BCM6225 Genome Editing

Genome editing

This is a type of genetic engineering in which DNA is inserted, replaced or removed using artificially engineering nucleases (molecular scissors)

double-strand breaks (DSBs)

Nucleases create specific ____ at specific positions and harness the cell's endogenous mechanisms to repair the induced break by natural processes (homologous directed repair/recombination).

Homologous recombination (HR)

A repair pathway for double-strand breaks involving the undamaged sister chromatid, which provides a template for high-fidelity repair; low efficiency but is still the preferred mechanism.

Non-homologous end joining (NHEJ)

A repair pathway for repairing double-strand breaks by joining nonhomologous DNA ends in a process that does not conserve the original sequence; high efficiency and prone to mutations.

Meganucleases

These nucleases are present in many microorganisms and plants; have large recognition sites which give them high specificity. They are involved in modifying recognition sequences.

Less toxicity and most specific naturally-occurring REs.

True.

Meganucleases are not widely used for genome editing because it's expensive.

T or F?

Zinc finger nucleases

These nucleases are DNA-binding domains; combination of different ZFNs and FokI (catalytic domains) engineered heterodimers.

Only has limited sites in the genome which can be targeted; is complex and high cost of protein domain construction.

Off-target mutagenesis (1:10)

3bp

1 zinc finger is to how many bp?

FokI

What restriction enzyme in Zinc Finger Nucleases is non-specific and induces dimerization?

Transcription Activator-Like Effector Nucleases (TALEN)

These nucleases are derived from Xanthomonas sp, a crop plant pathogen; mimicks eukaryotic transcription factors secreted into plants.

monomers

DNA binding domain of TALEN consists of ______ with each of them binding 1 nt in the target sequence.

34 amino acids

1 TALEN monomer has tandem repeats of how many amino acids?

2 of them (pos. 12 & 13) are highly variable = repeat variable diresidue (RVD); degenerate.

Nuclear Localizing Signal (NLS)

This TALEN feature looks for target sequences.

ZFN

Sequence encoding the DNA-binding domain from TALEN was cloned into plasmid vector previously used for creating?

Chimeric endonucleases

_____ containing DNA binding domain and catalytic domain of restriction endonuclease FokI.

artificial nucleases

This system allows ones, by combining monomers of the DNA-binding domain with different RVDs, to construct _______, the target of which can be any nucleotide sequence.

1. site selection may be made in most cases by varying the spacer sequence length

2. selection of mutation variants of the TALEN N-terminal domain that are capable of binding A, G, or C

2 limitations of TALEN:

Clustered Regularly Interspaced Short Palindromic Repeats/Cas Proteins (CRISPR/Cas)

This is a unique mechanism providing microorganism protection against foreign DNA penetration & acting along with the restriction modification system as a limiter of the horizontal gene transfer.

1. guide RNA
2. Cas9 (CRISPR-associated 9)
a. PAM (Protospacer Adjacent Motif)

2 essential components of CRISPR/Cas:

Francisco Mojica

A Spanish microbiologist, known for his research on the CRISPR gene editing technique and coining the name CRISPR.

True.

"spacer" DNA is homologous with phage DNA and plasmids.

T or F?

S. thermophilus

This bacteria has a sequence complementary with phage DNA that is resistant to phage infection.

87% in archaea; 48% in eubacteria

CRISPR/Cas is present in how many percent in Archaea? Eubacteria?

1-18 number of repeats

How many number of repeats are in CRISP/Cas?

23-37bp on average

What is the size of repeats in CRISPR/Cas?

17-84 bp

What is the number and size of spacers in CRISPR/Cas?

constant

In 1 CRISPR cassette, the length of repeats and spacers is ____ and the repeat sequence is almost identical.

1. adaptation (recognizes first the foreign DNA; creates spacer)
2. transcription (fast protein synthesis)
3. interference (targets DNA for degradation/cleavage)

CRISPR/Cas protection mechanism has 3 stages namely:

spacer

In the adaption stage of CRISPR/Cas, it involves the insertion of a small fragment of foreign DNA that entered a bacterial cell into the CRISPR locus of the host genome, forming a new ______.

PAM (protospacer adjacent motif)

[CRISPR/Cas]

In the viral genome, this fragment is present as a protospacer that is complementary to the spacer and flanked by a short (2-5 bp) sequence called?

This binds to 5'-NGG-3'.

AT-rich side

[CRISPR/Cas]

The new spacer is always inserted on the __-rich side of the leader sequence located before a CRISPR cassette that also contains promoter elements and landing sites for regulatory proteins.

This way, the targets of most of the CRISPR/Cas systems are formed.

pre-crRNA (polyspacer precursor crRNA)

In the transcription stage of CRISPR/Cas, the entire CRISPR locus is transcribed into a long?

This has a sequence that is 5' complementary to the UTR of the target RNA.

Cas6 endonuclease

In the transcription stage of CRISPR/Cas, the processing immature pre-crRNA to mature is performed by this enzyme which results in short crRNA.

short crRNA

This is a 39-45nt sequence which contains 1 spacer sequence and repeats involved in stem-loop creation.

last 8 nts = 5'-end;

loop = 3'-end

The last 8 nts of the short crRNA's repeats make stem at the __'-end and the hairpin structure forms the loop at the __'-end.

crRNA and Cas proteins: crRNA recognized complementarily the protospacer sequence and Cas proteins provide its degradation

The interference of foreign DNA or RNA is provided by the interaction between?

Guide RNA

A part of the CRISPR/Cas system that attaches to a complementary sequence of DNA and determines where the DNA is cut; a combination of crRNA and tracer RNA

Cas9

An RNA-guided DNA endonuclease enzyme that is extracted from Streptococcus pyogenes consist of 1368 aa.

1. REC lobe
2. NUC lobe

What are the 2 domains of Cas9?

REC lobe

This Cas9 domain is the DNA-binding domain/recognition lobe.

NUC lobe

This Cas9 domain is the nuclease lobe; composed RUV C (cleaves non-complementary strands) and HNH (cleaves complementary strands; introduces double strand breaks).

Class I

This major class of Cas9 consists of Type I, III, and IV.

Class II

This major class of Cas9 consists of Type II, V, and VI.

Type I

This type of CRISPR/Cas has Cas3 and a cascade responsible for CRISPR-associated complex antiviral defense.

long pre-crRNA

In CRISPR/Cas Type I, CRISPR is transcribed as long ____.

Cas9

In CRISPR/Cas Type I, ____ (part of cascade) cleaves the pre-crRNA.

DNA

In CRISPR/Cas Type I, crRNA+cascade complex binds to?

Cas3

In CRISPR/Cas Type I, ___ is recruited and cleaves DNA and degrades it with 3'-->5' processivity.

Type II

This type of CRISPR/Cas is the simplest; consists of RNA-guided endonuclease Cas9 and tracrRNA.

1. recognition
2. cleavage
3. repair

3 steps of CRISPR/Cas9:

tracrRNA

In CRISPR/Cas Type II, CRISPR loci produce a small trans-encoded crRNA, also known as _____, with a region of complementarity to repeat sequences.

Cas9

In CRISPR/Cas Type II, ______ binds to tracrRNA which anneals to the repeat sequences of the pre-crRNA.

RNAse III

In CRISPR/Cas Type II, the dsDNA structure formed by tracrRNA + precrRNA with the aid of Cas9 is cleaved by?

crRNA and tracrRNA

In CRISPR/Cas Type II, Cas9 loaded with (2) is generated.

This binds to PAM and creates an R-loop tat triggers cleavage by Cas9.

tracrRNA

In CRISPR/Cas Type II, this acts as a cofactor of Cas9 and is required for DNA cleavage.

Type III

This CRISPR/Cas type consists of Cas10, RAMP (repeat associated mysterious protein) Csm or Cmr, and Cas6.

Cas6

In CRISPR/Cas Type III, there is a precursor processing catalyzed by _____.

3'-end

In CRISPR/Cas Type III, there is crRNA maturation wherein crRNAs are trimmed at which end?

True.

In CRISPR/Cas Type III, there is cotranscriptional destruction of target DNA and its transcripts.

T or F?

1. transcription across the target req for immunity
2. both RNA and DNA targets are cleaved
3. no PAM requirements

Why is CRISPR/Cas Type III unique?

1. selection of target nt sequence in the genome
2. generation of a nuclease construct directed at the selected target
3. delivery of this construct to the cell nucleus (microinjections, vectors)
4. analysis of produced mutants

4 stages of genomic engineering using CRISPR/Cas: