Genome engineering using the CRISPR-Cas9 system

Abstract

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.

ERAD‐dependent control of the Wnt secretory factor Evi

“…CRISPR/Cas9 knockout cell lines were generated according to protocols described before in Ran et al (2013). In brief, 20‐nt single‐guide (sg) RNAs were designed using E‐CRISP (Heigwer et al , 2014).…”

“…Cells were harvested 48 h after transfection. The following expression constructs were used: pSpCas9(BB)‐2A‐Puro (px459; Addgene #48139; Ran et al , 2013), psPAX2 (Addgene #12260; D. Trono), pMD2.G‐VSVG (Addgene #12259; D. Trono), TCF4/Wnt‐Firefly Luciferase (Demir et al , 2013), Actin‐Renilla Luciferase (Nickles et al , 2012), pcDNA3‐Wnt11 (Addgene #35922; Najdi et al , 2012), pcDNA3‐Wnt10B, (Addgene #35921; Najdi et al , 2012), pcDNA3‐Wnt16 (Addgene #35923; Najdi et al , 2012), pcDNA3‐Wnt7B (Addgene #35915; Najdi et al , 2012), pcDNA3‐Wnt8 (Addgene #35916; Najdi et al , 2012), pcDNA3‐Wnt5A (Addgene #35911; Najdi et al , 2012), pcDNA3‐Wnt3A (Addgene #35908; Najdi et al , 2012), pcDNA3‐Wnt3 (Addgene #35909; Najdi et al , 2012), pcDNA3.2‐Wnt3A‐V5 (Addgene #43810; MacDonald et al , 2014), pcDNA3‐IGFBP5‐V5 (Addgene #11608; S. Johnson), pcDNA3‐EGF‐Myc (Addgene #11601; Nguyen et al , 2000), VCP(wt)‐EGFP (Addgene #23971; Tresse et al , 2010), VCP(DKO)‐EGFP (Addgene #23974; Tresse et al , 2010), pCMV6‐Myc‐DDK‐tagged PORCN (Origene # RC223764), sLuc (Katanaev et al , 2008), pCS2(+)‐mWnt3A (C. Niehrs), pCS2(+)‐Dvl3 (C. Niehrs), pCS2(+)‐mWnt3A‐S209A (Kumar et al , 2014), and β‐Catenin‐YFP (Stemmer et al , 2008). To generate C‐terminally FLAG‐HA‐tagged CGRRF1 and Hrd1, the FLAG‐HA sequence was inserted into the pcDNA5‐FRT/TO vector (Invitrogen).…”

Continuous signaling of CD 79b and CD 19 is required for the fitness of Burkitt lymphoma B cells

“…The sgRNAs targeting genes of mIg H chain, L chain, and CD19 were inserted into pSpCas9(BB)‐2A‐GFP (PX458, Addgene ID # 48138) following the Ran's protocol (Ran et al , 2013). The open reading frame of the Igβ and CD19 was cloned from Ramos cDNA and inserted into the pJET vector by CloneJET PCR Cloning Kit (Thermo Scientific).…”

Chromosome engineering in zygotes with CRISPR/Cas9

“…CRISPR/Cas9 target sites were identified using http://crispr.mit.edu/as described (Ran et al ., 2013). In addition the guide RNAs were picked following the guidelines from Doench et al .…”