CRISPR–Cas9-mediated functional dissection of 3′-UTRs

Zhao, Wenxue; Siegel, David; Biton, Anne; Tonquèze, Olivier Le; Zaitlen, Noah; Ahituv, N; Erle, David J.

doi:10.1093/nar/gkx675

Cited by 39 publications

(34 citation statements)

References 42 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In this case, a pair of guide RNAs is used to delete the 3 ′ UTR sequence between the stop codon and the polyadenylation signal. However, ∼100 nucleotides of sequence located upstream of the polyadenylation signal should be preserved to enable proper mRNA processing from the endogenous polyadenylation signal (Zhao et al 2017).…”

Section: Crispr Technology Facilitates Newmentioning

confidence: 99%

What Are 3′ UTRs Doing?

Mayr

2018

Cold Spring Harb Perspect Biol

356

278

View full text Add to dashboard Cite

SUMMARY3' untranslated regions (3' UTRs) of messenger RNAs (mRNAs) are best known to regulate mRNA-based processes, such as mRNA localization, mRNA stability, and translation. In addition, 3' UTRs can establish 3' UTR-mediated protein-protein interactions (PPIs), and thus can transmit genetic information encoded in 3' UTRs to proteins. This function has been shown to regulate diverse protein features, including protein complex formation or posttranslational modifications, but is also expected to alter protein conformations. Therefore, 3' UTR-mediated information transfer can regulate protein features that are not encoded in the amino acid sequence. This review summarizes both 3' UTR functions-the regulation of mRNA and protein-based processes-and highlights how each 3' UTR function was discovered with a focus on experimental approaches used and the concepts that were learned. This review also discusses novel approaches to study 3' UTR functions in the future by taking advantage of recent advances in technology.

show abstract

Section: Crispr Technology Facilitates Newmentioning

confidence: 99%

What Are 3′ UTRs Doing?

Mayr

2018

Cold Spring Harb Perspect Biol

356

278

View full text Add to dashboard Cite

show abstract

“…Integration of exogenous sequences may have unintended effects. For example, exogenous DNA sequences within splice sites, introns, and 5′ and 3′ non-coding regions can impact mRNA processing and/or protein translation ( Zhao et al., 2017 , Zhu et al., 2015 ). Thus, for precision disease-modeling applications, manipulation of only the desired sequence—without introduction of additional exogenous DNA or permanent integration of selection cassettes (herein referred to as “scarless editing”)—is desired.…”

Section: Introductionmentioning

confidence: 99%

Scarless Genome Editing of Human Pluripotent Stem Cells via Transient Puromycin Selection

Steyer

Cory

et al. 2018

Stem Cell Reports

View full text Add to dashboard Cite

SummaryGenome-edited human pluripotent stem cells (hPSCs) have broad applications in disease modeling, drug discovery, and regenerative medicine. We present and characterize a robust method for rapid, scarless introduction or correction of disease-associated variants in hPSCs using CRISPR/Cas9. Utilizing non-integrated plasmid vectors that express a puromycin N-acetyl-transferase (PAC) gene, whose expression and translation is linked to that of Cas9, we transiently select for cells based on their early levels of Cas9 protein. Under optimized conditions, co-delivery with single-stranded donor DNA enabled isolation of clonal cell populations containing both heterozygous and homozygous precise genome edits in as little as 2 weeks without requiring cell sorting or high-throughput sequencing. Edited cells isolated using this method did not contain any detectable off-target mutations and displayed expected functional phenotypes after directed differentiation. We apply the approach to a variety of genomic loci in five hPSC lines cultured using both feeder and feeder-free conditions.

show abstract

“…The med strategy, which results in deleting the sequence in between the proximal and distal polyA sites, has been previously implemented in cell-based systems (Zhao et al 2017). The major confounding factor for this strategy is that an ectopic, truncated mRNA slightly longer than Calm1-S is produced due to the distal polyA site remaining intact.…”

Section: Discussionmentioning

confidence: 99%

Precise removal ofCalm1long 3′ UTR isoform by CRISPR-Cas9 genome editing impairs dorsal root ganglion development in mice

Lynch

et al. 2019

Preprint

View full text Add to dashboard Cite

Most mammalian genes are subject to Alternative cleavage and PolyAdenylation (APA), often resulting in alternative length 3′ UTR isoforms. Thousands of extended or long 3′ UTR variants are preferentially expressed in neuron-enriched tissues of metazoans. However, the in vivo functions of these long 3′ UTR isoforms are largely unknown. Calmodulin 1 (Calm1) is a key integrator of calcium signaling that is required for correct neural development. Calm1 generates short (Calm1-S) and long 3′ UTR (Calm1-L) mRNA isoforms via APA. We found Calm1-S to be broadly expressed across mouse tissues, whereas Calm1-L expression was largely restricted to neural tissues, including the dorsal root ganglion (DRG). Using CRISPR-Cas9 genome editing, a series of mouse deletion lines were generated that successfully eliminated expression of Calm1-L while maintaining expression of Calm1-S. One of these lines, Calm1Δ3′ UTR, carried a 163 bp deletion surrounding the distal polyA site. Examination of Calm1Δ3′ UTR embryos revealed disrupted development of the DRG. In Calm1Δ3′ UTR DRG explant cultures undergoing axon outgrowth, we observed a dramatic increase in axon fasciculation. These results demonstrate a physiological role for Calm1-L in DRG development, and more generally, establish a genome-editing strategy to study in vivo functions of long 3′ UTR isoforms.Author SummaryMore than half of all human genes generate alternative mRNA isoforms which differ in the length of their 3’ Untranslated regions (3’ UTRs). Through a process called Alternative Cleavage and Polyadenylation thousands of broadly expressed genes preferentially express long 3’ UTR variants in brain tissues whereas their short 3’ UTR counterparts are more broadly expressed. A challenge to study the functions of these transcripts has been to generate loss of function mutant animals that lack a long 3’ UTR isoform but maintain expression of the corresponding short 3’ UTR isoform. Here, we used the precise, rapid, and efficient approach of CRISPR genome-editing to generate long 3’ UTR mutant mice. These mice, which do not express the long 3’ UTR of the Calmodulin 1 (Calm1) gene, exhibit impairment in the development of sensory neurons, including increased fasciculation of axons and aberrant cell body migration. This finding is important because it provides conclusive genetic evidence for a neural function of a long 3’ UTR isoform in an animal. The CRISPR genome-editing approach used here can be applied to the study of neuron-enriched long 3’ UTR isoforms, which number in the thousands and have largely unexplored functions.

show abstract

CRISPR–Cas9-mediated functional dissection of 3′-UTRs

Cited by 39 publications

References 42 publications

What Are 3′ UTRs Doing?

What Are 3′ UTRs Doing?

Scarless Genome Editing of Human Pluripotent Stem Cells via Transient Puromycin Selection

Precise removal ofCalm1long 3′ UTR isoform by CRISPR-Cas9 genome editing impairs dorsal root ganglion development in mice

Contact Info

Product

Resources

About