Genome engineering using the CRISPR-Cas9 system

By F. Ran, P. Hsu, Jason B Wright, Vineeta Agarwala, David A. Scott, Feng Zhang. Nature Protocols, 2013

7,437 citations recorded by the source corpus.

Targeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA. Here we describe 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. To minimize off-target cleavage, we further describe a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. This protocol provides experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1–2 weeks, and modified clonal cell lines can be derived within 2–3 weeks.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/86d0392dad4d83e0a044c17dbe0872fdb13221c6

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Citations in the corpus

Inference of CRISPR Edits from Sanger Trace Data (2019, neutral): “1,2 After a DSB occurs, non-homologous end joining (NHEJ) and/ or microhomology-mediated end joining (MMEJ) can introduce insertions or deletions (indels) at the DSB. 3,4 Alternatively, if a DNA donor template is provided, knocking in a sequence of interest is possible through homology-directed…”
Suppression of Exosomal PD-L1 Induces Systemic Anti-tumor Immunity and Memory (2019, methodology): “CRISPR–Cas9-mediated gene disruption—sgRNA oligonucleotides (ELIMBIO), were cloned into pSpCas9(BB)-2A-GFP (PX458, ADDGENE) according to the published protocol (Ran et al., 2013).”
Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery. (2019, positive): “130 ,138 Moreover, the ability to modify mRNA sequence to encode regulatory elements (e.g., K-turn motifs and microRNA [miRNA]- binding sites) could provide the means to control expression of gene-editing tools in a cell-speci ﬁ c manner.”
Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects (2020, background): “With the CRISPR/ Cas9 system, through pathways of NHEJ or high-fidelity HDR, targeted genomic modifications, including the introduction of small insertions and deletions (indels), can be made.(53) Known as the RNA‐guided system, CRISPR/Cas9 is more suitable for application compared to other gene edi”
Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018, background): “Meanwhile, different cell types or targeted genes can also affect the efficiency of the CRISPR/Cas9 system [93, 199].”