2014-Feng ZHANG-Book_Methods_Cas9
C hapter 17
R NA-Guided Genome Editing of Mammalian Cells
N eena K.P yzo cha,F.A nn R an,P atrick D.H su,and F eng Z hang
A bstract
T he microbial CRI SPR-Cas adaptive immune system can be harnessed to facilitate genome editing in eukaryotic cells (Cong L et al., Science 339, 819–823, 2013; Mali P et al., Science 339, 823–826, 2013). Here we describe a protocol for the use of the RNA-guided Cas9 nuclease from the S treptococcus pyogenes type II CRISPR system to achieve speci? c, scalable, and cost-ef? cient genome editing in mammalian cells.
K ey words C RISPR-Cas G enome editing D NA cleavage C as9 G uide RNA P AM sequence N HEJ
G ene knockout
1I ntro ductio n
T he ability to introduce targeted modi? cations into genomes and
engineer model organisms holds enormous promise for biomedical
and biotechnological applications. The development of program-
mable nucleases [ 1–9] has allowed targeting of speci? c genomic
loci to introduce double-strand breaks (DSBs) in the DNA. These
DSBs are subsequently repaired through either the error-prone
nonhomologous end-joining (NHEJ) pathway or the homology-
directed repair (HDR) pathway, allowing formation of indels or
precise editing of the genome, respectively [ 10]. These endonucle-
ases can be used for studies in basic biology, biotechnology, and
medicine, including the development of reporter cell lines [ 11],
transgenic organisms [ 12], disease models [ 13], and gene therapy
[ 14], among others. Although ZFNs and TALENs can be repro-
grammed to target speci? c DNA sequences, these tools still require
time-consuming engineering of proteins de novo for each target,
and there remains a de? cit for technologies that are easily custom-
izable, multiplexable, and affordable.
Francesca Storici (ed.), Gene Correction: Methods and Protocols, Methods in Molecular Biology, vol. 1114,
DOI 10.1007/978-1-62703-761-7_17, ? Springer Science+Business Media, LLC 2014
269
270 T
he microbial adaptive immune system CRI SPR (Clustered Regularly Interspaced Short Palindromic Repeats) consists of a set
of enzymes and noncoding RNA elements [
15 – 17 ]. Among the three types of CRISPR systems in bacteria and archaea [
15 , 16 ], type I I requires only a single protein, Cas9 (formerly Csn1), to
mediate DNA cleavage [
18 ]. Cas9 is targeted to speci? c DNA sequences by a pair of noncoding RNA elements: the CRI SPR
RNA (crRNA), which carries the target-specifying guide sequence
via Watson–Crick base pairing (Fig.
1 ), and the trans-activating crRNA (tracrRNA), which hybridizes with crRNA and is required
for loading onto Cas9 [
19 , 20 ]. T he type I I CRI SPR system of S treptococcus pyogenes can be
reconstituted in mammalian cells to mediate DNA cleavage with
three minimal components: Cas9, crRNA, and tracrRNA. The lat-
ter two components can further be truncated and fused into a single
chimeric guide RNA scaffold (Fig.
1 ) [ 18 ] for a target sequence selected from any genomic locus with its 3’ end followed by a NGG
trinucleotide motif [
19 ]. This protospacer-adjacent motif (PAM) is speci? c to each CRISPR system [
21 ]. Generation of speci? c guide RNAs for targeted genome editing only requires the purchase of
two short oligos and simple cloning that can take as little as two days.
T he wild-type S . pyogenes Cas9 (SpCas9) enzyme has multiple
endonuclease domains, two of which cleave DNA in a strand-
speci? c manner. Two catalytic residues, D10 or H840 [
18 ], can be mutated to convert the wild-type SpCas9 into a DNA-nicking
enzyme (SpCas9n) [ 1 , 18 ]. Given that single-stranded nicks in the F ig.1 T argeted DNA cleavage by SpCas9 in the human E MX1 locus. The SpCas9 enzyme ( y ellow ) interacts with its genomic target ( b lue ) with the help of a guide RNA. The genomic target is directly 5′ to the PAM sequence, which is -NGG- for SpCas9. The guide RNA is composed of the guide sequence ( b lue ), which anneals with the ge nomic targe t via Watson–Crick base pairing and a chime ric guide RNA scaffold consisting of a fusion between the crRNA ( g ray ) and the tracrRNA ( r ed )
Neena K. Pyzocha et al.
271
target DNA can also stimulate HDR, SpCas9n reduces the
l ikelihood of error-prone repair by NHEJ. Furthermore, both catalytic domains of SpCas9 can be mutated to convert SpCas9
into a RNA- g uided DNA-binding protein [
18 , 22 ]. This chapter describes a set of protocols for using the SpCas9 system for genome
editing in mammalian cells.
2M aterials
1. C loning plasmids: p X330 (CBh::SpCas9 + U6::chimeric guide
RNA) (Addgene) or p X335 (CBh::SpCas9n (D10A) + U6::
chimeric guide RNA) (Addgene) (Fig.
2 ). 2. O ligos for target sequence. S ee Subheading 3.1 for discussion
regarding locus selection and Subheading
3.2 on oligo design (Integrated DNA Technologies).
3. R estriction enzymes and phosphatase: FastDigest B bs I
(Fermentas), FastAP (Fermentas), 10× FastDigest Buffer
(Fermentas) ( s ee N ote 1 ).
4. Q IAquick Gel Extraction Kit (QIAGEN).
5. P hosphorylation, annealing, and ligation reagents: 10× T4
Ligation Buffer (NEB), T4 Polynucleotide Kinase (NEB), 2×
Quick Ligation Reaction Buffer (NEB), Quick Ligase (NEB).
6. P lasmid-Safe exonuclease (Epicentre Biotechnologies).
7. C ompetent cells and bacterial growth reagents. 2.1 M olecular Cloning Components F ig. 2 B icistronic expression vector for guide RNA and SpCas9 (or SpCas9n). A genomic target directly upstream to the PAM sequence can be cloned into the expression vector. After a target is selected, two DNA oligos can be designed based on the schematic showing the guide sequence insert. One oligo ( t op strand, written 5′–3′) contains ligation adapter sequences for cloning into the expression vector and G(N) 19
, which is the selected genomic target sequence. The other oligo ( b ottom strand, written 3′–5′) also contains ligation adapter sequences for cloning into the expression vector and the complementary bases to the genomic target sequence. Once annealed and phosphorylated, the oligos can be inserted into the vector digested with B bsI
RNA-Guided Genome Editing of Mammalian Cells
272 8. Q IAGEN Plasmid Midi Kit (QIAGEN).
9. S tandard gel electrophoresis reagents.
1. C ell line: For validation, human embryonic kidney (HEK) cell line 293FT (Life Technologies). For additional discussions on working with other cell lines, s ee N ote 2 .
2. C ell culture reagents for maintenance of 293FT cells:
Dulbecco’s Modi? ed Eagle’s Medium (DMEM) (Life
Technologies), 10 % fetal bovine serum (HyClone), 2 mM
GlutaMAX (Life Technologies), 100 U/mL penicillin, and
100 μg/mL streptomycin.
3. D issociation reagent: TrypLE? (Life Technologies).
4. T ransfection reagent: Lipofectamine 2000 (Life Technologies)
for HEK293FT or Neuro-2a cells (Sigma Aldrich) ( s ee N ote 3 ).
5. 24-well tissue culture plates (Corning).
6. T ransfection Control Plasmid: pMaxGFP (Lonza).
7. Q uickExtract? DNA extraction kit (Epicentre
Biotechnologies).
1. S URVEYOR Mutation Detection Kit (Transgenomic).
2. 4–20 % Novex TBE polyacrylamide gels (Life Technologies).
3. A mpli? cation primers speci? c to the targeted locus (Integrated
DNA Technologies).
4. H erculase II High Fidelity Polymerase (Agilent).
3M etho ds
F or use with the SpCas9 system, target sites must be followed by a NG
G trinucleotide motif on the 3′ end ( s ee N otes 4 and 5 ).
W e designed cloning vectors (pX330 for SpCas9 or pX335 for SpCas9n, a D10A nickase) to aid co-expression of SpCas9 and
guide RNA in mammalian cells (Fig.
2 ). In this vector, SpCas9 is driven by the CBh promoter [
23 ], and the guide RNA is driven by the human PolIII promoter U6. Phosphorylated and annealed oli-
gos (design indicated in Fig.
2 ) can be cloned into the B bsI digested plasmid containing the entire guide RNA scaffold. The oligos are
designed based on the target site sequence (20 bp sequence cor-
responding to the target site). The G(N) 19 refers to the sequence
selected upstream of the PAM sequence in the genomic DNA
( s ee N ote 6 ). Create oligos using the schematic in Fig. 2 . 2.2 T issue Culture,
Transfection, and DNA
Extraction
Components 2.3 C omponents for the Analysis of Genome Modi? cation 3.1 T arget Selection 3.2 C onstruct Design
Neena K. Pyzocha et al.
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1. D igest 1 μg of pX330 or pX335 with B bs I
for 30 min at 37 °C: 2. G el purify digested pX330 or pX335 using QI Aquick Gel
Extraction Kit and elute in EB.
3. P
hosphorylate and anneal each pair of oligos for the insert piece:
A
nneal in a thermocycler using the following parameters: 4. S et up ligation reaction and the negative control. Incubate at
room temperature for 10 min:
3.3 M olecular
Cloning: Oligo
Annealing and Cloning
into Backbone Vectors RNA-Guided Genome Editing of Mammalian Cells
274 5. (Optional but highly recommended) Treat ligation reaction with Plasmid-Safe exonuclease ( s ee N ote 7 )
: I ncubate reaction at 37 °C for 30 min. 6. T ransform 2 μL of reaction from s tep 5 into competent cells and plate on ampicillin selection plates. 7. P ick two colonies the following day and analyze for correct insertion of the target sequence oligos. T he CRISPR-Cas DNA cleavage system has been validated for use in a variety of mammalian cell lines [ 1 , 2 , 24 ] ( s ee N ote 2 ). The protocol below is for HEK239FT cells. 1. H EK293FT cells are maintained in DMEM supplemented with 10 % fetal bovine serum and passaged before reaching 70 % con? uency. Cells are maintained in an incubator set at 37 °C supplemented with 5 % CO 2 . 2. H EK293FT cells can be transfected using Lipofectamine 2000 according to the manufacturer’s protocol. 3. F or each well of a 24-well plate, a total of 500 ng of plasmid is transfected. One well should be a control to see the relative transfection ef? ciency using a plasmid such as pmaxGFP . 4. A fter 12 h of transfection, replace the medium with pre-warmed maintenance medium. After 72 h, genomic DNA can be iso-lated using the QuickExtract DNA extraction kit following the manufacturer’s protocol. Brie? y, cells are resuspended in QuickExtract solution (50 μL per 24 well) and incubated at 65 °C for 15 min followed by 98 °C for 10 min. 1. T he ef? ciency of cleavage can be detected by assessing the per-centage of cells containing indels in the target region ( s ee N ote 8 ). In order to detect indels in the DNA, follow the instructions provided in the SURVEYOR Mutation Detection Kit manual
( s ee N ote 9 ).
2. I t is recommended that the SURVEYOR Nuclease digestion
products are analyzed on a PAGE gel.
3.4 C ell Culture and
Transfection
3.5 A nalysis of
Genomic Modi? cation:
SURVEYOR and
Sequencing Neena K. Pyzocha et al.
275
3. T o calculate the percent cutting ef? ciency of a CRISPR locus,
use the following formula: %indel =-?è????÷÷1100 where a and b refer to the relative concentrations of the cut
bands and c equals the relative concentration of the full-length
PCR template. A representative SURVEYOR gel image and
quantitation is shown in Fig.
3 .4N o tes
1. C onventional restriction enzymes can be substituted for FastDigest
restriction enzymes. I n this case, adjust digestion reagents and
digestion times according to manufacturer’s protocol.
2. E xperimental conditions may need to be optimized for each
cell line. F ig.3 S URVEYOR assay comparing SpCas9-mediated DNA cleavage at two different targets in the same gene. ( a ) The third exon of the human E MX1 locus was targeted using guide RNAs at two unique sites. ( b )A re pre -sentative SURVEYOR assay gel image comparing the targeted cleavage ef? ciency by SpCas9 at the two targets in the human E MX1locus
a
b
RNA-Guided Genome Editing of Mammalian Cells
276
3. F or other cell lines, we suggest doing an initial comparison of
different transfection reagents (e.g., FuGENE HD, nucleofec-
tion, and TransIT).
4. A free computational resource maintained by the Zhang lab
(https://www.wendangku.net/doc/f310916832.html,) contains the most
up-to- d ate information relevant for Cas9 systems.
5. I t is ideal for these targets to be unique within the genome. We
also recommend testing multiple target sites for each gene and
selecting the most effective target.
6. S electing a target site with a 5′ G allows for ef? cient transcrip-
tion of the guide RNA from the U6 promoter.
7. P lasmid-Safe treatment is recommended because it degrades
linear dsDNA, helping to prevent unwanted recombination
products.
8. S pCas9-induced double-strand breaks in the target DNA are
usually repaired through the error-prone NHEJ process in
HEK293FT cells.
9. I t is important to make sure that the genomic PCR primers
yield a single amplicon for reliable quanti? cation of the percent
cutting ef? ciency. In the case that primers do not yield a single
amplicon, the PCR product needs to be gel puri?ed or new
primers should be designed.
A ckno wledgments
W e thank Randall Platt for comments and members of the Zhang
Lab for discussion, support, and advice. N.P. is supported by the
National Science Foundation Graduate Research Fellowship,
Primary Award #1122374. P.D.H. is a James Mills Pierce Fellow.
F.Z. is supported by the N H Transformative R01 Award
(R01-NS073124); the N I H Director’s Pioneer Award
(DP1-MH100706); the Keck, McKnight, Gates, Damon Runyon,
Searle Scholars, Merkin, Klingenstein, and Simons Foundations;
Bob Metcalfe; Mike Boylan; and Jane Pauley. Sequence and reagent
information are available through
h ttp://www.genome-
https://www.wendangku.net/doc/f310916832.html, .
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RNA-Guided Genome Editing of Mammalian Cells