Homology-independent multiallelic disruption via CRISPR/Cas9-based knock-in yields distinct functional outcomes in human cells

Abstract

BackgroundCultured human cells are pivotal models to study human gene functions, but introducing complete loss of function in diploid or aneuploid cells has been a challenge. The recently developed CRISPR/Cas9-mediated homology-independent knock-in approach permits targeted insertion of large DNA at high efficiency, providing a tool for insertional disruption of a selected gene. Pioneer studies have showed promising results, but the current methodology is still suboptimal and functional outcomes have not been well examined. Taking advantage of the promoterless fluorescence reporter systems established in our previous study, here, we further investigated potentials of this new insertional gene disruption approach and examined its functional outcomes.ResultsExemplified by using hyperploid LO2 cells, we demonstrated that simultaneous knock-in of dual fluorescence reporters through CRISPR/Cas9-induced homology-independent DNA repair permitted one-step generation of cells carrying complete disruption of target genes at multiple alleles. Through knocking-in at coding exons, we generated stable single-cell clones carrying complete disruption of ULK1 gene at all four alleles, lacking intact FAT10 in all three alleles, or devoid of intact CtIP at both alleles. We have confirmed the depletion of ULK1 and FAT10 transcripts as well as corresponding proteins in the obtained cell clones. Moreover, consistent with previous reports, we observed impaired mitophagy in ULK1−/− cells and attenuated cytokine-induced cell death in FAT10−/− clones. However, our analysis showed that single-cell clones carrying complete disruption of CtIP gene at both alleles preserved in-frame aberrant CtIP transcripts and produced proteins. Strikingly, the CtIP-disrupted clones raised through another two distinct targeting strategies also produced varied but in-frame aberrant CtIP transcripts. Sequencing analysis suggested that diverse DNA processing and alternative RNA splicing were involved in generating these in-frame aberrant CtIP transcripts, and some infrequent events were biasedly enriched among the CtIP-disrupted cell clones.ConclusionMultiallelic gene disruption could be readily introduced through CRISPR/Cas9-induced homology-independent knock-in of dual fluorescence reporters followed by direct tracing and cell isolation. Robust cellular mechanisms exist to spare essential genes from loss-of-function modifications, by generating partially functional transcripts through diverse DNA and RNA processing mechanisms.Electronic supplementary materialThe online version of this article (10.1186/s12915-018-0616-2) contains supplementary material, which is available to authorized users.

Gene Knock-Ins in Drosophila Using Homology-Independent Insertion of Universal Donor Plasmids

“…Large DNA elements can also be inserted in target genes without homology arms, known as homology-independent insertion (Cristea et al 2013;Maresca et al 2013;Katic et al 2015;Lackner et al 2015;Schmid-Burgk et al 2016;Suzuki et al 2016;Katoh et al 2017;Kumagai et al 2017;Zhang et al 2018;Gao et al 2019). In this method, simultaneous cutting of a circular donor plasmid and a genomic target site by a nuclease results in integration of linearized insert DNA into the genomic cut site by nonhomologous end joining (NHEJ).…”

“…For example, donor DNA can insert in two directions, insertion/deletion mutations (indels) at the integration site can affect the insert translation frame, the entire plasmid can integrate, and inserts can form concatemers. Nevertheless, for some targeting strategies such as C-terminal protein tagging or gene disruption, homology-independent insertion has been shown to be a fast, simple, and effective alternative to HDR in human cell lines (Cristea et al 2013;Maresca et al 2013;Lackner et al 2015;Schmid-Burgk et al 2016;Katoh et al 2017;Zhang et al 2018), mouse somatic cells (Suzuki et al 2016;Gao et al 2019), zebrafish , Caenorhabditis elegans (Katic et al 2015), and Daphnia (Kumagai et al 2017). However, this method has not yet been utilized in Drosophila.…”

Evolving AAV-delivered therapeutics towards ultimate cures

“…In 2014, Auer et al exploited the NHEJ mechanism to capture large DNA at Cas9-induced DSB sites and established a distinct homology-independent knock-in approach [ 110 ]. In 2016, He et al compared the NHEJ- and HDR-mediated knock-in side-by-side in various human cell types using a promoterless GFP reporter system and found that the homology-independent knock-in via NHEJ repair mechanism showed superior efficiency compared to HDR methods [ 111 , 112 ]. This is consistent with the understanding that HDR is associated with DNA replication, while the NHEJ mechanism adopts a more flexible process that is largely active throughout the cell cycle [ 113 ].…”

The rapidly advancing Class 2 CRISPR‐Cas technologies: A customizable toolbox for molecular manipulations

“…They found that the NHEJ pathway mediated homology‐independent knock‐in at previously unattainable high efficiencies, which was superior to commonly used HDR methods in all human cell lines examined . Subsequently, Zhang et al have applied this strategy to trace and enrich gene disruption in hyperploid somatic cell lines; and Suzuki et al applied this strategy for in vivo transgene integration, which has directed its application for genome editing‐based therapy. It is noteworthy that NHEJ‐mediated knock‐in introduces desired modifications as well as reversely oriented insertions, and it yields indels at integration junctions, which should be taken into consideration to prevent unwanted outcome in subsequent applications.…”

“…Up to now, sgRNA libraries have been constructed for both human and murine models, with target sites not only in the coding but also in the non‐coding regions . Future research might solve the systemic limitations such as restrictions due to PAM requirements, challenges for working with hyperploid cell models to generate loss‐of‐function phenotypes and compensatory effects introduced by survival stress when an essential gene is knocked out …”