Table 2 Comparison of genome-wide TMIM, CRISPR/Cas9 and shRNA/cDNA expression technologies
From: The utility of transposon mutagenesis for cancer studies in the era of genome editing
Feature | TMIM | CRISPR/Cas9 | shRNA and cDNA libraries |
|---|---|---|---|
Screen set-up | Two-component system (transposase and transposon) | Comprehensive delivery of libraries can be technically challenging | Comprehensive delivery of libraries can be technically challenging |
Possible types of mutation | Activating and disruptive mutations possible owing to transposon insertion or remobilization | Disruptive mutations (knockout libraries) | Knockdown or overexpression — potentially reversible |
Chromosomal deletions and translocations are possible (knockout libraries) | |||
Chromosomal deletions and rearrangements are rare | |||
Mutations either can (transcription repression [76, 77] and activation [79, 80] libraries) or cannot be reversed (knockout libraries) | |||
Mutations can be reversed following transposon remobilization | |||
Mutagenesis efficiency | Biallelic gene inactivation rare in diploid cells | Biallelic mutation achievable with knockout libraries [88] and 90–99 % knockdown efficiency achievable with repression libraries [89] | ≥70 % gene knockdown with validated shRNA clones [82–84] |
>2 standard deviation overexpression by 90Â % of cDNA expression vectors [90] | |||
Undesired and off-target effects | Local hopping effects [31], passenger insertions | ||
Genome coverage | Whole genome in principle, but affected by integration-site preferences, local hopping and chromatin accessibility | Dictated by library design | Dictated by library design |
Knockout libraries | ~8000 human, ~15,000 mouse genes (NKI shRNA library) [85]; >20,000 human and mouse genes (TRC shRNA library) [82, 83]; ~60,000 human and mouse genes (Hannon–Elledge shRNA library) [84]; >17,000 human genes (cDNA expression library) [90] | ||
GeCKOv2 [86]: ~20,000 genes, 1000–2000 miRNAs for human and mouse. Koike-Yusa et al . [87]: ~20,000 mouse genes. Wang et al . [88]: ~7000 human genes | |||
CRISPR-activation libraries | |||
CRISPRa [89]: ~16,000 human genes. SAM [78]: all human RefSeq genes | |||
CRISPR-inhibition library [89] | |||
~16,000 human genes |