CRISPR Technology and Gene Therapy

CRISPR-Cas

Clustered Regulatory Interspaced Palindromic Repeats and CRISPR-associated proteins

Adaptive Immunity System in Prokaryotes

• CRISPR-Cas evolved in bacteria to protect bacterial genomes from invading DNAs (ex. from viruses)

• CRISPR-Cas allows bacteria to recognizes and “remember” DNA from specific pathogens

Bacterial Immunity

1. Bacteriophage infects bacteria and injects viral DNA

2. Cas complex cleaves DNA

3. Inserts spacer DNA into Type II CRISPR locus

Record the outlaws into crime roster: copy a region of viral DNA known as Spacer into bacteria genome

CRISPR-Cas System Working Towards Immunity

1. Bacteriophage infects bacteria and injects viral DNA

2. RNase III and TracrRNA form complex with Cas9 and Pre-CRISPR RNA (crRNA-tracrRNA-Cas9 complex)

3. DNA targeting if PAM is present 

4. Viral DNA cleavage

Fights suspects by the roster: the integrated viral DNA can be transcribed into RNA to base pairing with future viral DNA that allows Cas nuclease to degrade viral DNA

Adaptation (Acquisition)

The Cas1-Cas2 complex selects protospacers next to a PAM in invading DNA and integrates the protospacer - excluding the PAM - at the leader end of the host CRISPR array

Expression

CRISPR array is transcribed into a long pre-crRNA that is processed by other Cas proteins. CRISPR RNA (crRNA) then partially base-pairing with tracrRNA and then join Cas protein to form effector complex

Interference

complementary base-pairing occurs between crRNAs and viral DNA and Cas protein cleaves the viral DNA

PAM

Protospacer adjacent motif (5’-NGG-3’ for Cas9) must be present in the target site

Should NOT be present on sgRNA

tracrRNA and crRNA in Prokaryotes

In prokaryotes, tracrRNA is transcribed from a gene located near the CRISPR array

Cas9 programmed by crRNA:tracrRNA duplex

• crRNA: DNA target specific sequence

• tracrRNA: constant (invariant) sequence

sgRNA in the Lab

Cas9 programmed by a single chimeric RNA (aka single guide RNA, sgRNA)

Jennifer Doudna’s lab discovered that a sgRNA can replace the tracrRNA+crRNA

CRISPR-Cas9 is a system for precisely editing a genome

• Recombinant Cas9 protein can be expressed in eukaryotic cells from an expression vector

• Synthetic sgRNA can be made to match a target sequence in any genome and is introduced into cells along with Cas9 protein

• Target DNA must have a PAM sequence located adjacent to where complementary base pairing occurs between sgRNA and target RNA 

• Effector complex associates with PAM sequence and unwinds nearby DNA

• A portion of the sgRNA binds to the target sequence → double strand breaks in target DNA 

Cas9

DNA endonuclease

Target DNA requirement

(5’ - 3’): Protospacer + PAM (NGG) sequence

sgRNA

single guide RNA: spacer sequence (20 nt, target specific) + scaffold sequence (76 nt, universal)

20 nt Protospacer

4²⁰ = 1.1 trillion combinations. Human genome contains 6.4 billion bp. Easy to find unique cut site within the genome

Two Mechanisms for Repair of Double Strand Breaks

Nonhomologous End-Joining (NHEJ)

Homology-Directed Repair (HDR)

Nonhomologous End-Joining

NHEJ: error-prone DNA repair pathway for DSBs creates indels at random

Homology-directed repair

HDR: DNA repair pathway for DSBs that uses another DNA molecule to repace damaged DNA by homologous recombination

Precise Edit of DSBs

Repair template has desired edit/mutation (missense sub, nonsense mutation, splice site mutation, etc.) 

Both are naturally-occuring mechanisms for DNA repair in eukaryotes

When tying to find crRNA sequences, you must…

1. Find PAM sequence NGG (5’ to 3’, N is any nucleotide) on both strands

2. Find the seed sequence: 20 nt adjacent to 5’ end of PAM

3. For cRNA: change all T in the seed sequence to U

Engineered CRISPR-Cas9 can be applied for genome editing of any species example

Edited pigementation gene tyrosinase (try) into lizards?

Treat Human Genetic Disease by CRISPR-Correction of Cultured iPSC Cells

In somatic cells (does not modifiy germline)

• Extraction of skin cells containing mutation within genome

• Form patient-specific iPSCs and use genome editing to repair disease causing mutation

• Allow in vitro differentiation of the iPSCs to the desired healthy cell

• Transplantion of genetically matched healthy cells back into the patient 

Duchenne Muscular Dystrophy (DMD)

• ~1 : 5,000 boys

• ~300,000 boys worldwide

• Affect both heart and skeletal muscles

Symptomes: walking problems, loss of ambulation, limited use of arms, eventually requiring ventilation and death

X chromosome linked gene: recessive mutation causes DMD, mostly boys

Mutations of Dystrophin gene causes DMD

2.4 megabases (0.08% of the human genome) is the Dystrophin gene

Absorbs energy during muscle contraction to protect myofiber tear

DMD gene is the largest gene in human genome

Common Mutations in DMD patients

Exon deletions or duplications in exons: 

• 6, 7, 8

• 43-46

• 50-53 and 55

Recombinant DNA technology

Set of techniques for locating, isolating, altering, and studying DNA segments

• Term recombinant is used because frequently the goal is to combine DNA from 2 distinct sources

• Also commonly called genetic engineering

Key reagents to generate recombinant DNA: restriction enzymes

Restriction Enzymes

Recognize a specific sequence of bases anywhere within the DNA

• Endonuclease cuts phosphodiester bonds of both strands

• Restriction fragments are generated by digestion of DNA with restriction enzymes

• Hundreds of restriction enzymes are now available, and each has a unique recognition site

Recognition Sites

Usually 4-8 bp of dsDNA

• Often palindromic - base sequences of each strand are identical when read 5’ - 3’

• Each enzyme cuts at the same place relative to its specific recognition sequence

Cohesive Ends

aka “sticky ends”

• 5’ or 3’ overhangs

Blunt Ends

Direct cut with no overhangs