TITLE: 3.4 Genome Editing with CRISPR/Cas9
AUTHORS: Evelyn J. Sauter, Lisa K. Kutsche, Simon D. Klapper, Volker Busskamp

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is part 3.4 of the "</span><a href="https://www.protocols.io/private/C65DB81838BC11EBA61A0A58A9FEAC2A/protocols" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Induced Neurons for the Study of Neurodegenerative and Neurodevelopmental Disorders</span></a><span>" collection of protocols.</span></div><br/><div class = "text-block"><span style = "font-weight:bold;">Collection Abstract:</span><span> Patient-derived or genomically modified human induced pluripotent stem cell s (iPSCs) offer the opportunity to study neurodevelopmental and neurodegenerative disorders. Overexpression of certain neurogenic transcription factors (TFs) in iPSCs can induce efficient differentiation into homogeneous populations of the disease-relevant neuronal cell types. Here we provide protocols for genomic manipulations of iPSCs by CRISPR/Cas9. We also introduce two methods, based on lentiviral delivery and the piggyBac transposon system, to stably integrate neurogenic TFs into human iPSCs. Furthermore, we describe the TF-mediated neuronal differentiation and maturation in combination with astrocyte cocultures.</span></div></div>

[STEPS]
?. [3.4 Genome Editing with CRISPR/Cas9]
Design a sgRNA for the locus of interest using designing tools (such as http://crispr.mit.edu/, see Note 23) [12].
?. [3.4 Genome Editing with CRISPR/Cas9]
Order two oligos representing the sgRNA with a design as follows: top oligo—CACC(G)[20 N of sgRNA], bottom oligo—AAAC[20 N reverse complement of sgRNA](C) [10] (see Note 24).
?. [3.4 Genome Editing with CRISPR/Cas9]
Anneal the oligos in a thermocycler using , , , T4 Polynucleotide Kinase, and  and program the thermocycler with  at ,  at , a ramp down to  (ramp rate of 5 °C/min) and hold at . The oligo hybrid can directly be used for cloning into the linearized PX459 or PX458 vector.
[top oligo]
[bottom oligo]
[10× T4 Ligation Buffer]
[ddH2O]
37 °C
95 °C
25 °C
4 °C
?. [3.4 Genome Editing with CRISPR/Cas9]
Cut and dephosphorylate the vector using  with  and fill up to 50 μl reaction volume with ddH2O (minimum of 0.5 μl BbsI for 5 μg of vector).
[BbsI]
[vector]
?. [3.4 Genome Editing with CRISPR/Cas9]
Ligate sgRNA insert and vector in a 10 μl reaction with , , , and ddH2O. Incubate the mixture for  at .
[vector]
[oligo hybrid (1:250 diluted)]
[Mighty Mix]
16 °C
?. [3.4 Genome Editing with CRISPR/Cas9]
Transform  into chemically competent bacteria, such as Stbl3. The rest of the reaction can be stored for up to 2 weeks.
[reaction product]
?. [3.4 Genome Editing with CRISPR/Cas9]
Check the construct for correctness using the respective sequencing primer and expand the DNA to a high concentration stock. Optionally: Test the cutting efficiency with a T7 endonuclease assay [13] (see Note 25).
?. [3.4 Genome Editing with CRISPR/Cas9]
If homologous recombination of a disease correction or knockin of a reporter is the aim of the gene editing, a donor construct has to be provided in addition to the Cas9-sgRNA vector(s). The tag or the gene for the knockin should be framed by regions of the locus in approximately the same length as the part that is transferred into the gene (such as ~1000 bp for incorporation of an antibiotic resistance with promoter) and provided as a linearized DNA fragment. Alternatively, 5–25 bp homology can be used with microhomologymediated end-joining [14].
?. [3.4 Genome Editing with CRISPR/Cas9]
Electroporate  encoding for the correct sgRNA and optionally ~ into iPSCs (see materials and steps in protocol 3.2). Seed the cells into two wells of a 6-well plate.
[vector ]
[ donor construct]
?. [3.4 Genome Editing with CRISPR/Cas9]
Using the PX459 vector, it is advisable to use – for – for the next night after electroporation in combination with ROCKi.
[puromycin]
?. [3.4 Genome Editing with CRISPR/Cas9]
Keep ROCKi for 2 days and check if you obtained single cells that grow to small colonies. Once the colonies are visible by eye, check half of a colony for integration or knockout of the target gene by (colony) PCR with primers binding around or in the locus that you tested for specificity before (using control DNA). Transfer the other part of each colony not used for PCR to a new well (of a 48-well plate). These are the monoclonal knockout lines usable if the PCR shows the respective shifts.
?. [3.4 Genome Editing with CRISPR/Cas9]
Once a potentially positive clone has been detected, sequence the locus using Sanger sequencing, optimally with subcloning of the PCR product into a carrier vector (for instance using TOPO cloning).
?. [3.4 Genome Editing with CRISPR/Cas9]
Incubate for  at  and optionally heat-inactivate the digestion for  at .
37 °C
65 °C
?. [3.4 Genome Editing with CRISPR/Cas9]
Gel-purify the vector by running on an agarose gel and extracting the band using a gel extraction kit, such as QIAquick ® Gel Extraction Kit (Qiagen), according to the manufacturer’s instructions. The vector can be stored at  for several months.
-20 °C
?. [3.4 Genome Editing with CRISPR/Cas9]
Thaw the bacteria for ~, add the ligation reaction, stir carefully and incubate  for –. Perform a heat shock for  at  transfer the sample back to ice for –. Add  and shake the bacteria at .
on ice
on ice
42 °C
[S.O.C. medium]
37 33
?. [3.4 Genome Editing with CRISPR/Cas9]
Plate the bacteria on LB-plates with  and incubate  at  and proceed with the plasmid preparation using a Miniprep kit such as the QIAprep® Spin Miniprep Kit (Qiagen) according to the manufacturer’s guidelines.
[ampicillin]
37 °C