Question

1) What is CRISPR? For what is it used? How does it work? What are some problems with using CRISPR in people? is this correct CRISPR stands for clustered regularly interspaced short palindromic repeats. CRISPR can be used to change DNA sequences and modify genes. Scientists can use CRISPR to turn genes off or replace genes that are causing diseases or change genes for a specific trait. It works by targeting a certain section of DNA. An enzyme cuts the DNA strand that is targeted or defective. The targeted strand can either be replaced with a different gene or the gene can be turned off or made nonfunctional. What are some problems with using CRISPR in people?

2) What is GenBank? Why might you need to use it?

3) In which cases is it better to use Sanger sequencing over second-generation sequencing? In which cases is it better to use second-generation sequencing over sanger sequencing?

Answer 1

1) CRISPR is an abbreviation of Clustered Regularly Interspaced Short Palindromic Repeats.. CRISPR are segments of DNA containing short, repetitive base sequences in a palindromic repeat (the sequence of nucleotides is the same in both directions). Each repetition is followed by short segments of spacer DNA from previous integration of foreign DNA from a virus or plasmid. Small clusters of cas (CRISPR-associated) genes are located next to CRISPR sequences.

Mostly CRISPR is used for gene editing,i.e. to change DNA sequences and modify genes.

Scientists can use it in association with cas genes, for example, in the CRISPR/Cas system, CRISPR/Cas9, has been modified to edit genomes. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added.

Problems of using CRISPR are:

Sometimes there can be many off target mutations when CRISPR-Cas9 cleaves other DNA sequences within the genome that are homologous to the target DNA sequences. These mutations can be deleterious. Off-target mutations can cause cell death or transformation.

The cost of germline editing technology by CRISPR method is very high to the extent that families coming from rich countries could afford it. The developing countries will not be in a position to afford the cost of this technology. This may confer an advantage to children born in developed countries.

Genome editing in human embryos using CRISPR-Cas9 could have unpredictable effects to the future generations. CRISPR-Cas9 technology could be used for non-therapeutic modifications . This procedure will open the door to the loss of human diversity and eugenics .

The nuclease may not be as efficient. The nuclease may not necessarily cleave both copies of the target gene or the cells may start dividing before the corrections are completed, resulting in genetic mosaic .

2) The GenBank sequence database is an open access, annotated collection of all publicly available nucleotide sequences and their protein translations. This database is produced and maintained by the National Center for Biotechnology Information (NCBI) as part of the International Nucleotide Sequence Database Collaboration (INSDC).

It is used by scientists to access all the known nucleotide sequences.

3) Sanger sequencing is used for

• Sequencing single genes
• Sequencing 1-100 amplicon targets at the lowest cost
• Sequencing up to 96 samples at a time without barcoding
• Microbial Identification
• Fragment analysis, high throughput genotyping using, for example, SNaPshot
• Microsatellite or STR analysis
• NGS confirmation

Next generation sequencing offers fast turnaround time and takes only about 4 hours to complete a run. NGS is used for:

• Interrogating >100 genes at a time cost effectively
• Finding novel variants by expanding the number of targets sequenced in a single run.
• Sequencing samples that have low input amounts of starting material, using, for example, Ion AmpliSeq library preparation, which requires as little as 10 ng of input DNA
• Sequencing microbial genomes for pathogen subtyping to enable research of critical outbreak situations