Lecture 5 and 6 microbial_sem_6_20180307

1. Gene$cally modified
microorganisms
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2. Random
mutagenesis
RNAi mediated
gene knockdown
Integra$on via
homologous
recombina$on
-Low efficiency and
laborious downstream
screening
First genera$on
genome edi$ng tool
-Zinc finger technology
- Transcrip$on-
ac$vator like
effector nucleases
(TALENs)
- Homing
meganucleases
Recent advances enable precision in genome edi$ng
CRISPR-Cas9
technology
Next genera$on tools:
No control, No precision Gain precision and control
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3. CRISPR: Clustered, Regularly Interspaced, Short Palindromic
Repeats and Cas: CRISPR Associated protein
What is CRISPR-Cas ?
• The CRISPR-Cas system is a prokaryoKc immune system that
confers resistance to foreign geneKc elements.
• CRISPRs were first discovered in archaea (and later in bacteria)
by Francisco Mojica, a scienKst at the University of Alicante in
Spain.
• Found in approximately 40% of sequenced bacteria genomes and
90% of sequenced archaea (CRISPR DATABASE, 2010).
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4. Mechanism of CRISPR-cas9 resistance
• Stage 1: CRISPR adap$on
- Foreign DNA is incorporated in the CRISPR array
----------------------------------------------------------------------------------------------------------------------------
• Stage 2: CRISPR expression
- CRISPR RNAs are transcribed from the CRISPR
locus. The tracrRNA is required for crRNA matura$on
from a primary transcript.
----------------------------------------------------------------------------------------------------------------------------
• Stage 3: CRISPR interference
- Foreign nucleic acid complementary to the crRNA
is neutralized
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Figure modified from Horizon Genomics GmbH
tracrRNA
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5. • 1987-These clustered repeats were first discovered in E.coli (Ishino et al., 1987).
• 2000- Similar clustered repeats were iden$fied in addi$onal bacteria and archaea and
were termed Short Regularly Spaced Repeats (SRSR) (Mojica et al., 2000).
• 2002- SRSR were renamed as CRISPR. Also a set of genes, called CRISPR-
associated genes were iden$fied (Jansen et al., 2002).
• 2007- Barrangou and colleagues (2007), demonstrated that S. thermophilus can
acquire resistance against a bacteriophage by integra$ng a genome fragment of an
infec$ous virus into its CRISPR locus.
• 2012- The poten$al of CRISPR-CAs9 as genome edi$ng tool was first revealed by Jinek
et al.
• It has since been used in a wide range of organisms including baker's yeast (S.
cerevisiae), zebra fish (D. rerio), flies (D. melanogaster), nematodes (C.
elegans), plants, mice, and several other organisms.
History of CRISPR-CAs9 as genome edi$ng tool
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6. GeneCopoeia, Inc
Any DNA sequence with the correct target sequence followed by the PAM sequence will
be bound by Cas9.
Cas9
PAM:
Protospacer
adjacent
moKf
guide RNA, a fusion of the crRNA and tracrRNA(trans-
acKvaKng crRNA) (Jinek et al, 2012)
Components of CRISPR-CAs9
SV40
nuclear
localizaKon
signal
5´ 3´
3´ 5´
5´ 3´
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7. Components of CRISPR-CAS9
guide RNA, a fusion of the crRNA
and tracrRNA (Jinek et al., 2012)
gRNA:
crRNA:
the endogenous bacterial RNA that
confers target specificity, requires
tracrRNA to bind to Cas9.
tracrRNA:
the endogenous bacterial RNA that links
the crRNA to the Cas9 nuclease, can bind
any crRNA.
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8. Components of CRISPR-CAS9
guide RNA, a fusion of the crRNA
and tracrRNA (Jinek et al., 2012)
gRNA:
• Jinek et al., 2012, combined tracrRNA and crRNA
into a single guide RNA (gRNA).
• gRNA programmed Cas9 was shown to be as
effecKve as Cas9 programmed with separate
tracrRNA and crRNA in guiding targeted gene
alteraKons.
• Components (gRNA, Cas9) derived from different
bacteria will not funcKon together.
• The AT content of Cas9 target sequences should
preferably be above 65% (Lin et al., 2014).
• Extension of guide sequence does not improve
Cas9 targeKng specificity (Ran et al., 2013)
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9. 9
Cas9
Components of CRISPR-CAs9
RuvC
HNH:
Cas9 domains
• Resembles to specific resolvase , which
cleaves the holiday juncKon during geneKc
recombinaKon.
• RuvC domain cleaves the non-complementary
strand
• Two pairs of conserved hisKdines
flanking a conserved asparagine
• The HNH domain cleaves the
complementary strand
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10. PAM:
Protospacer
adjacent moKf
Components of CRISPR-CAS9
Species PAM Sequence
Streptococcus pyogenes (SP) NGG
Neisseria meningi7dis (NM) NNNNGATT
Streptococcus thermophilus (ST) NNAGAA
Treponema den7cola (TD) NAAAAC
10
PAM: Protospacer adjacent mo$fs
• A short conserved sequence, (2–5 nts) needed
for double-stranded endonuclease ac$vity of
Cas9.
• Even fully complementary sequences are
ignored by Cas9-RNA in the absence of a PAM
sequence (Sternberg et al, 2014).
• The PAM sequence varies by the species of the
bacteria from which the Cas9 was derived.
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11. The resul$ng DSB is then repaired by one of two general repair
pathways:
• The efficient but error-prone non-homologous end joining
(NHEJ) pathway
• The less efficient but high-fidelity homology directed repair
(HDR) pathway
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12. Nickase system:
• The Cas9 nuclease is targeted to specific genomic loci by a 20-nt guide sequence,
which can tolerate certain mismatches to the DNA target and thereby promote
undesired off-target mutagenesis.
• Cong and colleagues (2013) created a mutated version of Cas9 known as
Cas9D10A, with only nickase acKvity.
• Paired nicking can be used to reduce off-target acKvity by 50–1,000 fold (Ran et al.,
2013)
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13. 13
Cas9-D10A Cas9 wild type MutaKon responsible for RuvC domain inacKvaKon
MutaKon responsible for HNH domain inacKvaKon
Taken from NEB

14. 14
Yeastric$on webtool
• Contains the genome and ORF sequences were downloaded from SGD
in GFF and FASTA file format.
• Contains set of 33 S. cerevisiae genomes.
• Extracts all possible Cas9 target sequences (20 base pairs followed by NGG).
• YeastricKon ranks potenKal Cas9 target sequences according to AT-content,
secondary structures and the presence of restricKon sites.
• A recent report indicates that the AT content of Cas9 target sequences should
preferably be above 65% (Lin et al., 2014).
• Sequences containing 6 or more consecKve Ts are discarded as this can terminate
transcripKon (Braglia et al., 2005; Wang et al., 2008).
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15. 15
OR
Donor DNA
TransformaKon
Mul$plex Genome Engineering with CRISPR
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16. 16
Cas9 vs. Cpf1
• Recognizes different PAMs, enabling new
targeKng possibiliKes.
• Creates 4-5 nt long sKcky ends, instead of blunt
ends produced by Cas9, enhancing the
efficiency of geneKc inserKons and specificity
during NHEJ or HDR.
• Cuts target DNA further away from PAM,
further away from the Cas9 culng site,
enabling new possibiliKes for cleaving the DNA.
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17. 17
Cas9 vs. Cpf1
Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. 2015.
Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS,
Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, Koonin EV, Zhang F. Cell.
163(3):759-71. PMID: 26422227
Engineered Cpf1 variants with altered PAM specificiKes. 2017. Gao L, Cox DBT,
Yan WX, Manteiga JC, Schneider MW, Yamano T, Nishimasu H, Nureki O,
Crosepo N, Zhang F. Nat Biotechnol. 35(8):789-792. PMID: 28581492
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