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Lecture 5 and 6 microbial_sem_6_20180307
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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). Microbial Biotechnology|Biotech-552|Dr. Zia|Lec 3
- 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 ---------------------------------------------------------------------------------------------------------------------- Figure modified from Horizon Genomics GmbH tracrRNA 4 Microbial Biotechnology|Biotech-552|Dr. Zia|Lec 3
- 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 Microbial Biotechnology|Biotech-552|Dr. Zia|Lec 3
- 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´ Microbial Biotechnology|Biotech-552|Dr. Zia|Lec 3
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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) Microbial Biotechnology|Biotech-552|Dr. Zia|Lec 3
- 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 Microbial Biotechnology|Biotech-552|Dr. Zia|Lec 3
- 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. Microbial Biotechnology|Biotech-552|Dr. Zia|Lec 3
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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) 12 (Ran et al., 2013) Microbial Biotechnology|Biotech-552|Dr. Zia|Lec 3
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- 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). Microbial Biotechnology|Biotech-552|Dr. Zia|Lec 3
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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. Microbial Biotechnology|Biotech-552|Dr. Zia|Lec 3
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