GENE EDITING
Random mutagenesis – introducing random changes in DNA in vivo. This can
be done
physical, biological or chemical. Ionizing radiation (UV, X-ray, etc.) causes
double stranded breaks which are incorrectly repaired. Viruses, bacteria
and transposons can insert DNA. Chemical methods often lead to
base(pair) transitions.
Cre-Lox recombination – targeted DNA editing. Cre recombinase recognizes
specific flox
sites in the target DNA and excises the DNA in between these sites. High
efficiency and specificity, but requires pre-introduced LoxP sites. Often
used in conditional gene knockouts, lineage tracing and transgenic
models.
TALENs – targeted gene editing. Engineered proteins consisting of a DNA-
binding domain
(TALE repeats) fused to a FokI nuclease that introduces double stranded
breaks at specific DNA sequences. Two amino-acids in the TALE repeat
(called RVD) correspond to one specific base. High specificity and
efficiency, but labor-intensive because of the complex protein assembly.
Results in relatively large complexes and works with protein-DNA
interactions. Used for gene knockouts, genome editing in plants and
animals and therapeutic research.
ZFNs – targeted gene editing. Zinc fingers recognize a specific 3 base sequence.
They are
fused with an endonuclease, usually FokI, which creates double stranded
breaks at the target site. It has a high specificity but can lead to off-target
effects and is difficult and expensive due the complexity. It was previously
used in research and gene therapy but is nowadays not really used
anymore.
CRISPR-Cas – targeted gene editing. Guide RNA, which is complementary to the
target
sequence, leads the Cas9 protein to the site. This then cuts the DNA and
leaves it, or adds in a donor sequence. This is the most popular gene
editing method, it is cheap, easy and highly specific. It is often used for
gene knockouts, knock-ins, epigenetic modifications, base editing and
gene therapy.
Meganucleases – targeted gene editing. Highly specific endonucleases that
recognize and
cut long DNA sequences (14-40 bp). They can be dimeric or monomeric
and they have a DNA binding site, an endonuclease site and assisting
metal ions. An advantage is their high specificity and small size, a
disadvantage is that they are difficult to use for new targets. It is used for
precise modification of the genome, creating GMOs and gene editing.
CLONING
Gibson seamless assembly – uses a mixture of T5 exonuclease, DNA
polymerase and DNA
ligase to join two DNA fragments with overlapping sequences. It is highly
efficient and does not need restriction enzymes. It does require designing
overlapping primers and PCR. It is used for large constructs, multiple
fragment assembly and cloning complex genes.
Gateway recombinational cloning – uses site-specific recombination (at ATT
sites)
, mediated by LR/BP clonase enzymes to transfer DNA between vectors.
This is very efficient for multi-step cloning, but is it limited to vectors
containing ATT sites and it leaves recombinational scars. It is used for
high-throughput cloning, gene expression studies and functional
genomics.
In-fusion seamless cloning – uses a 3’exonuclease (T4 DNA polymerase) to
generate single-
stranded overhangs, allowing seamless assembly of DNA fragments with
short homologous ends. This works best for single fragment cloning and no
restriction sites are required. It is used for seamless cloning, site-directed
mutagenesis and cloning into specific vectors.
Golden Gate cloning – uses type IIS restriction enzymes that cut outside their
recognition
site, allowing directional assembly of multiple fragments in one step. The
efficiency is very high, especially for modular cloning, but it does require
compatible restriction sites. It is used for high-throughput modular cloning
and assembly of multiple genes or pathways.
Segregation analysis – used to study how alleles segregate during the
formation of
gametes and how they are inherited from parents to offspring. It focusses
on analyzing the inheritance patterns of specific traits (usually controlled
by single genes).
DNA SEQUENCING
Plus-minus sequencing – an outdated method that uses DNA polymerase to
extend a
primer in the presence or absence of a specific dNTP. It has a low
efficiency because it can only sequence short fragments, is prone to errors
and is labor intensive. It is not used anymore.
Sanger sequencing – uses chain termination by incorporating fluorescent or
radiolabeled
ddNTPs during DNA polymerization. It can sequence up to 1000 bp per
reaction and is very accurate. It is mostly used for validation of variants
and sequencing of single genes.
Shotgun sequencing – randomly fragments the genome, sequences the
overlapping pieces
and assembles them computationally. This has a high efficiency and
accuracy, but requires a lot of computational power. It is used for whole-
genome sequencing, metagenomics and de novo genome assembly.
Maxam-Gilbert sequencing – uses chemical reactions to cleave DNA at
specific bases,
followed by gel electrophoresis to determine sequence. It is unsafe,
because of the chemical reactions and thus is not used anymore.
454 pyrosequencing – uses pyrophosphate release during DNA synthesis to
generate light
signals for nucleotide detection. This can generate long reads, but
struggles with homopolymer repeats. It has a high accuracy for short
reads. It is not really used anymore.
4-color sequencing (SOLiD) – uses ligation-based sequencing where
fluorescently labeled
probes interrogate each base twice. It has a high efficiency because it
allows parallel sequencing and a high accuracy. This does struggle with
homopolymer regions because of signal saturation and fluorescence
overlap. This can be solved by incorporating reversible terminator
Random mutagenesis – introducing random changes in DNA in vivo. This can
be done
physical, biological or chemical. Ionizing radiation (UV, X-ray, etc.) causes
double stranded breaks which are incorrectly repaired. Viruses, bacteria
and transposons can insert DNA. Chemical methods often lead to
base(pair) transitions.
Cre-Lox recombination – targeted DNA editing. Cre recombinase recognizes
specific flox
sites in the target DNA and excises the DNA in between these sites. High
efficiency and specificity, but requires pre-introduced LoxP sites. Often
used in conditional gene knockouts, lineage tracing and transgenic
models.
TALENs – targeted gene editing. Engineered proteins consisting of a DNA-
binding domain
(TALE repeats) fused to a FokI nuclease that introduces double stranded
breaks at specific DNA sequences. Two amino-acids in the TALE repeat
(called RVD) correspond to one specific base. High specificity and
efficiency, but labor-intensive because of the complex protein assembly.
Results in relatively large complexes and works with protein-DNA
interactions. Used for gene knockouts, genome editing in plants and
animals and therapeutic research.
ZFNs – targeted gene editing. Zinc fingers recognize a specific 3 base sequence.
They are
fused with an endonuclease, usually FokI, which creates double stranded
breaks at the target site. It has a high specificity but can lead to off-target
effects and is difficult and expensive due the complexity. It was previously
used in research and gene therapy but is nowadays not really used
anymore.
CRISPR-Cas – targeted gene editing. Guide RNA, which is complementary to the
target
sequence, leads the Cas9 protein to the site. This then cuts the DNA and
leaves it, or adds in a donor sequence. This is the most popular gene
editing method, it is cheap, easy and highly specific. It is often used for
gene knockouts, knock-ins, epigenetic modifications, base editing and
gene therapy.
Meganucleases – targeted gene editing. Highly specific endonucleases that
recognize and
cut long DNA sequences (14-40 bp). They can be dimeric or monomeric
and they have a DNA binding site, an endonuclease site and assisting
metal ions. An advantage is their high specificity and small size, a
disadvantage is that they are difficult to use for new targets. It is used for
precise modification of the genome, creating GMOs and gene editing.
CLONING
Gibson seamless assembly – uses a mixture of T5 exonuclease, DNA
polymerase and DNA
ligase to join two DNA fragments with overlapping sequences. It is highly
efficient and does not need restriction enzymes. It does require designing
overlapping primers and PCR. It is used for large constructs, multiple
fragment assembly and cloning complex genes.
Gateway recombinational cloning – uses site-specific recombination (at ATT
sites)
, mediated by LR/BP clonase enzymes to transfer DNA between vectors.
This is very efficient for multi-step cloning, but is it limited to vectors
containing ATT sites and it leaves recombinational scars. It is used for
high-throughput cloning, gene expression studies and functional
genomics.
In-fusion seamless cloning – uses a 3’exonuclease (T4 DNA polymerase) to
generate single-
stranded overhangs, allowing seamless assembly of DNA fragments with
short homologous ends. This works best for single fragment cloning and no
restriction sites are required. It is used for seamless cloning, site-directed
mutagenesis and cloning into specific vectors.
Golden Gate cloning – uses type IIS restriction enzymes that cut outside their
recognition
site, allowing directional assembly of multiple fragments in one step. The
efficiency is very high, especially for modular cloning, but it does require
compatible restriction sites. It is used for high-throughput modular cloning
and assembly of multiple genes or pathways.
Segregation analysis – used to study how alleles segregate during the
formation of
gametes and how they are inherited from parents to offspring. It focusses
on analyzing the inheritance patterns of specific traits (usually controlled
by single genes).
DNA SEQUENCING
Plus-minus sequencing – an outdated method that uses DNA polymerase to
extend a
primer in the presence or absence of a specific dNTP. It has a low
efficiency because it can only sequence short fragments, is prone to errors
and is labor intensive. It is not used anymore.
Sanger sequencing – uses chain termination by incorporating fluorescent or
radiolabeled
ddNTPs during DNA polymerization. It can sequence up to 1000 bp per
reaction and is very accurate. It is mostly used for validation of variants
and sequencing of single genes.
Shotgun sequencing – randomly fragments the genome, sequences the
overlapping pieces
and assembles them computationally. This has a high efficiency and
accuracy, but requires a lot of computational power. It is used for whole-
genome sequencing, metagenomics and de novo genome assembly.
Maxam-Gilbert sequencing – uses chemical reactions to cleave DNA at
specific bases,
followed by gel electrophoresis to determine sequence. It is unsafe,
because of the chemical reactions and thus is not used anymore.
454 pyrosequencing – uses pyrophosphate release during DNA synthesis to
generate light
signals for nucleotide detection. This can generate long reads, but
struggles with homopolymer repeats. It has a high accuracy for short
reads. It is not really used anymore.
4-color sequencing (SOLiD) – uses ligation-based sequencing where
fluorescently labeled
probes interrogate each base twice. It has a high efficiency because it
allows parallel sequencing and a high accuracy. This does struggle with
homopolymer regions because of signal saturation and fluorescence
overlap. This can be solved by incorporating reversible terminator
