This document discusses CRISPR/Cas9 genome engineering. It explains that CRISPR systems consist of repeats and spacers that capture viral sequences. Type II systems like Cas9 use both CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA) or a single chimeric RNA to cleave DNA at sites next to protospacer adjacent motifs (PAM). Double-strand breaks can be repaired by error-prone nonhomologous end joining or precise homology-directed repair with a repair template. The document also mentions using CRISPR nickase with two gRNAs, Cas9, and a DNA construct to reduce off-target effects and generate gene knock
This document discusses CRISPR/Cas9 genome engineering. It explains that CRISPR systems consist of repeats and spacers that capture viral sequences. Type II systems like Cas9 use both CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA) or a single chimeric RNA to cleave DNA at sites next to protospacer adjacent motifs (PAM). Double-strand breaks can be repaired by error-prone nonhomologous end joining or precise homology-directed repair with a repair template. The document also mentions using CRISPR nickase with two gRNAs, Cas9, and a DNA construct to reduce off-target effects and generate gene knock
This document discusses CRISPR/Cas9 genome engineering. It explains that CRISPR systems consist of repeats and spacers that capture viral sequences. Type II systems like Cas9 use both CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA) or a single chimeric RNA to cleave DNA at sites next to protospacer adjacent motifs (PAM). Double-strand breaks can be repaired by error-prone nonhomologous end joining or precise homology-directed repair with a repair template. The document also mentions using CRISPR nickase with two gRNAs, Cas9, and a DNA construct to reduce off-target effects and generate gene knock
This document discusses CRISPR/Cas9 genome engineering. It explains that CRISPR systems consist of repeats and spacers that capture viral sequences. Type II systems like Cas9 use both CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA) or a single chimeric RNA to cleave DNA at sites next to protospacer adjacent motifs (PAM). Double-strand breaks can be repaired by error-prone nonhomologous end joining or precise homology-directed repair with a repair template. The document also mentions using CRISPR nickase with two gRNAs, Cas9, and a DNA construct to reduce off-target effects and generate gene knock
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CRISPR/Cas9
CDK9, CDK12 and CDK13 knockout
CRISPR
Type I Type II Type III
Consist of several noncontiguous direct repeats (same sequence) separated by spacers of variable sequences (complementary to segments of captured viral and plasmid sequences).
** CRISPR (clustered regularly interspaced short palindromic repeats)
After infection of the bacteria:
Genome engineering
**The PAM (protospacer adjacent motifs) sequence is required
to immediately follow the target DNA locus, but that its not a part of the 20-nt guide sequence within the sgRNA.
Type II systems rely on a single protein, Cas9, for the cleavage
step.Cas9 requires both the crRNA and the tracrRNA to function (or chimeric sgRNA) and cleaves DNA using its domains: - HNH nuclease domain: cleaves the complementary strand. - RuvC-like domain: cleaves the non-complementary strand.
Both domains produce double-strand breaks, and separately they
can produce single-strand breaks (nickase)
DNA damage repair:
1 Nonhomologous end-joining (NHEJ) mechanism, which
is error prone and introduces frameshift indels,
resulting in a knockout inactivation of the gene. 2 High-fidelity HDR (homology-directed repair), which can
generate precise, defined modifications at a target
locus in the presence of an exogenously introduced repair template.
