Beyond basic CRISPR/Cas9 editing, genome engineering includes pooled functional screening and alternative nucleases for precise sequence modification.
CRISPR Screening
CRISPR screening is a high-throughput functional genomics method that uses pooled lentiviral libraries of single-guide RNAs (sgRNAs) to knock out every gene in the genome and identify those involved in a phenotype of interest.
A typical screen:
- A lentiviral sgRNA library (e.g., Brunello library: 4 sgRNAs per gene, 77,000 sgRNAs total) is transduced into Cas9-expressing cells at a low multiplicity of infection (~0.3) to ensure most cells receive one sgRNA.
- Cells are subjected to a selection pressure: drug treatment, toxin exposure, or a sorting-based phenotype (e.g., a reporter for a signaling pathway).
- Genomic DNA is extracted, and sgRNA sequences are amplified by PCR and quantified by next-generation sequencing.
- sgRNAs depleted or enriched in the treated group compared to the control identify genes essential for survival or resistance.
CRISPR screens are more sensitive and specific than shRNA screens because Cas9 creates complete knockouts and has fewer off-target effects.
TALENs
Transcription activator-like effector nucleases (TALENs) are artificial restriction enzymes made by fusing a TAL effector DNA-binding domain to the nuclease domain of FokI. Each TAL effector repeat recognizes a single DNA base pair, allowing modular assembly of binding domains that target any sequence.
The FokI domain must dimerize, so TALENs are used in pairs flanking the target site. The dimerization creates a double-strand break (DSB) at the target, which is repaired by non-homologous end joining (NHEJ) — producing small insertions or deletions — or by homology-directed repair (HDR) if a repair template is provided.
TALENs are more specific than early CRISPR systems but more laborious to construct because each new target requires assembling a new array of repeats.
Zinc Finger Nucleases (ZFNs)
ZFNs fuse zinc finger domains (each recognizing ~3 bp) to the FokI nuclease domain. Like TALENs, they require paired binding sites and FokI dimerization for activity. ZFNs were the first widely used targeted nucleases but have largely been supplanted by CRISPR and TALENs due to the difficulty of engineering zinc finger arrays with high specificity.
Choosing an Approach
- CRISPR is the simplest and most scalable — suited for genome-wide screens and routine editing.
- TALENs offer lower off-target editing and are preferred when precise single-base recognition is needed or when editing in organisms where CRISPR PAM constraints are limiting.
- ZFNs remain useful in specific contexts such as gene therapy (where smaller size aids viral packaging) and in organisms where the other tools are less developed.
