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1.
Proc Natl Acad Sci U S A ; 118(22)2021 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-34050017

RESUMEN

CRISPR-Cas9 nuclease-based gene drives have been developed toward the aim of control of the human malaria vector Anopheles gambiae Gene drives are based on an active source of Cas9 nuclease in the germline that promotes super-Mendelian inheritance of the transgene by homology-directed repair ("homing"). Understanding whether CRISPR-induced off-target mutations are generated in Anopheles mosquitoes is an important aspect of risk assessment before any potential field release of this technology. We compared the frequencies and the propensity of off-target events to occur in four different gene-drive strains, including a deliberately promiscuous set-up, using a nongermline restricted promoter for SpCas9 and a guide RNA with many closely related sites (two or more mismatches) across the mosquito genome. Under this scenario we observed off-target mutations at frequencies no greater than 1.42%. We witnessed no evidence that CRISPR-induced off-target mutations were able to accumulate (or drive) in a mosquito population, despite multiple generations' exposure to the CRISPR-Cas9 nuclease construct. Furthermore, judicious design of the guide RNA used for homing of the CRISPR construct, combined with tight temporal constriction of Cas9 expression to the germline, rendered off-target mutations undetectable. The findings of this study represent an important milestone for the understanding and managing of CRISPR-Cas9 specificity in mosquitoes, and demonstrates that CRISPR off-target editing in the context of a mosquito gene drive can be reduced to minimal levels.


Asunto(s)
Anopheles/genética , Sistemas CRISPR-Cas , Edición Génica , Genoma de los Insectos , Malaria , Mosquitos Vectores/genética , Animales , Humanos
2.
J Virol ; 89(14): 7428-32, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-25972561

RESUMEN

High-throughput integration site (IS) analysis of wild-type adeno-associated virus type 2 (wtAAV2) in human dermal fibroblasts (HDFs) and HeLa cells revealed that juxtaposition of a Rep binding site (RBS) and terminal resolution site (trs)-like motif leads to a 4-fold-increased probability of wtAAV integration. Electrophoretic mobility shift assays (EMSAs) confirmed binding of Rep to off-target RBSs. For the first time, we show Rep protein off-target nicking activity, highlighting the importance of the nicking substrate for Rep-mediated integration.


Asunto(s)
Secuencias de Aminoácidos , Proteínas de Unión al ADN/metabolismo , Dependovirus/fisiología , Proteínas Virales/metabolismo , Integración Viral , Línea Celular , Células Epiteliales/virología , Fibroblastos/virología , Humanos
3.
Elife ; 122024 Jun 07.
Artículo en Inglés | MEDLINE | ID: mdl-38847802

RESUMEN

CRISPR prime editing (PE) requires a Cas9 nickase-reverse transcriptase fusion protein (known as PE2) and a prime editing guide RNA (pegRNA), an extended version of a standard guide RNA (gRNA) that both specifies the intended target genomic sequence and encodes the desired genetic edit. Here, we show that sequence complementarity between the 5' and the 3' regions of a pegRNA can negatively impact its ability to complex with Cas9, thereby potentially reducing PE efficiency. We demonstrate this limitation can be overcome by a simple pegRNA refolding procedure, which improved ribonucleoprotein-mediated PE efficiencies in zebrafish embryos by up to nearly 25-fold. Further gains in PE efficiencies of as much as sixfold could also be achieved by introducing point mutations designed to disrupt internal interactions within the pegRNA. Our work defines simple strategies that can be implemented to improve the efficiency of PE.


Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , ARN Guía de Sistemas CRISPR-Cas , Pez Cebra , Pez Cebra/genética , Animales , Edición Génica/métodos , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismo , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genética , Embrión no Mamífero/metabolismo , Pliegue del ARN
4.
Nat Biotechnol ; 41(3): 337-343, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36163548

RESUMEN

The CRISPR prime editor PE2 consists of a Streptococcus pyogenes Cas9 nickase (nSpCas9) fused at its C-terminus to a Moloney murine leukemia virus reverse transcriptase (MMLV-RT). Here we show that separated nSpCas9 and MMLV-RT proteins function as efficiently as intact PE2 in human cells. We use this Split-PE system to rapidly identify and engineer more compact prime editor architectures that also broaden the types of RTs used for prime editing.


Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Virus de la Leucemia Murina de Moloney , ADN Polimerasa Dirigida por ARN , Streptococcus pyogenes , Animales , Humanos , Ratones , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Virus de la Leucemia Murina de Moloney/genética , ADN Polimerasa Dirigida por ARN/genética , Streptococcus pyogenes/genética , Desoxirribonucleasa I/genética
5.
bioRxiv ; 2023 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-37645936

RESUMEN

CRISPR prime editing (PE) requires a Cas9 nickase-reverse transcriptase fusion protein (known as PE2) and a prime editing guide RNA (pegRNA), an extended version of a standard guide RNA (gRNA) that both specifies the intended target genomic sequence and encodes the desired genetic edit. Here we show that sequence complementarity between the 5' and the 3' regions of a pegRNA can negatively impact its ability to complex with Cas9, thereby potentially reducing PE efficiency. We demonstrate this limitation can be overcome by a simple pegRNA refolding procedure, which improved ribonucleoprotein-mediated PE efficiencies in zebrafish embryos by up to nearly 25-fold. Further gains in PE efficiencies of as much as 6-fold could also be achieved by introducing point mutations designed to disrupt internal interactions within the pegRNA. Our work defines simple strategies that can be implemented to improve the efficiency of PE.

6.
bioRxiv ; 2023 May 27.
Artículo en Inglés | MEDLINE | ID: mdl-37292647

RESUMEN

Gene editing the BCL11A erythroid enhancer is a validated approach to fetal hemoglobin (HbF) induction for ß-hemoglobinopathy therapy, though heterogeneity in edit allele distribution and HbF response may impact its safety and efficacy. Here we compared combined CRISPR-Cas9 endonuclease editing of the BCL11A +58 and +55 enhancers with leading gene modification approaches under clinical investigation. We found that combined targeting of the BCL11A +58 and +55 enhancers with 3xNLS-SpCas9 and two sgRNAs resulted in superior HbF induction, including in engrafting erythroid cells from sickle cell disease (SCD) patient xenografts, attributable to simultaneous disruption of core half E-box/GATA motifs at both enhancers. We corroborated prior observations that double strand breaks (DSBs) could produce unintended on- target outcomes in hematopoietic stem and progenitor cells (HSPCs) such as long deletions and centromere-distal chromosome fragment loss. We show these unintended outcomes are a byproduct of cellular proliferation stimulated by ex vivo culture. Editing HSPCs without cytokine culture bypassed long deletion and micronuclei formation while preserving efficient on-target editing and engraftment function. These results indicate that nuclease editing of quiescent hematopoietic stem cells (HSCs) limits DSB genotoxicity while maintaining therapeutic potency and encourages efforts for in vivo delivery of nucleases to HSCs.

7.
Nat Biotechnol ; 40(2): 189-193, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-33927418

RESUMEN

Prime editors have been delivered using DNA or RNA vectors. Here we demonstrate prime editing with purified ribonucleoprotein complexes. We introduced somatic mutations in zebrafish embryos with frequencies as high as 30% and demonstrate germline transmission. We also observed unintended insertions, deletions and prime editing guide RNA (pegRNA) scaffold incorporations. In HEK293T and primary human T cells, prime editing with purified ribonucleoprotein complexes introduced desired edits with frequencies of up to 21 and 7.5%, respectively.


Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Pez Cebra , Animales , Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Edición Génica , Células HEK293 , Humanos , ARN Guía de Kinetoplastida/genética , Ribonucleoproteínas/genética , Pez Cebra/genética
8.
Mol Ther Nucleic Acids ; 28: 450-461, 2022 Jun 14.
Artículo en Inglés | MEDLINE | ID: mdl-35505961

RESUMEN

Presenilin 1 (PS1) is a central component of γ-secretase, an enzymatic complex involved in the generation of the amyloid-ß (Aß) peptide that deposits as plaques in the Alzheimer's disease (AD) brain. The M146L mutation in the PS1 gene (PSEN1) leads to an autosomal dominant form of early-onset AD by promoting a relative increase in the generation of the more aggregation-prone Aß42. This change is evident not only in the brain but also in peripheral cells of mutation carriers. In this study we used the CRISPR-Cas9 system from Streptococcus pyogenes to selectively disrupt the PSEN1 M146L allele in human fibroblasts. A disruption of more than 50% of mutant alleles was observed in all CRISPR-Cas9-treated samples, resulting in reduced extracellular Aß42/40 ratios. Fluorescence resonance energy transfer-based conformation and western blot analyses indicated that CRISPR-Cas9 treatment also affects the overall PS1 conformation and reduces PS1 levels. Moreover, our guide RNA did not lead to any detectable editing at the highest-ranking candidate off-target sites identified by ONE-seq and CIRCLE-seq. Overall, our data support the effectiveness of CRISPR-Cas9 in selectively targeting the PSEN1 M146L allele and counteracting the AD-associated phenotype. We believe that this system could be developed into a therapeutic strategy for patients with this and other dominant mutations leading to early-onset AD.

9.
Nat Commun ; 12(1): 1034, 2021 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-33589617

RESUMEN

Prime editing (PE) is a versatile genome editing technology, but design of the required guide RNAs is more complex than for standard CRISPR-based nucleases or base editors. Here we describe PrimeDesign, a user-friendly, end-to-end web application and command-line tool for the design of PE experiments. PrimeDesign can be used for single and combination editing applications, as well as genome-wide and saturation mutagenesis screens. Using PrimeDesign, we construct PrimeVar, a comprehensive and searchable database that includes candidate prime editing guide RNA (pegRNA) and nicking sgRNA (ngRNA) combinations for installing or correcting >68,500 pathogenic human genetic variants from the ClinVar database. Finally, we use PrimeDesign to design pegRNAs/ngRNAs to install a variety of human pathogenic variants in human cells.


Asunto(s)
Sistemas CRISPR-Cas , Edición Génica/métodos , Genoma Humano , ARN Guía de Kinetoplastida/genética , Emparejamiento Base , Secuencia de Bases , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Bases de Datos Genéticas , Enfermedad de Fabry/genética , Enfermedad de Fabry/metabolismo , Enfermedad de Fabry/patología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Hemofilia A/genética , Hemofilia A/metabolismo , Hemofilia A/patología , Humanos , Modelos Biológicos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patología , Mutación , Conformación de Ácido Nucleico , Plásmidos/química , Plásmidos/metabolismo , ARN Guía de Kinetoplastida/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo
10.
CRISPR J ; 4(1): 19-24, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33571044

RESUMEN

Gene drives hold promise for use in controlling insect vectors of diseases, agricultural pests, and for conservation of ecosystems against invasive species. At the same time, this technology comes with potential risks that include unknown downstream effects on entire ecosystems as well as the accidental or nefarious spread of organisms that carry the gene drive machinery. A code of ethics can be a useful tool for all parties involved in the development and regulation of gene drives and can be used to help ensure that a balanced analysis of risks, benefits, and values is taken into consideration in the interest of society and humanity. We have developed a code of ethics for gene drive research with the hope that this code will encourage the development of an international framework that includes ethical guidance of gene drive research and is incorporated into scientific practice by gaining broad agreement and adherence.


Asunto(s)
Códigos de Ética , Tecnología de Genética Dirigida , Ecosistema , Edición Génica , Humanos , Especies Introducidas , Principios Morales , Salud Pública
11.
Mol Ther Methods Clin Dev ; 4: 213-224, 2017 Mar 17.
Artículo en Inglés | MEDLINE | ID: mdl-28345006

RESUMEN

In T cells with transgenic high-avidity T cell receptors (TCRs), endogenous and transferred TCR chains compete for surface expression and may pair inappropriately, potentially causing autoimmunity. To knock out endogenous TCR expression, we assembled 12 transcription activator-like effector nucleases (TALENs) and five guide RNAs (gRNAs) from the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system. Using TALEN mRNA, TCR knockout was successful in up to 81% of T cells. Additionally, we were able to verify targeted gene addition of a GFP gene by homology-directed repair at the TALEN target site, using a donor suitable for replacement of the reporter transgene with therapeutic TCR chains. Remarkably, analysis of TALEN and CRISPR/Cas9 specificity using integrase-defective lentiviral vector capture revealed only one off-target site for one of the gRNAs and three off-target sites for both of the TALENs, indicating a high level of specificity. Collectively, our work shows highly efficient and specific nucleases for T cell engineering.

13.
Biotechniques ; 56(5): 269-73, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24806228

RESUMEN

The inverted terminal repeats (ITRs) of adeno-associated virus (AAV) are notoriously difficult to sequence owing to their high GC-content (70%) and palindromic sequences that result in the formation of a very stable, 125 bp long, T-shaped hairpin structure. Here we evaluate the performance of two widely used next-generation sequencing platforms, 454 GS FLX (Roche) and MiSeq Benchtop Sequencer (Illumina), in analyzing ITRs in comparatively sequencing linear amplification-meditated PCR (LAM-PCR) amplicons derived from AAV-concatemeric structures. While our data indicate that both platforms can sequence complete ITRs, efficiencies (MiSeq: 0.11% of sequence reads; 454: 0.02% of reads), frequencies (MiSeq: 171 full ITRs, 454: 3 full ITRs), and rates of deviation from the derived ITR consensus sequence (MiSeq: 0.8%-1.3%; 454: 0.5%) did differ. These results suggest that next-generation sequencing platforms can be used to specifically detect ITR mutations and sequence complete ITRs.


Asunto(s)
Dependovirus/genética , Análisis de Secuencia de ADN/métodos , Secuencias Repetidas Terminales , Células HeLa/virología , Humanos , Mutación , Reacción en Cadena de la Polimerasa/métodos
14.
CRISPR J ; 1: 209-211, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-31021254
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