CRISPR Mechanisms and Applications Quiz

Genome Editing by CRISPR-Cas

• Staphylococcus aureus Cas9 complex (SaCas9) crystal structure is shown
• 3D printed model of the Cas9 complex is also displayed
• Source: Innovative Genomics Institute

Arabidopsis CBF4 Gene Function Study

• Arabidopsis CBF4 gene encodes a transcription factor in the AP2/ERF family
• Designing sgRNA to target CBF4 in Arabidopsis is a part of the project
• Cloning sgRNA in CRISPR expression vector using Golden Gate cloning is required
• Transforming E. coli, isolating the plasmid and transforming Agrobacterium tumefaciens is part of the process
• Transforming Arabidopsis plants for stable expression
• Genotyping and phenotyping of cbf4-crispr mutants are needed (weeks 8-9)

CBF4-YFP Fusion Protein Generation

• Generating CBF4-YFP fusion protein by Gateway recombination cloning technology
• Transforming LR reaction into E. coli, colony PCR, plasmid isolation, and sequencing (Week 10) is required
• Transforming plasmid into Agrobacterium and infiltrating plant leaves (N. bentamiana) for transient expression
• Localizing protein through confocal microscopy (Week 11) is part of the process

CRISPR-Cas System Details

• CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA or trRNA) are components
• CRISPR-Cas9 used as in molecular biology tools
• Cas9 D10A variant has only nickase activity
• dCas9 variant lacks nuclease activity
• Applications include genome editing and genome targeting

Nobel Prize in Chemistry 2020

• Emmanuelle Charpentier and Jennifer Doudna jointly awarded for the development of a method for genome editing

CRISPR/Cas Function

• CRISPR (Cluster Regularly Interspaced Short Palindromic Repeats) and Cas genes essential for adaptive immunity in bacteria and archaea
• Enable these organisms to respond to and eliminate invading genetic material
• Foreign DNA incorporated into bacterial genome at CRISPR loci
• CRISPR loci transcribed into crRNA during crRNA biogenesis
• Cas9 endonuclease complexed with crRNA and tracrRNA cleaves foreign DNA with complementary sequence adjacent to PAM sequence

Type II CRISPR Mechanism

• One Cas protein (Cas9) required for gene silencing
• Three CRISPR mechanisms identified, type II most studied

Acquisition Phase

• Invading DNA, from viruses or plasmids, cut into small fragments, incorporated into CRISPR locus amidst repeats (around 20 bps) in bacteria genome

Interference Phase

• CRISPR loci transcribed and processed to generate small RNAs (e.g., CRISPR RNA or crRNA)
• crRNA guide Cas9 endonuclease, which cleaves invading DNA by sequence complementarity
• Cas9 must be complexed with crRNA and tracrRNA (or trRNA) to recognize and cleave DNA. tracrRNA partially complementary to crRNA
• Cas9 function depends on nuclease domains (RuvC-like and HNH-like) and PAM-interacting domain

Protospacer Associated Motif (PAM)

• Cas9 requires a short conserved sequence (NGG) to function as a protospacer-associated motif (PAM)
• PAM follows the crRNA sequence immediately
• Cas9-RNA ignores fully complementary sequences without the PAM sequence
• HNH and RuvC-like nuclease domains cut both DNA strands during cleavage, generating double-stranded breaks (DSBs) at defined sites in the target

CRISPR-Cas9 as Molecular Biology Tools

• The simplicity of the type II CRISPR nuclease, with only three components (Cas9, crRNA, and tracrRNA), allows for adaptation to genome editing
• Doudna and Charpentier labs simplified system, combining trRNA and crRNA into synthetic single guide RNA (sgRNA)
• sgRNA-programmed Cas9 effective in guiding targeted gene alterations
• Three Cas9 variants are used in genome editing protocols

Wild-Type Cas9

• Cleaves double-stranded DNA (dsDNA) site-specifically
• Cellular Non-Homologous End Joining (NHEJ) pathway repairs DSBs, leading to insertions and/or deletions (indels) impacting target gene
• Homology-directed repair (HDR) pathway allows precise replacement mutations if a donor DNA template is provided

Cas9 D10A Variant

• Mutant form (Cas9 D10A) has only nickase activity
• Cleaves only one DNA strand and does not activate Non-homologous end joining (NHEJ)
• High-fidelity homology-directed repair (HDR) pathway leads to reduced indel mutations

dCas9 Variant

• Nuclease-deficient (dCas9) variant lacking HNH and RuvC domains cannot cleave DNA; however, prevents DNA binding
• Used for gene silencing or activation, as well as visualization tools by fusing with effector domains (e.g., GFP)

Targeting Efficiency and Off-Target Mutations

• Cas9 system high efficiency (up to >70%) in zebrafish and plants
• Off-target mutations occur in sites that have slight nucleotide differences compared to original sequence, often adjacent to PAM sequence
• Difficult to detect off-target mutations; whole-genome sequencing required

Applications of CRISPR/Cas9

• Target important genes in various organisms (human, bacteria, zebrafish, etc.)
• Introducing single-point mutations, deletions, or insertions using single or multiple gRNAs
• Enabling rapid genome-wide interrogation of gene function through gRNA libraries

Week 6-9 Labs: Generating a cbf4-CRISPR Vector

• Designing single guide RNAs (gRNAs) targeting CBF4
• Cloning sgRNA in a CRISPR vector suitable for plant expression, containing Cas9
• Transforming bacteria with the vector, isolating plasmid, and transforming Arabidopsis plants
• Confirming deletion in CBF4 gene in cbf4-crispr mutants

Bioinformatics Lab 4

• Designing sgRNA using Chopchop
• Designing primers containing sgRNA and restriction sites

sgRNA Design Principles

• CRISPR/Cas9 targeting requires a custom sgRNA with a targeting sequence (crRNA) and Cas9 nuclease-recruiting sequence (tracrRNA)
• Targeting sequence (crRNA) is a 20-nucleotide sequence homologous to the region of interest
• Targeting sequence directs Cas9 nuclease activity

Guidelines for sgRNA design

• Identify PAM (NGG) sequence in target DNA region
• Determine 5' start of the 20 nucleotide sgRNA targeting sequence based on PAM (NGG)
• Determine the actual sgRNA targeting sequence
• Identify the actual sgRNA targeting sequence that does not include the PAM sequence

Cloning gRNA: CRISPR Vector & Expression Cassettes

• Cas9-sgRNA complex binds to DNA target and triggers double-strand break (DSB) when next to short PAM (NGG)
• U6 promoter (U6-prom) of RNA Pol III directs high sgRNA expression in the nucleus; repeated components (gRNA scaffold, and terminators) present
• Cas9 plant codon-optimized expressed using egg cell-specific promoter (EC1.2p) and terminated by rbcS-E9 terminator

Cloning 2 gRNA in plant expression vectors

• Selecting two gRNAs to create cuts in the target CBF4 gene
• Designing sgRNA primers for gRNAs (e.g., CBF4-sg11_For and CBF4-sg1_Rev)
• Amplifying a CRISPR cassette that includes the two gRNAs from pCBC-DT1T2 vector
• Cloning PCR product into a plant expression vector such as pHEE401E
• pCBC-DT1T2 plasmid serves as a template

Primer Design

• Primer CBF4-sg11_For and CBF4-sg1_Rev for cbf4 gRNAs
• Include overhang with Bsal sites (Bsal restriction sites) in the primers
• sgRNA sequences exclude the PAM sequence

Final CRISPR Plant Expression Vector

• PCR product cloned into a CRISPR-plant expression vector
• gRNA expression controlled by U6 RNA Pol III promoters and terminators
• Cas9 expression driven by egg cell-specific promoter and terminated
• Final cbf4-CRISPR vector resistant to Kanamycin but not Spectinomycin.