This pdf is about the gene knockin (KI) technique. For more details visit on YouTube; @SELF-EXPLANATORY; Gene Knockin: https://youtu.be/wuq6vwI9hag Thanks...!

1. Saba Parvin Haque
MSc in Neuroscience
from “Sophia College For
Women”, Mumbai. https://www.youtube.com/chann
el/UCAiarMZDNhe1A3Rnpr_WkzA

2. knockins

3. What Is a Knockin
and Why Is It Used?
https://d2jx2rerrg6sh3.cloudfront.net/image-handler/ts/20180110063242/ri/673/picture/2018/1/Gene_knockin_-_full.jpg
❑Gene knockin (KI) is the process of targeted insertion of
an exogenous gene at a specific locus in the genome.
❑It is used to modify the function of an endogenous gene in
order to study the detailed mutations of small nucleotide
in human disease.
https://images.contentstack.io/v3/assets/blte41c17d7f8dda379/blt90f7bb1e02791f40/5d37a1b9ffcdbf4f57a77623/CRISPR_Zebrafish_blog.jpg?format=webply&quality=90&width=560

4. Step 1: Know your gene.
General Procedure of Knockin
Step 2: Design sgRNAs.
Step 3: Validate sgRNAs.
Step 4: Design donor DNA
Source: ( Biswas & Enzmann 2021, & Gaj et al. 2017)
https://invivobiosystems.com/wp-content/uploads/2019/11/CRISPR-knock-in-technique.png

5. Step 5: Embryo injections.
Step 6: Rear your F0 zebrafish.
Step 7: Identify F0 founders.
Step 8: Sequence the transmitted edit.
Step 9: Rear your F1 heterozygotes.
Step 10: Finclip to identify F1
heterozygotes.
Step 11: Incross F1 heterozygotes to
find F2 homozygous fish.
https://invivobiosystems.com/view-from-the-bench/11-steps-to-a-successful-crispr-knock-in/

6. • It allows replacement of the gene-of-interest with mutated allele to
mimic a human disease.
• Site of integration is well known.
• This technique is more compatible with an inducible system that
allows the study of mutation effects that closely resemble the cause
of human disease.
• It allows study of the effects of genes with altered functions.
• The study of functional protein can be carried out in-depth.
Benefits of the gene knockin
method

7. • Development of complexity occurs due to the combination of
transgenes that lead to many side effects.
• The forced overexpression of the genes produces artifacts that
lead to unspecific protein–protein interaction.
• The knockin mouse generated does not entirely resemble the
pathology of human disease.
• Expensive method.
Limitations

8. • Nowogrodzki, A. (2019, March 4). The Challenge of Using CRISPR to Knock In Genes. The Scientist Magazine®. https://www.the-scientist.com/lab-
tools/jacking-up-gene-knock-ins-65504
• News Medical. (2019, February 26). Gene Knockin. News-Medical.Net. https://www.news-medical.net/life-sciences/Gene-Knockin.aspx
• I. (2020, October 22). Knock-In Mice Protocol. Ingenious Targeting Laboratory. https://www.genetargeting.com/knockin/knock-in-mice-protocol/
• Sharon, A. (n.d.). CRISPR Cas9 - Gene Knockout/Knock-in Case Studies. Abm Inc. https://info.abmgood.com/crispr-cas9-case-studies
• Nowogrodzki, A. (2019b, March 4). The Challenge of Using CRISPR to Knock In Genes. The Scientist Magazine®. https://www.the-scientist.com/lab-
tools/jacking-up-gene-knock-ins-65504
• ROCHA-MARTINS, M., CAVALHEIRO, G. R., MATOS-RODRIGUES, G. E., & MARTINS, R. A. (2015). From Gene Targeting to Genome Editing:
Transgenic animals applications and beyond. Anais Da Academia Brasileira de Ciências, 87(2 suppl), 1323–1348. https://doi.org/10.1590/0001-
3765201520140710
• Efficient gene knockins in iPS cells using long ssDNA templates for homology-directed repair. (n.d.). Y. https://www.takarabio.com/learning-
centers/gene-function/gene-editing/crispr/cas9-knockins/efficient-crispr/cas9-mediated-knockins-in-ips-cells
• I. (2021, May 4). Difference Between Knock In And Knockout. Ingenious Targeting Laboratory. https://www.genetargeting.com/knockin/difference-
between-knock-in-and-knockout/
• Hopkins, C. (2020, December 17). The Challenge of Getting CRISPR-based Knock-ins to Work in Zebrafish. InVivo Biosystems.
https://invivobiosystems.com/view-from-the-bench/the-challenge-of-getting-crispr-based-knock-ins-to-work-in-zebrafish/
• Thomas Gaj, Brett T. Staahl, Gonçalo M. C. Rodrigues, Prajit Limsirichai, Freja K. Ekman, Jennifer A. Doudna, David V. Schaffer, Targeted gene
knock-in by homology-directed genome editing using Cas9 ribonucleoprotein and AAV donor delivery, Nucleic Acids Research, Volume 45, Issue 11,
20 June 2017, Page e98, https://doi.org/10.1093/nar/gkx154
REFERENCES

9. Thank You