Large clumps will reduce transfection efficiency. U6-sgRNA PCR. Similarly to ZFNs and TALENs, Cas9 promotes genome editing by stimulating a DSB at a target genomic locus29,30. 10313-039), DMEM, high glucose, no phenol red (Life Technologies, cat. Biotechnol. Ding, Q. et al. To minimize error in amplifying sgRNAs, it is important to use a high-fidelity polymerase. Replace with normal mTeSR1 medium 24 h after plating. EF0654), Nextera XT index kit (Illumina, cat. Prepare the solution in ddH2O, divide it into aliquots and store them at −70 °C for up to 2 years. & Kucherlapati, R.S. To achieve high HDR efficiencies, ssODNs contain flanking sequences of at least 40 bp on each side that are homologous to the target region, and they can be oriented in either the sense or antisense direction relative to the target locus. Ran, F Ann, Patrick D Hsu, Jason Wright, Vineeta Agarwala, David A Scott, and Feng Zhang. Transform the PlasmidSafe-treated plasmid into a competent E. coli strain, according to the protocol supplied with the cells. Science 322, 1843–1845 (2008). 3). As cell types can vary greatly in their response to FACS, clonal-density dilution or other isolation procedures, literature specific to the cell type of interest should be consulted. Cellular Biol. Generation of FHL2 homozygous knockout lines from human embryonic stem cells by CRISPR/Cas9-mediated ablation. Transfections should be performed as follows: for sgRNAs cloned into pSpCas9(BB), transfect 500 ng of sequence-verified CRISPR plasmid (pSpCas9(sgRNA)); if you are transfecting more than one plasmid (Box 2), mix them at equimolar ratios and use no more han 500 ng of total DNA. (c) Schematic for scarless cloning of the guide sequence oligos into a plasmid containing Cas9 and the sgRNA scaffold (pSpCas9(BB)). Set up the annealing reaction as follows: Anneal the reaction by using the following conditions: SURVEYOR nuclease S digestion. 5c) as described below. Internet Explorer). M0273S), dNTP solution mix, 25 mM each (Enzymatics, cat. Some features of the site may not work correctly. Three types (I–III) of CRISPR systems have been identified across a wide range of bacterial and archaeal hosts, wherein each system comprises a cluster of CRISPR-associated (Cas) genes, noncoding RNAs and a distinctive array of repetitive elements (direct repeats). RNA-programmed genome editing in human cells. et al. Barrangou, R. et al. no. FC-131-1001), Standard microcentrifuge tubes, 1.5 ml (Eppendorf, cat. Add 1 ml of warm D10 medium into each well of a 12-well plate. Sapranauskas, R. et al. For culture of human embryonic stem cells (hESCs), prepare mTeSR1 medium by supplementing it with the supplement solution supplied with the medium and 100 μg ml−1 Normocin. By using a U6 promoter-containing PCR template and a fixed forward primer (U6-Fwd), sgRNA-encoding DNA can be appended onto the U6 reverse primer (U6-Rev) and synthesized as an extended DNA oligo (Ultramer oligos from IDT). no. CRISPR: From Prokaryotic Immune Systems to Plant Genome Editing Tools. Individual clones isolated from cell populations transfected with sgRNA 3, 4 or both are assayed by PCR (using the Out-Fwd and Out-Rev primers), reflecting a deletion of ∼270 bp long. To passage, remove the medium and rinse the cells once by gently adding DPBS to the side of the vessel, so as not to dislodge the cells. no. If you are using other ligases, substitute with the compatible buffer, T4 polynucleotide kinase (New England BioLabs, cat. Targeted mutagenesis in the silkworm Bombyx mori using zinc finger nuclease mRNA injection. Proc. of modified clones)/(no. Rev. These are detected by an RFLP analysis of the PCR amplicon: Run 10 μl of the digested product with loading dye on a 4–20% gradient polyacrylamide TBE gel until the xylene cyanol band has migrated to the bottom of the gel. Targeted nucleases are powerful tools for mediating genome alteration with high precision. HDR efficiency is estimated by using the following formula: (b + c)/(a + b + c), where a is the integrated intensity for the undigested HDR PCR product, and b and c are the integrated intensities for the HindIII-cut fragments.