CRISPR cas : Presented By: Pooya Rashvand Advised By: Dr. M.Aslanimehr

Transcription

1 Journal Club & MSc Seminar CRISPR cas : Presented By: Pooya Rashvand Advised By: Dr. M.Aslanimehr

2 CRISPR - cas : A New tool for Genetic Manipulations from Bacterial Immunity Systems Viral SS DNA RNA Guide CRISPR Cascade subunit

3 Content 1. Introduction 2. History 3. Components of CRISPR 4. Different Cas proteins and their function 5. Types of CRISPR cas system 6. Variants of Cas9 7. Targeted Genome editing in Mammalian cells 8. Applications in Microbiology 9. Conclusion

4 CRISPR C: Clustered R: regularly I: interspaced S: short P: palindromic R: repeat

5 CRISPR CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are DNA loci containing short repetitions of base sequences which separated by short "spacer DNA" from previous exposures to a virus or plasmid. It represents a family of DNA repeats in most archaeal (~90%) and bacterial (~40%) genomes provides acquired immunity against viruses and phages.

6 The size of CRISPR repeats and spacers varies between 23 to 47 base pairs (bp) and 21 to 72 bp, respectively. The number of spacers within a CRISPR locus vary widely ; from a few to several hundreds.

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8 HISTORY Researchers find CRISPR sequences in Escherichia coli, but do not characterize their function. CRISPR sequence are found to be common in other microbes Coined CRISPR name, defined signature Cas genes 2007 First experimental evidence for CRISPR adaptive immunity 2013 First demonstration of Cas9 genome engineering in eukaryotic cell

9 A 2010 study provided direct evidence that CRISPR-Cas cuts both strands of phage and plasmid DNA in S. thermophiles. Jennifer Doudna and Emmanuelle Charpentier ( ) engineered Cas9 endonuclease and "single-guide RNA". Jennifer Doudna Emmanuelle Charpentier

10 Components of CRISPR 1. Protospacer adjacent motif (PAM) 2. CRISPR-RNA (crrna) 3. trans-activating crrna (tracrrna) 4. Cas proteins

11 Stage 1: CRISPR spacer adaptation

12 Stage 2: CRISPR expression

13 Stage 3: interference Interference system rely on : -association of Cas protein with grnas and making ribonucleoprotein Complex (RNP)

14 Stage 3: interference RNP Complex

15 Stage 3: interference Interference system rely on : - PAM: protospacer adjacent motifs

16 Stage 3: interference 5'-NGG-3'

17 Stage 3: interference -Cas is an endonuclease enzyme which have two cleavage domain (DSB) - Viral target DNA will cleaving at 3 bp on PAM upstream - Multiple viral spacer acquisition = Multiple cleavage

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19 Different Cas proteins and their function Protein Distribution Process Function Cas1 public Spacer acquisition Cas2 public Spacer acquisition Cas3 Type I Target signature interference DNAse, not sequence specfic, can bind RNA; present in all Types specific to U-rich regions; present in all Types DNA helicase, endonuclease Cas4 Type I, II Spacer acquisition Cas5 Type I crrna expression RecB-like nuclease with exonuclease activity homologous to RecB RAMP protein, endoribonuclease involved in crrna biogenesis; part of CASCADE

20 Cas6 Type I, III crrna expression Cas7 Type I crrna expression Cas8 Type I crrna expression Cas9 Cas10 Type II signature Type III signature Target interference crrna expression and interference RAMP protein, endoribonuclease involved in crrna biogenesis; part of CASCADE RAMP protein, endoribonuclease involved in crrna biogenesis; part of CASCADE Large protein with McrA/HNH-nuclease domain and RuvC-like nuclease; part of CASCADE Large multidomain protein with McrA-HNH nuclease domain and RuvC-like nuclease domain; necessary for interference and target cleavage HD nuclease domain, palm domain, Zn ribbon; some homologies with CASCADE elements

21 Types of CRISPR CAS system There are three types of CRISPR-Cas systems, which vary in their specific target and mechanism of action. Type I systems cleave and degrade DNA, Type II systems cleave DNA, Type III systems cleave DNA or RNA.

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23 Type 1 CRISPR-Cas Systems Includes CRISPR associated complex for antiviral defense (cascade) and cas3 Cas3 = DNA helicase, endonuclease Six subtype (I-A / I-F)

24 Efficient immunity requires interaction between the first eight nucleotides of the target and the complementary sequence of the crrna guide. A second requirement for type I immunity is the presence the protospaceradjacent motif (PAM).

25 Type II CRISPR-Cas Systems defined by presence of the RNA-guided endonuclease Cas9 type II CRISPR loci produce a small trans-encoded crrna (tracrrna) with a region of complementarity to the repeat sequence grna = crrna + tracrrna

26 The Cas9 nuclease binds to tracrrna

27 The enzyme has two nuclease domains HNH and RuvC - cuts one target DNA strand. The tracrrna - cofactor of Cas9 - DNA cleavage Ruvc HNH

28 Type III CRISPR-Cas Systems Characterised by the presence of genes encoding Cas10 and repeatassociated mysterious protein (RAMP) modules Csm for type III-A and cmr for type III-B. 7

29 Type III CRISPR-Cas Systems unique feature of type III CRISPR-Cas systems : (a) both DNA and RNA targets are cleaved.

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31 CRISPR/Cas 9 engineering tool DNA cleavage is based on DNA pattern Change require only in the first 20 nucleotides of the grna Possibility of targeting multiple DNA sequences at once Much more easier to target DNA sequence

32 Some limitations: off-target Off-target: tolerance of Cas9 to mismatches in the RNA guide sequence. Limited by PAM motif

33 Variants of Cas9 Only one strand of DNA will be cut 15

34 CAS9-BASED APPLICATIONS IN MICROBIOLOGY Genetic Engineering programmed the human pathogen Streptococcus pneumoniae with CRISPR sequences that target capsule genes. CRISPR interference can prevent transformation of non encapsulated, avirulent pneumococci into capsulated, virulent strains during infection in mice.

35 CAS9-BASED APPLICATIONS IN MICROBIOLOGY This approach has been successfully applied for genetic manipulation in numerous bacterial species, including E. coli, Streptococcus pneumoniae, Lactobacillus, Clostridium and Streptomyces coelicolor as well as bacteriophages.

36 Sequence-Specific Antimicrobials Cas9 chromosomal cleavage leads to toxicity to cell. This property is used for the development of sequence specific antimicrobials by RNA guided nuclease. In contrast to conventional antibiotics, the Cas9 antimicrobial could be programmed with a guide RNA matching a specific virulence gene sequence to kill the bacterial pathogen harboring this gene but spare the rest of the microbiota.

37 Phagemids : (pdb114) Phages can be used to deliver cas9-containing plasmids harboring CRISPR RNA sequences that direct Cas9 to unique chromosomal loci to achieve sequence-specific killing. (Staphylococcus aureus) Only target cells with the cognate sequence are killed, whereas nontarget cells survive the phagemid treatment.

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39 Journal club Target grnas were designed against the enhanced green fluorescent protein (EGFP) coding region

40 Use of the CRISPR/Cas9 system as an intracellular defense against HIV-1 infection in human cells two possible mechanisms for the Cas9-mediated inactivation of viral gene expression. First, Cas9 could directly target the viral genomic DNA, when the virus enters the cell and reverse transcribes into dsdna before integration into the host genome. Second, Cas9 could disrupt the proviral elements already integrated in the host genome.

41 Anti-CRISPR mechanisms The most basic way for viruses to escape CRISPR-Cas activity is by random mutagenesis that affects key bases in the interaction with the crrna or the PAM recognition

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