so site selection is limited. Using open-source ZFP domains, sites could be targeted only every 200-bps in a random DNA sequence (Gupta & Musunuru, 2014).”

• Also stated in: § Zinc finger nucleases (ZFNs) · plain

  “Another significant challenge is off-target cutting (Gabriel et al., 2011; Pattanayak et al., 2011).”

• cites Ramirez et al., 2008Not checked yet

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• cites Gupta & Musunuru, 2014Not checked yet

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• cites Gabriel et al., 2011Not checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

CRISPR/Cas9 is a naturally occurring RNA-guided endonuclease.

“The most recently developed site-specific gene editing tool, CRISPR/Cas9, is a naturally occurring RNA-guided endonuclease.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Vehicles used to deliver CRISPR cargo can be classified into three general groups: physical delivery, viral vectors, and non-viral vectors.

“Vehicles used to deliver the gene editing system cargo (Table 1) can be classified into three general groups: physical delivery, viral vectors, and non-viral vectors.”

May combine two claims.

The dCas9-FokI nuclease dimer system increases specificity and reduces off-target cutting, but the number of potential target sites is lower due to PAM and sgRNA design constraints, and it significantly increases the size of the gene editing tool, constraining in vivo delivery.

“While these approaches increase specificity and reduce off-target cutting, the number of potential target sites is lower due to PAM and other sgRNA design constraints. This system also significantly increased the size of the gene editing tool, providing greater constraints on in vivo delivery approaches.”

• cites Guilinger et al., 2014Not checked yet

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• cites Tsai et al., 2014Not checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

Especially for in vivo work, viral vectors are the most common CRISPR/Cas9 delivery vectors.

“Especially for in vivo work, viral vectors have found favor and are the most common CRISPR/Cas9 delivery vectors.”

May combine two claims.

Cas9 subtypes II-A and II-C can recognize and cleave RNA in a directed manner independent of a PAM site, and this cleavage protected E. coli from MS2 bacteriophage, suggesting Cas9 can provide cellular defense against both DNA and RNA viruses.

“Strutt and colleagues demonstrated that Cas9 subtypes II-A and II-C can recognize and cleave RNA in a directed manner utilizing RNA–RNA interactions independent of the presence of a PAM site in the target RNA molecule (Strutt et al., 2018). This cleavage protected E. coli cells from infection with bacteriophage MS2 particles, suggesting that Cas9 can provide cellular defense against both DNA and R”

• cites Strutt et al., 2018Not checked yet

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May combine two claims.

Cas9 has low activity compared with other enzymes, with a single turnover rate of ∼0.3-1.0 min−1.

“Finally, in comparison with other enzymes, the activity of Cas9 is quite low, with a single turnover rate of ∼0.3–1.0 min−1(Jinek et al., 2012).”

• cites Jinek et al., 2012Not checked yet

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May combine two claims.

The delivery vehicle used often dictates which of the three cargos can be packaged and whether the system is usable in vitro and/or in vivo.

“The delivery vehicle used will often dictate which of these three cargos can be packaged, and whether the system is usable in vitro and/or in vivo.”

May combine two claims.

Haeussler et al. (2016) compared predictions from several sgRNA design tools with experimental results from eight SpCas9 off-target studies and showed evidence of algorithmic overfitting and the importance of using a model trained on data from the same guide RNA expression system.

“However, caution is still needed, as shown in a recent study by Haeussler et al. (2016) that compared predictions from several sgRNA design tools with experimental results published in eight SpCas9 off-target studies. The authors showed evidence of algorithmic overfitting and the importance of using a model trained on data from the same guide RNA expression system.”

• cites Haeussler et al., 2016Not checked yet

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May combine two claims.

Once bound to target DNA, displacement of Cas9 from the DNA strand is challenging; 1 nM Cas9 cleaved ∼2.5 nM plasmid DNA after 120 min.

“And, once bound to the target DNA sequence, displacement of Cas9 from the DNA strand, even after DSB formation, is challenging – 1 nM Cas9 cleaved ∼2.5 nM plasmid DNA after 120 min (Jinek et al., 2012).”

• cites Jinek et al., 2012Not checked yet

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May combine two claims.

Microinjection is considered the gold standard for introducing CRISPR components into cells, with efficiencies approaching 100%.

“Microinjection is considered the ‘gold standard’ for introducing CRISPR components into cells, with efficiencies approaching 100% (Yang et al., 2013; Horii et al., 2014).”

• cites Yang et al., 2013Not checked yet

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• cites Horii et al., 2014Not checked yet

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May combine two claims.

Scr7 targets the NHEJ component DNA ligase IV and has been reported to increase the efficiency of HDR from Cas9 editing by up to 19-fold.

“One of the most commonly used inhibitors, Scr7, targets the NHEJ component DNA ligase IV, and has been reported to increase efficiency of HDR from Cas9 editing by up to 19-fold (Srivastava et al., 2012; Chu et al., 2015; Vartak & Raghavan, 2015).”

• cites Srivastava et al., 2012Not checked yet

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• cites Chu et al., 2015Not checked yet

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• cites Vartak & Raghavan, 2015Not checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

One report suggests that CRISPR/Cas9 may be less specific than ZFNs or TALENs due to its relatively shorter targeting sequence.

“One report suggests that CRISPR/Cas9 may be less specific than ZFNs or TALENs due to the relatively shorter targeting sequence (Cradick et al., 2013).”

• cites Cradick et al., 2013Not checked yet

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May combine two claims.

Jinek et al. (2012) first used CRISPR/Cas9 to introduce double-stranded breaks in target DNA and showed that duo-tracrRNA:crRNA units could be engineered into a single guide RNA chimera that still directs efficient DNA cleavage.

“In 2012, Doudna and Charpentier showed the first use of CRISPR/Cas9 to introduce DSBs in target DNA (Jinek et al., 2012). Further, they showed that the duo-tracrRNA:crRNA units could be engineered into a single, truncated RNA chimera and still direct efficient DNA cleavage.”

• cites Jinek et al., 2012Not checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

The SpCas9 PAM occurs every eight bps on average.

“This motif has been reported to occur every eight bps, on an average, for the SpCas9 PAM (Ramakrishna et al., 2014).”

• cites Ramakrishna et al., 2014Not checked yet

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May combine two claims.

Cas9 protein is positively charged, while oligonucleotides and Cas9:sgRNA RNP are negatively charged.

“As an example, Cas9 protein is positively-charged, but oligonucleotides and Cas9:sgRNA RNP are negatively charged (Sun et al., 2015).”

• cites Sun et al., 2015Not checked yet

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May combine two claims.

Cas9-based gene editing in mice resulted in HDR repair efficiencies of 0.5-20%, while NHEJ-mediated repair occurred at 20-60%.

“For example, Cas9-based gene editing in mice resulted in HDR repair efficiencies of 0.5–20%, while NHEJ-mediated repair occurred at 20–60% (Maruyama et al., 2015).”

• cites Maruyama et al., 2015Not checked yet

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May combine two claims.

Many reports show no detectable off-target cleavage from CRISPR/Cas9 editing, with off-target effects being guide-RNA-specific.

“This contrasts with many reports that show no detectable off-target cleavage from CRISPR/Cas9 editing, with off-target effects being guide-RNA-specific (Cradick et al., 2013; Fu et al., 2013; Hsu et al., 2013; Pattanayak et al., 2013;Cho et al., 2014).”

• cites Cradick et al., 2013Not checked yet

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• cites Fu et al., 2013Not checked yet

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• cites Hsu et al., 2013Not checked yet

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May combine two claims.

Each Cas9 ortholog has differing PAM sequences and variable activity.

“Each has differing PAM sequences and variable activity.”

Classified as probably not a substantial claim. May combine two claims.

Factors that influence CRISPR/Cas9 efficacy include target DNA site selection, sgRNA design, off-target cutting, incidence/efficiency of HDR versus NHEJ, Cas9 activity, and method of delivery.

“These factors include target DNA site selection, sgRNA design, off-target cutting, incidence/efficiency of HDR vs. NHEJ, Cas9 activity, and the method of delivery.”

May combine two claims.

CRISPRi, using catalytically dead Cas9, represses expression of multiple genes simultaneously without altering the genome, as demonstrated in E. coli and human cells.

“They showed that this system, termed CRISPR interference (CRISPRi), could repress expression of multiple genes simultaneously without altering the genome. They demonstrated this gene repression in both Escherichia coli and human cells.”

• cites Qi et al., 2013Not checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

The PAM from Neisseria meningitidis Cas9 is 5′-NNNNGATT-3′.

“As an example, the PAM from Neisseria meningitidis Cas9 is reported to be 5′-NNNNGATT-3′ (Jiang et al., 2013; Ma et al., 2014).”

• cites Jiang et al., 2013Not checked yet

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• cites Ma et al., 2014Not checked yet

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May combine two claims.

Wang et al. (2016) could achieve approximately 70% in vitro modification efficiency in cells using lipid nanoparticles, but only after an intense screen to determine the most optimal lipids.

“While Wang et al. (2016) could achieve ∼70% in vitro modification efficiency in cells (see Figure 7(A)), that only came after an intense screen to determine the most optimal lipids with which to construct their liposomes for their system.”

• cites Wang et al., 2016Not checked yet

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May combine two claims.

Random insertion of the viral payload can result in an increase of expression and off-target effects, or even potential damage to the cell if the insertion randomly occurs within an important cellular protein.

“This can result in an increase of expression and off-target effects, or even potential damage to the cell if the insertion randomly occurs within an important cellular protein (Bestor, 2000; Papapetrou and Schambach, 2016).”

• cites Bestor, 2000Not checked yet

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• cites Papapetrou and Schambach, 2016Not checked yet

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May combine two claims.

SaCas9 is roughly 70% the size of SpCas9 while retaining the same potent cutting ability.

“This version of Cas9 is roughly 70% the size of SpCas9 while retaining the same potent cutting ability.”

• cites Ran et al., 2015Not checked yet

  Not checked in the free run.

May combine two claims.

AdV vector-associated immunotoxicity in the liver and humoral immunity against SpCas9, as well as a potential SpCas9-specific cellular immune response, were detected.

“Importantly, in addition to displaying AdV vector-associated immunotoxicity in the liver, humoral immunity against SpCas9 was detected, as was a potential SpCas9-specific cellular immune response.”

• cites Wang et al., 2015Not checked yet

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May combine two claims.

Immune responses are eventually seen to the AAV capsid, sometimes even causing CD-8 T-cell toxicity.

“Immune responses are eventually seen to the capsid, sometimes even causing CD-8 T-cell toxicity (Samulski and Muzyczka, 2014).”

• cites Samulski and Muzyczka, 2014Not checked yet

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May combine two claims.

LVs and AdVs are roughly 80-100 nm in diameter, compared with ∼20 nm for AAV, so larger insertions are better tolerated in these systems.

“The biggest difference between LV/AdV delivery and AAV delivery is the size of the particle; both LVs and AdVs are roughly 80–100 nm in diameter. Compared with the ∼20 nm diameter of AAV, larger insertions are better tolerated in these systems.”

May combine two claims.

Both LV and AdV can infect dividing and non-dividing cells; however, unlike LV, AdV does not integrate into the genome.

“Both LV and AdV can infect dividing and non-dividing cells; however, unlike LV, AdV does not integrate into the genome.”

May combine two claims.

Microinjection is not limited by the molecular weight of the cargo, which is a significant limiting factor with viral vector delivery systems.

“Further, microinjection is not limited by the molecular weight of the cargo, which is a significant limiting factor with viral vector delivery systems.”

May combine two claims.

AdV does not integrate into the genome, which is advantageous for limiting off-target effects in CRISPR/Cas9-based editing.

“This is advantageous in the case of CRISPR/Cas9-based editing for limiting off-target effects.”

May combine two claims.

Packaging SpCas9 and sgRNA into one AAV vector is challenging and makes it difficult to include other elements such as reporters, fluorescent tags, multiple sgRNAs, or DNA templates for HDR.

“This makes consistent packaging of this construct challenging, and it is also extremely difficult to include other elements (such as reporters, fluorescent tags, multiple sgRNAs, or DNA templates for HDR) to help ensure successful delivery of CRISPR/Cas9 components to cells and meet desired gene editing objectives.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Lipid nanoparticles can be utilized in vitro, ex vivo, and in vivo, allowing for extensive testing on a variety of scales of cell populations.

“They can also, like viral particles, be utilized in vitro, ex vivo, and in vivo, allowing for extensive testing on a variety of scales of cell populations.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

Electroporation is less dependent on cell type than other delivery techniques and can efficiently transfer cargo into cells that are traditionally difficult to manipulate.

“Electroporation is less dependent on cell type than other delivery techniques and can efficiently transfer cargo into cells that are traditionally difficult to manipulate.”

May combine two claims.

Single-stranded DNA is prone to random integration into the host genome, which may disrupt genes, result in constitutive expression of Cas9, and lead to greater off-target effects.

“However, single-stranded DNA is prone to random integration into the host genome, which may disrupt genes, result in constitutive expression of Cas9, and lead to greater off-target effects.”

• cites Yang et al., 2013Not checked yet

  Not checked in the free run.

May combine two claims.

Hydrodynamic delivery is significantly enriched in the liver, but also includes cells of the kidneys, lung, muscles, and heart.

“Delivery of cargo using this method is significantly enriched in the liver, but also includes cells of the kidneys, lung, muscles, and heart.”

May combine two claims.

Lipid nanoparticles do not contain any viral components, which helps minimize safety and immunogenicity concerns.

“Lipid nanoparticles do not contain any viral components, which helps minimize safety and immunogenicity concerns.”

May combine two claims.

Use of Cas9:sgRNA RNP reduces off-target effects versus plasmid transfection and is less stressful on cells, producing two-fold more embryonic stem cell colonies than plasmid transfection.

“The use of RNP was shown to reduce off-target effects versus plasmid transfection and be less stressful on the cells, producing two-fold more embryonic stem cell colonies than with plasmid transfection (Kim et al., 2014).”

• cites Kim et al., 2014Not checked yet

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May combine two claims.

AAV-delivered genomic material can exist indefinitely in cells either as exogenous DNA or, with some modification, be directly integrated into the host DNA.

“Finally, unlike some other methods, the use of AAV for gene therapy provides a persistent source of the provided DNA, as AAV-delivered genomic material can exist indefinitely in cells either as exogenous DNA or, with some modification, be directly integrated into the host DNA (Deyle and Russell, 2009).”

• cites Deyle and Russell, 2009Not checked yet

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May combine two claims.

Koike-Yusa et al. delivered 87,897 sgRNAs targeting 19,150 protein coding genes in mouse embryonic stem cells, both again constitutively expressing Cas9.

“Koike-Yusa et al. (2014) delivered 87,897 sgRNAs targeting 19,150 protein coding genes in mouse embryonic stem cells, both again constitutively expressing Cas9.”

• cites Koike-Yusa et al., 2014Not checked yet

  Not checked in the free run.

May combine two claims.

AAV efficiently infects cells while provoking little to no innate or adaptive immune response or associated toxicity, at least upon first treatment with a serotype.

“The virus itself is able to efficiently infect cells while provoking little to no innate or adaptive immune response or associated toxicity, at least upon first treatment with a serotype (Daya and Berns, 2008).”

• cites Daya and Berns, 2008Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

PLL has been used to complex with Cas9 plasmid DNA, forming a multifunctional envelope-type nanodevice (MEND).

“PLL has been used to complex with Cas9 plasmid DNA, forming a multifunctional envelope-type nanodevice (MEND), described in the Emerging Delivery Technologies section of this review.”

May combine two claims.

The nanoclew attained efficiencies on the order of other high-efficiency CRISPR/Cas9 delivery systems while still containing no viral components or any exogenous material besides repeating DNA and PEI.

“This allowed the nanoclew to attain efficiencies on the order of other high-efficiency CRISPR/Cas9 delivery systems, but still contain no viral components (or indeed, any exogenous material besides repeating DNA and PEI).”

Probably not a substantial claim: may not pass S3 (load-bearing).

Axford et al. (2017) demonstrated cellular and sub-cellular localization of CPP-delivered CRISPR/Cas9 RNP using confocal microscopy.

“Axford et al. (2017) in which the authors demonstrated cellular and sub-cellular localization of CPP-delivered CRISPR/Cas9 RNP using confocal microscopy.”

• cites Axford et al., 2017 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Mout et al. (2017) demonstrated that engineering Cas9:sgRNA RNP and AuNPs to associate creates a complex that can be efficiently delivered to cells and cause a desired mutation at a rate of roughly 30%.

“Mout et al. (2017) demonstrated that, by engineering Cas9:sgRNA RNP and AuNPs to associate with one another (Figure 8(B)), a complex is created that can be efficiently delivered to cells and cause a desired mutation at a rate of roughly 30%.”

• cites Mout et al., 2017Not checked yet

  Not checked in the free run.

May combine two claims.

SLO is a toxin produced by Group A streptococci that works by creating pores in mammalian cell membranes.

“SLO is a toxin produced by Group A streptococci that works by creating pores in mammalian cell membranes (Sierig et al., 2003).”

• cites Sierig et al., 2003Not checked yet

  Not checked in the free run.

May combine two claims.

AuNPs are inert and will not trigger an immune response to the nanoparticle itself.

“AuNPs are inert and will not trigger an immune response to the nanoparticle itself (Lee et al., 2017).”

• cites Lee et al., 2017Not checked yet

  Not checked in the free run.

May combine two claims.

ZALs complexed with Cas9 mRNA and sgRNA formed nanoparticles with approximately 15 nm diameter which showed effective transfection in mice, accumulating primarily in the liver, kidney, and lungs.

“ZALs were complexed with Cas9 mRNA and sgRNA, forming nanoparticles with ∼15 nm diameter which showed effective transfection in mice, accumulating primarily in the liver, kidney, and lungs.”

• cites Miller et al., 2017Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

As a newer delivery technique, applications of iTOP have been limited to in vitro settings at this time.

“As another newer delivery technique, applications of iTOP have been limited to in vitro settings at this time.”

May combine two claims.

Kennedy et al. used FuGENE-6 to deliver Cas9 and sgRNA encoding plasmid DNA, inactivating human papillomavirus E6 or E7 gene in cervical carcinoma cells, resulting in cell-cycle arrest and eventual cell death.

“Kennedy et al. used FuGENE-6 to deliver Cas9 and sgRNA encoding plasmid DNA, inactivating human papillomavirus E6 or E7 gene in cervical carcinoma cells, resulting in cell-cycle arrest and eventual cell death (Kennedy et al., 2014).”

• cites Kennedy et al., 2014Not checked yet

  Not checked in the free run.

May combine two claims.

The CRISPR construct targeted the provirus genome, blocking expression of viral components and removing internal viral genes from the host cell chromosome.

“The CRISPR construct targeted the provirus genome, blocking expression of viral components and removing internal viral genes from the host cell chromosome.”

• cites Ebina et al., 2013Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

AuNPs have been shown to stimulate immune cytokine production in general.

“Still, AuNPs have been shown to stimulate immune cytokine production in general (for a recent review, see Dykman and Khlebtsov, 2017).”

• cites Dykman and Khlebtsov, 2017Not checked yet

  Not checked in the free run.

May combine two claims.

Treated mice further showed partial restoration of muscle function and reduced levels of fibrosis.

“Treated mice further showed partial restoration of muscle function and reduced levels of fibrosis.”

• cites Lee et al., 2017Not checked yet

  Not checked in the free run.

May combine two claims.

A single injection of the AuNP-Cas9 conjugate corrected 5.4% of the mutated DMD-causing dystrophin gene and showed recovered dystrophin gene expression.

“A single injection of the AuNP-Cas9 conjugate corrected 5.4% of the mutated DMD-causing dystrophin gene and showed recovered dystrophin gene expression.”

• cites Lee et al., 2017Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

Branched PEI is cytotoxic.

“However, branched PEI is cytotoxic.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Branched PEI have high charge density, facilitating efficient plasmid DNA packing, and pH-buffering ability which enables escape from endosomes.

“Branched PEI have high charge density, facilitating efficient plasmid DNA packing, and pH-buffering ability which enables escape from endosomes.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

Efficiencies of CPPs themselves to penetrate cellular membranes vary with both attached cargo and cell type.

“as efficiencies of the CPP themselves to penetrate cellular membranes vary with both attached cargo and cell type.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

Each class of CPPs can facilitate uptake of different types of proteins into different cell types, and often different combinations of CPPs and the desired molecule for uptake will result in different uptake levels.

“Each class of CPPs can facilitate uptake of different types of proteins into different cell types, and often different combinations of CPPs and the desired molecule for uptake will result in different uptake levels.”

May combine two claims.

CPPs can be used for in vitro and ex vivo work quite readily, and extensive optimization for each cargo and cell type is usually required.

“CPPs can be used for in vitro and ex vivo work quite readily, and extensive optimization for each cargo and cell type is usually required.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The same challenges of translocating the Cas9:sgRNA complex into the nucleus once it is within the cell must also be overcome for CPP delivery.

“The same challenges of translocating the Cas9:sgRNA complex into the nucleus once it is within the cell must also be overcome.”

May combine two claims.

Cas9 nickases and mutants that reduce non-specific DNA binding have been engineered specifically to ameliorate off-target cutting, though they are an imperfect solution.

“Cas9 nickases and mutants that reduce non-specific DNA binding have been engineered specifically to ameliorate this problem, though they are an imperfect solution.”

May combine two claims.

Every CRISPR delivery method has both advantages and disadvantages, and some can be quite specific or ill-suited to certain types of delivery.

“Every method has both advantages and disadvantages, and some can be quite specific or ill-suited to certain types of delivery (e.g. delivery to cells in a flask vs. delivery to a living organism).”

Probably not a substantial claim: may not pass S4 (based).

There is currently limited information available on where various components of nanoparticle delivery systems end up in the body, how long they stay there, and whether there is any long-term toxicity associated with any component.

“There is currently limited information available on where various components of nanoparticle delivery systems end up in the body, how long they stay there, and whether there is any long-term toxicity associated with any”

May combine two claims.

Other groups have used SLO for delivery of siRNA and imaging agents for live-cell microscopy.

“Other groups have used SLO for delivery of siRNA (Brito et al., 2008) and imaging agents for live-cell microscopy (Teng et al., 2017).”

• cites Brito et al., 2008Not checked yet

  Not checked in the free run.

• cites Teng et al., 2017Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Much has been published on the altering of cell line genotypes and phenotypes using CRISPR/Cas9.

“Already, much has been published on the altering of cell line genotypes and phenotypes using this gene editing system.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

Extensive efforts have been made in understanding sgRNA binding and mismatch tolerance, leading to the development of several predictive software sgRNA design tools.

“Extensive efforts have been made in understanding sgRNA binding and mismatch tolerance, leading to the development of several predictive software sgRNA design tools;”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

Currently, there are more options for delivery of small-molecule cargo than for the relatively large protein-nucleic acid complex.

“Currently, there are more options for delivery of small-molecule cargo than for the relatively large protein–nucleic acid complex.”

Classified as probably not a substantial claim.

Ensuring that the chosen delivery system is both safe and specific is still another barrier for delivery systems.

“Still another barrier for delivery systems is ensuring that the chosen system is both safe and specific.”

May combine two claims.

The lipid coating of the particle can be tailored to maximize cargo loading, increase circulation times, and provide precise targeting and cargo release.

“The lipid coating of the particle can also be tailored to maximize cargo loading, increase circulation times, and provide precise targeting and cargo release.”

Probably not a substantial claim: may not pass S4 (based). May combine two claims.

A delivery vehicle that can target the desired cells with high-specificity will also limit off-target effects and improve safety.

“a delivery vehicle that can target the desired cells with high-specificity will also limit off-target effects and improve safety.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

The number of peer-reviewed scientific papers with the term CRISPR in the title or abstract has increased by 1,453% since 2011.

“Importantly, the CRISPR field is evolving at an incredible pace, with the number of peer-reviewed scientific papers with the term CRISPR in the title or abstract increasing by 1,453% since 2011.”

• AssumptionRests on an assumption: The number of peer-reviewed scientific papers with the term CRISPR in the title or abstract has increased by 1,453% since 2011.

Probably not a substantial claim: may not pass S2 (checkable).

The therapeutic potential of CRISPR/Cas9 is great.

“As evident through the many examples presented in the ‘Delivery Methods’ portion of this review, the therapeutic potential of CRISPR/Cas9 is great.”

May combine two claims.

A tetra-lamellar MEND targeted the cellular nucleus and mitochondria, and a PEG-peptide-DOPE-conjugated MEND targeted bladder cancer cells.

“Recently, a tetra-lamellar MEND (T-MEND) was developed that targeted the cellular nucleus and mitochondria, and a PEG-peptide-DOPE-conjugated MEND (PPD-MEND) was developed that targeted bladder cancer cells (Nakamura et al., 2012).”

• cites Nakamura et al., 2012Not checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S4 (based). May combine two claims.

Compared with viral and lipid/polymer based vectors, inorganic nanoparticles are simpler to generate, with reproducible composition, size, and size distribution, are simpler to characterize and chemically functionalize, and are more stable over time.

“However, when compared with viral and lipid/polymer based vectors, inorganic nanoparticles are simpler to generate, with reproducible composition, size, and size distribution, are simpler to characterize and chemically functionalize, and are more stable over time.”

May combine two claims.

Work has moved into animal models, and therapeutic effects are broad-ranging, including inhibition of viral infection, reversal of debilitating conditions such as muscular dystrophy, and elimination of tumors in cancer models.

“Work has even moved into animal models, and therapeutic effects are broad-ranging, including inhibition of viral infection, reversal of debilitating conditions such as muscular dystrophy, and elimination of tumors in cancer models.”