WEEK 6: Gene Delivery-CRISPR
Pre-lab Question:
1. Please check the Nobel Prize 2020 in Chemistry before coming to the lab.
Introduction
CRISPR (clustered regularly interspaced short palindromic repeat) technique, which is known
as a new and effective gene engineering tool, allows easy modification of the genome sequence.
The presence of CRISPR regions was first discovered in 1987 when the gene for the Apoptosis
inhibitor protein (IAP) gene was studied in Escherichia coli1. However, the main function
emerged in 2007. With this study, it has been proven that CRISPR regions are used as an
immune system to defend genomes of prokaryotes against viruses2. In virus attack, survivor
bacteria arrays integration of a short invader nucleic acid which is called as a spacer into the
CRISPR locus. Protospacer adjacent motif (PAM) which is found in the invasive genome is
required for selection of this spacer3. The spacer which has been the PAM sequence is inserted
into the CRISPR region along with the repeating sequence. The region into which the target
sequence in the Viral DNA is inserted into the CRISPR locus is transcribed to precursor
CRISPR RNAs (pre-crRNA) and the resulting pre-crRNA transcripts are transformed into
small crRNAs by Cas proteins. Invasive nucleic acids are targeted with crRNA and are
degraded by cas nucleases4 (Figure 1a-b). Three types are defined as CRISPR-Cas systems,
CRISPR-Cas-I, II and III5. The CRISPR-Cas Type II system is a mostly studied system and it
is separated from other systems by the ease of development in gene modification. After the
adaptation of Streptococcus pyogenes CRISPR Type II system for genomic editing in 2012,
many genomic editing studies were performed using the CRISPR/Cas9 technique. A single
guide RNA (sgRNA) was generated and the resulting sgRNA/Cas9 enzyme complexes were
targeted to specific genomic regions for genome editing6 (Figure 1c). DSBs which are created
by the CRISPR/Cas9 system are repaired by using homologous recombination and nonhomologous end joining DNA repair mechanisms. While NHEJ involves direct linking of the
broken ends and can cause insertions and deletions, the HDR uses single stranded donor
sequence to repair and HDR introduces precise genome edit7 (Figure 1d).
Figure 1: a) A CRISPR Locus b) Mechanism of CRISPR in Bacteria c) sgRNA/Cas9 enzyme complex
d) DNA repair mechanisms
Figure 2: (A) Shown is the schematic workflow of sgRNA cloning and screening strategy.
Annealed oligos with overhangs and empty sgRNA vector (pRB1017) containing U6 promoter
followed by two BsaI recognition sites and sgRNA scaffold are placed into the cloning reaction
buffer containing BsaI and T4 ligase. pRB1017 includes the kanamycin resistance gene (Kan)
and the forward and reverse sites of universal M13. Following transformation, colony PCR
was performed using the universal M13 forward and reverse sgRNA oligonucleotide with 4 nt
overhang (red). (B) A total of 12 random colonies were chosen, and PCR reactions were
carried out. The control included an empty sgRNA expression vector and a cloned sgRNA
(OK132: sgRNA3) containing different sgRNA sequences. Each sample was placed into
separate wells followed by separation of DNA molecules in 1 % agarose gels. If the 21-nt was
successfully cloned into pRB1017, the predicted band size was 580 bp. A strong single band
near 600 bp was observed for all randomly selected colonies (1-12) while controls (empty
sgRNA and OK132) showed no band around 600 bp, indicating successful cloning of 12
sgRNAs into the plasmid (8).
Figure 2. Generation of a C40H1.3 (an orthologue of human Joubert syndrome associated
CEP104) mutant mediated by CRISPR/Cas9. A) ConVarT search using human CEP104
protein sequence revealed that C. elegans T06D8.2 and C40H1.3 are likely orthologues of
human Joubert syndrome associated CEP104. CEP104 amino sequences from C. elegans
C40H1.3 (NP_499053.1), C. elegans T06D8.2 (NP_496400.3), H. sapiens CEP104
(NP_055519.1),
P
(XP_001084851.1),
(NP_808341.1),
R.
troglodytes
CEP104
B.
CEP104
taurus
norvegicus
(XP_001152852.1),
(XP_002694164.1)
Cep104
(NP_659550.2),
M.
M.
X.
mulatta
CEP104
musculus
Cep104
tropicalis_CEP104
(XP_002937508.2), D. rerio cep104 (XP_005167010.1), D. melanogaster CG10137
(NP_609981.1) were obtained followed by generation of a phylogenetic tree. Shown is the
phylogenetic tree for CEP104, with bootstrap values shown at nodes (high percentage
represents support for them). B) sgRNA sequences (sgRNA1 and sgRNA2) for CRISPR/Cas9
targeting C40H1.3 are displayed. These two sgRNA were cloned into vectors followed by an
introduction into C. elegans. F1 screening was performed with PCRs. Homozygous F2
containing 585 bp deletion (some sequences in the promoter and exon I and some sequences
in Exon II). The tur012 deletion in C40H1.3 likely results in a null allele of C40H1.3 because of
a frameshift. C) PCR amplification products of wild type and C40H1.3(tur012) on a 1%
(weight/vol) DNA agarose gel; lane L: DNA marker (100 bp plus); lane 1: C40H1.3(tur012);
lane 2: wild type D) Shown is the Sanger sequencing of a PCR product from C40H1.3(tur012).
Sanger analysis revealed a 585-bps deletion that included exon I, intron I, and some exon II
sequences, as well as the first 16 base pairs of the C40H1.3 promoter (8).
Material/Method:
sgRNA Cloning Protocol
1. Annealing:
1 ul----Forward Oligo
1 ul----Rev Oligo
2 ul----Ligase Buffer
6 ul----ddH20
2. Cloning:
1 ug—Vector
1:1-5:1---Annealed Oligos
2 ul T4 ligase
0.5 ul BsaI
X ul ddH20
Total: 20 ul
Incubation Conditions:
1 hr 37 0C
5 min 50 0C
20 min 65 0C
Transformation:
Transform 100 ul competent cells with all of the ligation products.
Plate the cells with kan+ plates.
Checking for Insert:
PCR:
10 ng Plasmid
0.5 ul dNTP
0.5 ul M13 fwd primer
0.5 ul sgRNA rev primer
0.125 ul taq pol.
_________________________________________
Total: 25 ul
Post-Lab Questions:
1. Please list 3 different gene editing models and compare them.
2. Please propose a method to treat a genetic disease with the help of CRISPR gene editing
method.
References:
1. Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A. Nucleotide sequence of the
iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli,
and identification of the gene product. J Bacteriol 1987;169(12):5429-33.
2. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, et al. CRISPR
provides
acquired
resistance
against
viruses
in
prokaryotes.
Science
2007;315(5819):170912.
3. H. Deveau, R. Barrangou, J.E. Garneau, J. Labonté, C. Fremaux, P. Boyaval, D.A.
Romero, P. Horvath, S. Moineau Phage response to CRISPR-encoded resistance in
Streptococcus thermophilus J. Bacteriol., 190 (2008), pp. 1390-1400
4. K.A. Datsenko, K. Pougach, A. Tikhonov, B.L. Wanner, K. Severinov, E. Semenova
Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial
immunity system Nat. Commun., 3 (2012), p. 945
5. Jiang, F., Doudna, J.A. (2015). The structural biology of CRISPR-Cas systems. Current
Opinion in Structural Biology, 30, 100- 111
6. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A., Charpentier, E. (2012).
A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.
Science 337 (6096), 816-21.
7. Wilkinson, R., & Wiedenheft, B. (2014). A CRISPR method for genome
engineering. F1000Prime Reports, 6, 3. http://doi.org/10.12703/P6-3
8. Ferhan Yenisert, Merve Gül Turan, Sebiha Cevik, Oktay I. Kaplan, PCR-based, costefficient and fast screening strategy for sgRNA inserts, Rare Disease Laboratory, School
of Life and Natural Sciences, Abdullah Gul University, Kayseri, Turkey.