Lentiviral Capsid-Mediated <i>Streptococcus pyogenes</i> Cas9 Ribonucleoprotein Delivery for Efficient and Safe Multiplex Genome Editing

Lu, Zuyan; Yao, Xingang; Lyu, Pin; Yadav, Manish Kumar; Yoo, Kyung Whan; Atala, Anthony

doi:10.1089/crispr.2020.0106

Cited by 22 publications

(25 citation statements)

References 47 publications

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“…Compared with the VLP-based Cas9 mRNA and sgRNA co-delivery method reported recently [33, 34], our method reported here modified Gag with near-normal capsid assembly efficiency [24, 25, 28]. The resulting VLPs co-packaged with Cas9 mRNA and sgRNA-expressing LV genomic RNA could be used without further concentration to achieve efficient genome editing.…”

Section: Discussionmentioning

confidence: 99%

“…Although VLPs can be developed to deliver Cas9 RNPs [20][21][22][23][24][25][26][27], delivering Cas9 mRNA may achieve short-term yet strong Cas9 expression to increase genome editing efficiency. This is especially relevant when targeting loci in heterochromatin regions.…”

Section: Discussionmentioning

confidence: 99%

“…We took advantage of interactions between RNA aptamers and ABPs to package and deliver Cas9 mRNA or RNPs by VLPs [24-26, 28, 29]. Contrary to fusing ABP to the N-terminus of Gag [33, 34] or removing NC [31], which affected particle assembly [20, 36], we inserted ABPs after the second zinc finger domain of the NC and observed minimal effects on particle assembly [24, 25, 28]. Here we report the development of all-in-one VLPs for co-delivery of Cas9 mRNA and sgRNA, and modified LVs to improve LV genomic RNA packaging and gene delivery.…”

Section: Introductionmentioning

confidence: 99%

“…Delivering Cas9 ribonucleoprotein (Cas9 RNP) [18] or Cas9 mRNA [19] enabled shortterm expression of CRISPR/Cas9 and greatly improved safety. Based on these observations, viral vectors have been modified for delivering Cas9 RNPs [20][21][22][23][24][25][26][27] or Cas9 mRNAs [28][29][30][31][32][33][34] to improve safety.…”

Section: Introductionmentioning

confidence: 99%

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Developing all-in-one virus-like particles for Cas9 mRNA/single guide RNA co-delivery and aptamer-containing lentiviral vectors for improved gene expression

Yadav

Atala

2022

Preprint

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Lentiviral vectors (LVs) are widely used for delivering foreign genes for long-term expression. Normal LVs contain the RNA genome, which is reverse transcribed into DNA and integrates into the host genome to mediate long-term gene expression. Recently, virus-like particles (VLPs) were developed for mRNA delivery. In these VLPs, packaging mRNA into the particles is achieved via interactions between the aptamer and aptamer-binding protein (ABP), and mRNA is not reverse transcribed or integrated. These VLPs are useful for delivering Cas9 mRNA for short-term endonuclease expression. Generating high-titer normal integrating LVs is challenging. Until recently, VLPs were not efficient for co-delivering Cas9 mRNA and single guide RNA (sgRNA). By fusing ABP to the N-terminus of HIV Gag protein, hybrid particles were developed to co-deliver Cas9 mRNA and sgRNA. But the method for modifying Gag impaired particle assembly. Previously we found that adding an ABP after the second zinc finger domain of nucleocapsid (NC) protein had minimal effects on particle assembly. Based on this observation, we developed hybrid particles to co-deliver Cas9 mRNA and sgRNA. We further improved LVs for integrated gene expression by including an aptamer sequence in lentiviral transfer plasmids, which improved lentiviral particle production and enhanced LV genomic RNA packaging. In summary, we describe development of new all-in-one VLPs for co-delivery of Cas9 mRNA and sgRNA and new LVs for enhanced vector production and gene expression.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Developing all-in-one virus-like particles for Cas9 mRNA/single guide RNA co-delivery and aptamer-containing lentiviral vectors for improved gene expression

Yadav

Atala

2022

Preprint

Self Cite

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“…An RNA segment of the mom gene of bacteriophage Mu (com) was strongly and specifically associated with a phage Com protein (COM) [120,121]. The com sequence inserted into the tetraloop of sgRNA had little or no influence on the package and activity of the Cas9-sgRNA RNP complex and interacted with the COM protein fused to the Gag polyprotein in the 3 -part of NC [122][123][124]. Besides Gag polyprotein, the G3C, V153L, and G177E gene-modified NEF protein (1100 copies per capsid) [125,126] and VPR protein (550 copies per capsid) [127,128] could be used as a carrier for the aptamer binding proteins [129].…”

Section: Delivery Of Cas9/sgrna Rnp With Virus-like Particlesmentioning

confidence: 99%

Points of View on the Tools for Genome/Gene Editing

Chuang

Lin

2021

IJMS

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Theoretically, a DNA sequence-specific recognition protein that can distinguish a DNA sequence equal to or more than 16 bp could be unique to mammalian genomes. Long-sequence-specific nucleases, such as naturally occurring Homing Endonucleases and artificially engineered ZFN, TALEN, and Cas9-sgRNA, have been developed and widely applied in genome editing. In contrast to other counterparts, which recognize DNA target sites by the protein moieties themselves, Cas9 uses a single-guide RNA (sgRNA) as a template for DNA target recognition. Due to the simplicity in designing and synthesizing a sgRNA for a target site, Cas9-sgRNA has become the most current tool for genome editing. Moreover, the RNA-guided DNA recognition activity of Cas9-sgRNA is independent of both of the nuclease activities of it on the complementary strand by the HNH domain and the non-complementary strand by the RuvC domain, and HNH nuclease activity null mutant (H840A) and RuvC nuclease activity null mutant (D10A) were identified. In accompaniment with the sgRNA, Cas9, Cas9(D10A), Cas9(H840A), and Cas9(D10A, H840A) can be used to achieve double strand breakage, complementary strand breakage, non-complementary strand breakage, and no breakage on-target site, respectively. Based on such unique characteristics, many engineered enzyme activities, such as DNA methylation, histone methylation, histone acetylation, cytidine deamination, adenine deamination, and primer-directed mutation, could be introduced within or around the target site. In order to prevent off-targeting by the lasting expression of Cas9 derivatives, a lot of transient expression methods, including the direct delivery of Cas9-sgRNA riboprotein, were developed. The issue of biosafety is indispensable in in vivo applications; Cas9-sgRNA packaged into virus-like particles or extracellular vesicles have been designed and some in vivo therapeutic trials have been reported.

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Engineered extracellular vesicles as versatile ribonucleoprotein delivery vehicles for efficient and safe CRISPR genome editing

Yao

Lyu

Yoo

et al. 2021

J of Extracellular Vesicle

132

112

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Transient delivery of CRISPR-based genome editing effectors is important to reduce off-target effects and immune responses. Recently extracellular vesicles (EVs) have been explored for Cas9 ribonucleoprotein (RNP) delivery. However, lack of mechanisms to enrich RNPs into EVs limited the efficiency of EVs as a RNP delivery vehicle. Here we describe a mechanism to actively enrich RNPs into EVs. We used the specific interaction between RNA aptamer and aptamer-binding protein (ABP) to enrich RNPs into EVs. We inserted RNA aptamer com into single guide RNA (sgRNA), and fused com-binding ABP Com to both termini of tetraspan protein CD63 that is abundant in exosomes. We found that the Com/com interaction enriched Cas9 and adenine base editor (ABE) RNPs into EVs, via forming a three-component complex including CD63-Com fusion protein, com-modified sgRNA and Cas9 or ABE. The RNP enriched EVs are efficient in genome editing and transiently expressed. The system is capable of delivering RNPs targeting multiple loci for multiplex genome editing. In addition, Cas9 from different species can be used together. The EV-delivered RNPs are active in vivo. The data show that the aptamer and ABP interactions can be utilized to actively enrich RNPs into EVs for improved genome editing efficiency and safety. K E Y WO R D S adenine base editor, aptamer, aptamer-binding protein, CD63, CRISPR/Cas9, delivery, extracellular vesicle, ribonucleoprotein  INTRODUCTION Extracellular vesicles (EVs) can be broadly divided into two main categories, exosomes and microvesicles, based on the mechanisms of generation. Exosomes are heterogeneous membranous vesicles released by various cells via inward budding of multivesicular bodies and subsequent fusion of the multivesicular body membranes with the plasma membrane (Heijnen et al., 1999). Exosomes play important roles in intercellular crosstalk and disease pathogenesis, and are believed to function by transporting RNAs, proteins and lipids from one cell to the other (Ratajczak et al., 2006). Microvesicles are generated by the outward budding and fission of the plasma membrane and the subsequent release of vesicles into the extracellular space. Exosomes and microvesicles overlap in sizes and currently it is difficult to separate the two types of vesicles in preparation. The transportation capability of EVs prompted the exploration of using EVs as drug delivery vehicles (Alvarez-Erviti et al.

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Lentiviral Capsid-Mediated Streptococcus pyogenes Cas9 Ribonucleoprotein Delivery for Efficient and Safe Multiplex Genome Editing

Cited by 22 publications

References 47 publications

Developing all-in-one virus-like particles for Cas9 mRNA/single guide RNA co-delivery and aptamer-containing lentiviral vectors for improved gene expression

Developing all-in-one virus-like particles for Cas9 mRNA/single guide RNA co-delivery and aptamer-containing lentiviral vectors for improved gene expression

Points of View on the Tools for Genome/Gene Editing

Engineered extracellular vesicles as versatile ribonucleoprotein delivery vehicles for efficient and safe CRISPR genome editing

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