Systematic evaluation of CRISPR-Cas systems reveals design principles for genome editing in human cells

Wang, Yuanming; Liu, Kaiwen Ivy; Sutrisnoh, Norfala-Aliah Binte; Srinivasan, Harini; Zhang, Junyi; Li, Jia; Fan, Zhengqiu; Richard, John Lalith Charles; Xing, Heyun; Shanmugam, Raghuvaran; Foo, Jia Nee; Yeo, Hwee Ting; Ooi, Kim Tiow; Bleckwehl, Tore; Par, Yi Yun Rachel; Lee, Shi Mun; Ismail, Nur Nadiah Binte; Sanwari, Nur Aidah Binti; Lee, Si Ting Vanessa; Lew, Jan; Tan, Meng How

doi:10.1186/s13059-018-1445-x

Cited by 80 publications

(79 citation statements)

References 47 publications

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“…Accounting also for ‘CC’ (representing the PAM site on the complementary strand) will give on average one PAM site within a 10 base distance from the mutation site of interest. Although PAM site recognized by Cas9 from S pyogenes is relatively simple and abundant, it might be advantageous to use CRISPR systems from other bacteria, which recognize other PAM sites . It could potentially allow cutting of DNA closer to the target site, and increase the efficiency of HDR.…”

Section: Discussionmentioning

confidence: 99%

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A simple and efficient workflow for generation of knock‐in mutations in Jurkat T cells using CRISPR/Cas9

et al. 2020

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CRISPR/Cas9 is a powerful gene‐editing tool allowing for specific gene manipulation at targeted sites in the genome. Here, we used CRISPR/Cas9‐mediated gene editing to introduce single amino acid mutations into proteins involved in T cell receptor signalling pathways. Knock‐in mutations were introduced in Jurkat T cells by homologous directed repair using single‐stranded oligodeoxynucleotides. Specifically, we aimed to create targeted mutations at two loci within LCK, a constitutively expressed gene, and at three loci within SH2D2A, whose expression is induced upon T cell activation. Here, we present a simple workflow that can be applied by any laboratory equipped for cell culture work, utilizing basic flow cytometry, Western blotting and PCR techniques. Our data reveal that gene editing may be locus‐dependent and can vary between target sites, also within a gene. In our two targeted genes, on average 2% of the clones harboured homozygous mutations as assessed by allele‐specific PCR and subsequent sequencing. We highlight the importance of decreasing the clonal heterogeneity and developing robust screening methods to accurately select for correct knock‐in mutations. Our workflow may be employed in other immune cell lines and acts as a useful approach for decoding functional mechanisms of proteins of interest.

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Section: Discussionmentioning

confidence: 99%

“…Although PAM site recognized by Cas9 from S pyogenes is relatively simple and abundant, it might be advantageous to use CRISPR systems from other bacteria, which recognize other PAM sites. 35 It could potentially allow cutting of DNA closer to the target site, and increase the efficiency of HDR.…”

Section: Discussionmentioning

confidence: 99%

A simple and efficient workflow for generation of knock‐in mutations in Jurkat T cells using CRISPR/Cas9

et al. 2020

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“…At this stage, genetic knockouts are facile, but replacing sequences by HDR remains inefficient and technically challenging, although great strides are being made to improve the technique. [65][66][67][68][69][70][71][72] CRISPR systems have generated an extraordinary level of excitement for the clinical potential for curing human disease. In this nascent stage of CRISPR in the clinic, initial applications are treating diseases with the most accessible cells (immune cells and HSPCs edited ex vivo), which avoids the ongoing challenge of tissue-specific delivery in vivo.…”

Section: Discussionmentioning

confidence: 99%

“…54 The donor is typically a singleor double-stranded DNA bearing "homology arms" that match either side of the Cas9 cut site. 65 The likelihood of successful insertion can be improved using a few different strategies, but the general inefficiency of this gene replacement approach represents a substantial challenge in the field. [65][66][67][68][69][70][71][72] As our ability to carefully manipulate DNA grows, so will the clinical potential of CRISPR technologies.…”

Section: Repurposing Crispr Proteins For Genome Editingmentioning

confidence: 99%

“…65 The likelihood of successful insertion can be improved using a few different strategies, but the general inefficiency of this gene replacement approach represents a substantial challenge in the field. [65][66][67][68][69][70][71][72] As our ability to carefully manipulate DNA grows, so will the clinical potential of CRISPR technologies. 17 CURRENT AND NEAR-TERM THERAPEUTIC APPLICATIONS Genetic therapies offer hope for treating and effectively curing monogenic diseases by reversing the underlying genetic cause.…”

Section: Repurposing Crispr Proteins For Genome Editingmentioning

confidence: 99%

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Clinical applications of CRISPR‐based genome editing and diagnostics

2019

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Clustered regularly interspaced short palindromic repeats (CRISPR)‐driven genome editing has rapidly transformed preclinical biomedical research by eliminating the underlying genetic basis of many diseases in model systems and facilitating the study of disease etiology. Translation to the clinic is under way, with announced or impending clinical trials utilizing ex vivo strategies for anticancer immunotherapy or correction of hemoglobinopathies. These exciting applications represent just a fraction of what is theoretically possible for this emerging technology, but many technical hurdles must be overcome before CRISPR‐based genome editing technology can reach its full potential. One exciting recent development is the use of CRISPR systems for diagnostic detection of genetic sequences associated with pathogens or cancer. We review the biologic origins and functional mechanism of CRISPR systems and highlight several current and future clinical applications of genome editing.

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CRISPR/Cas Systems in Genome Editing: Methodologies and Tools for sgRNA Design, Off‐Target Evaluation, and Strategies to Mitigate Off‐Target Effects

et al. 2020

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Life sciences have been revolutionized by genome editing (GE) tools, including zinc finger nucleases, transcription activator‐Like effector nucleases, and CRISPR (clustered regulatory interspaced short palindromic repeats)/Cas (CRISPR‐associated) systems, which make the targeted modification of genomic DNA of all organisms possible. CRISPR/Cas systems are being widely used because of their accuracy, efficiency, and cost‐effectiveness. Various classes of CRISPR/Cas systems have been developed, but their extensive use may be hindered by off‐target effects. Efforts are being made to reduce the off‐target effects of CRISPR/Cas9 by generating various CRISPR/Cas systems with high fidelity and accuracy. Several approaches have been applied to detect and evaluate the off‐target effects. Here, the current GE tools, the off‐target effects generated by GE technology, types of off‐target effects, mechanisms of off‐target effects, major concerns, and outcomes of off‐target effects in plants and animals are summarized. The methods to detect off‐target effects, tools for single‐guide RNA (sgRNA) design, evaluation and prediction of off‐target effects, and strategies to increase the on‐target efficiency and mitigate the off‐target impact on intended genome‐editing outcomes are summarized.

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Systematic evaluation of CRISPR-Cas systems reveals design principles for genome editing in human cells

Cited by 80 publications

References 47 publications

A simple and efficient workflow for generation of knock‐in mutations in Jurkat T cells using CRISPR/Cas9

A simple and efficient workflow for generation of knock‐in mutations in Jurkat T cells using CRISPR/Cas9

Clinical applications of CRISPR‐based genome editing and diagnostics

CRISPR/Cas Systems in Genome Editing: Methodologies and Tools for sgRNA Design, Off‐Target Evaluation, and Strategies to Mitigate Off‐Target Effects

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