Abstract
This protocol describes the application of the CRISPR-Trap from designing of the gene targeting strategy to validation of successfully edited clones that was validated on various human cell lines, among them human induced pluripotent stem cells (hiPSCs). The advantage of CRISPR-Trap over conventional approaches is the complete removal of any endogenous full-length transcript from the target gene. CRISPR-Trap is applicable for any target gene with no or little coding sequence in its first exon. Several human cell lines and different genes have so far been edited successfully with CRISPR-Trap.
Keywords: CRISPR, CRISPR-Trap, Gene knockout, Gene replacement, Gene editing, hiPSCs
Background
The advent of CRISPR/Cas9 technology facilitated the genomic targeting for the generation of gene knockouts and gene editing. The conventional method to perform a knockout relies on the introduction of a frameshift leading to premature termination codons (PTCs), truncating the open reading frame (ORF) and subsequent degradation of the transcript of the targeted gene by nonsense-mediated mRNA decay (NMD). A possible pitfall of this approach is full-length transcripts which may escape NMD and give rise to C-terminal truncated proteins harboring residual or even dominant negative functions. This protocol presents the CRISPR-Trap, a method we recently established (Reber et al., 2018), which upon successful editing will prevent the expression of any full-length transcript from the target gene locus (Figure 1). Simply put, this approach targets the first intron of the gene of interest with CRISPR/Cas9. Using homology-directed repair (HDR) a customizable cassette flanked by a strong 3’-splice site and a strong polyadenylation signal is introduced in the first intron, thereby generating an artificial second and effectively last exon. Since transcription is terminated by the introduced polyadenylation signal, only the first endogenous exon and the inserted cassette is transcribed. The customizable cassette can be used to introduce a selection marker, thereby enabling easy selection for at least heterozygous edited clones. If a gene replacement is wanted, the customizable cassette can be used to introduce the replacement gene, followed by an internal ribosomal entry site (IRES) and a selection marker (Reber et al., 2016 and 2018).Figure 1. Schematic of the application of the CRISPR-Trap. The first intron of the target gene is cleaved using the CRISPR/Cas9 system and template DNA for homology-directed repair is provided. The template DNA contains a strong 3’ prime splice signal (dark green), a customizable cassette (light green) and a strong polyadenylation signal (turquoise). The customizable cassette can be utilized to either knockout (left) or replace the target gene (right). The cassette contains a selection marker that will be under the control of the endogenous promoter of the target gene upon successful editing. For gene replacements, an IRES is introduced in between the replacement gene and the selection marker. Figure adapted from Reber et al. (2018).
Materials and Reagents
Equipment
Software
Procedure
Notes:
Notes
Homozygous clones should then finally be analyzed by Western Blotting to demonstrate the absence of the targeted endogenous protein. An example for cell harvesting, extract preparation and immunoblotting is described in the original CRISPR-trap publication (Reber et al., 2018). In our hands, CRISPR-trap yielded no homozygous clones when targeting essential genes (lethal knockout). In this case, a gene replacement with a degron-tagged cDNA (see pHDR-TDP43 repl-Puro as example) should be considered to allow an inducible depletion of the protein of interest. The success rate of CRISPR-trap will depend on cell line and target gene. It is therefore difficult to predict the number of clones that need to be sequenced before a homozygous clone is detected. In our hands, the success rate for homozygous clones ranged from 10 % to 90 % of the analyzed clones.
Acknowledgments
This work was made possible by the support of the NOMIS Foundation, the National Centre of Competence in Research (NCCR) RNA & Disease funded by the Swiss National Science Foundation, and the support of the UK Dementia Research Institute. The protocol is based on the publications (Reber et al., 2016 and 2018) in The EMBO Journal and Molecular Biology of the Cell.
Competing interests
The authors declare that they have no competing or conflicting interests.
References
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