Question

can someone give an easy and simple explantion of all the cas proteins involved in the steps (adaption, biogenesis, interference) of bacterial immunity by crispr? i know that the cas9 protein cuts the DNA, but what are all the other proteins involved before that and what do they do? please make it easy to understand. thank you.

Answer 1

CAS 9 is an enzyme that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence. Cas9 enzymes together with CRISPR sequences form the basis of a technology known as CRISPR-Cas9 that can be used to edit genes within organisms. This editing process has a wide variety of applications including basic biological research, development of biotechnology products, and treatment of diseases

The CRISPR-Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages that provides a form of acquired immunity. RNA harboring the spacer sequence helps Cas (CRISPR-associated) proteins recognize and cut foreign pathogenic DNA. Other RNA-guided Cas proteins cut foreign RNA. CRISPR are found in approximately 50% of sequenced bacterial genomes and nearly 90% of sequenced archaea bacteria

Acquisition

When a microbe is invaded by a bacteriophage, the first stage of the immune response is to capture phage DNA and insert it into a CRISPR locus in the form of a spacer. Cas1 and Cas2 are found in both types of CRISPR-Cas immune systems, which indicates that they are involved in spacer acquisition. Mutation studies confirmed this hypothesis, showing that removal of cas1 or cas2 stopped spacer acquisition, without affecting CRISPR immune response

Biogenesis

CRISPR-RNA (crRNA),later guides the Cas nuclease to the target during the interference step, must be generated from the CRISPR sequence. The crRNA is initially transcribed as part of a single long transcript encompassing much of the CRISPR array. This transcript is then cleaved by Cas proteins to form crRNAs. The mechanism to produce crRNAs differs among CRISPR/Cas systems. In type I-E and type I-F systems, the proteins Cas6e and Cas6f respectively, recognise stem-loops[1 created by the pairing of identical repeats that flank the crRNA.These Cas proteins cleave the longer transcript at the edge of the paired region, leaving a single crRNA along with a small remnant of the paired repeat region.

Type III systems also use Cas6, however their repeats do not produce stem-loops. Cleavage instead occurs by the longer transcript wrapping around the Cas6 to allow cleavage just upstream of the repeat sequence.

Type II systems lack the Cas6 gene and instead utilize RNaseIII for cleavage. Functional type II systems encode an extra small RNA that is complementary to the repeat sequence, known as a trans-activating crRNA (tracrRNA).Transcription of the tracrRNA and the primary CRISPR transcript results in base pairing and the formation of dsRNA at the repeat sequence, which is subsequently targeted by RNaseIII to produce crRNAs. Unlike the other two systems the crRNA does not contain the full spacer, which is instead truncated at one end.

CrRNAs associate with Cas proteins to form ribonucleotide complexes that recognize foreign nucleic acids. CrRNAs show no preference between the coding and non-coding strands, which is indicative of an RNA-guided DNA-targeting system.The type I-E complex (commonly referred to as Cascade) requires five Cas proteins bound to a single crRNA.

Interference

During interference stage in type I systems the PAM sequence is recognized on the crRNA-complementary strand and is required along with crRNA annealing. In type I systems correct base pairing between the crRNA and the protospacer signals a conformational change in Cascade that recruits Cas3 for DNA degradation.

Type II systems rely on a single multifunctional protein, Cas9, for the interference step. Cas9 requires both the crRNA and the tracrRNA to function and cleaves DNA using its dual HNH and RuvC/RNaseH-like endonuclease domains. Basepairing between the PAM and the phage genome is required in type II systems. However, the PAM is recognized on the same strand as the crRNA (the opposite strand to type I systems).

Type III systems, like type I require six or seven Cas proteins binding to crRNAs.The type III systems analysed from S. solfataricus and P. furiosus both target the mRNA of phages rather than phage DNA genome, which may make these systems uniquely capable of targeting RNA-based phage genomes.Type III systems were also found to target DNA in addition to RNA using a different Cas protein in the complex, Cas10. The DNA cleavage was shown to be transcription dependent.

The mechanism for distinguishing self from foreign DNA during interference is built into the crRNAs and is therefore likely common to all three systems. Throughout the distinctive maturation process of each major type, all crRNAs contain a spacer sequence and some portion of the repeat at one or both ends. It is the partial repeat sequence that prevents the CRISPR-Cas system from targeting the chromosome as base pairing beyond the spacer sequence signals self and prevents DNA cleavage. RNA-guided CRISPR enzymes are classified as type V restriction enzymes.