Marker-free coselection for CRISPR-driven genome editing in human cells

Agudelo, Daniel; Duringer, Alexis; Bozoyan, Lusiné; Huard, Caroline; Carter, Sophie; Loehr, Jérémy; Synodinou, Dafni; Drouin, Mathieu; Salsman, Jayme; Dellaire, Graham; Laganière, Josée; Doyon, Yannick

doi:10.1038/nmeth.4265

Cited by 149 publications

(187 citation statements)

References 57 publications

(69 reference statements)

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“…This led us to investigate whether we could further boost its activity. First, we added an N-terminal nuclear localization signal (NLS) to a previously described human codon-optimized expression construct 35 and established a K562 cell line stably expressing St1Cas9 (S. thermophilus strain LMD-9) from the AAVS1 safe harbor locus 36,37 ( Fig. 1a and Supplementary Fig.…”

Section: Identification Of An Sgrna Architecture Directing Robust Dnamentioning

confidence: 99%

“…Cells (2E5 per transfection) were transfected using the Amaxa 4D-Nucleofector (Lonza) per manufacturer's recommendations. K562 cell lines expressing SaCas9 and St1Cas9 from the AAVS1 safe harbor locus were generated as described 36,37 . Briefly, simultaneous selection and cloning was performed for 10 days in methylcellulose-based semi-solid RPMI medium supplemented with 0.5 µg/ml puromycin starting 3 days post-transfection.…”

Section: Cell Culture and Transfectionmentioning

confidence: 99%

“…The various loci were amplified by PCR using the primers described in Supplementary Table 8. Assays were performed with the Surveyor mutation detection kit (Transgenomics) as described 36,38 . Samples were separated on 10% PAGE gels in TBE buffer.…”

Section: Surveyor Nuclease and Tide Assaysmentioning

confidence: 99%

“…1). St1Cas9-expressing human cells were transfected with increasing amounts of each sgRNA construct and the Surveyor nuclease assay was used to determine the frequency of indels characteristic of imprecise DSB repair by NHEJ 36,38 ( Fig. 1d).…”

mentioning

confidence: 99%

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Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

Agudelo

Carter

Velimirovic

et al. 2018

Preprint

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The broad spectrum of activities displayed by CRISPR-Cas systems has led to biotechnological innovations that are poised to transform human therapeutics. Therefore, the comprehensive characterization of distinct Cas proteins is highly desirable. Here we expand the repertoire of nucleases for mammalian genome editing using the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9). We define functional protospacer adjacent motif (PAM) sequences and variables required for robust and efficient editing in vitro. Expression of holoSt1Cas9 from a single adeno-associated viral (rAAV) vector in the neonatal liver rescued lethality and metabolic defects in a mouse model of hereditary tyrosinemia type I demonstrating effective cleavage activity in vivo. Furthermore, we identified potent anti-CRISPR proteins to regulate the activity of both St1Cas9 and the related type II-A Staphylococcus aureus Cas9 (SaCas9). This work expands the targeting range and versatility of CRISPR-associated enzymes and should encourage studies to determine its structure, genome-wide specificity profile and sgRNA design rules. INTRODUCTIONClustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins form a prokaryotic adaptive immune system and some of its components have been harnessed for robust genome editing 1 . Type II-based editing tools rely on a large multidomain endonuclease, Cas9, guided to its DNA target by an engineered single-guide RNA (sgRNA) chimera 2 (See 3, 4 for a classification of CRISPR-Cas systems). The Cas9-sgRNA binary complex finds its target through recognition of a short sequence called the protospacer adjacent motif (PAM) and subsequent base pairing of the guide RNA with the DNA to generate a specific doublestrand break (DSB) 1,5 . While Streptococcus pyogenes (SpCas9) remains the most widely used Cas9 variant for genome engineering, the diversity of naturally occurring RNA-guided nucleases is astonishing 4 . Hence, Cas9 enzymes from different microbial species can contribute to the expansion of the CRISPR toolset by increasing targeting density, improving activity and specificity as well as easing delivery 1,6 .In principle, engineering complementary CRISPRCas systems from distinct bacterial species should be relatively straightforward, as they have been minimized to only two components. However, many such enzymes were found inactive in human cells despite being accurately reprogrammed for DNA binding and cleavage in vitro [7][8][9][10] . Nevertheless, the full potential of selected enzymes can be unleashed using machine learning to establish sgRNA design rules 11,12 .Perhaps the most striking example of the value of alternative Cas9 enzymes is the implementation of the type II-A Cas9 from Staphylococcus aureus (SaCas9) for in vivo editing using recombinant adeno-associated virus (rAAV) vectors 7,13, 14 . More recently, Campylobacter jejuni and Neisseria meningitidis Cas9s from the type II-C 15 CRISPRCas systems have been added to this repertoire 16,17 .In vivo genome edi...

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Section: Identification Of An Sgrna Architecture Directing Robust Dnamentioning

confidence: 99%

Section: Cell Culture and Transfectionmentioning

confidence: 99%

Section: Surveyor Nuclease and Tide Assaysmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

Agudelo

Carter

Velimirovic

et al. 2018

Preprint

Self Cite

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“…We noted that this modus operandi is preferable to the pooling of preparations of two particle types, each of them programmed with a single sgRNA. Multiplexing of sgRNAs may also allow the introduction of an additional sgRNA targeting a specific gene that will allow selection of cells efficiently edited by Nanoblademediated CRISPR 28 . This versatility allows any laboratory equipped with BSL2 facilities to generate its own batches of particles.…”

Section: Targeted Transcriptional Activation Through Nanobladesmentioning

confidence: 99%

Efficient genome editing in primary cells andin vivousing viral-derived “Nanoblades” loaded with Cas9/sgRNA ribonucleoproteins

Mangeot

Rissons

Fusil

et al. 2017

Preprint

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Programmable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. However, their delivery into target cells can be technically challenging when working with primary cells or in vivo. Using engineered murine leukemia virus-like particles loaded with Cas9/sgRNA ribonucleoproteins ("Nanoblades"), we were able to induce efficient genome-editing in cell lines and primary cells including human induced pluripotent stem cells, human hematopoietic stem cells and mouse bone-marrow cells. Transgene-free Nanoblades were also capable of in vivo genome-editing in mouse embryos and in the liver of injected mice. Nanoblades can be complexed with donor DNA for "all-in-one" homology-directed repair or programmed with modified Cas9 variants to mediate transcriptional up-regulation of target genes. Nanoblades preparation process is simple, relatively inexpensive and can be easily implemented in any laboratory equipped for cellular biology.

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Manipulating and studying gene function in human pluripotent stem cell models

Balmas,

Sozza,

Bottini

et al. 2023

FEBS Letters

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Human pluripotent stem cells (hPSCs) are uniquely suited to study human development and disease and promise to revolutionize regenerative medicine. These applications rely on robust methods to manipulate gene function in hPSC models. This comprehensive review aims to both empower scientists approaching the field and update experienced stem cell biologists. We begin by highlighting challenges with manipulating gene expression in hPSCs and their differentiated derivatives, and relevant solutions (transfection, transduction, transposition, and genomic safe harbor editing). We then outline how to perform robust constitutive or inducible loss‐, gain‐, and change‐of‐function experiments in hPSCs models, both using historical methods (RNA interference, transgenesis, and homologous recombination) and modern programmable nucleases (particularly CRISPR/Cas9 and its derivatives, i.e., CRISPR interference, activation, base editing, and prime editing). We further describe extension of these approaches for arrayed or pooled functional studies, including emerging single‐cell genomic methods, and the related design and analytical bioinformatic tools. Finally, we suggest some directions for future advancements in all of these areas. Mastering the combination of these transformative technologies will empower unprecedented advances in human biology and medicine.

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Marker-free coselection for CRISPR-driven genome editing in human cells

Cited by 149 publications

References 57 publications

Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

Efficient genome editing in primary cells andin vivousing viral-derived “Nanoblades” loaded with Cas9/sgRNA ribonucleoproteins

Manipulating and studying gene function in human pluripotent stem cell models

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