Glossary

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Base

The four “letters” of the genetic code (A, C, T, and G) are chemical groups called bases or nucleobases. A = adenine, C = cytosine, T = thymine, and G = guanine. Instead of thymine, RNA contains a based called uracil (U).

Base pair

Different chemicals known as bases or nucleobases are found on each strand of DNA. Each base has a chemical attraction for a particular partner base, known as its complement. C matches up with G, while A pairs with T or U. These bonded genetic letters are called base pairs. Two strands of DNA can zip together to form a double-helix shape when complementary bases match up to form base pairs.

Cas

Abbreviation of CRISPR-associated, may refer to genes (cas) or proteins (Cas) that protect bacteria and archaea from viral infection.

Cas9

A protein derived from the CRISPR-Cas bacterial immune system that has been co-opted for genome engineering. Uses an RNA molecule as a guide to find a complementary DNA sequence. Once the target DNA is identified, Cas9 cuts both strands. Has been compared to “molecular scissors” or a “genetic scalpel.” In CRISPR immunity, cutting viral DNA prevents it from destroying the host cell. In genome engineering, cutting genomic DNA initiates a repair process that ends up making a change or “edit” to its sequence.

Cell

The basic unit of life. The number of cells in a living organism ranges from one (e.g. yeast) to quadrillions (e.g. blue whale). A cell is composed of four key macromolecules that allow it to function (protein, lipids, carbohydrates, and nucleic acids). Among other things, cells can build and break down molecules, move, grow, divide, and die.

Cleave

The scientific term for cut or break apart. Typically refers to splitting apart a long polymeric molecule like DNA, RNA, or protein. For example, a nuclease like Cas9 can be directed to cleave DNA at a specific location.

Complementary

Describes any two DNA or RNA sequences that can form a series of base pairs with each other. Each base forms a bond with a complementary partner. T (DNA) and U (RNA) bond with A, and C complements G. For example, in CRISPR immunity, the spacer sequence in a guide RNA is complementary to a sequence found in a viral genome. When the RNA bases pair with complementary DNA bases from an invading virus, the Cas9 protein will cut the target to stop the viral infection.

CRISPR

Pronounced “crisper.” An adaptive immune system found in bacteria and archaea, co-opted as a genome-engineering tool. Acronym of “clustered regularly interspaced short palindromic repeats,” which refers to a section of the host genome containing alternating repetitive sequences and unique snippets of foreign DNA. CRISPR-associated surveillance proteins use these unique sequences as molecular mugshots as they seek out and destroy viral DNA to protect the cell.

crRNA

Abbreviation of CRISPR RNA. In CRISPR immunity, the host cell generates crRNA molecules, each containing one spacer that is complementary to a portion of a viral genome. crRNAs guide CRISPR immune proteins to find and destroy matching invader sequences.

dCas9

Catalytically inactive, or “dead,” Cas9. This mutated version of the Cas9 protein cannot cut, but still binds tightly to a particular DNA sequence specified by the guide RNA. Can be used to physically block the process of transcription, turning off a specific gene, or to shuttle other proteins to a particular site in the genome.

DNA

Abbreviation of deoxyribonucleic acid, a long molecule that encodes the information needed for a cell to function or a virus to replicate. Forms a double-helix shape that resembles a twisted ladder. Different chemicals called bases, abbreviated as A, C, T, and G, are found on each side of the ladder, or strand. The bases have an attraction for each other, making A stick to T while C sticks to G. These rungs of the ladder are called base pairs. The sequence of these letters is called the genetic code.

Double-strand break (DSB)

When both strands of DNA are broken, two free ends are created. May be made intentionally by a tool such as Cas9. Cells repair their DNA to prevent cell death, sometimes changing the DNA sequence at the site of the break. Initiating or controlling this process with the intent to alter a DNA sequence is known as genome engineering.

Enzyme

A molecule, typically a protein, that causes or catalyzes a chemical change. Usually an enzyme’s name describes a molecule involved in the activity it performs and ends with the suffix -ase. For example, lactase is a well-known enzyme that breaks down lactose, a sugar found in milk. Cas9 is a nuclease, an enzyme that breaks apart the backbone of nucleic acids (RNA or DNA).

Gene

A segment of DNA that encodes the information used to make a protein. Each gene is a set of instructions for making a particular molecular machine that helps a cell, organism, or virus function.

Gene editing / genome engineering

Intentionally altering the genetic code of a living organism. Can be done with ZFNs, TALENs, or CRISPR. These systems are used to create a double-strand break at a specific DNA site. When the cell repairs the break, the sequence is changed. Can be used to remove, change, or add DNA.

Genome

The entire DNA sequence of an organism or virus. The genome is essentially a huge set of instructions for making individual parts of a cell and directing how everything should run.

Guide RNA (gRNA)

A two-piece molecule that Cas9 binds and uses to identify a complementary DNA sequence. Composed of the CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA). Cas9 uses the tracrRNA portion of the guide as a handle, while the crRNA spacer sequence directs the complex to a matching DNA sequence.

Scientists have also formed a version of the guide RNA that consists of a single molecule, the single-guide RNA (sgRNA).

Homology-directed repair (HDR)

A way for a cell to repair a break in its DNA by “patching” it with a piece of donor DNA. The donor DNA must contain similar sequences, or homology, to the broken DNA ends for it to be incorporated. HDR is a more precise repair pathway than non-homologous end joining. In genome engineering, a researcher designs and adds in the donor DNA, potentially allowing scientists to replace a disease-causing gene with a healthy copy.

Indel

Abbreviation for insertion or deletion. Refers to the random removal or addition of nucleotides from a DNA sequence. This can be enough to stop a gene from functioning (imagine removing a page from the middle of an instruction manual). Indels occur when DNA is broken and “sloppily” repaired by the cell in a process called non-homologous end joining (NHEJ).

Microbe / microorganism

A microscopic organism. Can be single-celled or multicellular, and is sometimes used to refer to viruses, although they are not considered to be alive. Examples include bacteria, yeast, and algae.

Mutation

A change from one genetic letter (nucleotide) to another. Variation in DNA sequence gives rise to the incredible diversity of species in the world, and even occurs between different organisms of the same species. While some mutations have no consequence at all, certain mutations can directly cause disease. Mutations may be caused by DNA-damaging agents such as UV light or may arise from errors that occur when DNA is copied by cellular enzymes. They can also be made deliberately via genome engineering methods.

Nick

When only one strand of DNA is broken, there is a gap called a nick in the backbone, but the DNA does not separate. A tool like CRISPR-Cas9 may be used to generate a nick.

Non-homologous end joining (NHEJ)

A way for a cell to repair a break in its DNA by attaching the free DNA ends. This pathway is “sloppier” than homology-directed repair, and often results in the random addition or removal of nucleotides around the site of the DNA break, causing insertions or deletions in the genetic code. In genome engineering, this allows scientists to stop a gene from working (similar to removing a page from the middle of an instruction manual).

 

Nuclease

An enzyme that breaks apart the backbone of RNA or DNA. Breaking one strand generates a nick and breaking both strands generates a double-strand break. Genome engineering tools like Cas9 are nucleases.

Nucleic acid

A term for DNA and RNA. Refers to nucleotides, the basic chemical units that are strung together to make DNA or RNA. One of the four macromolecules that make up all living things (protein, lipids, carbohydrates, and nucleic acids).

Nucleotide

One of the basic chemical units strung together to make DNA or RNA. Consists of a base, a sugar, and a phosphate group. The phosphates can link with sugars to form a string called the DNA/RNA backbone, while the bases can bind to their complementary partners to form base pairs.

Off-target effects

When a genome engineering enzyme cuts DNA at an unintended site that is similar to the intended target, it is referred to as an off-target effect.

PAM

Abbreviation of protospacer adjacent motif, a short sequence that must be present next to a DNA target sequence for Cas9 to bind and cut. Prevents cleavage of host CRISPR array, where PAM is not present.

Protein

A string of amino acids folded into a three-dimensional structure. Proteins are each specialized to perform a specific role to help cells grow, divide, and function. One of the four macromolecules that make up all living things (protein, lipids, carbohydrates, and nucleic acids).

RNA

Abbreviation of ribonucleic acid. Transcribed from a DNA template and typically used to direct the synthesis of proteins. CRISPR-associated proteins use RNAs as guides to find matching target sequences in DNA.

Single-guide RNA (sgRNA)

A version of the naturally occurring two-piece guide RNA complex engineered into a single, continuous sequence. The simplified single-guide RNA is used to direct the Cas9 protein to bind and cleave a particular DNA sequence for genome editing.

Strand

A string of connected nucleotides; can be DNA or RNA. Two strands of DNA can zip together when complementary, bases match up to form base pairs. DNA typically exists in this double-stranded form, which takes the shape of a twisted ladder or double helix. RNA is typically composed of just a single strand, though it can fold up into complex shapes.

tracrRNA

Abbreviation for trans-activating CRISPR RNA, pronounced “tracer RNA.” In the CRISPR-Cas9 system, the tracrRNA base pairs with the crRNA to form a functional guide RNA (gRNA). Cas9 uses the tracrRNA portion of the guide as a handle, while the crRNA spacer sequence directs the complex to a matching viral sequence.

Transcription

The process by which DNA information is copied into a strand of RNA; performed by an enzyme called RNA polymerase.

Transcription activator-like effector nuclease (TALEN)

A genetic engineering tool wherein one portion of the protein recognizes a specific DNA sequence and another part cuts DNA. Made by attaching a series of smaller DNA-binding domains together to recognize a longer DNA sequence. This DNA binding domain is fused to a nuclease that will cut nearby DNA. Like CRISPR-Cas9 and ZFNs, it can be used to alter DNA sequences.

Translation

The process by which proteins are made based on instructions encoded in an RNA molecule. Performed by a molecular machine called the ribosome, which links together a series of amino acid building blocks. The resulting polypeptide chain folds up into a particular 3D shape, known as a protein.

Virus

An infectious entity that can only persist by hijacking a host organism to replicate itself. Has its own genome, but is technically not considered a living organism. Viruses infect all organisms, from humans to plants to microbes. A virus that infects bacteria is called a bacteriophage. Multicellular organisms have sophisticated immune systems that combat viruses, while CRISPR systems evolved to stop viral infection in bacteria and archaea.

Zinc-finger nuclease (ZFN)

A genetic engineering tool wherein one portion of the protein recognizes a specific DNA sequence and another part cuts DNA. Made by attaching a series of smaller DNA-binding domains together to recognize a longer DNA sequence. This DNA binding domain is fused to a nuclease that will cut nearby DNA. Like CRISPR-Cas9 and TALENs, it can be used to alter DNA sequences.