However, while it is considered as one of the most precise gene-editing tools, CRISPR suffers problems of off-target effects that can affect non-targeted DNA sequences elsewhere in the genome. This is a big problem for the modification of human cells, and can result in the creation of doub...
such as in the case of inherited forms of cancer caused by BRCA1 and BRCA2 mutations [37]. The CRISPR-Cas9 system is highly specific in targeting genetic mutations due to the guide RNA's ability to recognize and bind to a particular DNA sequence [38]. However, undesirable site effects...
Base editors (BEs) are groundbreaking tools developed for precise nucleotide conversion, which is achieved by combining catalytically modified Cas proteins (such as Cas9-D10A nickases) with deaminases41. When a Cas protein and its gRNA recognize target DNA sequences, they form a single-stranded ...
Here we reconstitute CRISPR sequence capture, processing and integration by a naturally occurringMegasphaeraNM10-related Cas2 and DEDDh fusion protein (Cas2/DEDDh) in complex with Cas1 (Cas1–Cas2/DEDDh). We show that Cas1–Cas2/DEDDh preserves the PAM during prespacer processing and the f...
naturally manipulate the proliferative state of cells. Large libraries of cell lines have been established for researchers to study the effects of genes of interest on cell proliferation or survival. Such as Depmap (https://depmap.org/portal/), PICKLES (https://pickles.hart-lab.org), etc. [...
In 2016, Burstein et al.identified CasX and CasYfrom genome sequences of uncultivated bacteria from soil, water, biofilms, and other microbial communities. CasX and CasY are two of the most compact naturally occurring CRISPR variants and could have interesting applications as Cas9 substitutes in bio...
for the sgRNA targeting promoter sequences, gene silencing occurs regardless of whether the target is the template or nontemplate strand. In July 2013, another study by Qi et al. revealed that dCas9 interacts with effectors related to transcriptional regulation, such as VP64 and KRAB, to coreg...
AsCas12a [43,44]. Chimeric Cas proteins created by exchanging PAM-interacting domains between naturally occurring Cas orthologs have also been applied to expand PAM recognition. Substituting the loop sequence of Cas9 fromStreptococcus anginosus, together with the T1227K mutation, into the open ...
In addition, defending mechanisms in the form of naturally occurring host proteins can resist the CRISPR/Cas system. New opportunities for gene editing and bioengineering have emerged as a result of the discovery of anti-CRISPR proteins [189]. Researchers have utilized these proteins’ power to inc...
sequence recognition. The challenge however presents itself in engineering of the nucleases, which is only done by a number of laboratories. TALENs, as the follower of ZFNs, have a simpler design, where the designed sequences can recognize individual sequences by discrete nucleotide matching. This...