#Cas9

Gene Tar getting

Which mutation type is created by NHEJ repair after Cas9 cut?

Cas9 induced double strand break activates cellular repair pathways. Homology directed repair uses donor template for precise correction but operates mainly in S phase. Predominant pathway in mammalian somatic cells is non-homologous end joining which directly ligates broken ends without template, often introducing small errors. Ku proteins and DNA ligase IV mediated NHEJ results in insertion or deletion of few nucleotides at cut site, causing frameshift or premature stop codon. This error-prone repair underlies knockout generation by CRISPR through disruptive indels.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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The Cas9-based CRISPR mechanism in type II system uses:

Type II CRISPR system of Streptococcus pyogenes minimal interference module requires trans-activating crRNA tracrRNA. Pre-crRNA containing repeats is annealed to tracrRNA forming duplex recognized by RNase III for processing into mature guide RNAs. TracrRNA also hybridizes to crRNA to form dual guide structure that activates Cas9 conformational change enabling PAM scanning and DNA cleavage. Unlike type I requiring Cas3 helicase nuclease or type V using Cas12 alone, type II canonical system depends on tracrRNA for biogenesis and Cas9 loading, later fused as single guide RNA.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Cas9 endonuclease cuts the DNA and creates:

Cas9 is dual nuclease utilizing HNH domain to cut target strand complementary to guide RNA and RuvC domain to cut non-target strand simultaneously. Both nicks occur three base pairs upstream of PAM on opposite strands, generating predominantly blunt ended double strand breaks without single stranded overhangs. Such blunt breaks are predominantly repaired by non-homologous end joining causing small indels, or by homology directed repair if donor template provided. Blunt nature facilitates efficient ligation mediated insertion and predictable editing outcomes in genome engineering laboratories worldwide.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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In CRISPR-Cas9, what sequence is essential for Cas9 to recognize the target?

Streptococcus pyogenes Cas9 requires a protospacer adjacent motif PAM downstream of target for binding and cleavage. The canonical PAM is 5'-NGG-3' on non-target strand. Guide RNA pairs with 20 nucleotides upstream of PAM, but Cas9 first scans DNA for PAM, triggering DNA unwinding and R-loop formation. Without PAM, even perfect guide complementarity fails to induce double strand break. PAM ensures self versus non-self discrimination since bacterial CRISPR array lacks PAM. Engineering higher fidelity Cas9 variants often alters PAM recognition profiles.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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