The mainstream genome editing tools include clustered regularly interspaced short palindromic repeat (CRISPR)/CAS9 RNA-guided nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and helper dependent adenoviral vectors (HDAdVs). The process and protocols of mainstream genome editing methods from literature:
1). the CRISPR-Cas9 system:
– Genome engineering of mammalian haploid embryonic stem cells using the Cas9/RNA system.
The stem stable cell line development method uses the CRISPR system in combination with haploid embryonic stem cells (ESCs) to manipulate the mammalian genome.
– CasOT: a genome-wide Cas9/gRNA off-target searching tool.
A local tool designed to find potential off-target sites.
– Cas9 effector-mediated regulation of transcription and differentiation in human pluripotent stem cells.
A method to influence the differentiation state of human pluripotent stem cells (hPSCs) with a CRISPR-associated catalytically inactive dCas9 fused to an effector domain.
– Genome-scale CRISPR-Cas9 knockout screening in human cells.
Lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells.
– Genetic screens in human cells using the CRISPR-Cas9 system.
A pooled, loss-of-function genetic screening approach suitable for both positive and negative selection that uses a genome-scale lentiviral single-guide RNA (sgRNA) library containing 73,000 sgRNAs to generate knockout collections and performed screens in two human cell lines.
– Genome engineering using the CRISPR-Cas9 system.
A set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies. It takes 1-2 weeks to do target design and gene modifications, and 2-3 weeks to obtain modified clonal cell lines.
– CRISPR Genome Engineering Resources
Comprehensive CRISPR genome engineering resources organized by Zhang’s lab.
– Cas9 as a versatile tool for engineering biology.
Advances of the Cas9 targeting methodology, and potential applications ranging from basic science to the clinic.
2). zinc finger nucleases (ZFNs):
3). transcription activator-like effector nucleases (TALENs):
4). helper dependent adenoviral vectors (HDAdVs):
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