CRISPR and Targeted Genome Editing (CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats) Dr Nemat Sokhandan Bashir Associate Professor University of Tabriz

Current Circumstance Lots of sequencing data are available Relating of these data to phenotype is a major challenge Reverse genetics Forward genetics

Protein-based Approaches Protein directed Recombinases Integrases ZFNs (zinc finger nucleases) TALENs (transcription-activator-like effector nucleases)

Shortcomes of Protein-based Approaches Harder to customize Recombinases and integrases require suitable preexisting recognition sites in the genome -often have some inherent application limitations Difficult and expensive to customize ZFs or TALs by protein engineering ZFN and TALEN activities are affected by many factors ZFN and TALEN difficult for multiple mutations in a single genome

Nucleotide-based Approaches RNA interference (RNAi), group II intron retrotransposition, widely applied to inactivate genes in bacteria Cas9-based platforms (CRISPR)

RNAi Need for long target sites Amplification of small RNAi -severe off-target effects Repress gene expression instead of knocking them out

History of CRISPR First noticed in bacterial genomes (1987) Japanese examining E. coli iap gene Could not attach biological significance to such “unusual structure[s]” Nearly a decade later, a group from Spain recognized that these repeating sequences were a common feature in microbial genomes.

characteristic of CRISPR A series of palindromic DNA repeats (20-50 bp) separated by spacer sequences of ~ 20-50 Precise function (2005): spacer sequences were often consistent with nt sequences found in phages suggesting a role in bacterial innate immunity

characteristic of CRISPR 2007: resistance to a phage could be altered by modifying bacterial spacer DNA Spacers between palindromic repeats correspond to phage DNA sequences Spacers as “memory” of prior infection “memory” alone is not enough to protect bacteria from subsequent exposure to phage

exposure to a previously-exposed phage -> targeted DNA-cutting function Triggers transcription of the corresponding spacer sequence into a short guide CRISPR-RNA (crRNA) The CRISPR-associated (“Cas”) enzyme (Cas-9) is then guided to the target phage by a two-part RNA structure bears a short region of homology to the original phage DNA. Cas-9 identify and cleave the phage DNA, creating a dsDNA cut at a specific site.

Classification of CRISPR/Cas system On sequence and structure of Cas protein Types I, II, and III crRNA-guided surveillance complexes in types I and III need multiple Cas subunits Type II requires only Cas9

CRISPR/Cas type II Studied in Streptococcus and Neisseria Distinct in each species pre-crRNA transcript features pre-crRNA processing nucleoprotein complexes A promising programmable tool three crucial components: RNA-guided Cas9 nuclease crRNA (CRISPR RNA) a partially complementary trans-acting crRNA (tracrRNA)

crRNA biogenesis pre-crRNA processing and length varies in species Streptococcus pyogenes produces only one form of full-length primary pre-crRNA of 511 nt, consisting a leader region and a number of repeat-spacer-repeat units

tracrRNA a non-protein-coding RNA for crRNA maturation and subsequent DNA cleavage In S. pyogenes, the tracrRNA gene is transcribed from two start sites producing two primary species of 171 nt and 89 nt, both are processed into75-nt RNA species tracrRNA precursors have almost perfect (one mismatch) complementarity with each of the pre-crRNA repeats. Base-pairing RNA duplex is important for tracrRNA precursor trimming and crRNA maturation

CRISPR/Cas-based method highly flexible and programmable All of the essential components can be expressed by delivering Plasmids linear DNA expression cassettes RNA transcripts most genes or exons can be targeted specifically in Arabidopsis, rice and yeast ((N21GG))

Genomic target can be any ∼20 nucleotide DNA, if: Sequence is unique compared to the rest of the genome. Target is present immediately upstream of a Protospacer Adjacent Motif (PAM).

Cas9 nuclease (formerly Csn1 or Csx12) Cleaves dsDNA (sequence-specific) RuvC-like nuclease domain at N-terminus Named for an E. coli DNA repair protein HNH (or McrA-like) nuclease domain in the middle Named for histidine and asparagine residues Each cuts opposite DNA strand to give DSBs

Generating a Knock-out Using CRISPR/Cas9 S. pyogenes Cas9 most widely used Once expressed, Cas9 protein and gRNA form a riboprotein complex through: Interactions between the gRNA “scaffold” domain and surface-exposed positively-charged grooves on Cas9 Cas9 undergoes a conformational change Shifts from an inactive, non-DNA binding conformation, into an active DNA-binding conformation “spacer” sequence of the gRNA remains free to interact with target DNA

DSB is 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

NHEJ repair The most active repair mechanism of DSBs But frequently gives small nt InDels at DSB site This randomness has practical implications: because a population of cells expressing Cas9 and a gRNA will result in a diverse array of mutations

NHEJ repair Mostly results in small InDels in the target DNA in-frame amino acid deletions, insertions, or frameshift mutations leading to premature stop codons within the open reading frame (ORF) of the targeted gene. Ideally, the end result is a loss-of-function mutation within the targeted gene;

Increase specificity More specific gRNA with no off-targets dual nickase approach

dual nickase approach for more specificity Cas9 generates DSBs by combined activity of RuvC and HNH. Exact AA residues within each domain critical for endonuclease activity are known D10A for HNH and H840A for RuvC in S. pyogenes Cas9 modified versions of the Cas9 enzyme containing only one active catalytic domain (“Cas9 nickase”)

Cas9 nickases Still bind DNA based on gRNA specificity, Cuts only one strand Nicks are rapidly repaired by HDR Two nickases targeting opposite strands are required Unlikely that two off-target nicks will be generated within close enough proximity to cause a DSB. The nickase system can also be combined with HDR-mediated gene editing for highly specific gene edits.

Increasing efficiency of HDR HDR efficiency is generally low (<10% of modified alleles) synchronizing the cells within the cell cycle stage when HDR is most active, or by chemically or genetically inhibiting genes involved in NHEJ

implications of low efficiency of HDR efficiency of Cas9 cleavage is relatively high efficiency of HDR is relatively low, a portion of the Cas9-induced DSBs will be repaired via NHEJ. In other words, resulting cell population will contain: combination of wild-type alleles, NHEJ-repaired alleles, and/or the desired HDR-edited allele

Applications

Know your cell line and genome sequence Select gene and genetic element to be manipulated Select gRNAs based on predicted “on-target” and “off-target” activity Synthesize and clone desired gRNAs Deliver Cas9 and gRNA Validate genetic modification

An Example of Genome Editing Assay

a chromosomal deletion by targeting two adjacent sequences

Targeting virus

Pooled Lentivirus CRISPR libraries

CRISPR/Cas9 and Plant Viruses Plant viruses can deliver CRISPR/Cas9 components Manipulation and improvement of R gene Rx of potato is a fast HR- a potential use

Purification of genomic region by Chromatin immunoprecipitation

Cas9-based Forward Genetic Screen

Fungi to produce diverse enzymes or metabolites Trichoderma reesei, Aspergillus niger, A. oryzae, Penicillium chrysogenum,

To obtain hyper-producers classical mutagenesis Genetic engineering well developed These are not efficient due to: additional complexity such as multicellular morphology, cellular differentiation, thick chitinous cell walls, and the lack of suitable plasmids

Prospects and Challenges Discover new gene functions with high sensitivity and precision 1000s of guides target all coding genes in genome genome-scale gain- and loss of function genetic screens Identify novel disease-protective mutations such as loss-of-function mutations in CCR5 protection to HIV Direct treatment of genetic diseases via genome editing of somatic cells

Treatment of eye and hearing disorders actively evaluated in animal models Many groups striving to make CRISPR/Cas9-based therapies a reality Raises certain societal challenges and brings a sense of uncertainty and fear of catastrophic misuse One thing is certain—nature will never cease to inspire us with its biological toolbox The tools themselves do not pose a threat Lets hope CRISPR/Cas9 technology will live up to its promise by being used responsibly and carefully

References CRISPR Guide, Addgene,https://www.addgene.org/crispr/guide/. Accessed 7 Decebmer 2015. Xu,T., Li,L., D. Van Nostrand,J., He,Z., Zhou, J. 2014. Cas9-Based Tools for Targeted Genome Editing and Transcriptional Control. Applied and Environmental Microbiology, 80(5):1544–1552. Peng, C., Lu, M., Yang, D. 2015. CRISPR/Cas9-based tools for targeted genome editing and replication control of HBV. VIROLOGICA SINICA 2015, 30 (5): 317-325 Feng, Z. CRISPR/Cas9: Prospects and Challenges. HUMAN GENE THERAPY, 26(&): 409-410. Belhaj, K., Chaparro-Garcia, A., Kamoun, S., Nekrasov, V. 2013. Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system. Belhaj et al. Plant Methods, 9:39.