2020
DOI: 10.1038/s41467-020-15053-x
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Multiplexed CRISPR technologies for gene editing and transcriptional regulation

Abstract: Multiplexed CRISPR technologies, in which numerous gRNAs or Cas enzymes are expressed at once, have facilitated powerful biological engineering applications, vastly enhancing the scope and efficiencies of genetic editing and transcriptional regulation. In this review, we discuss multiplexed CRISPR technologies and describe methods for the assembly, expression and processing of synthetic guide RNA arrays in vivo. Applications that benefit from multiplexed CRISPR technologies, including cellular recorders, genet… Show more

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Cited by 372 publications
(279 citation statements)
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“…The ability to rapidly and automatically validate plasmids by Illumina sequencing will be especially applicable to large-scale plasmid production projects associated with synthetic biology (42). The rapidly decreasing cost of DNA synthesis and sophisticated computer-aided design tools (43,44) have facilitated disciplined factorial experiments involving large libraries of plasmids with various genetic parts (45) for applications such as improved genetic circuit design (46), multiplexed CRISPR-Cas (47) or metabolic engineering (48). These workflows, which are often automated (49,50), necessitate an automated sequence verification pipeline like the one we've described.…”
Section: Discussionmentioning
confidence: 99%
“…The ability to rapidly and automatically validate plasmids by Illumina sequencing will be especially applicable to large-scale plasmid production projects associated with synthetic biology (42). The rapidly decreasing cost of DNA synthesis and sophisticated computer-aided design tools (43,44) have facilitated disciplined factorial experiments involving large libraries of plasmids with various genetic parts (45) for applications such as improved genetic circuit design (46), multiplexed CRISPR-Cas (47) or metabolic engineering (48). These workflows, which are often automated (49,50), necessitate an automated sequence verification pipeline like the one we've described.…”
Section: Discussionmentioning
confidence: 99%
“…For instance, the transcriptional activation of many plant-dominant resistance genes by CRISPR-Act2.0 would enhance the plant immunity to confer a broad range of resistance to different kinds of pathogens (Lowder et al, 2018), and the primer editor is also an interesting alternative applied into the antiviral engineering (Anzalone et al, 2019). The multiplexed CRISPR technologies, in which numerous gRNAs or Cas proteins are expressed at once, have vastly enhanced the scope and efficiency of genetic editing and transcriptional regulation (McCarty et al, 2020). However, the application of these multiplexed CRISPR technologies into the field to control plant viruses is still on the way, mainly because of the complexity of experimental manipulation, the limitations of the genetic transformation of several simultaneous Cas proteins into one plant, and the lack of validated and effective endogenous targets against different viruses, among other things (McCarty et al, 2020).…”
Section: Conclusion and Future Perspectivesmentioning
confidence: 99%
“…In addition, given the potential for CRISPRi for multiplexed gene silencing [25], it would be interesting to knockdown TMEM97 together with other AMD-associated genes to determine if these genes have synergistic contribution to retinal degeneration and pathogenesis of AMD.…”
Section: To Enablementioning
confidence: 99%